Search results: Max Planck Institute of Molecular Cell Biology and Genetics

Alexandra A Baumann^*, Lisanne I Knol^*, Marie Arlt, Tim Hutschenreiter, Anja Richter, Thomas Widmann, Marcus Franke, Karl Hackmann, Sylke Winkler, Daniela Richter, Isabel Spier, Stefan Aretz, Daniela Aust, Joseph Porrmann, Doreen William, Evelin Schröck, Hanno Glimm, Arne Jahn
Long-read genome and RNA sequencing resolve a pathogenic intronic germline LINE-1 insertion in APC.
NPJ Genom Med, 10(1) Art. No. 30 (2025)
Open Access DOI

Familial adenomatous polyposis (FAP) is caused by pathogenic germline variants in the tumor suppressor gene APC. Confirmation of diagnosis was not achieved by cancer gene panel and exome sequencing or custom array-CGH in a family with suspected FAP across five generations. Long-read genome sequencing (PacBio), short-read genome sequencing (Illumina), short-read RNA sequencing, and further validations were performed in different tissues of multiple family members. Long-read genome sequencing resolved a 6 kb full-length intronic insertion of a heterozygous LINE-1 element between exons 7 and 8 of APC that could be detected but not fully resolved by short-read genome sequencing. Targeted RNA analysis revealed aberrant splicing resulting in the formation of a pseudo-exon with a premature stop codon. The variant segregated with the phenotype in several family members allowing its evaluation as likely pathogenic. This study supports the utility of long-read DNA sequencing and complementary RNA approaches to tackle unsolved cases of hereditary disease.

Adrian Pascal Nievergelt
Genome editing in the green alga Chlamydomonas: past, present practice and future prospects.
Plant J, 122(1) Art. No. e70140 (2025)
Open Access DOI

The green alga Chlamydomonas is an important and versatile model organism for research topics ranging from photosynthesis and metabolism, cilia, and basal bodies to cellular communication and the cellular cycle and is of significant interest for green bioengineering processes. The genome in this unicellular green alga is contained in 17 haploid chromosomes and codes for 16 883 protein coding genes. Functional genomics, as well as biotechnological applications, rely on the ability to remove, add, and change these genes in a controlled and efficient manner. In this review, the history of gene editing in Chlamydomonas is put in the context of the wider developments in genetics to demonstrate how many of the key developments to engineer these algae follow the global trends and the availability of technology. Building on this background, an overview of the state of the art in Chlamydomonas engineering is given, focusing primarily on the practical aspects while giving examples of recent applications. Commonly encountered Chlamydomonas-specific challenges, recent developments, and community resources are presented, and finally, a comprehensive discussion on the emergence and evolution of CRISPR/Cas-based precision gene editing is given. An outline of possible future paths for gene editing based on current global trends in genetic engineering and tools for gene editing is presented.

Benjamin R Sabari^#, Anthony Hyman^#, Denes Hnisz
Functional specificity in biomolecular condensates revealed by genetic complementation.
Nat Rev Genet, 26(4) 279-290 (2025)
DOI

Biomolecular condensates are thought to create subcellular microenvironments that regulate specific biochemical activities. Extensive in vitro work has helped link condensate formation to a wide range of cellular processes, including gene expression, nuclear transport, signalling and stress responses. However, testing the relationship between condensate formation and function in cells is more challenging. In particular, the extent to which the cellular functions of condensates depend on the nature of the molecular interactions through which the condensates form is a major outstanding question. Here, we review results from recent genetic complementation experiments in cells, and highlight how genetic complementation provides important insights into cellular functions and functional specificity of biomolecular condensates. Combined with observations from human genetic disease, these experiments suggest that diverse condensate-promoting regions within cellular proteins confer different condensate compositions, biophysical properties and functions.

Ian Seim^#, Stephan W. Grill^#
Empirical methods that provide physical descriptions of dynamic cellular processes.
Biophys J, 124(6) 861-875 (2025)
Open Access DOI

We review empirical methods that can be used to provide physical descriptions of dynamic cellular processes during development and disease. Our focus will be nonspatial descriptions and the inference of underlying interaction networks including cell-state lineages, gene regulatory networks, and molecular interactions in living cells. Our overarching questions are: How much can we learn from just observing? To what degree is it possible to infer causal and/or precise mathematical relationships from observations? We restrict ourselves to data sets arising from only observations, or experiments in which minimal perturbations have taken place to facilitate observation of the systems as they naturally occur. We discuss analysis perspectives in order from those offering the least descriptive power but requiring the least assumptions such as statistical associations. We end with those that are most descriptive, but require stricter assumptions and more previous knowledge of the systems such as causal inference and dynamical systems approaches. We hope to provide and encourage the use of a wide array of options for quantitative cell biologists to learn as much as possible from their observations at all stages of understanding of their system of interest. Finally, we provide our own recipe of how to empirically determine quantitative relationships and growth laws from live-cell microscopy data, the resultant predictions of which can then be verified with perturbation experiments. We also include an extended supplement that describes further inference algorithms and theory for the interested reader.

Michela Milani, Francesco Starinieri, Anna Fabiano, Stefano Beretta, Tiziana Plati, Cesare Canepari, Mauro Biffi, Fabio Russo, Valeria Berno, Rossana Norata, Francesca Sanvito, Ivan Merelli, Luigi Aloia, Meritxell Huch, Luigi Naldini, Alessio Cantore
Identification of hepatocyte-primed cholangiocytes in the homeostatic liver by in vivo lentiviral gene transfer to mice and non-human primates.
Cell Rep, 44(3) Art. No. 115341 (2025)
Open Access DOI

Liver regeneration is supported by hepatocytes and, in certain conditions, biliary epithelial cells (BECs). BECs are facultative liver stem cells that form organoids in culture and engraft in damaged livers. However, BEC heterogeneity in the homeostatic liver remains to be fully elucidated. Here, we exploit systemic lentiviral vector (LV) administration to achieve efficient and lifelong gene transfer to BECs in mice. We find that LV-marked BECs retain organoid formation potential and predominantly respond to liver damage; however, they are less clonogenic and display a hepatocyte-primed transcriptome compared to untransduced BECs. We thus identify a BEC subset committed to hepatocyte lineage in the absence of liver damage, characterized by a transcriptional network orchestrated by hepatocyte nuclear factor 4α. We also report in vivo targeting of such BECs in non-human primates. This work highlights intrinsic BEC heterogeneity and that in vivo LV gene transfer to the liver may persist following BEC-mediated repair of hepatic damage.

Ariadna E Morales, Yue Dong, Thomas Brown, Kaushal Baid, Dimitrios-Georgios Kontopoulos, Victoria Gonzalez, Zixia Huang, Alexis-Walid Ahmed, Arkadeb Bhuinya, Leon Hilgers, Sylke Winkler, Graham M Hughes, Xiaomeng Li, Ping Lu, Yixin Yang, Bogdan Kirilenko, Paolo Devanna, Tanya M Lama, Yomiran Nissan, Martin Pippel, Liliana M Dávalos, Sonja Vernes, Sébastien J Puechmaille, Stephen J Rossiter, Yossi Yovel, Joseph B Prescott, Andreas Kurth, David A Ray, Burton K Lim, Eugene W Myers, Emma Teeling, Arinjay Banerjee, Aaron T Irving^#, Michael Hiller^#
Bat genomes illuminate adaptations to viral tolerance and disease resistance.
Nature, 638(8050) 449-458 (2025)
Open Access DOI

Zoonoses are infectious diseases transmitted from animals to humans. Bats have been suggested to harbour more zoonotic viruses than any other mammalian order1. Infections in bats are largely asymptomatic2,3, indicating limited tissue-damaging inflammation and immunopathology. To investigate the genomic basis of disease resistance, the Bat1K project generated reference-quality genomes of ten bat species, including potential viral reservoirs. Here we describe a systematic analysis covering 115 mammalian genomes that revealed that signatures of selection in immune genes are more prevalent in bats than in other mammalian orders. We found an excess of immune gene adaptations in the ancestral chiropteran branch and in many descending bat lineages, highlighting viral entry and detection factors, and regulators of antiviral and inflammatory responses. ISG15, which is an antiviral gene contributing to hyperinflammation during COVID-19 (refs. 4,5), exhibits key residue changes in rhinolophid and hipposiderid bats. Cellular infection experiments show species-specific antiviral differences and an essential role of protein conjugation in antiviral function of bat ISG15, separate from its role in secretion and inflammation in humans. Furthermore, in contrast to humans, ISG15 in most rhinolophid and hipposiderid bats has strong anti-SARS-CoV-2 activity. Our work reveals molecular mechanisms that contribute to viral tolerance and disease resistance in bats.

Yohan Kim^*, Minseok Kang^*, Michael Girma Mamo, Michael Adisasmita, Meritxell Huch, Dongho Choi
Liver organoids: Current advances and future applications for hepatology.
Clin Mol Hepatol, 31(Suppl) 327-348 (2025)
Open Access DOI

The creation of self-organizing liver organoids represents a significant, although modest, step toward addressing the ongoing organ shortage crisis in allogeneic liver transplantation. However, researchers have recognized that achieving a fully functional whole liver remains a distant goal, and the original ambition of organoid-based liver generation has been temporarily put on hold. Instead, liver organoids have revolutionized the field of hepatology, extending their influence into various domains of precision and molecular medicine. These 3D cultures, capable of replicating key features of human liver function and pathology, have opened new avenues for human-relevant disease modeling, CRISPR gene editing, and high-throughput drug screening that animal models cannot accomplish. Moreover, advancements in creating more complex systems have led to the development of multicellular assembloids, dynamic organoid-on-chip systems, and 3D bioprinting technologies. These innovations enable detailed modeling of liver microenvironments and complex tissue interactions. Progress in regenerative medicine and transplantation applications continues to evolve and strives to overcome the obstacles of biocompatibility and tumorigenecity. In this review, we examine the current state of liver organoid research by offering insights into where the field currently stands, and the pivotal developments that are shaping its future.

Srustidhar Das, S Martina Parigi, Xinxin Luo, Jennifer Fransson, Bianca C Kern, Ali Okhovat, Oscar E Diaz, Chiara Sorini, Paulo Czarnewski, Anna T Webb, Rodrigo A Morales, Sacha Lebon, Gustavo Monasterio, Francisca Castillo, Kumar P Tripathi, Ning He, Penelope Pelczar, Nicola Schaltenberg, Marjorie De la Fuente, Francisco López-Köstner, Susanne Nylén, Hjalte List Larsen, Raoul Kuiper, Per Antonson, Marcela A Hermoso, Samuel Huber, Moshe Biton, Sandra Scharaw, Jan-Åke Gustafsson, Pekka Katajisto, Eduardo J Villablanca
Liver X receptor unlinks intestinal regeneration and tumorigenesis.
Nature, 637(8048) 1198-1206 (2025)
Open Access DOI

Uncontrolled regeneration leads to neoplastic transformation1-3. The intestinal epithelium requires precise regulation during continuous homeostatic and damage-induced tissue renewal to prevent neoplastic transformation, suggesting that pathways unlinking tumour growth from regenerative processes must exist. Here, by mining RNA-sequencing datasets from two intestinal damage models4,5 and using pharmacological, transcriptomics and genetic tools, we identified liver X receptor (LXR) pathway activation as a tissue adaptation to damage that reciprocally regulates intestinal regeneration and tumorigenesis. Using single-cell RNA sequencing, intestinal organoids, and gain- and loss-of-function experiments, we demonstrate that LXR activation in intestinal epithelial cells induces amphiregulin (Areg), enhancing regenerative responses. This response is coordinated by the LXR-ligand-producing enzyme CYP27A1, which was upregulated in damaged intestinal crypt niches. Deletion of Cyp27a1 impaired intestinal regeneration, which was rescued by exogenous LXR agonists. Notably, in tumour models, Cyp27a1 deficiency led to increased tumour growth, whereas LXR activation elicited anti-tumour responses dependent on adaptive immunity. Consistently, human colorectal cancer specimens exhibited reduced levels of CYP27A1, LXR target genes, and B and CD8 T cell gene signatures. We therefore identify an epithelial adaptation mechanism to damage, whereby LXR functions as a rheostat, promoting tissue repair while limiting tumorigenesis.

Mitsuhiro Matsuda^#, Jorge Lázaro, Miki Ebisuya^#
Metabolic activities are selective modulators for individual segmentation clock processes.
Nat Commun, 16(1) Art. No. 845 (2025)
Open Access DOI

Numerous cellular and molecular processes during embryonic development prompt the fundamental question of how their tempos are coordinated and whether a common global modulator exists. While the segmentation clock tempo scales with the kinetics of gene expression and degradation processes of the core clock gene Hes7 across mammals, the coordination of these processes remains unclear. This study examines whether metabolic activities serve as a global modulator for the segmentation clock, finding them to be selective instead. Several metabolic inhibitions extend the clock period but affect key processes differently: glycolysis inhibition slows Hes7 protein degradation and production delay without altering intron delay, while electron transport chain inhibition extends intron delay without influencing the other processes. Combinations of distinct metabolic inhibitions exhibit synergistic effects. We propose that the scaled kinetics of segmentation clock processes across species may result from combined selective modulators shaped by evolutionary constraints, rather than a single global modulator.

A Sophie Brumm, Afshan McCarthy, Claudia Gerri, Todd Fallesen, Laura Woods, Riley McMahon, Athanasios Papathanasiou, Kay Elder, Phil Snell, Leila Christie, Patricia Garcia, Valerie Shaikly, Mohamed Taranissi, Paul Serhal, Rabi A Odia, Mina Vasilic, Anna Osnato, Peter J Rugg-Gunn, Ludovic Vallier, Caroline S Hill, Kathy K Niakan
Initiation and maintenance of the pluripotent epiblast in pre-implantation human development is independent of NODAL signaling.
Dev Cell, 60(2) 174-185 (2025)
Open Access DOI

The human blastocyst contains the pluripotent epiblast from which human embryonic stem cells (hESCs) can be derived. ACTIVIN/NODAL signaling maintains expression of the transcription factor NANOG and in vitro propagation of hESCs. It is unknown whether this reflects a functional requirement for epiblast development in human embryos. Here, we characterized NODAL signaling activity during pre-implantation human development. We showed that NANOG is an early molecular marker restricted to the nascent human pluripotent epiblast and was initiated prior to the onset of NODAL signaling. We further demonstrated that expression of pluripotency-associated transcription factors NANOG, SOX2, OCT4, and KLF17 were maintained in the epiblast in the absence of NODAL signaling activity. Genome-wide transcriptional analysis showed that NODAL signaling inhibition did not decrease NANOG transcription or impact the wider pluripotency-associated gene regulatory network. These data suggest differences in the signaling requirements regulating pluripotency in the pre-implantation human epiblast compared with existing hESC culture.

Wieland Huttner
Human-specific gene ARHGAP11B-potentially an additional tool in the treatment of neurodegenerative diseases?
Front Mol Med, 4 Art. No. 1465647 (2024)
Open Access DOI

Maarten P Bebelman^*, Lenka Belicova^*, Elzbieta Gralinska, Tobias Jumel, Aparajita Lahree, Sarah Sommer, Andrej Shevchenko, Timofei Zatsepin, Yannis Kalaidzidis, Martin Vingron, Marino Zerial
Hepatocyte differentiation requires anisotropic expansion of bile canaliculi.
Development, 151(22) Art. No. dev202777 (2024)
Open Access DOI

During liver development, bipotential progenitor cells called hepatoblasts differentiate into hepatocytes or cholangiocytes. Hepatocyte differentiation is uniquely associated with multi-axial polarity, enabling the anisotropic expansion of apical lumina between adjacent cells and formation of a three-dimensional network of bile canaliculi. Cholangiocytes, the cells forming the bile ducts, exhibit the vectorial polarity characteristic of epithelial cells. Whether cell polarization feeds back on the gene regulatory pathways governing hepatoblast differentiation is unknown. Here, we used primary mouse hepatoblasts to investigate the contribution of anisotropic apical expansion to hepatocyte differentiation. Silencing of the small GTPase Rab35 caused isotropic lumen expansion and formation of multicellular cysts with the vectorial polarity of cholangiocytes. Gene expression profiling revealed that these cells express reduced levels of hepatocyte markers and upregulate genes associated with cholangiocyte identity. Timecourse RNA sequencing demonstrated that loss of lumen anisotropy precedes these transcriptional changes. Independent alterations in apical lumen morphology induced either by modulation of the subapical actomyosin cortex or by increased intraluminal pressure caused similar transcriptional changes. These findings suggest that cell polarity and lumen morphogenesis feed back to hepatoblast-to-hepatocyte differentiation.

David Thomas Gonzales, Naresh Yandrapalli, Tom Robinson, Christoph Zechner^#, T-Y Dora Tang^#
Correction to "Cell-Free Gene Expression Dynamics in Synthetic Cell Populations".
ACS Synth Biol, 13(10) 3471-3484 (2024)
Open Access DOI

Bassam Aljani, Annette I Garbe, Eva-Maria Sedlmeier, Ramona Lickert, Fabian Rost, Anette-Gabriele Ziegler, Enzio Bonifacio, Anne Eugster
Gene expression profiles in placenta and their association with anesthesia, delivery mode and maternal diabetes.
Placenta, 158 126-135 (2024)
Open Access DOI

Fetal development is dependent on placenta and affected by multiple factors including maternal diabetes. Here we aimed to identify maternal diabetes-associated changes in placentas and analyzed placental gene expression to understand its modulation by maternal diabetes and birth mode.

Alice Spadea, Annalisa Tirella, Julio Manuel Rios de la Rosa, Enrique Lallana, Manal Mehibel, Brian Telfer, Nicola Tirelli, Margaret Jayne Lawrence, Kaye J Williams, Ian J Stratford, Marianne Ashford
Targeting Hypoxia-Inducible Factor-1α in Pancreatic Cancer: siRNA Delivery Using Hyaluronic Acid-Displaying Nanoparticles.
Pharmaceutics, 16(10) Art. No. 1286 (2024)
Open Access DOI

Background/Objectives: Conventional anticancer therapies often lack specificity, targeting both cancerous and normal cells, which reduces efficacy and leads to undesired off-target effects. An additional challenge is the presence of hypoxic regions in tumors, where the Hypoxia Inducible Factor (HIF) transcriptional system drives the expression of pro-survival and drug resistance genes, leading to radio- and chemo-resistance. This study aims to explore the efficacy of targeted nanoparticle (NP)-based small interfering RNA (siRNA) therapies in downregulating these genes to enhance treatment outcomes in pancreatic cancer, a tumor type characterized by high CD44 expression and hypoxia. Methods: We utilized hyaluronic acid (HA)-displaying nanoparticles composed of positively charged chitosan (CS) complexed with siRNA to target and knock down HIF-1α in pancreatic cancer cells. Two NP formulations were prepared using either low molecular weight (LMW) or high molecular weight (HMW) CS. These formulations were evaluated for their internalization by cells and their effectiveness in gene silencing, both in vitro and in vivo. Results: The study found that the molecular weight (MW) of CS influenced the interaction between HA and CD44, as well as the release of siRNA upon internalization. The LMW CS formulation shows faster uptake kinetics, while HMW CS is more effective in gene knockdown across different cell lines in vitro. In vivo, both were able to significantly knockdown HIF-1α and some of its downstream genes. Conclusions: The results suggest that HMW and LMW CS-based NPs exhibit distinct characteristics, showing that both MWs have potential for targeted pancreatic cancer therapy by influencing different aspects of delivery and gene silencing, particularly in the hypoxic tumor microenvironment.

Mario Ivanković^*, Jeremias N Brand^*, Luca Pandolfini, Thomas Brown, Martin Pippel, Andrei Rozanski, Til Schubert, Markus Grohme, Sylke Winkler, Laura Robledillo, Meng Zhang, Azzurra Codino, Stefano Gustincich, Miquel Vila-Farré, Shu Zhang, Argyris Papantonis, André Marques, Jochen Rink
A comparative analysis of planarian genomes reveals regulatory conservation in the face of rapid structural divergence.
Nat Commun, 15(1) Art. No. 8215 (2024)
Open Access DOI

The planarian Schmidtea mediterranea is being studied as a model species for regeneration, but the assembly of planarian genomes remains challenging. Here, we report a high-quality haplotype-phased, chromosome-scale genome assembly of the sexual S2 strain of S. mediterranea and high-quality chromosome-scale assemblies of its three close relatives, S. polychroa, S. nova, and S. lugubris. Using hybrid gene annotations and optimized ATAC-seq and ChIP-seq protocols for regulatory element annotation, we provide valuable genome resources for the planarian research community and a first comparative perspective on planarian genome evolution. Our analyses reveal substantial divergence in protein-coding sequences and regulatory regions but considerable conservation within promoter and enhancer annotations. We also find frequent retrotransposon-associated chromosomal inversions and interchromosomal translocations within the genus Schmidtea and, remarkably, independent and nearly complete losses of ancestral metazoan synteny in Schmidtea and two other flatworm groups. Overall, our results suggest that platyhelminth genomes can evolve without syntenic constraints.

Jan Gerwin^*, Julián Torres-Dowdall^*^#, Thomas Brown, Axel Meyer^#
Expansion and Functional Diversification of Long-Wavelength-Sensitive Opsin in Anabantoid Fishes.
J Mol Evol, 92(4) 432-448 (2024)
Open Access DOI

Gene duplication is one of the most important sources of novel genotypic diversity and the subsequent evolution of phenotypic diversity. Determining the evolutionary history and functional changes of duplicated genes is crucial for a comprehensive understanding of adaptive evolution. The evolutionary history of visual opsin genes is very dynamic, with repeated duplication events followed by sub- or neofunctionalization. While duplication of the green-sensitive opsins rh2 is common in teleost fish, fewer cases of multiple duplication events of the red-sensitive opsin lws are known. In this study, we investigate the visual opsin gene repertoire of the anabantoid fishes, focusing on the five lws opsin genes found in the genus Betta. We determine the evolutionary history of the lws opsin gene by taking advantage of whole-genome sequences of nine anabantoid species, including the newly assembled genome of Betta imbellis. Our results show that at least two independent duplications of lws occurred in the Betta lineage. The analysis of amino acid sequences of the lws paralogs of Betta revealed high levels of diversification in four of the seven transmembrane regions of the lws protein. Amino acid substitutions at two key-tuning sites are predicted to lead to differentiation of absorption maxima (λmax) between the paralogs within Betta. Finally, eye transcriptomics of B. splendens at different developmental stages revealed expression shifts between paralogs for all cone opsin classes. The lws genes are expressed according to their relative position in the lws opsin cluster throughout ontogeny. We conclude that temporal collinearity of lws expression might have facilitated subfunctionalization of lws in Betta and teleost opsins in general.

Arun Pal, Dajana Grossmann, Hannes Glaß, Vitaly Zimyanin, René Günther, Marica Catinozzi, Tobias M Boeckers, Jared Sterneckert, Erik Storkebaum, Susanne Petri, Florian Wegner, Stephan W. Grill, Francisco Pan-Montojo^#, Andreas Hermann^#
Glycolic acid and D-lactate-putative products of DJ-1-restore neurodegeneration in FUS - and SOD1-ALS.
Life Sci Alliance, 7(8) Art. No. e202302535 (2024)
Open Access DOI

Amyotrophic lateral sclerosis (ALS) leads to death within 2-5 yr. Currently, available drugs only slightly prolong survival. We present novel insights into the pathophysiology of Superoxide Dismutase 1 (SOD1)- and in particular Fused In Sarcoma (FUS)-ALS by revealing a supposedly central role of glycolic acid (GA) and D-lactic acid (DL)-both putative products of the Parkinson's disease associated glyoxylase DJ-1. Combined, not single, treatment with GA/DL restored axonal organelle phenotypes of mitochondria and lysosomes in FUS- and SOD1-ALS patient-derived motoneurons (MNs). This was not only accompanied by restoration of mitochondrial membrane potential but even dependent on it. Despite presenting an axonal transport deficiency as well, TDP43 patient-derived MNs did not share mitochondrial depolarization and did not respond to GA/DL treatment. GA and DL also restored cytoplasmic mislocalization of FUS and FUS recruitment to DNA damage sites, recently reported being upstream of the mitochondrial phenotypes in FUS-ALS. Whereas these data point towards the necessity of individualized (gene-) specific therapy stratification, it also suggests common therapeutic targets across different neurodegenerative diseases characterized by mitochondrial depolarization.

Leila Haj Abdullah Alieh^*, Beatriz C de Toledo^*, Anna Hadarovich, Agnes Toth-Petroczy, Federico Calegari
Characterization of alternative splicing during mammalian brain development reveals the extent of isoform diversity and potential effects on protein structural changes.
Biol Open, 13(10) Art. No. bio061721 (2024)
Open Access DOI

Regulation of gene expression is critical for fate commitment of stem and progenitor cells during tissue formation. In the context of mammalian brain development, a plethora of studies have described how changes in the expression of individual genes characterize cell types across ontogeny and phylogeny. However, little attention has been paid to the fact that different transcripts can arise from any given gene through alternative splicing (AS). Considered a key mechanism expanding transcriptome diversity during evolution, assessing the full potential of AS on isoform diversity and protein function has been notoriously difficult. Here, we capitalize on the use of a validated reporter mouse line to isolate neural stem cells, neurogenic progenitors and neurons during corticogenesis and combine the use of short- and long-read sequencing to reconstruct the full transcriptome diversity characterizing neurogenic commitment. Extending available transcriptional profiles of the mammalian brain by nearly 50,000 new isoforms, we found that neurogenic commitment is characterized by a progressive increase in exon inclusion resulting in the profound remodeling of the transcriptional profile of specific cortical cell types. Most importantly, we computationally infer the biological significance of AS on protein structure by using AlphaFold2, revealing how radical protein conformational changes can arise from subtle changes in isoforms sequence. Together, our study reveals that AS has a greater potential to impact protein diversity and function than previously thought, independently from changes in gene expression.

Julia Flock^*, Yexin Xie^*, Regis P. Lemaitre, Karine Lapouge, Kim Remans
The Use of Baculovirus-Mediated Gene Expression in Mammalian Cells for Recombinant Protein Production.
Methods Mol Biol, 2810 29-53 (2024)
DOI

Baculovirus-mediated gene expression in mammalian cells, BacMam, is a useful alternative to transient transfection for recombinant protein production in various types of mammalian cell lines. We decided to establish BacMam in our lab in order to streamline our workflows for gene expression in insect and mammalian cells, as it is straightforward to parallelize the baculovirus generation for both types of eukaryotic cells. This chapter provides a step-by-step description of the protocols we use for the generation of the recombinant BacMam viruses, the transduction of mammalian cell cultures, and optimization of the protein production conditions through small-scale expression and purification tests.

Alexandra M Garfinkel^*, Efe Ilker^*, Hidenobu Miyazawa^*, Kathrin Schmeisser^*, Jason M Tennessen^*
Historic obstacles and emerging opportunities in the field of developmental metabolism - lessons from Heidelberg.
Development, 151(12) Art. No. dev202937 (2024)
DOI

The field of developmental metabolism is experiencing a technological revolution that is opening entirely new fields of inquiry. Advances in metabolomics, small-molecule sensors, single-cell RNA sequencing and computational modeling present new opportunities for exploring cell-specific and tissue-specific metabolic networks, interorgan metabolic communication, and gene-by-metabolite interactions in time and space. Together, these advances not only present a means by which developmental biologists can tackle questions that have challenged the field for centuries, but also present young scientists with opportunities to define new areas of inquiry. These emerging frontiers of developmental metabolism were at the center of a highly interactive 2023 EMBO workshop 'Developmental metabolism: flows of energy, matter, and information'. Here, we summarize key discussions from this forum, emphasizing modern developmental biology's challenges and opportunities.

Rebecca A. Green^*^#, Renat N Khaliullin^*, Zhiling Zhao, Stacy D Ochoa, Jeffrey M Hendel, Tiffany-Lynn Chow, HongKee Moon, Ronald J Biggs, Arshad Desai, Karen Oegema^#
Automated profiling of gene function during embryonic development.
Cell, 187(12) 3141-3160 (2024)
DOI

Systematic functional profiling of the gene set that directs embryonic development is an important challenge. To tackle this challenge, we used 4D imaging of C. elegans embryogenesis to capture the effects of 500 gene knockdowns and developed an automated approach to compare developmental phenotypes. The automated approach quantifies features-including germ layer cell numbers, tissue position, and tissue shape-to generate temporal curves whose parameterization yields numerical phenotypic signatures. In conjunction with a new similarity metric that operates across phenotypic space, these signatures enabled the generation of ranked lists of genes predicted to have similar functions, accessible in the PhenoBank web portal, for ∼25% of essential development genes. The approach identified new gene and pathway relationships in cell fate specification and morphogenesis and highlighted the utilization of specialized energy generation pathways during embryogenesis. Collectively, the effort establishes the foundation for comprehensive analysis of the gene set that builds a multicellular organism.

Hendrik Frentzel, Marco Kraemer, Ylanna Kelner-Burgos, Laura Uelze, Dorina Bodi
Cereulide production capacities and genetic properties of 31 emetic Bacillus cereus group strains.
Int J Food Microbiol, 417 Art. No. 110694 (2024)
Open Access DOI

The highly potent toxin cereulide is a frequent cause of foodborne intoxications. This extremely resistant toxin is produced by Bacillus cereus group strains carrying the plasmid encoded cesHPTABCD gene cluster. It is known that the capacities to produce cereulide vary greatly between different strains but the genetic background of these variations is not clear. In this study, cereulide production capacities were associated with genetic characteristics. For this, cereulide levels in cultures of 31 strains were determined after incubation in tryptic soy broth for 24 h at 24 °C, 30 °C and 37 °C. Whole genome sequencing based data were used for an in-depth characterization of gene sequences related to cereulide production. The taxonomy, population structure and phylogenetic relationships of the strains were evaluated based on average nucleotide identity, multi-locus sequence typing (MLST), core genome MLST and single nucleotide polymorphism analyses. Despite a limited strain number, the approach of a genome wide association study (GWAS) was tested to link genetic variation with cereulide quantities. Our study confirms strain-dependent differences in cereulide production. For most strains, these differences were not explainable by sequence variations in the cesHPTABCD gene cluster or the regulatory genes abrB, spo0A, codY and pagRBc. Likewise, the population structure and phylogeny of the tested strains did not comprehensively reflect the cereulide production capacities. GWAS yielded first hints for associated proteins, while their possible effect on cereulide synthesis remains to be further investigated.

Katherine Benjamin^*, Aneesha Bhandari^*, Jessica D Kepple, Rui Qi, Zhouchun Shang, Yanan Xing, Yanru An, Nannan Zhang, Yong Hou, Tanya L Crockford, Oliver McCallion, Fadi Issa, Joanna Hester, Ulrike Tillmann, Heather A Harrington^#, Katherine R Bull^#
Multiscale topology classifies cells in subcellular spatial transcriptomics.
Nature, 630(8018) 943-949 (2024)
Open Access DOI

Spatial transcriptomics measures in situ gene expression at millions of locations within a tissue1, hitherto with some trade-off between transcriptome depth, spatial resolution and sample size2. Although integration of image-based segmentation has enabled impactful work in this context, it is limited by imaging quality and tissue heterogeneity. By contrast, recent array-based technologies offer the ability to measure the entire transcriptome at subcellular resolution across large samples3-6. Presently, there exist no approaches for cell type identification that directly leverage this information to annotate individual cells. Here we propose a multiscale approach to automatically classify cell types at this subcellular level, using both transcriptomic information and spatial context. We showcase this on both targeted and whole-transcriptome spatial platforms, improving cell classification and morphology for human kidney tissue and pinpointing individual sparsely distributed renal mouse immune cells without reliance on image data. By integrating these predictions into a topological pipeline based on multiparameter persistent homology7-9, we identify cell spatial relationships characteristic of a mouse model of lupus nephritis, which we validate experimentally by immunofluorescence. The proposed framework readily generalizes to new platforms, providing a comprehensive pipeline bridging different levels of biological organization from genes through to tissues.

Andy Göbel, Sophie Pählig, Anja Motz, Dorit Breining, Sofia Traikov, Lorenz C Hofbauer, Tilman D Rachner
Overcoming statin resistance in prostate cancer cells by targeting the 3-hydroxy-3-methylglutaryl-CoA-reductase.
Biochem Biophys Res Commun, 710 Art. No. 149841 (2024)
DOI

Prostate cancer is the most prevalent malignancy in men. While diagnostic and therapeutic interventions have substantially improved in recent years, disease relapse, treatment resistance, and metastasis remain significant contributors to prostate cancer-related mortality. Therefore, novel therapeutic approaches are needed. Statins are inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway which plays an essential role in cholesterol homeostasis. Numerous preclinical studies have provided evidence for the pleiotropic antitumor effects of statins. However, results from clinical studies remain controversial and have shown substantial benefits to even no effects on human malignancies including prostate cancer. Potential statin resistance mechanisms of tumor cells may account for such discrepancies. In our study, we treated human prostate cancer cell lines (PC3, C4-2B, DU-145, LNCaP) with simvastatin, atorvastatin, and rosuvastatin. PC3 cells demonstrated high statin sensitivity, resulting in a significant loss of vitality and clonogenic potential (up to - 70%; p < 0.001) along with an activation of caspases (up to 4-fold; p < 0.001). In contrast, C4-2B and DU-145 cells were statin-resistant. Statin treatment induced a restorative feedback in statin-resistant C4-2B and DU-145 cells through upregulation of the HMGCR gene and protein expression (up to 3-folds; p < 0.01) and its transcription factor sterol-regulatory element binding protein 2 (SREBP-2). This feedback was absent in PC3 cells. Blocking the feedback using HMGCR-specific small-interfering (si)RNA, the SREBP-2 activation inhibitor dipyridamole or the HMGCR degrader SR12813 abolished statin resistance in C4-2B and DU-145 and induced significant activation of caspases by statin treatment (up to 10-fold; p < 0.001). Consistently, long-term treatment with sublethal concentrations of simvastatin established a stable statin resistance of a PC3SIM subclone accompanied by a significant upregulation of both baseline as well as post-statin HMGCR protein (gene expression up to 70-fold; p < 0.001). Importantly, the statin-resistant phenotype of PC3SIM cells was reversible by HMGCR-specific siRNA and dipyridamole. Our investigations reveal a key role of a restorative feedback driven by the HMGCR/SREBP-2 axis in statin resistance mechanisms of prostate cancer cells.

Maria Luisa Romero Romero^#, Jonas Poehls, Anastasiia Kirilenko, Doris Richter, Tobias Jumel, Anna Shevchenko, Agnes Toth-Petroczy^#
Environment modulates protein heterogeneity through transcriptional and translational stop codon readthrough.
Nat Commun, 15(1) Art. No. 4446 (2024)
Open Access DOI

Stop codon readthrough events give rise to longer proteins, which may alter the protein's function, thereby generating short-lasting phenotypic variability from a single gene. In order to systematically assess the frequency and origin of stop codon readthrough events, we designed a library of reporters. We introduced premature stop codons into mScarlet, which enabled high-throughput quantification of protein synthesis termination errors in E. coli using fluorescent microscopy. We found that under stress conditions, stop codon readthrough may occur at rates as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon readthrough events. The analysis of selected reporters by mass spectrometry and RNA-seq showed that not only translation but also transcription errors contribute to stop codon readthrough. The RNA polymerase was more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon readthrough by mass spectrometry revealed that temperature regulated the expression of cryptic sequences generated by stop codon readthrough in E. coli. Overall, our findings suggest that the environment affects the accuracy of protein production, which increases protein heterogeneity when the organisms need to adapt to new conditions.

Lei Xing^#, Vasiliki Gkini, Anni I Nieminen, Hui-Chao Zhou, Matilde Aquilino, Ronald Naumann, Katrin Reppe, Kohichi Tanaka, Peter Carmeliet, Oskari Heikinheimo, Svante Pääbo, Wieland Huttner^#, Takashi Namba^#
Functional synergy of a human-specific and an ape-specific metabolic regulator in human neocortex development.
Nat Commun, 15(1) Art. No. 3468 (2024)
Open Access DOI

Metabolism has recently emerged as a major target of genes implicated in the evolutionary expansion of human neocortex. One such gene is the human-specific gene ARHGAP11B. During human neocortex development, ARHGAP11B increases the abundance of basal radial glia, key progenitors for neocortex expansion, by stimulating glutaminolysis (glutamine-to-glutamate-to-alpha-ketoglutarate) in mitochondria. Here we show that the ape-specific protein GLUD2 (glutamate dehydrogenase 2), which also operates in mitochondria and converts glutamate-to-αKG, enhances ARHGAP11B's ability to increase basal radial glia abundance. ARHGAP11B + GLUD2 double-transgenic bRG show increased production of aspartate, a metabolite essential for cell proliferation, from glutamate via alpha-ketoglutarate and the TCA cycle. Hence, during human evolution, a human-specific gene exploited the existence of another gene that emerged during ape evolution, to increase, via concerted changes in metabolism, progenitor abundance and neocortex size.

Martino Ugolini, Maciej A Kerlin, Ksenia Kuznetsova, Haruka Oda, Hiroshi Kimura, Nadine Vastenhouw
Transcription bodies regulate gene expression by sequestering CDK9.
Nat Cell Biol, 26(4) 604-612 (2024)
Open Access DOI

The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. It remains unclear, however, if and how transcription bodies affect gene expression. Here we disrupted the formation of two prominent endogenous transcription bodies that mark the onset of zygotic transcription in zebrafish embryos and analysed the effect on gene expression using enriched SLAM-seq and live-cell imaging. We find that the disruption of transcription bodies results in the misregulation of hundreds of genes. Here we focus on genes that are upregulated. These genes have accessible chromatin and are poised to be transcribed in the presence of the two transcription bodies, but they do not go into elongation. Live-cell imaging shows that disruption of the two large transcription bodies enables these poised genes to be transcribed in ectopic transcription bodies, suggesting that the large transcription bodies sequester a pause release factor. Supporting this hypothesis, we find that CDK9-the kinase that releases paused polymerase II-is highly enriched in the two large transcription bodies. Overexpression of CDK9 in wild-type embryos results in the formation of ectopic transcription bodies and thus phenocopies the removal of the two large transcription bodies. Taken together, our results show that transcription bodies regulate transcription by sequestering machinery, thereby preventing genes elsewhere in the nucleus from being transcribed.

Wieland Huttner, Michael Heide, Felipe Mora-Bermúdez, Takashi Namba
Neocortical neurogenesis in development and evolution-Human-specific features.
J Comp Neurol, 532(2) Art. No. e25576 (2024)
Open Access DOI

In this review, we focus on human-specific features of neocortical neurogenesis in development and evolution. Two distinct topics will be addressed. In the first section, we discuss the expansion of the neocortex during human evolution and concentrate on the human-specific gene ARHGAP11B. We review the ability of ARHGAP11B to amplify basal progenitors and to expand a primate neocortex. We discuss the contribution of ARHGAP11B to neocortex expansion during human evolution and its potential implications for neurodevelopmental disorders and brain tumors. We then review the action of ARHGAP11B in mitochondria as a regulator of basal progenitor metabolism, and how it promotes glutaminolysis and basal progenitor proliferation. Finally, we discuss the increase in cognitive performance due to the ARHGAP11B-induced neocortical expansion. In the second section, we focus on neocortical development in modern humans versus Neanderthals. Specifically, we discuss two recent findings pointing to differences in neocortical neurogenesis between these two hominins that are due to a small number of amino acid substitutions in certain key proteins. One set of such proteins are the kinetochore-associated proteins KIF18a and KNL1, where three modern human-specific amino acid substitutions underlie the prolongation of metaphase during apical progenitor mitosis. This prolongation in turn is associated with an increased fidelity of chromosome segregation to the apical progenitor progeny during modern human neocortical development, with implications for the proper formation of radial units. Another such key protein is transketolase-like 1 (TKTL1), where a single modern human-specific amino acid substitution endows TKTL1 with the ability to amplify basal radial glia, resulting in an increase in upper-layer neuron generation. TKTL1's ability is based on its action in the pentose phosphate pathway, resulting in increased fatty acid synthesis. The data imply greater neurogenesis during neocortical development in modern humans than Neanderthals due to TKTL1, in particular in the developing frontal lobe.

Said El Bouhaddani^*, Matthias Höllerhage^*, Hae-Won Uh, Claudia Moebius, Marc Bickle, Günter U Höglinger, Jeanine Houwing-Duistermaat
Statistical integration of multi-omics and drug screening data from cell lines.
PLoS Comput Biol, 20(1) Art. No. e1011809 (2024)
Open Access DOI

Data integration methods are used to obtain a unified summary of multiple datasets. For multi-modal data, we propose a computational workflow to jointly analyze datasets from cell lines. The workflow comprises a novel probabilistic data integration method, named POPLS-DA, for multi-omics data. The workflow is motivated by a study on synucleinopathies where transcriptomics, proteomics, and drug screening data are measured in affected LUHMES cell lines and controls. The aim is to highlight potentially druggable pathways and genes involved in synucleinopathies. First, POPLS-DA is used to prioritize genes and proteins that best distinguish cases and controls. For these genes, an integrated interaction network is constructed where the drug screen data is incorporated to highlight druggable genes and pathways in the network. Finally, functional enrichment analyses are performed to identify clusters of synaptic and lysosome-related genes and proteins targeted by the protective drugs. POPLS-DA is compared to other single- and multi-omics approaches. We found that HSPA5, a member of the heat shock protein 70 family, was one of the most targeted genes by the validated drugs, in particular by AT1-blockers. HSPA5 and AT1-blockers have been previously linked to α-synuclein pathology and Parkinson's disease, showing the relevance of our findings. Our computational workflow identified new directions for therapeutic targets for synucleinopathies. POPLS-DA provided a larger interpretable gene set than other single- and multi-omic approaches. An implementation based on R and markdown is freely available online.

Benjamin Seelbinder, Susan Wagner, Manavi Jain, Elena Erben, Sergei Klykov, Iliya D. Stoev, Venkat Raghavan Krishnaswamy, Moritz Kreysing
Probe-free optical chromatin deformation and measurement of differential mechanical properties in the nucleus.
Elife, 13 Art. No. e76421 (2024)
Open Access DOI

The nucleus is highly organized to facilitate coordinated gene transcription. Measuring the rheological properties of the nucleus and its sub-compartments will be crucial to understand the principles underlying nuclear organization. Here, we show that strongly localized temperature gradients (approaching 1°C/µm) can lead to substantial intra-nuclear chromatin displacements (>1 µm), while nuclear area and lamina shape remain unaffected. Using particle image velocimetry (PIV), intra-nuclear displacement fields can be calculated and converted into spatio-temporally resolved maps of various strain components. Using this approach, we show that chromatin displacements are highly reversible, indicating that elastic contributions are dominant in maintaining nuclear organization on the time scale of seconds. In genetically inverted nuclei, centrally compacted heterochromatin displays high resistance to deformation, giving a rigid, solid-like appearance. Correlating spatially resolved strain maps with fluorescent reporters in conventional interphase nuclei reveals that various nuclear compartments possess distinct mechanical identities. Surprisingly, both densely and loosely packed chromatin showed high resistance to deformation, compared to medium dense chromatin. Equally, nucleoli display particularly high resistance and strong local anchoring to heterochromatin. Our results establish how localized temperature gradients can be used to drive nuclear compartments out of mechanical equilibrium to obtain spatial maps of their material responses.

Dennis Schifferl, Manuela Scholze-Wittler, Alba Villaronga Luque, Milena Pustet, Lars Wittler, Jesse V Veenvliet, Frederic Koch^#, Bernhard G Herrmann^#
Genome-wide identification of notochord enhancers comprising the regulatory landscape of the brachyury locus in mouse.
Development, 150(22) Art. No. dev202111 (2023)
Open Access DOI

The node and notochord are important signaling centers organizing the dorso-ventral patterning of cells arising from neuro-mesodermal progenitors forming the embryonic body anlage. Owing to the scarcity of notochord progenitors and notochord cells, a comprehensive identification of regulatory elements driving notochord-specific gene expression has been lacking. Here, we have used ATAC-seq analysis of FACS-purified notochord cells from Theiler stage 12-13 mouse embryos to identify 8921 putative notochord enhancers. In addition, we established a new model for generating notochord-like cells in culture, and found 3728 of these enhancers occupied by the essential notochord control factors brachyury (T) and/or Foxa2. We describe the regulatory landscape of the T locus, comprising ten putative enhancers occupied by these factors, and confirmed the regulatory activity of three of these elements. Moreover, we characterized seven new elements by knockout analysis in embryos and identified one new notochord enhancer, termed TNE2. TNE2 cooperates with TNE in the trunk notochord, and is essential for notochord differentiation in the tail. Our data reveal an essential role of Foxa2 in directing T-expressing cells towards the notochord lineage.

Belin Selcen Beydag-Tasöz, Joyson Verner D'Costa, Lena Hersemann, Byung Ho Lee, Federica Luppino, Yung Hae Kim, Christoph Zechner, Anne Grapin-Botton
Integrating single-cell imaging and RNA sequencing datasets links differentiation and morphogenetic dynamics of human pancreatic endocrine progenitors.
Dev Cell, 58(21) 2292-2308 (2023)
Open Access DOI

Basic helix-loop-helix genes, particularly proneural genes, are well-described triggers of cell differentiation, yet information on their dynamics is limited, notably in human development. Here, we focus on Neurogenin 3 (NEUROG3), which is crucial for pancreatic endocrine lineage initiation. By monitoring both NEUROG3 gene expression and protein in single cells using a knockin dual reporter in 2D and 3D models of human pancreas development, we show an approximately 2-fold slower expression of human NEUROG3 than that of the mouse. We observe heterogeneous peak levels of NEUROG3 expression and reveal through long-term live imaging that both low and high NEUROG3 peak levels can trigger differentiation into hormone-expressing cells. Based on fluorescence intensity, we statistically integrate single-cell transcriptome with dynamic behaviors of live cells and propose a data-mapping methodology applicable to other contexts. Using this methodology, we identify a role for KLK12 in motility at the onset of NEUROG3 expression.

Cassie L Kemmler, Jana Smolikova, Hannah R Moran, Brandon J Mannion, Dunja Knapp, Fabian Lim, Anna Czarkwiani, Viviana Hermosilla Aguayo, Vincent Rapp, Olivia E Fitch, Seraina Bötschi, Licia Selleri, Emma Farley, Ingo Braasch, Maximina H Yun, Axel Visel, Marco Osterwalder, Christian Mosimann, Zbynek Kozmik^#, Alexa Burger^#
Conserved enhancers control notochord expression of vertebrate Brachyury.
Nat Commun, 14(1) Art. No. 6594 (2023)
Open Access DOI

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development.

Jirina Zackova Suchanova, Gust Bilcke, Beata Romanowska, Ali Fatlawi, Martin Pippel, Alastair W Skeffington, Michael Schroeder, Wim Vyverman, Klaas Vandepoele, Nils Kröger^#, Nicole Poulsen^#
Diatom adhesive trail proteins acquired by horizontal gene transfer from bacteria serve as primers for marine biofilm formation.
New Phytol, 240(2) 770-783 (2023)
Open Access DOI

Biofilm-forming benthic diatoms are key primary producers in coastal habitats, where they frequently dominate sunlit intertidal substrata. The development of gliding motility in raphid diatoms was a key molecular adaptation that contributed to their evolutionary success. However, the structure-function correlation between diatom adhesives utilized for gliding and their relationship to the extracellular matrix that constitutes the diatom biofilm is unknown. Here, we have used proteomics, immunolocalization, comparative genomics, phylogenetics and structural homology analysis to investigate the evolutionary history and function of diatom adhesive proteins. Our study identified eight proteins from the adhesive trails of Craspedostauros australis, of which four form a new protein family called Trailins that contain an enigmatic Choice-of-Anchor A (CAA) domain, which was acquired through horizontal gene transfer from bacteria. Notably, the CAA-domain shares a striking structural similarity with one of the most widespread domains found in ice-binding proteins (IPR021884). Our work offers new insights into the molecular basis for diatom biofilm formation, shedding light on the function and evolution of diatom adhesive proteins. This discovery suggests that there is a transition in the composition of biomolecules required for initial surface colonization and those utilized for 3D biofilm matrix formation.

Adrian Pascal Nievergelt, Dennis R Diener, Aliona Bogdanova, Thomas Brown, Gaia Pigino
Efficient precision editing of endogenous Chlamydomonas reinhardtii genes with CRISPR-Cas.
Cell Rep Methods, 3(8) Art. No. 100562 (2023)
Open Access DOI

CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until now been primarily applied to targeted gene disruption, whereas scarless knockin transgenesis has generally been considered difficult in practice. We have developed an efficient homology-directed method for knockin mutagenesis in Chlamydomonas by delivering CRISPR-Cas ribonucleoproteins and a linear double-stranded DNA (dsDNA) donor into cells by electroporation. Our method allows scarless integration of fusion tags and sequence modifications of proteins without the need for a preceding mutant line. We also present methods for high-throughput crossing of transformants and a custom quantitative PCR (qPCR)-based high-throughput screening of mutants as well as meiotic progeny. We demonstrate how to use this pipeline to facilitate the generation of mutant lines without residual selectable markers by co-targeted insertion. Finally, we describe how insertional cassettes can be erroneously mutated during insertion and suggest strategies to select for lines that are modified as designed.

David Thomas Gonzales, Surased Suraritdechachai, Christoph Zechner, T-Y Dora Tang
Bidirectional Communication between Droplet Interface Bilayers Driven by Cell-Free Quorum Sensing Gene Circuits.
ChemSystemsChem, 5 Art. No. e20230002 (2023)
Open Access DOI

Building synthetic multicellular systems using non-living molecular components is a grand challenge in the field of bottom-up synthetic biology. Towards this goal, a diverse range of chemistries have been developed to provide mechanisms of intercellular communication and methods to assemble multicellular compartments. However, building bottom-up synthetic multicellular systems is still challenging because it requires the integration of intercellular reaction networks with compatible cellular compartment properties. In this study, we encapsulated cell-free expression systems (CFES) expressing two quorum sensing genetic circuits into droplet interface bilayer (DIB) synthetic cells to demonstrate bidirectional communication. We further develop a method of generating custom DIB multicellular structures by acoustic liquid handling to automatically dispense the CFES droplets and show the potential for multiplexing compartmentalized gene circuits for generating heterogeneous populations of cells. Our work provides a step towards building more complex multicellular systems with intercellular communication from the bottom-up to study and experimentally model biological multiscalar processes.

Bismark Appiah^*, Camila L Fullio^*, Chiara Ossola, Ilaria Bertani, Elena Restelli, Arquimedes Cheffer, Martina Polenghi, Christiane Haffner, Marta Garcia-Miralles, Patrice Zeis, Martin Treppner, Patrick Bovio, Laura Schlichtholz, Aina Mas-Sanchez, Lea Zografidou, Jennifer Winter, Harald Binder, Dominic Grün, Nereo Kalebic, Elena Taverna, Tanja Vogel
DOT1L activity affects neural stem cell division mode and reduces differentiation and ASNS expression.
EMBO Rep, 24(8) Art. No. e56233 (2023)
Open Access DOI

Cortical neurogenesis depends on the balance between self-renewal and differentiation of apical progenitors (APs). Here, we study the epigenetic control of AP's division mode by focusing on the enzymatic activity of the histone methyltransferase DOT1L. Combining lineage tracing with single-cell RNA sequencing of clonally related cells, we show at the cellular level that DOT1L inhibition increases neurogenesis driven by a shift of APs from asymmetric self-renewing to symmetric neurogenic consumptive divisions. At the molecular level, DOT1L activity prevents AP differentiation by promoting transcription of metabolic genes. Mechanistically, DOT1L inhibition reduces activity of an EZH2/PRC2 pathway, converging on increased expression of asparagine synthetase (ASNS), a microcephaly associated gene. Overexpression of ASNS in APs phenocopies DOT1L inhibition, and also increases neuronal differentiation of APs. Our data suggest that DOT1L activity/PRC2 crosstalk controls AP lineage progression by regulating asparagine metabolism.

Elisa Nerli^#, Jenny Kretzschmar, Tommaso Bianucci, Mauricio Rocha-Martins, Christoph Zechner, Caren Norden^#
Deterministic and probabilistic fate decisions co-exist in a single retinal lineage.
EMBO J, 42(14) Art. No. e112657 (2023)
DOI

Correct nervous system development depends on the timely differentiation of progenitor cells into neurons. While the output of progenitor differentiation is well investigated at the population and clonal level, how stereotypic or variable fate decisions are during development is still more elusive. To fill this gap, we here follow the fate outcome of single neurogenic progenitors in the zebrafish retina over time using live imaging. We find that neurogenic progenitor divisions produce two daughter cells, one of deterministic and one of probabilistic fate. Interference with the deterministic branch of the lineage affects lineage progression. In contrast, interference with fate probabilities of the probabilistic branch results in a broader range of fate possibilities than in wild-type and involves the production of any neuronal cell type even at non-canonical developmental stages. Combining the interference data with stochastic modelling of fate probabilities revealed that a simple gene regulatory network is able to predict the observed fate decision probabilities during wild-type development. These findings unveil unexpected lineage flexibility that could ensure robust development of the retina and other tissues.

Bogdan Kirilenko, Chetan Munegowda, Ekaterina Osipova, David Jebb, Virag Sharma, Moritz Blumer, Ariadna E Morales, Alexis-Walid Ahmed, Dimitrios-Georgios Kontopoulos, Leon Hilgers, Kerstin Lindblad-Toh, Elinor K Karlsson, Zoonomia Consortium, Michael Hiller
Integrating gene annotation with orthology inference at scale.
Science, 380(6643) Art. No. eabn3107 (2023)
DOI

Annotating coding genes and inferring orthologs are two classical challenges in genomics and evolutionary biology that have traditionally been approached separately, limiting scalability. We present TOGA (Tool to infer Orthologs from Genome Alignments), a method that integrates structural gene annotation and orthology inference. TOGA implements a different paradigm to infer orthologous loci, improves ortholog detection and annotation of conserved genes compared with state-of-the-art methods, and handles even highly fragmented assemblies. TOGA scales to hundreds of genomes, which we demonstrate by applying it to 488 placental mammal and 501 bird assemblies, creating the largest comparative gene resources so far. Additionally, TOGA detects gene losses, enables selection screens, and automatically provides a superior measure of mammalian genome quality. TOGA is a powerful and scalable method to annotate and compare genes in the genomic era.

Lidiia Tynianskaia^*, Nesil Eşiyok^*, Wieland Huttner^#, Michael Heide^#
Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification.
J Vis Exp, (193) Art. No. e65176 (2023)
DOI

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders. As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

Blair P Bentley^#, Tomás Carrasco-Valenzuela, Elisa K S Ramos, Harvinder Pawar, Larissa Souza Arantes, Alana Alexander, Shreya M Banerjee, Patrick Masterson, Martin Kuhlwilm, Martin Pippel, Jacquelyn Mountcastle, Bettina Haase, Marcela Uliano-Silva, Giulio Formenti, Kerstin Howe, William Chow, Alan Tracey, Ying Sims, Sarah Pelan, Jonathan Wood, Kelsey Yetsko, Justin R Perrault, Kelly Stewart, Scott R Benson, Yaniv Levy, Erica V Todd, H Bradley Shaffer, Peter Scott, Brian T Henen, Robert F Murphy, David W Mohr, Alan F Scott, D. Duffy, Neil J Gemmell, Alexander Suh, Sylke Winkler, Francoise Thibaud-Nissen, Mariana F Nery, Tomas Marques-Bonet, Agostinho Antunes, Yaron Tikochinski, Peter H Dutton, Olivier Fedrigo, Eugene W Myers, Erich D Jarvis, Camila J Mazzoni^#, Lisa M Komoroske^#
Divergent sensory and immune gene evolution in sea turtles with contrasting demographic and life histories.
Proc Natl Acad Sci U.S.A., 120(7) Art. No. e2201076120 (2023)
Open Access DOI

Sea turtles represent an ancient lineage of marine vertebrates that evolved from terrestrial ancestors over 100 Mya. The genomic basis of the unique physiological and ecological traits enabling these species to thrive in diverse marine habitats remains largely unknown. Additionally, many populations have drastically declined due to anthropogenic activities over the past two centuries, and their recovery is a high global conservation priority. We generated and analyzed high-quality reference genomes for the leatherback (Dermochelys coriacea) and green (Chelonia mydas) turtles, representing the two extant sea turtle families. These genomes are highly syntenic and homologous, but localized regions of noncollinearity were associated with higher copy numbers of immune, zinc-finger, and olfactory receptor (OR) genes in green turtles, with ORs related to waterborne odorants greatly expanded in green turtles. Our findings suggest that divergent evolution of these key gene families may underlie immunological and sensory adaptations assisting navigation, occupancy of neritic versus pelagic environments, and diet specialization. Reduced collinearity was especially prevalent in microchromosomes, with greater gene content, heterozygosity, and genetic distances between species, supporting their critical role in vertebrate evolutionary adaptation. Finally, diversity and demographic histories starkly contrasted between species, indicating that leatherback turtles have had a low yet stable effective population size, exhibit extremely low diversity compared with other reptiles, and harbor a higher genetic load compared with green turtles, reinforcing concern over their persistence under future climate scenarios. These genomes provide invaluable resources for advancing our understanding of evolution and conservation best practices in an imperiled vertebrate lineage.

Franziska Hettler^*, Christina Schreck^*, Sandra Romero Marquez, Thomas Engleitner, Baiba Vilne, Theresa Landspersky, Heike Weidner, Renate Hausinger, Ritu Mishra, Rupert Oellinger, Martina Rauner, Ronald Naumann, Christian Peschel, Florian Bassermann, Roland Rad, Rouzanna Istvanffy, Robert A J Oostendorp
Osteoprogenitor SFRP1 prevents exhaustion of hematopoietic stem cells via PP2A-PR72/130-mediated regulation of p300.
Haematologica, 108(2) 490-501 (2023)
Open Access DOI

Remodeling of the bone marrow microenvironment in chronic inflammation and in aging reduces hematopoietic stem cell (HSC) function. To assess the mechanisms of this functional decline of HSC and find strategies to counteract it, we established a model in which the Sfrp1 gene was deleted in Osterix+ osteolineage cells (OS1Δ/Δ mice). HSC from these mice showed severely diminished repopulating activity with associated DNA damage, enriched expression of the reactive oxygen species pathway and reduced single-cell proliferation. Interestingly, not only was the protein level of Catenin beta-1 (bcatenin) elevated, but so was its association with the phosphorylated co-activator p300 in the nucleus. Since these two proteins play a key role in promotion of differentiation and senescence, we inhibited in vivo phosphorylation of p300 through PP2A-PR72/130 by administration of IQ-1 in OS1Δ/Δ mice. This treatment not only reduced the b-catenin/phosphop300 association, but also decreased nuclear p300. More importantly, in vivo IQ-1 treatment fully restored HSC repopulating activity of the OS1Δ/Δ mice. Our findings show that the osteoprogenitor Sfrp1 is essential for maintaining HSC function. Furthermore, pharmacological downregulation of the nuclear b-catenin/phospho-p300 association is a new strategy to restore poor HSC function.

Kaiqing Zhang^*, Fabio Da Silva^*, Carina Seidl, Michaela Wilsch-Bräuninger, Jessica Herbst, Wieland Huttner, Christof Niehrs
Primary cilia are WNT-transducing organelles whose biogenesis is controlled by a WNT-PP1 axis.
Dev Cell, 58(2) 139-154 (2023)
DOI

WNT signaling is important in development, stem cell maintenance, and disease. WNT ligands typically signal via receptor activation across the plasma membrane to induce β-catenin-dependent gene activation. Here, we show that in mammalian primary cilia, WNT receptors relay a WNT/GSK3 signal that β-catenin-independently promotes ciliogenesis. Characterization of a LRP6 ciliary targeting sequence and monitoring of acute WNT co-receptor activation (phospho-LRP6) support this conclusion. Ciliary WNT signaling inhibits protein phosphatase 1 (PP1) activity, a negative regulator of ciliogenesis, by preventing GSK3-mediated phosphorylation of the PP1 regulatory inhibitor subunit PPP1R2. Concordantly, deficiency of WNT/GSK3 signaling by depletion of cyclin Y and cyclin-Y-like protein 1 induces primary cilia defects in mouse embryonic neuronal precursors, kidney proximal tubules, and adult mice preadipocytes.

Ksenia Kuznetsova, Noémie M Chabot, Martino Ugolini, Edlyn Wu, Manan Lalit, Haruka Oda, Yuko Sato, Hiroshi Kimura, Florian Jug, Nadine Vastenhouw
Nanog organizes transcription bodies.
Curr Biol, 33(1) 164-173 (2023)
DOI

The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells.1-3 How proteins of the transcriptional machinery come together to form such bodies, however, is unclear. Here, we take advantage of two large, isolated, and long-lived transcription bodies that reproducibly form during early zebrafish embryogenesis to characterize the dynamics of transcription body formation. Once formed, these transcription bodies are enriched for initiating and elongating RNA polymerase II, as well as the transcription factors Nanog and Sox19b. Analyzing the events leading up to transcription, we find that Nanog and Sox19b cluster prior to transcription. The clustering of transcription factors is sequential; Nanog clusters first, and this is required for the clustering of Sox19b and the initiation of transcription. Mutant analysis revealed that both the DNA-binding domain as well as one of the two intrinsically disordered regions of Nanog are required to organize the two bodies of transcriptional activity. Taken together, our data suggest that the clustering of transcription factors dictates the formation of transcription bodies.

Angela L Caipa Garcia, Jill E Kucab, Halh Al-Serori, Rebekah S S Beck, Franziska Fischer, Matthias Hufnagel, Andrea Hartwig, Andrew Floeder, Silvia Balbo, Hayley E Francies, Mathew J Garnett, Meritxell Huch, Jarno Drost, Matthias Zilbauer, Volker M Arlt, David H Phillips
Metabolic Activation of Benzo[a]pyrene by Human Tissue Organoid Cultures.
Int J Mol Sci, 24(1) Art. No. 606 (2022)
Open Access DOI

Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[a]pyrene (BaP) was investigated. While organoids from the different tissues showed varied cytotoxic responses to BaP, with gastric and colon organoids being the most susceptible, the xenobiotic-metabolising enzyme (XME) genes, CYP1A1 and NQO1, were highly upregulated in all organoid types, with kidney organoids having the highest levels. Furthermore, the presence of two key metabolites, BaP-t-7,8-dihydrodiol and BaP-tetrol-l-1, was detected in all organoid types, confirming their ability to metabolise BaP. BaP bioactivation was confirmed both by the activation of the DNA damage response pathway (induction of p-p53, pCHK2, p21 and γ-H2AX) and by DNA adduct formation. Overall, pancreatic and undifferentiated liver organoids formed the highest levels of DNA adducts. Colon organoids had the lowest responses in DNA adduct and metabolite formation, as well as XME expression. Additionally, high-throughput RT-qPCR explored differences in gene expression between organoid types after BaP treatment. The results demonstrate the potential usefulness of organoids for studying environmental carcinogenesis and genetic toxicology.

Takeshi Takeuchi^*, Yoshihiko Suzuki^*, Shugo Watabe, Kosuke Nagai, Tetsuji Masaoka, Manabu Fujie, Mayumi Kawamitsu, Noriyuki Satoh, Eugene W Myers
A high-quality, haplotype-phased genome reconstruction reveals unexpected haplotype diversity in a pearl oyster.
DNA Res, 29(6) Art. No. dsac035 (2022)
Open Access DOI

Homologous chromosomes in the diploid genome are thought to contain equivalent genetic information, but this common concept has not been fully verified in animal genomes with high heterozygosity. Here we report a near-complete, haplotype-phased, genome assembly of the pearl oyster, Pinctada fucata, using hi-fidelity (HiFi) long reads and chromosome conformation capture data. This assembly includes 14 pairs of long scaffolds (>38 Mb) corresponding to chromosomes (2n = 28). The accuracy of the assembly, as measured by an analysis of k-mers, is estimated to be 99.99997%. Moreover, the haplotypes contain 95.2% and 95.9%, respectively, complete and single-copy BUSCO genes, demonstrating the high quality of the assembly. Transposons comprise 53.3% of the assembly and are a major contributor to structural variations. Despite overall collinearity between haplotypes, one of the chromosomal scaffolds contains megabase-scale non-syntenic regions, which necessarily have never been detected and resolved in conventional haplotype-merged assemblies. These regions encode expanded gene families of NACHT, DZIP3/hRUL138-like HEPN, and immunoglobulin domains, multiplying the immunity gene repertoire, which we hypothesize is important for the innate immune capability of pearl oysters. The pearl oyster genome provides insight into remarkable haplotype diversity in animals.

Jan Fischer^*, Eduardo Fernández Ortuño^*, Fabio Marsoner^*, Annasara Artioli, Jula Peters, Takashi Namba, Christina Eugster Oegema, Wieland Huttner^#, Julia Ladewig^#, Michael Heide^#
Human-specific ARHGAP11B ensures human-like basal progenitor levels in hominid cerebral organoids.
EMBO Rep, 23(11) Art. No. e54728 (2022)
Open Access DOI

The human-specific gene ARHGAP11B has been implicated in human neocortex expansion. However, the extent of ARHGAP11B's contribution to this expansion during hominid evolution is unknown. Here we address this issue by genetic manipulation of ARHGAP11B levels and function in chimpanzee and human cerebral organoids. ARHGAP11B expression in chimpanzee cerebral organoids doubles basal progenitor levels, the class of cortical progenitors with a key role in neocortex expansion. Conversely, interference with ARHGAP11B's function in human cerebral organoids decreases basal progenitors down to the chimpanzee level. Moreover, ARHGAP11A or ARHGAP11B rescue experiments in ARHGAP11A plus ARHGAP11B double-knockout human forebrain organoids indicate that lack of ARHGAP11B, but not of ARHGAP11A, decreases the abundance of basal radial glia-the basal progenitor type thought to be of particular relevance for neocortex expansion. Taken together, our findings demonstrate that ARHGAP11B is necessary and sufficient to ensure the elevated basal progenitor levels that characterize the fetal human neocortex, suggesting that this human-specific gene was a major contributor to neocortex expansion during human evolution.

Sean P A Ritter, Logan A Brand, Shelby L Vincent, Albert Remus R Rosana, Allison Lewis, Denise S Whitford, George W Owttrim
Multiple Light-Dark Signals Regulate Expression of the DEAD-Box RNA Helicase CrhR in Synechocystis PCC 6803.
Cells, 11(21) Art. No. 3397 (2022)
Open Access DOI

Since oxygenic photosynthesis evolved in the common ancestor of cyanobacteria during the Archean, a range of sensing and response strategies evolved to allow efficient acclimation to the fluctuating light conditions experienced in the diverse environments they inhabit. However, how these regulatory mechanisms are assimilated at the molecular level to coordinate individual gene expression is still being elucidated. Here, we demonstrate that integration of a series of three distinct light signals generate an unexpectedly complex network regulating expression of the sole DEAD-box RNA helicase, CrhR, encoded in Synechocystis sp. PCC 6803. The mechanisms function at the transcriptional, translational and post-translation levels, fine-tuning CrhR abundance to permit rapid acclimation to fluctuating light and temperature regimes. CrhR abundance is enhanced 15-fold by low temperature stress. We initially confirmed that the primary mechanism controlling crhR transcript accumulation at 20 °C requires a light quantity-driven reduction of the redox poise in the vicinity of the plastoquinone pool. Once transcribed, a specific light quality cue, a red light signal, was required for crhR translation, far-red reversal of which indicates a phytochrome-mediated mechanism. Examination of CrhR repression at 30 °C revealed that a redox- and light quality-independent light signal was required to initiate CrhR degradation. The crucial role of light was further revealed by the observation that dark conditions superseded the light signals required to initiate each of these regulatory processes. The findings reveal an unexpected complexity of light-dark sensing and signaling that regulate expression of an individual gene in cyanobacteria, an integrated mechanism of environmental perception not previously reported.

Keiichi Katsumoto^#, Siham Yennek, Chunguang Chen, Luis Fernando Delgadillo Silva, Sofia Traikov, Dror Sever, Ajuna Azad, Jingdong Shan, Seppo Vainio, Nikolay Ninov, Stephan Speier, Anne Grapin-Botton^#
Wnt4 is heterogeneously activated in maturing β-cells to control calcium signaling, metabolism and function.
Nat Commun, 13(1) Art. No. 6255 (2022)
Open Access DOI

Diabetes is a multifactorial disorder characterized by loss or dysfunction of pancreatic β-cells. β-cells are heterogeneous, exhibiting different glucose sensing, insulin secretion and gene expression. They communicate with other endocrine cell types via paracrine signals and between β-cells via gap junctions. Here, we identify the importance of signaling between β-cells via the extracellular signal WNT4. We show heterogeneity in Wnt4 expression, most strikingly in the postnatal maturation period, Wnt4-positive cells, being more mature while Wnt4-negative cells are more proliferative. Knock-out in adult β-cells shows that WNT4 controls the activation of calcium signaling in response to a glucose challenge, as well as metabolic pathways converging to lower ATP/ADP ratios, thereby reducing insulin secretion. These results reveal that paracrine signaling between β-cells is important in addition to gap junctions in controling insulin secretion. Together with previous reports of WNT4 up-regulation in obesity our observations suggest an adaptive insulin response coordinating β-cells.

Shovamayee Maharana^*^#, Stefanie Kretschmer^*^#, Susan Hunger, Xiao Yan, David Kuster, Sofia Traikov, Thomas Zillinger, Marc Gentzel, Shobha Elangovan, Padmanava Dasgupta, Nagaraja Chappidi, Nadja Lucas, Katharina Isabell Maser, Henrike Maatz, Alexander Rapp, Virginie Marchand, Young-Tae Chang, Yuri Motorin, Norbert Hubner, Gunther Hartmann, Anthony Hyman, Simon Alberti, Min Ae Lee-Kirsch^#
SAMHD1 controls innate immunity by regulating condensation of immunogenic self RNA.
Mol Cell, 82(19) 3712-3728 (2022)
DOI

Recognition of pathogen-derived foreign nucleic acids is central to innate immune defense. This requires discrimination between structurally highly similar self and nonself nucleic acids to avoid aberrant inflammatory responses as in the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). How vast amounts of self RNA are shielded from immune recognition to prevent autoinflammation is not fully understood. Here, we show that human SAM-domain- and HD-domain-containing protein 1 (SAMHD1), one of the AGS-causing genes, functions as a single-stranded RNA (ssRNA) 3'exonuclease, the lack of which causes cellular RNA accumulation. Increased ssRNA in cells leads to dissolution of RNA-protein condensates, which sequester immunogenic double-stranded RNA (dsRNA). Release of sequestered dsRNA from condensates triggers activation of antiviral type I interferon via retinoic-acid-inducible gene I-like receptors. Our results establish SAMHD1 as a key regulator of cellular RNA homeostasis and demonstrate that buffering of immunogenic self RNA by condensates regulates innate immune responses.

Ifeanyi Jude Ezeonwumelu, Edurne García-Vidal, Eudald Felip, Maria C Puertas, Bruna Oriol-Tordera, Lucía Gutiérrez-Chamorro, André Gohr, Marta Ruiz-Riol, Marta Massanella, Bonaventura Clotet, Javier Martinez-Picado, Roger Badia, Eva Riveira-Muñoz^#, Ester Ballana^#
IRF7 expression correlates with HIV latency reversal upon specific blockade of immune activation.
Front Immunol, 13 Art. No. 1001068 (2022)
Open Access DOI

The persistence of latent HIV reservoirs allows for viral rebound upon antiretroviral therapy interruption, hindering effective HIV-1 cure. Emerging evidence suggests that modulation of innate immune stimulation could impact viral latency and contribute to the clearing of HIV reservoir. Here, the latency reactivation capacity of a subclass of selective JAK2 inhibitors was characterized as a potential novel therapeutic strategy for HIV-1 cure. Notably, JAK2 inhibitors reversed HIV-1 latency in non-clonal lymphoid and myeloid in vitro models of HIV-1 latency and also ex vivo in CD4+ T cells from ART+ PWH, albeit its function was not dependent on JAK2 expression. Immunophenotypic characterization and whole transcriptomic profiling supported reactivation data, showing common gene expression signatures between latency reactivating agents (LRA; JAK2i fedratinib and PMA) in contrast to other JAK inhibitors, but with significantly fewer affected gene sets in the pathway analysis. In depth evaluation of differentially expressed genes, identified a significant upregulation of IRF7 expression despite the blockade of the JAK-STAT pathway and downregulation of proinflammatory cytokines and chemokines. Moreover, IRF7 expression levels positively correlated with HIV latency reactivation capacity of JAK2 inhibitors and also other common LRAs. Collectively, these results represent a promising step towards HIV eradication by demonstrating the potential of innate immune modulation for reducing the viral reservoir through a novel pathway driven by IRF7.

Aleksandra Spiegel, Chris Lauber, Mandy Bachmann, Anne-Kristin Heninger, Christian Klose, Kai Simons, Mihail Sarov, Mathias J. Gerl
A set of gene knockouts as a resource for global lipidomic changes.
Sci Rep, 12(1) Art. No. 10533 (2022)
Open Access DOI

Enzyme specificity in lipid metabolic pathways often remains unresolved at the lipid species level, which is needed to link lipidomic molecular phenotypes with their protein counterparts to construct functional pathway maps. We created lipidomic profiles of 23 gene knockouts in a proof-of-concept study based on a CRISPR/Cas9 knockout screen in mammalian cells. This results in a lipidomic resource across 24 lipid classes. We highlight lipid species phenotypes of multiple knockout cell lines compared to a control, created by targeting the human safe-harbor locus AAVS1 using up to 1228 lipid species and subspecies, charting lipid metabolism at the molecular level. Lipid species changes are found in all knockout cell lines, however, some are most apparent on the lipid class level (e.g., SGMS1 and CEPT1), while others are most apparent on the fatty acid level (e.g., DECR2 and ACOT7). We find lipidomic phenotypes to be reproducible across different clones of the same knockout and we observed similar phenotypes when two enzymes that catalyze subsequent steps of the long-chain fatty acid elongation cycle were targeted.

Henrike Indrischek, John A Hammer, Anja Machate, Nikolai Hecker, Bogdan Kirilenko, Juliana G. Roscito, Stefan Hans, Caren Norden, Michael Brand^#, Michael Hiller^#
Vision-related convergent gene losses reveal SERPINE3's unknown role in the eye.
Elife, 11 Art. No. e77999 (2022)
Open Access DOI

Despite decades of research, knowledge about the genes that are important for development and function of the mammalian eye and are involved in human eye disorders remains incomplete. During mammalian evolution, mammals that naturally exhibit poor vision or regressive eye phenotypes have independently lost many eye-related genes. This provides an opportunity to predict novel eye-related genes based on specific evolutionary gene loss signatures. Building on these observations, we performed a genome-wide screen across 49 mammals for functionally uncharacterized genes that are preferentially lost in species exhibiting lower visual acuity values. The screen uncovered several genes, including SERPINE3, a putative serine proteinase inhibitor. A detailed investigation of 381 additional mammals revealed that SERPINE3 is independently lost in 18 lineages that typically do not primarily rely on vision, predicting a vision-related function for this gene. To test this, we show that SERPINE3 has the highest expression in eyes of zebrafish and mouse. In the zebrafish retina, serpine3 is expressed in Müller glia cells, a cell type essential for survival and maintenance of the retina. A CRISPR-mediated knockout of serpine3 in zebrafish resulted in alterations in eye shape and defects in retinal layering. Furthermore, two human polymorphisms that are in linkage with SERPINE3 are associated with eye-related traits. Together, these results suggest that SERPINE3 has a role in vertebrate eyes. More generally, by integrating comparative genomics with experiments in model organisms, we show that screens for specific phenotype-associated gene signatures can predict functions of uncharacterized genes.

Maximina H Yun^#, Toshinori Hayashi^#, Andras Simon
Standardized gene and genetic nomenclature for the newt Pleurodeles waltl.
Dev Dyn, 251(6) 911-912 (2022)
Open Access DOI

Tina Schubert, Nicole Reisch, Ronald Naumann, Ilka Reichardt, Dana Landgraf, Friederike Quitter, Shamini Ramkumar Thirumalasetty, Anne-Kristin Heninger, Mihail Sarov, M Peitzsch, Angela Huebner, Katrin Koehler
CYP21A2 Gene Expression in a Humanized 21-Hydroxylase Mouse Model Does Not Affect Adrenocortical Morphology and Function.
J Endocr Soc, 6(6) Art. No. bvac062 (2022)
Open Access DOI

Steroid 21-hydroxylase is an enzyme of the steroid pathway that is involved in the biosynthesis of cortisol and aldosterone by hydroxylation of 17α-hydroxyprogesterone and progesterone at the C21 position. Mutations in CYP21A2, the gene encoding 21-hydroxylase, cause the most frequent form of the autosomal recessive disorder congenital adrenal hyperplasia (CAH). In this study, we generated a humanized 21-hydroxylase mouse model as the first step to the generation of mutant mice with different CAH-causing mutations. We replaced the mouse Cyp21a1 gene with the human CYP21A2 gene using homologous recombination in combination with CRISPR/Cas9 technique. The aim of this study was to characterize the new humanized mouse model. All results described are related to the homozygous animals in comparison with wild-type mice. We show analogous expression patterns of human 21-hydroxylase by the murine promoter and regulatory elements in comparison to murine 21-hydroxylase in wild-type animals. As expected, no Cyp21a1 transcript was detected in homozygous CYP21A2 adrenal glands. Alterations in adrenal gene expression were observed for Cyp11a1, Star, and Cyb11b1. These differences, however, were not pathological. Outward appearance, viability, growth, and fertility were not affected in the humanized CYP21A2 mice. Plasma steroid levels of corticosterone and aldosterone showed no pathological reduction. In addition, adrenal gland morphology and zonation were similar in both the humanized and the wild-type mice. In conclusion, humanized homozygous CYP21A2 mice developed normally and showed no differences in histological analyses, no reduction in adrenal and gonadal gene expression, or in plasma steroids in comparison with wild-type littermates.

Sergey Nurk^*, Sergey Koren^*, Arang Rhie^*, Mikko Rautiainen^*, Andrey V Bzikadze, Alla Mikheenko, Mitchell R Vollger, Nicolas Altemose, Lev Uralsky, Ariel Gershman, Sergey Aganezov, Savannah J Hoyt, Mark Diekhans, Glennis A Logsdon, Michael Alonge, Stylianos E Antonarakis, Matthew Borchers, Gerard G Bouffard, Shelise Y Brooks, Gina V Caldas, Nae-Chyun Chen, Haoyu Cheng, Chen-Shan Chin, William Chow, Leonardo G de Lima, Philip C Dishuck, Richard Durbin, Tatiana Dvorkina, Ian T Fiddes, Giulio Formenti, Robert S Fulton, Arkarachai Fungtammasan, Erik Garrison, Patrick G S Grady, Tina A Graves-Lindsay, Ira M Hall, Nancy F Hansen, Gabrielle A Hartley, Marina Haukness, Kerstin Howe, Michael W Hunkapiller, Chirag Jain, Miten Jain, Erich D Jarvis, Peter Kerpedjiev, Melanie Kirsche, Mikhail Kolmogorov, Jonas Korlach, Milinn Kremitzki, Heng Li, Valerie V Maduro, Tobias Marschall, Ann M McCartney, J McDaniel, Danny E Miller, James C Mullikin, Eugene W Myers, Nathan D Olson, Benedict Paten, Paul Peluso, Pavel Pevzner, David Porubsky, Tamara Potapova, Evgeny I Rogaev, Jeffrey A Rosenfeld, Steven L Salzberg, Valerie A Schneider, Fritz J Sedlazeck, Kishwar Shafin, Colin J Shew, Alaina Shumate, Ying Sims, Arian F A Smit, Daniela C Soto, Ivan Sović, Jessica M Storer, Aaron Streets, Beth A Sullivan, Francoise Thibaud-Nissen, James Torrance, Justin Wagner, Brian Walenz, Aaron M Wenger, Jonathan Wood, Chunlin Xiao, Stephanie M Yan, Alice C Young, Samantha Zarate, Urvashi Surti, Rajiv C McCoy, Megan Y Dennis, Ivan A Alexandrov, Jennifer L Gerton, Rachel J O'Neill, Winston Timp, Justin M Zook, Michael C Schatz, Evan E Eichler^#, Karen H Miga^#, Adam M Phillippy^#
The complete sequence of a human genome.
Science, 376(6588) 44-53 (2022)
DOI

Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion-base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies.

Karin D Prummel, Helena L Crowell, Susan Nieuwenhuize, Eline C Brombacher, Stephan Daetwyler, Charlotte Soneson, Jelena Kresoja-Rakic, Agnese Kocere, Manuel Ronner, Alexander Ernst, Zahra Labbaf, David E Clouthier, Anthony B Firulli, Héctor Sánchez-Iranzo, Sundar Naganathan, Rebecca O'Rourke, Erez Raz, Nadia Mercader, Alexa Burger, Emanuela Felley-Bosco, Jan Huisken, Mark D Robinson, Christian Mosimann
Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma.
Nat Commun, 13(1) Art. No. 1677 (2022)
Open Access DOI

The mesothelium lines body cavities and surrounds internal organs, widely contributing to homeostasis and regeneration. Mesothelium disruptions cause visceral anomalies and mesothelioma tumors. Nonetheless, the embryonic emergence of mesothelia remains incompletely understood. Here, we track mesothelial origins in the lateral plate mesoderm (LPM) using zebrafish. Single-cell transcriptomics uncovers a post-gastrulation gene expression signature centered on hand2 in distinct LPM progenitor cells. We map mesothelial progenitors to lateral-most, hand2-expressing LPM and confirm conservation in mouse. Time-lapse imaging of zebrafish hand2 reporter embryos captures mesothelium formation including pericardium, visceral, and parietal peritoneum. We find primordial germ cells migrate with the forming mesothelium as ventral migration boundary. Functionally, hand2 loss disrupts mesothelium formation with reduced progenitor cells and perturbed migration. In mouse and human mesothelioma, we document expression of LPM-associated transcription factors including Hand2, suggesting re-initiation of a developmental program. Our data connects mesothelium development to Hand2, expanding our understanding of mesothelial pathologies.

Moritz Blumer, Tom Brown, Mariella Bontempo Freitas, Ana Luiza Destro, Juraci A Oliveira, Ariadna E Morales, Tilman Schell, Carola Greve, Martin Pippel, David Jebb, Nikolai Hecker, Alexis-Walid Ahmed, Bogdan Kirilenko, Maddy Foote, Axel Janke, Burton K Lim, Michael Hiller
Gene losses in the common vampire bat illuminate molecular adaptations to blood feeding.
Sci Adv, 8(12) Art. No. eabm6494 (2022)
Open Access DOI

Vampire bats are the only mammals that feed exclusively on blood. To uncover genomic changes associated with this dietary adaptation, we generated a haplotype-resolved genome of the common vampire bat and screened 27 bat species for genes that were specifically lost in the vampire bat lineage. We found previously unknown gene losses that relate to reduced insulin secretion (FFAR1 and SLC30A8), limited glycogen stores (PPP1R3E), and a unique gastric physiology (CTSE). Other gene losses likely reflect the biased nutrient composition (ERN2 and CTRL) and distinct pathogen diversity of blood (RNASE7) and predict the complete lack of cone-based vision in these strictly nocturnal bats (PDE6H and PDE6C). Notably, REP15 loss likely helped vampire bats adapt to high dietary iron levels by enhancing iron excretion, and the loss of CYP39A1 could have contributed to their exceptional cognitive abilities. These findings enhance our understanding of vampire bat biology and the genomic underpinnings of adaptations to blood feeding.

Kang Du, Martin Pippel, Susanne Kneitz, Romain Feron, Irene da Cruz, Sylke Winkler, Brigitta Wilde, Edgar G Avila Luna, Gene Myers, Yann Guiguen, Constantino Macias Garcia, Manfred Schartl
Genome biology of the darkedged splitfin, Girardinichthys multiradiatus, and the evolution of sex chromosomes and placentation.
Genome Res, 32(3) 583-594 (2022)
DOI

Viviparity evolved independently about 150 times in vertebrates and more than 20 times in fish. Several lineages added to the protection of the embryo inside the body of the mother, the provisioning of nutrients, and physiological exchange. This often led to the evolution of a placenta. Among fish, one of the most complex systems serving the function of the placenta is the embryonal trophotaenia/ovarian luminal epithelium of the goodeid fishes. For a better understanding of this feature and others of this group of fishes, high-quality genomic resources are essential. We have sequenced the genome of the darkedged splitfin, Girardinichthys multiradiatus The assembly is chromosome level and includes the X and Y Chromosomes. A large male-specific region on the Y was identified covering 80% of Chromosome 20, allowing some first inferences on the recent origin and a candidate male sex determining gene. Genome-wide transcriptomics uncovered sex-specific differences in brain gene expression with an enrichment for neurosteroidogenesis and testis genes in males. The expression signatures of the splitfin embryonal and maternal placenta showed overlap with homologous tissues including human placenta, the ovarian follicle epithelium of matrotrophic poeciliid fish species and the brood pouch epithelium of the seahorse. Our comparative analyses on the evolution of embryonal and maternal placenta indicate that the evolutionary novelty of maternal provisioning development repeatedly made use of genes that already had the same function in other tissues. In this way, preexisting modules are assembled and repurposed to provide the molecular changes for this novel trait.

Dora Tang
Cell scientist to watch - Dora Tang.
J Cell Sci, 135(5) Art. No. jcs259851 (2022)
DOI

Dora Tang studied chemistry and then completed her PhD in membrane physics at Imperial College London, UK, under the supervision of John Seddon and Richard Templer. She then received Knowledge Transfer Secondment funding from Imperial College and worked for a year at Diamond Light Source, Oxfordshire, the UK's national synchrotron science facility. In 2011, Dora joined the lab of Stephen Mann at the University of Bristol, where she worked in the areas of the origin of life and bottom-up synthetic biology. Her research there included developing a hybrid protocell model based on the self-assembly of fatty acid membrane on coacervate microdroplets, as well as showing that coacervates support cell-free gene expression and building communication networks between two different populations of synthetic cells. In 2016, she started her own group at the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden, Germany, as part of the MaxSynBio consortium. Her research aims to understand the chemical and physical processes that drive molecular organisation in lipids, polymers and proteins in order to rationally control their self-assembly and understand how molecular ensembles make life.

Franziska Wagner^*, Irina Ruf^*, Thomas Lehmann^*, Rebecca Hofmann, Sylvia Ortmann, Christian Schiffmann, Michael Hiller, Clara Stefen, Heiko Stuckas
Reconstruction of evolutionary changes in fat and toxin consumption reveals associations with gene losses in mammals: A case study for the lipase inhibitor PNLIPRP1 and the xenobiotic receptor NR1I3.
J Evol Biol, 35(2) 225-239 (2022)
Open Access DOI

The inactivation of ancestral protein-coding genes (gene loss) can be associated with phenotypic modifications. Within placental mammals, repeated losses of PNLIPRP1 (gene inhibiting fat digestion) occurred preferentially in strictly herbivorous species, whereas repeated NR1I3 losses (gene involved in detoxification) occurred preferentially in strictly carnivorous species. It was hypothesized that lower fat contents of herbivorous diets and lower toxin contents of carnivorous diets cause relaxed selection pressure on these genes, resulting in the accumulation of mutations and ultimately to convergent gene losses. However, because herbivorous and carnivorous diets differ vastly in their composition, a fine-grained analysis is required for hypothesis testing. We generated a trait matrix recording diet and semi-quantitative estimates of fat and toxin consumption for 52 placental species. By including data from 31 fossil taxa, we reconstructed the ancestral diets in major lineages (grundplan reconstruction). We found support that PNLIPRP1 loss is primarily associated with low levels of fat intake and not simply with herbivory/carnivory. In particular, PNLIPRP1 loss also occurred in carnivorous lineages feeding on a fat-poor diet, suggesting that the loss of this gene may be beneficial for occupying ecological niches characterized by fat-poor food resources. Similarly, we demonstrated that carnivorous species are indeed less exposed to diet-related toxins, suggesting that the loss of NR1I3 and related genes (NR1I2 and UGT1A6) resulted from relaxed selection pressure. This study illustrates the need of detailed phenotype studies to obtain a deeper understanding of factors underlying gene losses and to progress in understanding genomic causes of phenotypic variation in mammals.

Giulia Furesi, Antonio Domingues, Dimitra Alexopoulou, Andreas Dahl, Matthias Hackl, Johannes R Schmidt, Stefan Kalkhof, Thomas Kurth, Hanna Taipaleenmäki, Stefanie Conrad, Christine Hofbauer, Martina Rauner, Lorenz C Hofbauer
Exosomal miRNAs from Prostate Cancer Impair Osteoblast Function in Mice.
Int J Mol Sci, 23(3) Art. No. 1285 (2022)
Open Access DOI

Prostate cancer (PCa) is the most frequent malignancy in older men with a high propensity for bone metastases. Characteristically, PCa causes osteosclerotic lesions as a result of disrupted bone remodeling. Extracellular vesicles (EVs) participate in PCa progression by conditioning the pre-metastatic niche. However, how EVs mediate the cross-talk between PCa cells and osteoprogenitors in the bone microenvironment remains poorly understood. We found that EVs derived from murine PCa cell line RM1-BM increased metabolic activity, vitality, and cell proliferation of osteoblast precursors by >60%, while significantly impairing mineral deposition (-37%). The latter was further confirmed in two complementary in vivo models of ossification. Accordingly, gene and protein set enrichments of osteoprogenitors exposed to EVs displayed significant downregulation of osteogenic markers and upregulation of proinflammatory factors. Additionally, transcriptomic profiling of PCa-EVs revealed the abundance of three microRNAs, miR-26a-5p, miR-27a-3p, and miR-30e-5p involved in the suppression of BMP-2-induced osteogenesis in vivo, suggesting the critical role of these EV-derived miRNAs in PCa-mediated suppression of osteoblast activity. Taken together, our results indicate the importance of EV cargo in cancer-bone cross-talk in vitro and in vivo and suggest that exosomal miRNAs may contribute to the onset of osteosclerotic bone lesions in PCa.

Aditya Saxena, Virag Sharma, Pushpanathan Muthuirulan, Stanley J Neufeld, Mai P Tran, Haydee L Gutierrez, Kevin D Chen, Joel M Erberich, Amanda Birmingham, Terence D Capellini, John Cobb, Michael Hiller, Kimberly L Cooper
Interspecies transcriptomics identify genes that underlie disproportionate foot growth in jerboas.
Curr Biol, 32(2) 289-303 (2022)
DOI

Despite the great diversity of vertebrate limb proportion and our deep understanding of the genetic mechanisms that drive skeletal elongation, little is known about how individual bones reach different lengths in any species. Here, we directly compare the transcriptomes of homologous growth cartilages of the mouse (Mus musculus) and bipedal jerboa (Jaculus jaculus), the latter of which has "mouse-like" arms but extremely long metatarsals of the feet. Intersecting gene-expression differences in metatarsals and forearms of the two species revealed that about 10% of orthologous genes are associated with the disproportionately rapid elongation of neonatal jerboa feet. These include genes and enriched pathways not previously associated with endochondral elongation as well as those that might diversify skeletal proportion in addition to their known requirements for bone growth throughout the skeleton. We also identified transcription regulators that might act as "nodes" for sweeping differences in genome expression between species. Among these, Shox2, which is necessary for proximal limb elongation, has gained expression in jerboa metatarsals where it has not been detected in other vertebrates. We show that Shox2 is sufficient to increase mouse distal limb length, and a nearby putative cis-regulatory region is preferentially accessible in jerboa metatarsals. In addition to mechanisms that might directly promote growth, we found evidence that jerboa foot elongation may occur in part by de-repressing latent growth potential. The genes and pathways that we identified here provide a framework to understand the modular genetic control of skeletal growth and the remarkable malleability of vertebrate limb proportion.

David Thomas Gonzales, Naresh Yandrapalli, Tom Robinson, Christoph Zechner^#, T-Y Dora Tang^#
Cell-Free Gene Expression Dynamics in Synthetic Cell Populations.
ACS Synth Biol, 11(1) 205-215 (2022)
Open Access DOI

The ability to build synthetic cellular populations from the bottom-up provides the groundwork to realize minimal living tissues comprising single cells which can communicate and bridge scales into multicellular systems. Engineered systems made of synthetic micron-sized compartments and integrated reaction networks coupled with mathematical modeling can facilitate the design and construction of complex and multiscale chemical systems from the bottom-up. Toward this goal, we generated populations of monodisperse liposomes encapsulating cell-free expression systems (CFESs) using double-emulsion microfluidics and quantified transcription and translation dynamics within individual synthetic cells of the population using a fluorescent Broccoli RNA aptamer and mCherry protein reporter. CFE dynamics in bulk reactions were used to test different coarse-grained resource-limited gene expression models using model selection to obtain transcription and translation rate parameters by likelihood-based parameter estimation. The selected model was then applied to quantify cell-free gene expression dynamics in populations of synthetic cells. In combination, our experimental and theoretical approaches provide a statistically robust analysis of CFE dynamics in bulk and monodisperse synthetic cell populations. We demonstrate that compartmentalization of CFESs leads to different transcription and translation rates compared to bulk CFE and show that this is due to the semipermeable lipid membrane that allows the exchange of materials between the synthetic cells and the external environment.

Zahra Ghezelayagh^*, Mahsa Zabihi^*, Ibrahim Zarkesh, Carla A C Gonçalves, Michael Larsen, Newsha Hagh-Parast, Mohammad Pakzad, Massoud Vosough, Babak Arjmand, Hossein Baharvand, Banafshé Larijani, Anne Grapin-Botton, Hamid Reza Aghayan^#, Yaser Tahamtani^#
Improved Differentiation of hESC-Derived Pancreatic Progenitors by Using Human Fetal Pancreatic Mesenchymal Cells in a Micro-scalable Three-Dimensional Co-culture System.
Stem Cell Rev Rep, 18(1) 360-377 (2022)
DOI

Mesenchymal cells of diverse origins differ in gene and protein expression besides producing varying effects on their organ-matched epithelial cells' maintenance and differentiation capacity. Co-culture with rodent's tissue-specific pancreatic mesenchyme accelerates proliferation, self-renewal, and differentiation of pancreatic epithelial progenitors. Therefore, in our study, the impact of three-dimensional (3D) co-culture of human fetal pancreatic-derived mesenchymal cells (hFP-MCs) with human embryonic stem cell-derived pancreatic progenitors (hESC-PPs) development towards endocrine and beta cells was assessed. Besides, the ability to maintain scalable cultures combining hFP-MCs and hESC-PPs was investigated. hFP-MCs expressed many markers in common with bone marrow-derived mesenchymal stem cells (BM-MSCs). However, they showed higher expression of DESMIN compared to BM-MSCs. After co-culture of hESC-PPs with hFP-MCs, the pancreatic progenitor (PP) spheroids generated in Matrigel had higher expression of NGN3 and INSULIN than BM-MSCs co-culture group, which shows an inductive impact of pancreatic mesenchyme on hESC-PPs beta-cells maturation. Pancreatic aggregates generated by forced aggregation through scalable AggreWell system showed similar features compared to the spheroids. These aggregates, a combination of hFP-MCs and hESC-PPs, can be applied as an appropriate tool for assessing endocrine-niche interactions and developmental processes by mimicking the pancreatic tissue.

Yuhan Wang, Mark Eddison, Greg Fleishman, Martin Weigert, Shengjin Xu, Tim Wang, Konrad Rokicki, Cristian Goina, Fredrick E Henry, Andrew L Lemire, Uwe Schmidt, Hui Yang, Karel Svoboda, Eugene W Myers, Stephan Saalfeld, Wyatt Korff, Scott M Sternson^#, Paul W Tillberg^#
EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization.
Cell, 184(26) 6361-6377 (2021)
Open Access DOI

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 μm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.

Benjamin Seelbinder, Manavi Jain, Elena Erben, Sergei Klykov, Iliya D. Stoev, Moritz Kreysing
Non-invasive Chromatin Deformation and Measurement of Differential Mechanical Properties in the Nucleus.
bioRxiv, Art. No. https://doi.org/10.1101/2021.12.15.472786 (2021)
Open Access PDF DOI

The nucleus is highly organized to facilitate coordinated gene transcription. Measuring the rheological properties of the nucleus and its sub-compartments will be crucial to understand the principles underlying nuclear organization. Here, we show that strongly localized temperature gradients (approaching 1°C /μm) can lead to substantial intra-nuclear chromatin displacements (>1 μm), while nuclear area and lamina shape remain unaffected. Using particle image velocimetry (PIV), intra-nuclear displacement fields can be calculated and converted into spatio-temporally resolved maps of various strain components. Using this approach, we show that chromatin displacements are highly reversible, indicating that elastic contributions are dominant in maintaining nuclear organization on the time scale of seconds. In genetically inverted nuclei, centrally compacted heterochromatin displays high resistance to deformation, giving a rigid, solid-like appearance. Correlating spatially resolved strain maps with fluorescent reporters in conventional interphase nuclei reveals that various nuclear compartments possess distinct mechanical identities. Surprisingly, both densely and loosely packed chromatin showed high resistance to deformation, compared to medium dense chromatin. Equally, nucleoli display particularly high rigidity and strong local anchoring to heterochromatin. Our results establish how localized temperature gradients can be used to drive nuclear compartments out of mechanical equilibrium to obtain spatial maps of their material responses.

Diana D Moreno Santillán, Tanya M Lama, Yocelyn T Gutierrez Guerrero, Alexis M Brown, Paul Donat, Huabin Zhao, Stephen J Rossiter, Laurel R Yohe, Joshua H Potter, Emma Teeling, Sonja Vernes, Kalina T J Davies, Eugene W Myers, Graham M Hughes, Zixia Huang, Federico Hoffmann, Angelique P Corthals, David A Ray^#, Liliana M Dávalos^#
Large-scale genome sampling reveals unique immunity and metabolic adaptations in bats.
Mol Ecol, 30(23) 6449-6467 (2021)
DOI

Comprising more than 1,400 species, bats possess adaptations unique among mammals including powered flight, unexpected longevity, and extraordinary immunity. Some of the molecular mechanisms underlying these unique adaptations includes DNA repair, metabolism and immunity. However, analyses have been limited to a few divergent lineages, reducing the scope of inferences on gene family evolution across the Order Chiroptera. We conducted an exhaustive comparative genomic study of 37 bat species, one generated in this study, encompassing a large number of lineages, with a particular emphasis on multi-gene family evolution across immune and metabolic genes. In agreement with previous analyses, we found lineage-specific expansions of the APOBEC3 and MHC-I gene families, and loss of the proinflammatory PYHIN gene family. We inferred more than 1,000 gene losses unique to bats, including genes involved in the regulation of inflammasome pathways such as epithelial defence receptors, the natural killer gene complex and the interferon-gamma induced pathway. Gene set enrichment analyses revealed genes lost in bats are involved in defence response against pathogen-associated molecular patterns and damage-associated molecular patterns. Gene family evolution and selection analyses indicate bats have evolved fundamental functional differences compared to other mammals in both innate and adaptive immune system, with the potential to enhance antiviral immune response while dampening inflammatory signalling. In addition, metabolic genes have experienced repeated expansions related to convergent shifts to plant-based diets. Our analyses support the hypothesis that, in tandem with flight, ancestral bats had evolved a unique set of immune adaptations whose functional implications remain to be explored.

Dipannita Sarkar, Mohammad Shariq, Deepanjali Dwivedi, Nirmal Krishnan, Ronald Naumann, Upinder Singh Bhalla, Hiyaa Singhee Ghosh
Adult brain neurons require continual expression of the schizophrenia-risk gene Tcf4 for structural and functional integrity.
Transl Psychiatry, 11(1) Art. No. 494 (2021)
Open Access DOI

The schizophrenia-risk gene Tcf4 has been widely studied in the context of brain development using mouse models of haploinsufficiency, in utero knockdown and embryonic deletion. However, Tcf4 continues to be abundantly expressed in adult brain neurons where its functions remain unknown. Given the importance of Tcf4 in psychiatric diseases, we investigated its role in adult neurons using cell-specific deletion and genetic tracing in adult animals. Acute loss of Tcf4 in adult excitatory neurons in vivo caused hyperexcitability and increased dendritic complexity of neurons, effects that were distinct from previously observed effects in embryonic-deficiency models. Interestingly, transcriptomic analysis of genetically traced adult-deleted FACS-sorted Tcf4-knockout neurons revealed that Tcf4 targets in adult neurons are distinct from those in the embryonic brain. Meta-analysis of the adult-deleted neuronal transcriptome from our study with the existing datasets of embryonic Tcf4 deficiencies revealed plasma membrane and ciliary genes to underlie Tcf4-mediated structure-function regulation specifically in adult neurons. The profound changes both in the structure and excitability of adult neurons upon acute loss of Tcf4 indicates that proactive regulation of membrane-related processes underlies the functional and structural integrity of adult neurons. These findings not only provide insights for the functional relevance of continual expression of a psychiatric disease-risk gene in the adult brain but also identify previously unappreciated gene networks underpinning mature neuronal regulation during the adult lifespan.

Cemil Kerimoglu^*, Linh Pham^*, Anton B Tonchev^*, M Sadman Sakib^*, Yuanbin Xie, Godwin Sokpor, Pauline Antonie Ulmke, Lalit Kaurani, Eman Abbas, Huong Nguyen, Joachim Rosenbusch, Alexandra Michurina, Vincenzo Capece, Meglena Angelova, Nenad Maricic, Beate Brand-Saberi, Miriam Esgleas, Mareike Albert, Radoslav Minkov, Emil Kovachev, Ulrike Teichmann, Rho H Seong, Wieland Huttner, Huu Phuc Nguyen, Anastassia Stoykova, Jochen F Staiger, Andre Fischer^#, Tran Tuoc^#
H3 acetylation selectively promotes basal progenitor proliferation and neocortex expansion.
Sci Adv, 7(38) Art. No. eabc6792 (2021)
Open Access DOI

Increase in the size of human neocortex―acquired in evolution―accounts for the unique cognitive capacity of humans. This expansion reflects the evolutionarily enhanced proliferative ability of basal progenitors (BPs), including the basal radial glia and basal intermediate progenitors (bIPs) in mammalian cortex, which may have been acquired through epigenetic alterations in BPs. However, how the epigenome in BPs differs across species is not known. Here, we report that histone H3 acetylation is a key epigenetic regulation in bIP amplification and cortical expansion. Through epigenetic profiling of sorted bIPs, we show that histone H3 lysine 9 acetylation (H3K9ac) is low in murine bIPs and high in human bIPs. Elevated H3K9ac preferentially increases bIP proliferation, increasing the size and folding of the normally smooth mouse neocortex. H3K9ac drives bIP amplification by increasing expression of the evolutionarily regulated gene, Trnp1, in developing cortex. Our findings demonstrate a previously unknown mechanism that controls cortical architecture.

Stefano Suzzi, Reiner Ahrendt, Stefan Hans, Svetlana A Semenova, Avinash Chekuru, Paul Wirsching, Volker Kroehne, Saygın Bilican, Shady Sayed, Sylke Winkler, Sandra Spieß, Anja Machate, Jan Kaslin, Pertti Panula, Michael Brand
Deletion of lrrk2 causes early developmental abnormalities and age-dependent increase of monoamine catabolism in the zebrafish brain.
PLoS Genet, 17(9) Art. No. 1009794 (2021)
Open Access DOI

LRRK2 gain-of-function is considered a major cause of Parkinson's disease (PD) in humans. However, pathogenicity of LRRK2 loss-of-function in animal models is controversial. Here we show that deletion of the entire zebrafish lrrk2 locus elicits a pleomorphic transient brain phenotype in maternal-zygotic mutant embryos (mzLrrk2). In contrast to lrrk2, the paralog gene lrrk1 is virtually not expressed in the brain of both wild-type and mzLrrk2 fish at different developmental stages. Notably, we found reduced catecholaminergic neurons, the main target of PD, in specific cell populations in the brains of mzLrrk2 larvae, but not adult fish. Strikingly, age-dependent accumulation of monoamine oxidase (MAO)-dependent catabolic signatures within mzLrrk2 brains revealed a previously undescribed interaction between LRRK2 and MAO biological activities. Our results highlight mzLrrk2 zebrafish as a tractable tool to study LRRK2 loss-of-function in vivo, and suggest a link between LRRK2 and MAO, potentially of relevance in the prodromic stages of PD.

Oliver J Bower^*, Afshan McCarthy^*, Rebecca A Lea, Gregorio Alanis-Lobato, Jasmin Zohren, Claudia Gerri, James M A Turner, Kathy K Niakan
Generating CRISPR-Cas9-Mediated Null Mutations and Screening Targeting Efficiency in Human Pluripotent Stem Cells.
Curr Protoc, 1(8) Art. No. e232 (2021)
Open Access DOI

CRISPR-Cas9 mutagenesis facilitates the investigation of gene function in a number of developmental and cellular contexts. Human pluripotent stem cells (hPSCs), either embryonic or induced, are a tractable cellular model to investigate molecular mechanisms involved in early human development and cell fate decisions. hPSCs also have broad potential in regenerative medicine to model, investigate, and ameliorate diseases. Here, we provide an optimized protocol for efficient CRISPR-Cas9 genome editing of hPSCs to investigate the functional role of genes by engineering null mutations. We emphasize the importance of screening single guide RNAs (sgRNAs) to identify those with high targeting efficiency for generation of clonally derived null mutant hPSC lines. We provide important considerations for targeting genes that may have a role in hPSC maintenance. We also present methods to evaluate the on-target mutation spectrum and unintended karyotypic changes. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Selecting and ligating sgRNAs into expression plasmids Basic Protocol 2: Validation of sgRNA via in vitro transcription and cleavage assay Basic Protocol 3: Nucleofection of primed human embryonic stem cells Basic Protocol 4: MiSeq analysis of indel mutations Basic Protocol 5: Single cell cloning of targeted hPSCs Basic Protocol 6: Karyotyping of targeted hPSCs.

Elisa De Crignis, Tanvir Hossain, Shahla Romal, Fabrizia Carofiglio, Panagiotis Moulos, Mir Mubashir Khalid, Shringar Rao, Ameneh Bazrafshan, Monique Ma Verstegen, Farzin Pourfarzad, Christina Koutsothanassis, Helmuth Gehart, Tsung Wai Kan, Robert-Jan Palstra, Charles Boucher, Jan N M IJzermans, Meritxell Huch, Sylvia F Boj, Robert R G Vries, Hans Clevers, Luc J W van der Laan, Pantelis Hatzis, Tokameh Mahmoudi
Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma.
Elife, 10 Art. No. e60747 (2021)
Open Access DOI

The molecular events that drive Hepatitis B virus (HBV)-mediated transformation and tumorigenesis have remained largely unclear, due to the absence of a relevant primary model system. Here we propose the use of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. We first describe a primary ex vivo HBV-infection model derived from healthy donor liver organoids after challenge with recombinant virus or HBV-infected patient serum. HBV infected organoids produced cccDNA, HBeAg, expressed intracellular HBV RNA and proteins, and produced infectious HBV. This ex vivo HBV infected primary differentiated hepatocyte organoid platform was amenable to drug screening for both anti-HBV activity as well as for drug-induced toxicity. We also studied HBV replication in transgenically modified organoids; liver organoids exogenously overexpressing the HBV receptor NTCP after lentiviral transduction were not more susceptible to HBV, suggesting the necessity for additional host factors for efficient infection. We also generated transgenic organoids harboring integrated HBV, representing a long-term culture system also suitable for viral production and the study of HBV transcription. Finally, we generated HBV-infected patient-derived liver organoids from non-tumor cirrhotic tissue of explants from liver transplant patients. Interestingly, transcriptomic analysis of patient-derived liver organoids indicated the presence of an aberrant early cancer gene signature, which clustered with the HCC cohort on the TCGA LIHC dataset and away from healthy liver tissue, and may provide invaluable novel biomarkers for the development of HCC and surveillance in HBV infected patients.

Lei Xing, Agnieszka Kubik-Zahorodna, Takashi Namba, Anneline Pinson, Marta Florio, Jan Prochazka, Mihail Sarov, Radislav Sedlacek, Wieland Huttner
Expression of human-specific ARHGAP11B in mice leads to neocortex expansion and increased memory flexibility.
EMBO J, 40(13) Art. No. 107093 (2021)
Open Access DOI

Neocortex expansion during human evolution provides a basis for our enhanced cognitive abilities. Yet, which genes implicated in neocortex expansion are actually responsible for higher cognitive abilities is unknown. The expression of human-specific ARHGAP11B in embryonic/foetal mouse, ferret and marmoset neocortex was previously found to promote basal progenitor proliferation, upper-layer neuron generation and neocortex expansion during development, features commonly thought to contribute to increased cognitive abilities. However, a key question is whether this phenotype persists into adulthood and if so, whether cognitive abilities are indeed increased. Here, we generated a transgenic mouse line with physiological ARHGAP11B expression that exhibits increased neocortical size and upper-layer neuron numbers persisting into adulthood. Adult ARHGAP11B-transgenic mice showed altered neurobehaviour, notably increased memory flexibility and a reduced anxiety level. Our data are consistent with the notion that neocortex expansion by ARHGAP11B, a gene implicated in human evolution, underlies some of the altered neurobehavioural features observed in the transgenic mice, such as the increased memory flexibility, a neocortex-associated trait, with implications for the increase in cognitive abilities during human evolution.

Emiel P C van der Vorst^*^#, Mario A A Pepe^*, Linsey J F Peters, Markus Haberbosch, Yvonne Jansen, Ronald Naumann, Georgios T Stathopoulos, Christian Weber, Kiril Bidzhekov^#
Transcriptome signature of miRNA-26b KO mouse model suggests novel targets.
BMC Genom Data, 22(1) Art. No. 23 (2021)
Open Access DOI

MicroRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. One of the miRNAs that has been shown to play a role in various pathologies like cancer, neurological disorders and cardiovascular diseases is miRNA-26b. However, these studies only demonstrated rather ambiguous associations without revealing a causal relationship. Therefore, the aim of this study is to establish and validate a mouse model which enables the elucidation of the exact role of miRNA-26b in various pathologies.

Anneline Pinson^#, Wieland Huttner^#
Neocortex expansion in development and evolution-from genes to progenitor cell biology.
Curr Opin Cell Biol, 73 9-18 (2021)
DOI

The evolutionary expansion of the neocortex, the seat of higher cognitive functions in humans, is primarily due to an increased and prolonged proliferation of neural progenitor cells during development. Basal progenitors, and in particular basal radial glial cells, are thought to have a key role in the increased generation of neurons that constitutes a foundation of neocortex expansion. Recent studies have identified primate-specific and human-specific genes and changes in gene expression that promote increased proliferative capacity of cortical progenitors. In many cases, the cell biological basis underlying this increase has been uncovered. Model systems such as mouse, ferret, nonhuman primates, and cerebral organoids have been used to establish the relevance of these genes for neocortex expansion.

Michael Heide^#, Wieland Huttner^#
Human-Specific Genes, Cortical Progenitor Cells, and Microcephaly.
Cells, 10(5) Art. No. 1209 (2021)
Open Access DOI

Over the past few years, human-specific genes have received increasing attention as potential major contributors responsible for the 3-fold difference in brain size between human and chimpanzee. Accordingly, mutations affecting these genes may lead to a reduction in human brain size and therefore, may cause or contribute to microcephaly. In this review, we will concentrate, within the brain, on the cerebral cortex, the seat of our higher cognitive abilities, and focus on the human-specific gene ARHGAP11B and on the gene family comprising the three human-specific genes NOTCH2NLA, -B, and -C. These genes are thought to have significantly contributed to the expansion of the cerebral cortex during human evolution. We will summarize the evolution of these genes, as well as their expression and functional role during human cortical development, and discuss their potential relevance for microcephaly. Furthermore, we will give an overview of other human-specific genes that are expressed during fetal human cortical development. We will discuss the potential involvement of these genes in microcephaly and how these genes could be studied functionally to identify a possible role in microcephaly.

Giulio Formenti^#, Arang Rhie, Jennifer Balacco, Bettina Haase, Jacquelyn Mountcastle, Olivier Fedrigo, Samara Brown, Marco Rosario Capodiferro, Farooq O Al-Ajli, Roberto Ambrosini, Peter Houde, Sergey Koren, Karen Oliver, Michelle Smith, Jason Skelton, Emma Betteridge, Jale Dolucan, Craig Corton, Iliana Bista, James Torrance, Alan Tracey, Jonathan Wood, Marcela Uliano-Silva, Kerstin Howe, Shane A McCarthy, Sylke Winkler, Woori Kwak, Jonas Korlach, Arkarachai Fungtammasan, Daniel Fordham, Vania Costa, Simon Mayes, Matteo Chiara, David S Horner, Eugene W Myers, Richard Durbin, Alessandro Achilli, Edward L Braun, Adam M Phillippy, Erich D Jarvis^#, Erich D null
Complete vertebrate mitogenomes reveal widespread repeats and gene duplications.
Genome Biol, 22(1) Art. No. 120 (2021)
Open Access DOI

Modern sequencing technologies should make the assembly of the relatively small mitochondrial genomes an easy undertaking. However, few tools exist that address mitochondrial assembly directly.

Arang Rhie, Shane A McCarthy, Olivier Fedrigo, Joana Damas, Giulio Formenti, Sergey Koren, Marcela Uliano-Silva, William Chow, Arkarachai Fungtammasan, Juwan Kim, Chul Lee, Byung June Ko, Mark Chaisson, Gregory L Gedman, Lindsey J Cantin, Francoise Thibaud-Nissen, Leanne Haggerty, Iliana Bista, Michelle Smith, Bettina Haase, Jacquelyn Mountcastle, Sylke Winkler, Sadye Paez, Jonathon Howard, Sonja Vernes, Tanya M Lama, Frank Grutzner, Wesley C Warren, Christopher N Balakrishnan, Dave Burt, Julia M George, Matthew T Biegler, David Iorns, Andrew Digby, Daryl Eason, Bruce Robertson, Taylor Edwards, Mark Wilkinson, George Turner, Axel Meyer, Andreas F Kautt, Paolo Franchini, H William Detrich, Hannes Svardal, Maximilian Wagner, Gavin J P Naylor, Martin Pippel, Milan Malinsky, Mark Mooney, Maria Simbirsky, Brett T Hannigan, Trevor Pesout, Marlys Houck, Ann Misuraca, Sarah B Kingan, Richard J Hall, Zev Kronenberg, Ivan Sović, Christopher Dunn, Zemin Ning, Alex R Hastie, Joyce Lee, Siddarth Selvaraj, Richard E Green, Nicholas H Putnam, Ivo Gut, Jay Ghurye, Erik Garrison, Ying Sims, Joanna Collins, Sarah Pelan, James Torrance, Alan Tracey, Jonathan Wood, Robel E Dagnew, Dengfeng Guan, Sarah E London, David F Clayton, Claudio V Mello, Samantha R Friedrich, Peter V Lovell, Ekaterina Osipova, Farooq O Al-Ajli, Simona Secomandi, Heebal Kim, Constantina Theofanopoulou, Michael Hiller, Yang Zhou, Robert S Harris, Kateryna D Makova, Paul Medvedev, Jinna Hoffman, Patrick Masterson, Karen Clark, Fergal Martin, Kerstin Howe^#, Paul Flicek, Brian Walenz, Woori Kwak, Hiram Clawson, Mark Diekhans, Luis Nassar, Benedict Paten, Robert H S Kraus, Andrew J Crawford, M Thomas P Gilbert, Guojie Zhang, Byrappa Venkatesh, Robert F Murphy, Klaus-Peter Koepfli, Beth Shapiro, Warren E Johnson, Federica Di Palma, Tomas Marques-Bonet, Emma Teeling, Tandy Warnow, Jennifer Marshall Graves, Oliver A Ryder, David Haussler, Stephen J O'Brien, Jonas Korlach, Harris A Lewin, Kerstin Howe^#, Eugene W Myers^#, Richard Durbin^#, Adam M Phillippy^#, Erich D Jarvis^#
Towards complete and error-free genome assemblies of all vertebrate species.
Nature, 592(7856) 737-746 (2021)
Open Access DOI

High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1-4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

Krishan Gupta, Manan Lalit, Aditya Biswas, Chad D Sanada, Cassandra Greene, Kyle Hukari, Ujjwal Maulik, Sanghamitra Bandyopadhyay, Naveen Ramalingam, Gaurav Ahuja, Abhik Ghosh, Debarka Sengupta
Modeling expression ranks for noise-tolerant differential expression analysis of scRNA-seq data.
Genome Res, 31(4) 689-697 (2021)
DOI

Systematic delineation of complex biological systems is an ever-challenging and resource-intensive process. Single-cell transcriptomics allows us to study cell-to-cell variability in complex tissues at an unprecedented resolution. Accurate modeling of gene expression plays a critical role in the statistical determination of tissue-specific gene expression patterns. In the past few years, considerable efforts have been made to identify appropriate parametric models for single-cell expression data. The zero-inflated version of Poisson/negative binomial and log-normal distributions have emerged as the most popular alternatives owing to their ability to accommodate high dropout rates, as commonly observed in single-cell data. Although the majority of the parametric approaches directly model expression estimates, we explore the potential of modeling expression ranks, as robust surrogates for transcript abundance. Here we examined the performance of the discrete generalized beta distribution (DGBD) on real data and devised a Wald-type test for comparing gene expression across two phenotypically divergent groups of single cells. We performed a comprehensive assessment of the proposed method to understand its advantages compared with some of the existing best-practice approaches. We concluded that besides striking a reasonable balance between Type I and Type II errors, ROSeq, the proposed differential expression test, is exceptionally robust to expression noise and scales rapidly with increasing sample size. For wider dissemination and adoption of the method, we created an R package called ROSeq and made it available on the Bioconductor platform.

Andreas Lackner^*, Robert Sehlke^*, Marius Garmhausen^*, Giuliano Giuseppe Stirparo^*, Michelle Huth, Fabian Titz-Teixeira, Petra van der Lelij, Julia Ramesmayer, Henry F Thomas, Meryem Ralser, Laura Santini, Elena Galimberti, Mihail Sarov, A F Stewart, Austin Smith, Andreas Beyer, Martin Leeb
Cooperative genetic networks drive embryonic stem cell transition from naïve to formative pluripotency.
EMBO J, 40(8) Art. No. e105776 (2021)
Open Access DOI

In the mammalian embryo, epiblast cells must exit the naïve state and acquire formative pluripotency. This cell state transition is recapitulated by mouse embryonic stem cells (ESCs), which undergo pluripotency progression in defined conditions in vitro. However, our understanding of the molecular cascades and gene networks involved in the exit from naïve pluripotency remains fragmentary. Here, we employed a combination of genetic screens in haploid ESCs, CRISPR/Cas9 gene disruption, large-scale transcriptomics and computational systems biology to delineate the regulatory circuits governing naïve state exit. Transcriptome profiles for 73 ESC lines deficient for regulators of the exit from naïve pluripotency predominantly manifest delays on the trajectory from naïve to formative epiblast. We find that gene networks operative in ESCs are also active during transition from pre- to post-implantation epiblast in utero. We identified 496 naïve state-associated genes tightly connected to the in vivo epiblast state transition and largely conserved in primate embryos. Integrated analysis of mutant transcriptomes revealed funnelling of multiple gene activities into discrete regulatory modules. Finally, we delineate how intersections with signalling pathways direct this pivotal mammalian cell state transition.

Szabolcs Horvát^*, Adeeba Fathima^*, Stefan Goerlich, Michael Schlierf^#, Carl D. Modes^#, Nils Kroeger^#
Computational analysis of the effects of nitrogen source and sin1 knockout on biosilica morphology in the model diatom Thalassiosira pseudonana.
Discover Materials, 1 Art. No. 8 (2021)
Open Access DOI

Morphogenesis of the silica based cell walls of diatoms, a large group of microalgae, is a paradigm for the self-assembly of complex 3D nano- and microscale patterned inorganic materials. In recent years, loss-of-function studies using genetic manipulation were successfully applied for the identification of genes that guide silica morphogenesis in diatoms. These studies revealed that the loss of one gene can affect multiple morphological parameters, and the morphological changes can be rather subtle being blurred by natural variations in morphology even within the same clone. Both factors severely hamper the identification of morphological mutants using subjective by-eye inspection of electron micrographs. Here we have developed automated image analysis for objectively quantifying the morphology of ridge networks and pore densities from numerous electron micrographs of diatom biosilica. This study demonstrated differences in ridge network morphology and pore density in diatoms growing on ammonium rather than nitrate as the sole nitrogen source. Fur- thermore, it revealed shortcomings in previous by-eye evaluation of the biosilica phenotype of the silicanin-1 knockout mutant. We anticipate that the computational methods established in the present work will be invaluable for unraveling genotype–phenotype correlations in diatom biosilica morphogenesis.

Anne Seifert, Hauke Drechsler, Julia Japtok, Till Korten, Stefan Diez^#, Andreas Hermann^#
The ALS-Associated FUS (P525L) Variant Does Not Directly Interfere with Microtubule-Dependent Kinesin-1 Motility.
Int J Mol Sci, 22(5) Art. No. 2422 (2021)
Open Access DOI

Deficient intracellular transport is a common pathological hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the fused-in-sarcoma (FUS) gene are one of the most common genetic causes for familial ALS. Motor neurons carrying a mutation in the nuclear localization sequence of FUS (P525L) show impaired axonal transport of several organelles, suggesting that mislocalized cytoplasmic FUS might directly interfere with the transport machinery. To test this hypothesis, we studied the effect of FUS on kinesin-1 motility in vitro. Using a modified microtubule gliding motility assay on surfaces coated with kinesin-1 motor proteins, we showed that neither recombinant wildtype and P525L FUS variants nor lysates from isogenic ALS-patient-specific iPSC-derived spinal motor neurons expressing those FUS variants significantly affected gliding velocities. We hence conclude that during ALS pathogenesis the initial negative effect of FUS (P525L) on axonal transport is an indirect nature and requires additional factors or mechanisms.

Koichiro Uriu^*, Bo-Kai Liao^*, Andrew C. Oates, Luis G Morelli
From local resynchronization to global pattern recovery in the zebrafish segmentation clock.
Elife, 10 Art. No. e61358 (2021)
Open Access DOI

Integrity of rhythmic spatial gene expression patterns in the vertebrate segmentation clock requires local synchronization between neighboring cells by Delta-Notch signaling and its inhibition causes defective segment boundaries. Whether deformation of the oscillating tissue complements local synchronization during patterning and segment formation is not understood. We combine theory and experiment to investigate this question in the zebrafish segmentation clock. We remove a Notch inhibitor, allowing resynchronization, and analyze embryonic segment recovery. We observe unexpected intermingling of normal and defective segments, and capture this with a new model combining coupled oscillators and tissue mechanics. Intermingled segments are explained in the theory by advection of persistent phase vortices of oscillators. Experimentally observed changes in recovery patterns are predicted in the theory by temporal changes in tissue length and cell advection pattern. Thus, segmental pattern recovery occurs at two length and time scales: rapid local synchronization between neighboring cells, and the slower transport of the resulting patterns across the tissue through morphogenesis.

Anders S Hansen^#, Christoph Zechner^#
Promoters adopt distinct dynamic manifestations depending on transcription factor context.
Mol Syst Biol, 17(2) Art. No. e9821 (2021)
Open Access DOI

Cells respond to external signals and stresses by activating transcription factors (TF), which induce gene expression changes. Prior work suggests that signal-specific gene expression changes are partly achieved because different gene promoters exhibit distinct induction dynamics in response to the same TF input signal. Here, using high-throughput quantitative single-cell measurements and a novel statistical method, we systematically analyzed transcriptional responses to a large number of dynamic TF inputs. In particular, we quantified the scaling behavior among different transcriptional features extracted from the measured trajectories such as the gene activation delay or duration of promoter activity. Surprisingly, we found that even the same gene promoter can exhibit qualitatively distinct induction and scaling behaviors when exposed to different dynamic TF contexts. While it was previously known that promoters fall into distinct classes, here we show that the same promoter can switch between different classes depending on context. Thus, promoters can adopt context-dependent "manifestations". Our analysis suggests that the full complexity of signal processing by genetic circuits may be significantly underestimated when studied in only specific contexts.

Arnaud Rondelet^*, Andrei I. Pozniakovsky^*, Devika Namboodiri, Richard Cardoso da Silva, Divya Singh, Marit Leuschner, Ina Poser, Andrea Ssykor, Julian Berlitz, Nadine Schmidt, Lea Röhder, Gerben Vader, Anthony Hyman, Alexander W. Bird
ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes.
Life Sci Alliance, 4(2) Art. No. e202000836 (2021)
Open Access DOI

Bacterial artificial chromosome (BAC)-based transgenes have emerged as a powerful tool for controlled and conditional interrogation of protein function in higher eukaryotes. Although homologous recombination-based recombineering methods have streamlined the efficient integration of protein tags onto BAC transgenes, generating precise point mutations has remained less efficient and time-consuming. Here, we present a simplified method for inserting point mutations into BAC transgenes requiring a single recombineering step followed by antibiotic selection. This technique, which we call exogenous/synthetic intronization (ESI) mutagenesis, relies on co-integration of a mutation of interest along with a selectable marker gene, the latter of which is harboured in an artificial intron adjacent to the mutation site. Cell lines generated from ESI-mutated BACs express the transgenes equivalently to the endogenous gene, and all cells efficiently splice out the synthetic intron. Thus, ESI mutagenesis provides a robust and effective single-step method with high precision and high efficiency for mutating BAC transgenes.

Sarita Hebbar^*, Malte Lehmann^*, Sarah Behrens, Catrin Hälsig, Weihua Leng, Michaela Yuan, Sylke Winkler, Elisabeth Knust
Mutations in the splicing regulator Prp31 lead to retinal degeneration in Drosophila.
Biol Open, 10(1) Art. No. bio052332 (2021)
Open Access DOI

Retinitis pigmentosa (RP) is a clinically heterogeneous disease affecting 1.6 million people worldwide. The second-largest group of genes causing autosomal dominant RP in human encodes regulators of the splicing machinery. Yet, how defects in splicing factor genes are linked to the aetiology of the disease remains largely elusive. To explore possible mechanisms underlying retinal degeneration caused by mutations in regulators of the splicing machinery, we induced mutations in Drosophila Prp31, the orthologue of human PRPF31, mutations in which are associated with RP11. Flies heterozygous mutant for Prp31 are viable and develop normal eyes and retina. However, photoreceptors degenerate under light stress, thus resembling the human disease phenotype. Degeneration is associated with increased accumulation of the visual pigment rhodopsin 1 and increased mRNA levels of twinfilin, a gene associated with rhodopsin trafficking. Reducing rhodopsin levels by raising animals in a carotenoid-free medium not only attenuates rhodopsin accumulation, but also retinal degeneration. Given a similar importance of proper rhodopsin trafficking for photoreceptor homeostasis in human, results obtained in flies presented here will also contribute to further unravel molecular mechanisms underlying the human disease.This paper has an associated First Person interview with the co-first authors of the article.

Vladimir M Jovanovic, Melanie Sarfert, Carlos S Reyna-Blanco, Henrike Indrischek, Dulce I Valdivia, Ekaterina Shelest, Katja Nowick
Positive Selection in Gene Regulatory Factors Suggests Adaptive Pleiotropic Changes During Human Evolution.
Front Genet, 12 Art. No. 662239 (2021)
Open Access DOI

Gene regulatory factors (GRFs), such as transcription factors, co-factors and histone-modifying enzymes, play many important roles in modifying gene expression in biological processes. They have also been proposed to underlie speciation and adaptation. To investigate potential contributions of GRFs to primate evolution, we analyzed GRF genes in 27 publicly available primate genomes. Genes coding for zinc finger (ZNF) proteins, especially ZNFs with a Krüppel-associated box (KRAB) domain were the most abundant TFs in all genomes. Gene numbers per TF family differed between all species. To detect signs of positive selection in GRF genes we investigated more than 3,000 human GRFs with their more than 70,000 orthologs in 26 non-human primates. We implemented two independent tests for positive selection, the branch-site-model of the PAML suite and aBSREL of the HyPhy suite, focusing on the human and great ape branch. Our workflow included rigorous procedures to reduce the number of false positives: excluding distantly similar orthologs, manual corrections of alignments, and considering only genes and sites detected by both tests for positive selection. Furthermore, we verified the candidate sites for selection by investigating their variation within human and non-human great ape population data. In order to approximately assign a date to positively selected sites in the human lineage, we analyzed archaic human genomes. Our work revealed with high confidence five GRFs that have been positively selected on the human lineage and one GRF that has been positively selected on the great ape lineage. These GRFs are scattered on different chromosomes and have been previously linked to diverse functions. For some of them a role in speciation and/or adaptation can be proposed based on the expression pattern or association with human diseases, but it seems that they all contributed independently to human evolution. Four of the positively selected GRFs are KRAB-ZNF proteins, that induce changes in target genes co-expression and/or through arms race with transposable elements. Since each positively selected GRF contains several sites with evidence for positive selection, we suggest that these GRFs participated pleiotropically to phenotypic adaptations in humans.

Kaushikaram Subramanian^*, Heike Petzold^*, Benjamin Seelbinder^*, Lena Hersemann, Ina Nüsslein, Moritz Kreysing
Optical plasticity of mammalian cells.
J Biophotonics, 14(4) Art. No. e202000457 (2020)
Open Access PDF DOI

Transparency is widespread in nature, ranging from transparent insect wings to ocular tissues that enable you to read this text, and transparent marine vertebrates. And yet, cells and tissue models in biology are usually strongly light scattering and optically opaque, precluding deep optical microscopy. Here we describe the directed evolution of cultured mammalian cells toward increased transparency. We find that mutations greatly diversify the optical phenotype of Chinese Hamster Ovary cells, a cultured mammalian cell line. Furthermore, only three rounds of high-throughput optical selection and competitive growth are required to yield fit cells with greatly improved transparency. Based on 15 monoclonal cell lines derived from this directed evolution experiment, we find that the evolved transparency frequently goes along with a reduction of nuclear granularity and physiological shifts in gene expression profiles. In the future this optical plasticity of mammalian cells may facilitate genetic clearance of living tissues for in vivo microscopy.

Jesse V Veenvliet^*^#, Adriano Bolondi^*, Helene Kretzmer, Leah Haut, Manuela Scholze-Wittler, Dennis Schifferl, Frederic Koch, Léo Guignard, Abhishek Sampath Kumar, Milena Pustet, Simon Heimann, René Buschow, Lars Wittler, Bernd Timmermann, Alexander Meissner^#, Bernhard G Herrmann^#
Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites.
Science, 370(6522) Art. No. eaba4937 (2020)
DOI

Post-implantation embryogenesis is a highly dynamic process comprising multiple lineage decisions and morphogenetic changes that are inaccessible to deep analysis in vivo. We found that pluripotent mouse embryonic stem cells (mESCs) form aggregates that upon embedding in an extracellular matrix compound induce the formation of highly organized "trunk-like structures" (TLSs) comprising the neural tube and somites. Comparative single-cell RNA sequencing analysis confirmed that this process is highly analogous to mouse development and follows the same stepwise gene-regulatory program. Tbx6 knockout TLSs developed additional neural tubes mirroring the embryonic mutant phenotype, and chemical modulation could induce excess somite formation. TLSs thus reveal an advanced level of self-organization and provide a powerful platform for investigating post-implantation embryogenesis in a dish.

Peiwen Xiong, C Darrin Hulsey, Carmelo Fruciano, Wai Y Wong, Alexander Nater, Andreas F Kautt, Oleg Simakov, Martin Pippel, Shigehiro Kuraku, Axel Meyer, Paolo Franchini
The comparative genomic landscape of adaptive radiation in crater lake cichlid fishes.
Mol Ecol, 30(4) 955-972 (2020)
Open Access DOI

Factors ranging from ecological opportunity to genome composition might explain why only some lineages form adaptive radiations. While being rare, particular systems can provide natural experiments within an identical ecological setting where species numbers and phenotypic divergence in two closely related lineages are notably different. We investigated one such natural experiment using two de novo assembled and 40 resequenced genomes and asked why two closely related Neotropical cichlid fish lineages, the Amphilophus citrinellus species complex (Midas cichlids; radiating) and Archocentrus centrarchus (Flyer cichlid; nonradiating), have resulted in such disparate evolutionary outcomes. Although both lineages inhabit many of the same Nicaraguan lakes, whole-genome inferred demography suggests that priority effects are not likely to be the cause of the dissimilarities. Also, genome-wide levels of selection, transposable element dynamics, gene family expansion, major chromosomal rearrangements and the number of genes under positive selection were not markedly different between the two lineages. To more finely investigate particular subsets of the genome that have undergone adaptive divergence in Midas cichlids, we also examined if there was evidence for 'molecular pre-adaptation' in regions identified by QTL mapping of repeatedly diverging adaptive traits. Although most of our analyses failed to pinpoint substantial genomic differences, we did identify functional categories containing many genes under positive selection that provide candidates for future studies on the propensity of Midas cichlids to radiate. Our results point to a disproportionate role of local, rather than genome-wide factors underlying the propensity for these cichlid fishes to adaptively radiate.

José M Martín-Durán, Bruno C Vellutini, Ferdinand Marlétaz, Viviana Cetrangolo, Nevena Cvetesic, Daniel Thiel, Simon Henriet, Xavier Grau-Bové, Allan M Carrillo-Baltodano, Wenjia Gu, Alexandra Kerbl, Yamile Marquez, Nicolas Bekkouche, Daniel Chourrout, Jose Luis Gómez-Skarmeta, Manuel Irimia, Boris Lenhard, Katrine Worsaae, Andreas Hejnol
Conservative route to genome compaction in a miniature annelid.
Nat Ecol Evol, 5(2) 231-242 (2020)
Open Access DOI

The causes and consequences of genome reduction in animals are unclear because our understanding of this process mostly relies on lineages with often exceptionally high rates of evolution. Here, we decode the compact 73.8-megabase genome of Dimorphilus gyrociliatus, a meiobenthic segmented worm. The D. gyrociliatus genome retains traits classically associated with larger and slower-evolving genomes, such as an ordered, intact Hox cluster, a generally conserved developmental toolkit and traces of ancestral bilaterian linkage. Unlike some other animals with small genomes, the analysis of the D. gyrociliatus epigenome revealed canonical features of genome regulation, excluding the presence of operons and trans-splicing. Instead, the gene-dense D. gyrociliatus genome presents a divergent Myc pathway, a key physiological regulator of growth, proliferation and genome stability in animals. Altogether, our results uncover a conservative route to genome compaction in annelids, reminiscent of that observed in the vertebrate Takifugu rubripes.

Claudia Gerri, Afshan McCarthy, Gregorio Alanis-Lobato, Andrej Demtschenko, Alexandre Bruneau, Sophie Loubersac, Norah M E Fogarty, Daniel Hampshire, Kay Elder, Phil Snell, Leila Christie, Laurent David, Hilde Van de Velde, Ali A Fouladi-Nashta, Kathy K Niakan
Initiation of a conserved trophectoderm program in human, cow and mouse embryos.
Nature, 587(7834) 443-447 (2020)
DOI

Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac1. Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human2-5 and cow6. Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical-basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos.

Andreas F Kautt, Claudius F Kratochwil, Alexander Nater, Gonzalo Machado-Schiaffino, Melisa Olave, Frederico Henning, Julián Torres-Dowdall, Andreas Härer, C Darrin Hulsey, Paolo Franchini, Martin Pippel, Eugene W Myers, Axel Meyer
Contrasting signatures of genomic divergence during sympatric speciation.
Nature, 588(7836) 106-111 (2020)
Open Access DOI

The transition from 'well-marked varieties' of a single species into 'well-defined species'-especially in the absence of geographic barriers to gene flow (sympatric speciation)-has puzzled evolutionary biologists ever since Darwin1,2. Gene flow counteracts the buildup of genome-wide differentiation, which is a hallmark of speciation and increases the likelihood of the evolution of irreversible reproductive barriers (incompatibilities) that complete the speciation process3. Theory predicts that the genetic architecture of divergently selected traits can influence whether sympatric speciation occurs4, but empirical tests of this theory are scant because comprehensive data are difficult to collect and synthesize across species, owing to their unique biologies and evolutionary histories5. Here, within a young species complex of neotropical cichlid fishes (Amphilophus spp.), we analysed genomic divergence among populations and species. By generating a new genome assembly and re-sequencing 453 genomes, we uncovered the genetic architecture of traits that have been suggested to be important for divergence. Species that differ in monogenic or oligogenic traits that affect ecological performance and/or mate choice show remarkably localized genomic differentiation. By contrast, differentiation among species that have diverged in polygenic traits is genomically widespread and much higher overall, consistent with the evolution of effective and stable genome-wide barriers to gene flow. Thus, we conclude that simple trait architectures are not always as conducive to speciation with gene flow as previously suggested, whereas polygenic architectures can promote rapid and stable speciation in sympatry.

Donato Santovito, Virginia Egea, Kiril Bidzhekov, Lucia Natarelli, André Mourão, Xavier Blanchet, Kanin Wichapong, Maria Aslani, Coy Brunßen, Michael Horckmans, Michael Hristov, Arie Geerlof, Esther Lutgens, Mat J A P Daemen, Tilman M Hackeng, Christian Ries, Trian Chavakis, Henning Morawietz, Ronald Naumann, Philipp von Hundelshausen, Sabine Steffens, Johan Duchêne, Remco T A Megens, Michael Sattler, Christian Weber
Autophagy unleashes noncanonical microRNA functions.
Autophagy, 16(12) 2294-2296 (2020)
DOI

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression which act by guiding AGO (argonaute) proteins to target RNA transcripts in the RNA-induced silencing complex (RISC). This macromolecular complex includes multiple additional components (e.g., TNRC6A) that allow for interaction with enzymes mediating inhibition of translation or RNA decay. However, miRNAs also reside in low-molecular weight complexes without being engaged in target repression, and their function in this context is largely unknown. Our recent findings show that endothelial cells exposed to protective high-shear stress or MTORC inhibition activate the macroautophagy/autophagy machinery to sustain viability by promoting differential trafficking of MIR126 strands and by enabling unconventional features of MIR126-5p. Whereas MIR126-3p is degraded upon autophagy activation, MIR126-5p interacts with the RNA-binding protein MEX3A to form a ternary complex with AGO2. This complex forms on the autophagosomal surface and facilitates its nuclear localization. Once in the nucleus, MIR126-5p dissociates from AGO2 and establishes aptamer-like interactions with the effector CASP3 (caspase 3). The binding to MIR126-5p prevents dimerization and proper active site formation of CASP3, thus inhibiting proteolytic activity and limiting apoptosis. Disrupting this pathway in vivo by genetic deletion of Mex3a or by specific deficiency of endothelial autophagy aggravates endothelial apoptosis and exacerbates the progression of atherosclerosis. The direct inhibition of CASP3 by MIR126-5p reveals a non-canonical mechanism by which miRNAs can modulate protein function and mediate the autophagy-apoptosis crosstalk.

Francisca M Real, Stefan A Haas, Paolo Franchini, Peiwen Xiong, Oleg Simakov, Heiner Kuhl, Robert Schöpflin, David Heller, M-Hossein Moeinzadeh, Verena Heinrich, Thomas Krannich, Annkatrin Bressin, Michaela F Hartmann, Stefan A Wudy, Dina K N Dechmann, Alicia Hurtado, Francisco J Barrionuevo, Magdalena Schindler, Izabela Harabula, Marco Osterwalder, Michael Hiller, Lars Wittler, Axel Visel, Bernd Timmermann, Axel Meyer, Martin Vingron, Rafael Jiménez, Stefan Mundlos^#, Darío G Lupiáñez^#
The mole genome reveals regulatory rearrangements associated with adaptive intersexuality.
Science, 370(6513) 208-214 (2020)
DOI

Linking genomic variation to phenotypical traits remains a major challenge in evolutionary genetics. In this study, we use phylogenomic strategies to investigate a distinctive trait among mammals: the development of masculinizing ovotestes in female moles. By combining a chromosome-scale genome assembly of the Iberian mole, Talpa occidentalis, with transcriptomic, epigenetic, and chromatin interaction datasets, we identify rearrangements altering the regulatory landscape of genes with distinct gonadal expression patterns. These include a tandem triplication involving CYP17A1, a gene controlling androgen synthesis, and an intrachromosomal inversion involving the pro-testicular growth factor gene FGF9, which is heterochronically expressed in mole ovotestes. Transgenic mice with a knock-in mole CYP17A1 enhancer or overexpressing FGF9 showed phenotypes recapitulating mole sexual features. Our results highlight how integrative genomic approaches can reveal the phenotypic impact of noncoding sequence changes.

Claudia Gerri^*, Sergio Menchero^*, Shantha K Mahadevaiah, James M A Turner^#, Kathy K Niakan^#
Human Embryogenesis: A Comparative Perspective.
Annu Rev Cell Dev Biol, 36 411-440 (2020)
DOI

Understanding human embryology has historically relied on comparative approaches using mammalian model organisms. With the advent of low-input methods to investigate genetic and epigenetic mechanisms and efficient techniques to assess gene function, we can now study the human embryo directly. These advances have transformed the investigation of early embryogenesis in nonrodent species, thereby providing a broader understanding of conserved and divergent mechanisms. Here, we present an overview of the major events in human preimplantation development and place them in the context of mammalian evolution by comparing these events in other eutherian and metatherian species. We describe the advances of studies on postimplantation development and discuss stem cell models that mimic postimplantation embryos. A comparative perspective highlights the importance of analyzing different organisms with molecular characterization and functional studies to reveal the principles of early development. This growing field has a fundamental impact in regenerative medicine and raises important ethical considerations.

Gaelle R Carrat, Elizabeth Haythorne, Alejandra Tomas, Leena Haataja, Andreas Müller, Peter Arvan, Alexandra Piunti, Kaiying Cheng, Mutian Huang, Timothy J Pullen, Eleni Georgiadou, Theodoros Stylianides, Nur Shabrina Amirruddin, Victoria Salem, Walter Distaso, Andrew Cakebread, Kate J Heesom, Philip A Lewis, David J Hodson, Linford J Briant, Annie C H Fung, Richard B Sessions, Fabien Alpy, Alice P S Kong, Peter I Benke, Federico Torta, Adrian Kee Keong Teo, Isabelle Leclerc, Michele Solimena, Dale B Wigley, Guy A Rutter
The type 2 diabetes gene product STARD10 is a phosphoinositide-binding protein that controls insulin secretory granule biogenesis.
Mol Metab, 40 Art. No. 101015 (2020)
Open Access DOI

Risk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the β-cell, impaired glucose-induced insulin secretion, and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids and thus the pathways through which STARD10 regulates β-cell function are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the β-cell and the role of the protein in controlling proinsulin processing and insulin granule biogenesis and maturation.

Giulia Serafini, Giorgia Giordani, Luca Grillini, Davide Andrenacci, Giuseppe Gargiulo, Valeria Cavaliere
The Impact of Drosophila Awd/NME1/2 Levels on Notch and Wg Signaling Pathways.
Int J Mol Sci, 21(19) Art. No. 7257 (2020)
Open Access DOI

Awd, the Drosophila homologue of NME1/2 metastasis suppressors, plays key roles in many signaling pathways. Mosaic analysis of the null awdJ2A4 allele showed that loss of awd gene function blocks Notch signaling and the expression of its target genes including the Wingless (Wg/Wnt1) morphogen. We also showed that RNA interference (RNAi)-mediated awd silencing (awdi) in larval wing disc leads to chromosomal instability (CIN) and to Jun amino-terminal kinases (JNK)-mediated cell death. Here we show that this cell death is independent of p53 activity. Based on our previous finding showing that forced survival of awdi-CIN cells leads to aneuploidy without the hyperproliferative effect, we investigated the Wg expression in awdi wing disc cells. Interestingly, the Wg protein is expressed in its correct dorso-ventral domain but shows an altered cellular distribution which impairs its signaling. Further, we show that RNAi-mediated knock down of awd in wing discs does not affect Notch signaling. Thus, our analysis of the hypomorphic phenotype arising from awd downregulation uncovers a dose-dependent effect of Awd in Notch and Wg signaling.

Racha Chouaib, Adham Safieddine, Xavier Pichon, Arthur Imbert, Oh Sung Kwon, Aubin Samacoits, Abdel-Meneem Traboulsi, Marie-Cécile Robert, Nikolay Tsanov, Emeline Coleno, Ina Poser, Christophe Zimmer, Anthony Hyman, Hervé Le Hir, Kazem Zibara, Matthias Peter, Florian Mueller, Thomas Walter, Edouard Bertrand
A Dual Protein-mRNA Localization Screen Reveals Compartmentalized Translation and Widespread Co-translational RNA Targeting.
Dev Cell, 54(6) 773-791 (2020)
DOI

Local translation allows spatial control of gene expression. Here, we performed a dual protein-mRNA localization screen, using smFISH on 523 human cell lines expressing GFP-tagged genes. 32 mRNAs displayed specific cytoplasmic localizations with local translation at unexpected locations, including cytoplasmic protrusions, cell edges, endosomes, Golgi, the nuclear envelope, and centrosomes, the latter being cell-cycle-dependent. Automated classification of mRNA localization patterns revealed a high degree of intercellular heterogeneity. Surprisingly, mRNA localization frequently required ongoing translation, indicating widespread co-translational RNA targeting. Interestingly, while P-body accumulation was frequent (15 mRNAs), four mRNAs accumulated in foci that were distinct structures. These foci lacked the mature protein, but nascent polypeptide imaging showed that they were specialized translation factories. For β-catenin, foci formation was regulated by Wnt, relied on APC-dependent polysome aggregation, and led to nascent protein degradation. Thus, translation factories uniquely regulate nascent protein metabolism and create a fine granular compartmentalization of translation.

Manan Lalit, Mette Handberg-Thorsager, Yu-Wen Hsieh, Florian Jug^#, Pavel Tomancak^#
Registration of Multi-modal Volumetric Images by Establishing Cell Correspondence.
In: Computer vision - ECCV 2020 workshops : Glasgow, UK, August 23-28, 2020 : proceedings : Part 1 (2020)(Eds.) Adrien Bartoli (Lecture Notes in Computer Science ; 12535), Cham, Springer International Publishing (2020), 458-473
DOI

Early development of an animal from an egg involves a rapid increase in cell number and several cell fate specification events accompanied by dynamic morphogenetic changes. In order to correlate the morphological changes with the genetic events, one typically needs to monitor the living system with several imaging modalities offering different spatial and temporal resolution. Live imaging allows monitoring the embryo at a high temporal resolution and observing the morphological changes. On the other hand, confocal images of specimens fixed and stained for the expression of certain genes enable observing the transcription states of an embryo at specific time points during development with high spatial resolution. The two imaging modalities cannot, by definition, be applied to the same specimen and thus, separately obtained images of different specimens need to be registered. Biologically, the most meaningful way to register the images is by identifying cellular correspondences between these two imaging modalities. In this way, one can bring the two sources of information into a single domain and combine dynamic information on morphogenesis with static gene expression data. Here we propose a new computational pipeline for identifying cell-to-cell correspondences between images from multiple modalities and for using these correspondences to register 3D images within and across imaging modalities. We demonstrate this pipeline by combining four-dimensional recording of embryogenesis of Spiralian annelid ragworm Platynereis dumerilii with three-dimensional scans of fixed Platynereis dumerilii embryos stained for the expression of a variety of important developmental genes. We compare our approach with methods for aligning point clouds and show that we match the accuracy of these state-of-the-art registration pipelines on synthetic data. We show that our approach outperforms these methods on real biological imaging datasets. Importantly, our approach uniquely provides, in addition to the registration, also the non-redundant matching of corresponding, biologically meaningful entities within the registered specimen which is the prerequisite for generating biological insights from the combined datasets. The complete pipeline is available for public use through a Fiji plugin.

Juliana G. Roscito, Kaushikaram Subramanian, Ronald Naumann, Mihail Sarov, Anna Shevchenko, Aliona Bogdanova, Thomas Kurth, Leo Foerster, Moritz Kreysing, Michael Hiller
Recapitulating evolutionary divergence in a single cis-regulatory element is sufficient to cause expression changes of the lens gene Tdrd7.
Mol Biol Evol, 38(2) 380-392 (2020)
Open Access PDF DOI

Mutations in cis-regulatory elements play important roles for phenotypic changes during evolution. Eye degeneration in the blind mole rat (BMR; Nannospalax galili) and other subterranean mammals is significantly associated with widespread divergence of eye regulatory elements, but the effect of these regulatory mutations on eye development and function has not been explored. Here, we investigate the effect of mutations observed in the BMR sequence of a conserved non-coding element upstream of Tdrd7, a pleiotropic gene required for lens development and spermatogenesis. We first show that this conserved element is a transcriptional repressor in lens cells and that the BMR sequence partially lost repressor activity. Next, we recapitulated evolutionary changes in this element by precisely replacing the endogenous regulatory element in a mouse line by the orthologous BMR sequence with CRISPR-Cas9. Strikingly, this repressor replacement caused a more than two-fold up-regulation of Tdrd7 in the developing lens; however, increased mRNA level does not result in a corresponding increase in TDRD7 protein nor an obvious lens phenotype, possibly explained by buffering at the posttranscriptional level. Our results are consistent with eye degeneration in subterranean mammals having a polygenic basis where many small-effect mutations in different eye-regulatory elements collectively contribute to phenotypic differences.

Michael Heide^#, Christiane Haffner, Ayako Y Murayama, Yoko Kurotaki, Haruka Shinohara, Hideyuki Okano, Erika Sasaki, Wieland Huttner^#
Human-specific ARHGAP11B increases size and folding of primate neocortex in the fetal marmoset.
Science, 369(6503) 546-550 (2020)
DOI

The neocortex has expanded during mammalian evolution. Overexpression studies in developing mouse and ferret neocortex have implicated the human-specific gene ARHGAP11B in neocortical expansion, but the relevance for primate evolution has been unclear. Here, we provide functional evidence that ARHGAP11B causes expansion of the primate neocortex. ARHGAP11B expressed in fetal neocortex of the common marmoset under control of the gene's own (human) promoter increased the numbers of basal radial glia progenitors in the marmoset outer subventricular zone, increased the numbers of upper-layer neurons, enlarged the neocortex, and induced its folding. Thus, the human-specific ARHGAP11B drives changes in development in the nonhuman primate marmoset that reflect the changes in evolution that characterize human neocortical development.

David Jebb^*, Zixia Huang^*, Martin Pippel^*, Graham M Hughes, Ksenia Lavrichenko, Paolo Devanna, Sylke Winkler, Lars S Jermiin, Emilia C Skirmuntt, Aris Katzourakis, Lucy Burkitt-Gray, David A Ray, Kevin F. Sullivan, Juliana G. Roscito, Bogdan Kirilenko, Liliana M Dávalos, Angelique P Corthals, Megan L Power, Gareth Jones, Roger D Ransome, Dina K N Dechmann, Andrea G Locatelli, Sébastien J Puechmaille, Olivier Fedrigo, Erich D Jarvis, Michael Hiller^#, Sonja Vernes^#, Eugene W Myers^#, Emma Teeling^#
Six reference-quality genomes reveal evolution of bat adaptations.
Nature, 583(7817) 578-584 (2020)
Open Access DOI

Bats possess extraordinary adaptations, including flight, echolocation, extreme longevity and unique immunity. High-quality genomes are crucial for understanding the molecular basis and evolution of these traits. Here we incorporated long-read sequencing and state-of-the-art scaffolding protocols1 to generate, to our knowledge, the first reference-quality genomes of six bat species (Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pipistrellus kuhlii and Molossus molossus). We integrated gene projections from our 'Tool to infer Orthologs from Genome Alignments' (TOGA) software with de novo and homology gene predictions as well as short- and long-read transcriptomics to generate highly complete gene annotations. To resolve the phylogenetic position of bats within Laurasiatheria, we applied several phylogenetic methods to comprehensive sets of orthologous protein-coding and noncoding regions of the genome, and identified a basal origin for bats within Scrotifera. Our genome-wide screens revealed positive selection on hearing-related genes in the ancestral branch of bats, which is indicative of laryngeal echolocation being an ancestral trait in this clade. We found selection and loss of immunity-related genes (including pro-inflammatory NF-κB regulators) and expansions of anti-viral APOBEC3 genes, which highlights molecular mechanisms that may contribute to the exceptional immunity of bats. Genomic integrations of diverse viruses provide a genomic record of historical tolerance to viral infection in bats. Finally, we found and experimentally validated bat-specific variation in microRNAs, which may regulate bat-specific gene-expression programs. Our reference-quality bat genomes provide the resources required to uncover and validate the genomic basis of adaptations of bats, and stimulate new avenues of research that are directly relevant to human health and disease1.

Bharath Kumar Raghuraman^*, Sarita Hebbar^*, Mukesh Kumar, HongKee Moon, Ian Henry, Elisabeth Knust, Andrej Shevchenko
Absolute Quantification of Proteins in the Eye of Drosophila melanogaster.
Proteomics, 20(23) Art. No. e1900049 (2020)
Open Access DOI

Absolute (molar) quantification of proteins determines their molar ratios in complexes, networks and metabolic pathways. We employed MS Western workflow to determine molar abundances of proteins potentially critical for morphogenesis and phototransduction (PT) in eyes of Drosophila melanogaster. We used a single chimeric 264 kDa protein standard that covers, in total, 197 peptides from 43 proteins. The majority of proteins were independently quantified with 2 to 4 proteotypic peptides with the coefficient of variation of less than 15%, better than 1000-fold dynamic range and sub-femtomole sensitivity. We determined molar abundances of the components of the PT machinery and the rhabdomere, the photosensitive organelle of the fly eye, and how they changed when rhabdomere morphogenesis is perturbed by genetic manipulation of the evolutionary conserved gene crumbs (crb). Data are available via ProteomeXchange with identifier PXD018001 This article is protected by copyright. All rights reserved.

Samir Vaid^#, Wieland Huttner^#
Transcriptional Regulators and Human-Specific/Primate-Specific Genes in Neocortical Neurogenesis.
Int J Mol Sci, 21(13) Art. No. 4614 (2020)
Open Access DOI

During development, starting from a pool of pluripotent stem cells, tissue-specific genetic programs help to shape and develop functional organs. To understand the development of an organ and its disorders, it is important to understand the spatio-temporal dynamics of the gene expression profiles that occur during its development. Modifications in existing genes, the de-novo appearance of new genes, or, occasionally, even the loss of genes, can greatly affect the gene expression profile of any given tissue and contribute to the evolution of organs or of parts of organs. The neocortex is evolutionarily the most recent part of the brain, it is unique to mammals, and is the seat of our higher cognitive abilities. Progenitors that give rise to this tissue undergo sequential waves of differentiation to produce the complete sets of neurons and glial cells that make up a functional neocortex. We will review herein our understanding of the transcriptional regulators that control the neural precursor cells (NPCs) during the generation of the most abundant class of neocortical neurons, the glutametergic neurons. In addition, we will discuss the roles of recently-identified human- and primate-specific genes in promoting neurogenesis, leading to neocortical expansion.

Deepthi Ashokkumar^*, Qinyu Zhang^*, Christian Much, Anita S. Bledau, Ronald Naumann, Dimitra Alexopoulou, Andreas Dahl, Neha Goveas, Jun Fu, Konstantinos Anastassiadis, A F Stewart^#, Andrea Kranz^#
MLL4 is required after implantation, whereas MLL3 becomes essential during late gestation.
Development, 147(12) Art. No. dev186999 (2020)
DOI

Methylation of histone 3 lysine 4 (H3K4) is a major epigenetic system associated with gene expression. In mammals there are six H3K4 methyltransferases related to yeast Set1 and fly Trithorax, including two orthologs of fly Trithorax-related: MLL3 and MLL4. Exome sequencing has documented high frequencies of MLL3 and MLL4 mutations in many types of human cancer. Despite this emerging importance, the requirements of these paralogs in mammalian development have only been incompletely reported. Here, we examined the null phenotypes to establish that MLL3 is first required for lung maturation, whereas MLL4 is first required for migration of the anterior visceral endoderm that initiates gastrulation in the mouse. This collective cell migration is preceded by a columnar-to-squamous transition in visceral endoderm cells that depends on MLL4. Furthermore, Mll4 mutants display incompletely penetrant, sex-distorted, embryonic haploinsufficiency and adult heterozygous mutants show aspects of Kabuki syndrome, indicating that MLL4 action, unlike MLL3, is dosage dependent. The highly specific and discordant functions of these paralogs in mouse development argues against their action as general enhancer factors.

Alexandra Lewis, Ahmet C Berkyurek, Andre Greiner, Ahilya N Sawh, Ajay A Vashisht, Stephanie Merrett, Mathieu N Flamand, James Wohlschlegel, Mihail Sarov, Eric A Miska, Thomas F Duchaine
A Family of Argonaute-Interacting Proteins Gates Nuclear RNAi.
Mol Cell, 78(5) 862-875 (2020)
DOI

Nuclear RNA interference (RNAi) pathways work together with histone modifications to regulate gene expression and enact an adaptive response to transposable RNA elements. In the germline, nuclear RNAi can lead to trans-generational epigenetic inheritance (TEI) of gene silencing. We identified and characterized a family of nuclear Argonaute-interacting proteins (ENRIs) that control the strength and target specificity of nuclear RNAi in C. elegans, ensuring faithful inheritance of epigenetic memories. ENRI-1/2 prevent misloading of the nuclear Argonaute NRDE-3 with small RNAs that normally effect maternal piRNAs, which prevents precocious nuclear translocation of NRDE-3 in the early embryo. Additionally, they are negative regulators of nuclear RNAi triggered from exogenous sources. Loss of ENRI-3, an unstable protein expressed mostly in the male germline, misdirects the RNAi response to transposable elements and impairs TEI. The ENRIs determine the potency and specificity of nuclear RNAi responses by gating small RNAs into specific nuclear Argonautes.

Donato Santovito, Virginia Egea, Kiril Bidzhekov, Lucia Natarelli, André Mourão, Xavier Blanchet, Kanin Wichapong, Maria Aslani, Coy Brunßen, Michael Horckmans, Michael Hristov, Arie Geerlof, Esther Lutgens, Mat J A P Daemen, Tilman M Hackeng, Christian Ries, Trian Chavakis, Henning Morawietz, Ronald Naumann, Philipp von Hundelshausen, Sabine Steffens, Johan Duchêne, Remco T A Megens, Michael Sattler, Christian Weber
Noncanonical inhibition of caspase-3 by a nuclear microRNA confers endothelial protection by autophagy in atherosclerosis.
Sci Transl Med, 12(546) Art. No. eaaz2294 (2020)
DOI

MicroRNAs (miRNAs) are versatile regulators of gene expression with profound implications for human disease including atherosclerosis, but whether they can exert posttranslational functions to control cell adaptation and whether such noncanonical features harbor pathophysiological relevance is unknown. Here, we show that miR-126-5p sustains endothelial integrity in the context of high shear stress and autophagy. Bound to argonaute-2 (Ago2), miR-126-5p forms a complex with Mex3a, which occurs on the surface of autophagic vesicles and guides its transport into the nucleus. Mutational studies and biophysical measurements demonstrate that Mex3a binds to the central U- and G-rich regions of miR-126-5p with nanomolar affinity via its two K homology domains. In the nucleus, miR-126-5p dissociates from Ago2 and binds to caspase-3 in an aptamer-like fashion with its seed sequence, preventing dimerization of the caspase and inhibiting its activity to limit apoptosis. The antiapoptotic effect of miR-126-5p outside of the RNA-induced silencing complex is important for endothelial integrity under conditions of high shear stress promoting autophagy: ablation of Mex3a or ATG5 in vivo attenuates nuclear import of miR-126-5p, aggravates endothelial apoptosis, and exacerbates atherosclerosis. In human plaques, we found reduced nuclear miR-126-5p and active caspase-3 in areas of disturbed flow. The direct inhibition of caspase-3 by nuclear miR-126-5p reveals a noncanonical mechanism by which miRNAs can modulate protein function.

Christiane Iserman, Christine Desroches Altamirano, Ceciel Jegers, Ulrike Friedrich, Taraneh Zarin, Anatol Fritsch, Matthäus Mittasch, Antonio Domingues, Lena Hersemann, Marcus Jahnel, Doris Richter, Ulf-Peter Guenther, Matthias W. Hentze, Alan M Moses, Anthony Hyman, Günter Kramer, Moritz Kreysing, Titus Franzmann, Simon Alberti
Condensation of Ded1p Promotes a Translational Switch from Housekeeping to Stress Protein Production.
Cell, 181(4) 818-831 (2020)
Open Access PDF DOI

Cells sense elevated temperatures and mount an adaptive heat shock response that involves changes in gene expression, but the underlying mechanisms, particularly on the level of translation, remain unknown. Here we report that, in budding yeast, the essential translation initiation factor Ded1p undergoes heat-induced phase separation into gel-like condensates. Using ribosome profiling and an in vitro translation assay, we reveal that condensate formation inactivates Ded1p and represses translation of housekeeping mRNAs while promoting translation of stress mRNAs. Testing a variant of Ded1p with altered phase behavior as well as Ded1p homologs from diverse species, we demonstrate that Ded1p condensation is adaptive and fine-tuned to the maximum growth temperature of the respective organism. We conclude that Ded1p condensation is an integral part of an extended heat shock response that selectively represses translation of housekeeping mRNAs to promote survival under conditions of severe heat stress.

Nereo Kalebic^#, Barbara Langen, Jussi Helppi, Hiroshi Kawasaki, Wieland Huttner^#
In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation.
J Vis Exp, (159) Art. No. e61171 (2020)
DOI

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.

József Jászai^#, Kristina Thamm, Jana Karbanová, Peggy Janich, Christine A. Fargeas, Wieland Huttner, Denis Corbeil^#
Prominins control ciliary length throughout the animal kingdom: New lessons from human prominin-1 and zebrafish prominin-3.
J Biol Chem, 295(18) 6007-6022 (2020)
Open Access DOI

Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin-Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor-like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies.

Elliott W Abrams, Ricardo Fuentes, Florence L Marlow, Manami Kobayashi, Hong Zhang, Sumei Lu, Lee Kapp, Shai Joseph, Amy Kugath, Tripti Gupta, Virginia Lemon, Greg Runke, Amanda A Amodeo, Nadine Vastenhouw, Mary C Mullins
Molecular genetics of maternally-controlled cell divisions.
PLoS Genet, 16(4) Art. No. e1008652 (2020)
Open Access DOI

Forward genetic screens remain at the forefront of biology as an unbiased approach for discovering and elucidating gene function at the organismal and molecular level. Past mutagenesis screens targeting maternal-effect genes identified a broad spectrum of phenotypes ranging from defects in oocyte development to embryonic patterning. However, earlier vertebrate screens did not reach saturation, anticipated classes of phenotypes were not uncovered, and technological limitations made it difficult to pinpoint the causal gene. In this study, we performed a chemically-induced maternal-effect mutagenesis screen in zebrafish and identified eight distinct mutants specifically affecting the cleavage stage of development and one cleavage stage mutant that is also male sterile. The cleavage-stage phenotypes fell into three separate classes: developmental arrest proximal to the mid blastula transition (MBT), irregular cleavage, and cytokinesis mutants. We mapped each mutation to narrow genetic intervals and determined the molecular basis for two of the developmental arrest mutants, and a mutation causing male sterility and a maternal-effect mutant phenotype. One developmental arrest mutant gene encodes a maternal specific Stem Loop Binding Protein, which is required to maintain maternal histone levels. The other developmental arrest mutant encodes a maternal-specific subunit of the Minichromosome Maintenance Protein Complex, which is essential for maintaining normal chromosome integrity in the early blastomeres. Finally, we identify a hypomorphic allele of Polo-like kinase-1 (Plk-1), which results in a male sterile and maternal-effect phenotype. Collectively, these mutants expand our molecular-genetic understanding of the maternal regulation of early embryonic development in vertebrates.

Siamak Redhai^*, Clare Pilgrim^*, Pedro Gaspar, Lena van Giesen, Tatiana Lopes, Olena Riabinina, Théodore Grenier, Alexandra Milona, Bhavna Chanana, Jacob B Swadling, Yi-Fang Wang, Farah Dahalan, Michaela Yuan, Michaela Wilsch-Brauninger, Wei-Hsiang Lin, Nathan Dennison, Paolo Capriotti, Mara K N Lawniczak, Richard A Baines, Tobias Warnecke, Nikolai Windbichler, Francois Leulier, Nicholas W Bellono, Irene Miguel-Aliaga
An intestinal zinc sensor regulates food intake and developmental growth.
Nature, 580(7802) 263-268 (2020)
DOI

In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment1. In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes1. Here we use a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes-interstitial cells-by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR-Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. Our findings highlight the need to consider the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis.

Takashi Namba^#, Judit Dóczi, Anneline Pinson, Lei Xing, Nereo Kalebic, Michaela Wilsch-Bräuninger, Katherine S. Long, Samir Vaid, Janelle Lauer, Aliona Bogdanova, Barbara Borgonovo, Anna Shevchenko, Patrick Keller, David N. Drechsel, Teymuras V. Kurzchalia, Pauline Wimberger, Christos Chinopoulos, Wieland Huttner^#
Human-Specific ARHGAP11B Acts in Mitochondria to Expand Neocortical Progenitors by Glutaminolysis.
Neuron, 105(5) 867-881 (2020)
DOI

The human-specific gene ARHGAP11B is preferentially expressed in neural progenitors of fetal human neocortex and increases abundance and proliferation of basal progenitors (BPs), which have a key role in neocortex expansion. ARHGAP11B has therefore been implicated in the evolutionary expansion of the human neocortex, but its mode of action has been unknown. Here, we show that ARHGAP11B is imported into mitochondria, where it interacts with the adenine nucleotide translocase (ANT) and inhibits the mitochondrial permeability transition pore (mPTP). BP expansion by ARHGAP11B requires its presence in mitochondria, and pharmacological inhibition of ANT function or mPTP opening mimic BP expansion by ARHGAP11B. Searching for the underlying metabolic basis, we find that BP expansion by ARHGAP11B requires glutaminolysis, the conversion of glutamine to glutamate for the tricarboxylic acid (TCA) cycle. Hence, an ARHGAP11B-induced, mitochondria-based effect on BP metabolism that is a hallmark of highly mitotically active cells appears to underlie its role in neocortex expansion.

Ritika Giri, Dimitrios Papadopoulos, Diana M Posadas, Hemanth K Potluri, Pavel Tomancak, Madhav Mani^#, Richard W Carthew^#
Ordered patterning of the sensory system is susceptible to stochastic features of gene expression.
Elife, 9 Art. No. e53638 (2020)
Open Access DOI

Sensory neuron numbers and positions are precisely organized to accurately map environmental signals in the brain. This precision emerges from biochemical processes within and between cells that are inherently stochastic. We investigated impact of stochastic gene expression on pattern formation, focusing on senseless (sens), a key determinant of sensory fate in Drosophila. Perturbing microRNA regulation or genomic location of sens produced distinct noise signatures. Noise was greatly enhanced when both sens alleles were present in homologous loci such that each allele was regulated in trans by the other allele. This led to disordered patterning. In contrast, loss of microRNA repression of sens increased protein abundance but not sensory pattern disorder. This suggests that gene expression stochasticity is a critical feature that must be constrained during development to allow rapid yet accurate cell fate resolution.

Lillian Garrett, Yoon Jeung Chang, Kristina M Niedermeier, Tamara Heermann, Wolfgang Enard, Helmut Fuchs, Valérie Gailus-Durner, Martin Hrabe de Angelis, Wieland Huttner, Wolfgang Wurst, Sabine M Hölter
A truncating Aspm allele leads to a complex cognitive phenotype and region-specific reductions in parvalbuminergic neurons.
Transl Psychiatry, 10(1) 66-66 (2020)
Open Access DOI

Neurodevelopmental disorders are heterogeneous and identifying shared genetic aetiologies and converging signalling pathways affected could improve disease diagnosis and treatment. Truncating mutations of the abnormal spindle-like microcephaly associated (ASPM) gene cause autosomal recessive primary microcephaly (MCPH) in humans. ASPM is a positive regulator of Wnt/β-Catenin signalling and controls symmetric to asymmetric cell division. This process balances neural progenitor proliferation with differentiation during embryogenesis, the malfunction of which could interfere with normal brain development. ASPM mutations may play a role also in other neurodevelopmental disorders, nevertheless, we lack the details of how or to what extent. We therefore assessed neurodevelopmental disease and circuit endophenotypes in mice with a truncating Aspm1-7 mutation. Aspm1-7 mice exhibited impaired short- and long-term object recognition memory and markedly enhanced place learning in the IntelliCage®. This behaviour pattern is reminiscent of a cognitive phenotype seen in mouse models and patients with a rare form of autism spectrum disorder (ASD) as well as in mouse models of altered Wnt signalling. These alterations were accompanied by ventriculomegaly, corpus callosum dysgenesis and decreased parvalbumin (PV)+ interneuron numbers in the hippocampal Cornu Ammonis (CA) region and thalamic reticular nucleus (TRN). PV+ cell number correlated to object recognition (CA and TRN) and place learning (TRN). This opens the possibility that, as well as causing MCPH, mutant ASPM potentially contributes to other neurodevelopmental disorders such as ASD through altered parvalbuminergic interneuron development affecting cognitive behaviour. These findings provide important information for understanding the genetic overlap and improved treatment of neurodevelopmental disorders associated with ASPM.

Masin Abo-Rady^*, Norman Kalmbach^*, Arun Pal, Carina Schludi, Antje Janosch, Tanja Richter, Petra Freitag, Marc Bickle, Anne-Karin Kahlert, Susanne Petri, Stefan Stefanov, Hannes Glass, Selma Staege, Walter Just, Rajat Bhatnagar, Dieter Edbauer, Andreas Hermann, Florian Wegner^#, Jared Sterneckert^#
Knocking out C9ORF72 Exacerbates Axonal Trafficking Defects Associated with Hexanucleotide Repeat Expansion and Reduces Levels of Heat Shock Proteins.
Stem Cell Rep, 14(3) 390-405 (2020)
Open Access DOI

In amyotrophic lateral sclerosis (ALS) motor neurons (MNs) undergo dying-back, where the distal axon degenerates before the soma. The hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of ALS, but the mechanism of pathogenesis is largely unknown with both gain- and loss-of-function mechanisms being proposed. To better understand C9ORF72-ALS pathogenesis, we generated isogenic induced pluripotent stem cells. MNs with HRE in C9ORF72 showed decreased axonal trafficking compared with gene corrected MNs. However, knocking out C9ORF72 did not recapitulate these changes in MNs from healthy controls, suggesting a gain-of-function mechanism. In contrast, knocking out C9ORF72 in MNs with HRE exacerbated axonal trafficking defects and increased apoptosis as well as decreased levels of HSP70 and HSP40, and inhibition of HSPs exacerbated ALS phenotypes in MNs with HRE. Therefore, we propose that the HRE in C9ORF72 induces ALS pathogenesis via a combination of gain- and loss-of-function mechanisms.

Gema Gómez-Mariano, Nerea Matamala, Selene Martínez, Iago Justo, Alberto Marcacuzco, Carlos Jimenez, Sara Monzón, Isabel Cuesta, Cristina Garfia, María Teresa Martínez, Meritxell Huch, Ignacio Pérez de Castro, Manuel Posada, Sabina Janciauskiene, Beatriz Martínez-Delgado
Liver organoids reproduce alpha-1 antitrypsin deficiency-related liver disease.
Hepatol Int, 14(1) 127-137 (2020)
Open Access DOI

Alpha-1 antitrypsin (AAT) is a product of SERPINA1 gene mainly expressed by hepatocytes. Clinically relevant mutations in the SERPINA1 gene, such as Z (Glu342Lys), results in an expression of misfolded AAT protein having high propensity to polymerize, accumulate in hepatocytes and thus to enhance a risk for hepatocyte damage and subsequent liver disease. So far, the relationship between the Z-AAT accumulation and liver cell damage remains not completely understood. We present three-dimensional organoid culture systems, as a novel tool for modeling Z-AAT-related liver diseases.

Sean P A Ritter^*, Allison Lewis^*^#, Shelby L Vincent, Li Ling Lo, Ana Margarida da Conceicao Cunha, Danuta Chamot, Ingo Ensminger, George S Espie, George W Owttrim^#
Evidence for convergent sensing of multiple abiotic stresses in cyanobacteria.
Biochim Biophys Acta Gen Subj, 1864(1) Art. No. 129462 (2020)
DOI

Bacteria routinely utilize two-component signal transduction pathways to sense and alter gene expression in response to environmental cues. While cyanobacteria express numerous two-component systems, these pathways do not regulate all of the genes within many of the identified abiotic stress-induced regulons.

Lei Xing^#, Wieland Huttner^#
Neurotransmitters as Modulators of Neural Progenitor Cell Proliferation During Mammalian Neocortex Development.
Front Cell Dev Biol, 8 Art. No. 391 (2020)
Open Access DOI

Neural progenitor cells (NPCs) play a central role during the development and evolution of the mammalian neocortex. Precise temporal and spatial control of NPC proliferation by a concert of cell-intrinsic and cell-extrinsic factors is essential for the correct formation and proper function of the neocortex. In this review, we focus on the regulation of NPC proliferation by neurotransmitters, which act as a group of cell-extrinsic factors during mammalian neocortex development. We first summarize, from both in vivo and in vitro studies, our current knowledge on how γ-aminobutyric acid (GABA), glutamate and serotonin modulate NPC proliferation in the developing neocortex and the potential involvements of different receptors in the underlying mechanisms. Another focus of this review is to discuss future perspectives using conditionally gene-modified mice and human brain organoids as model systems to further our understanding on the contribution of neurotransmitters to the development of a normal neocortex, as well as how dysregulated neurotransmitter signaling leads to developmental and psychiatric disorders.

Yuting Wang, Sebastian Hinz, Ortrud Uckermann, Pia Hönscheid, Witigo von Schönfels, Greta Burmeister, Alexander Hendricks, Jacobo Miranda Ackerman, Gustavo Baretton, Jochen Hampe, Mario Brosch, Clemens Schafmayer, Andrej Shevchenko^#, Sebastian Zeissig^#
Shotgun lipidomics-based characterization of the landscape of lipid metabolism in colorectal cancer.
Biochim Biophys Acta Mol Cell Biol Lipids, 1865(3) Art. No. 158579 (2020)
DOI

Solid tumors are characterized by global metabolic alterations which contribute to their growth and progression. Altered gene expression profiles and plasma lipid composition suggested a role for metabolic reprogramming in colorectal cancer (CRC) development. However, a conclusive picture of CRC-associated lipidome alterations in the tumor tissue has not emerged. Here, we determined molar abundances of 342 species from 20 lipid classes in matched biopsies of CRC and adjacent normal mucosa. We demonstrate that in contrast to previous reports, CRC shows a largely preserved lipidome composition that resembles that of normal colonic mucosa. Important exceptions include increased levels of lyso-phosphatidylinositols in CRC and reduced abundance of ether phospholipids in advanced stages of CRC. As such, our observations challenge the concept of widespread alterations in lipid metabolism in CRC and rather suggest changes in the cellular lipid profile that are limited to selected lipids involved in signaling and the scavenging of reactive oxygen species.

Jessica Kobylka, Miriam S Kuth, Reinke T Müller, Eric R Geertsma, Klaas M Pos
AcrB: a mean, keen, drug efflux machine.
Ann N Y Acad Sci, 1459(1) 38-68 (2020)
DOI

Gram-negative bacteria are intrinsically resistant against cytotoxic substances by means of their outer membrane and a network of multidrug efflux systems, acting in synergy. Efflux pumps from various superfamilies with broad substrate preferences sequester and pump drugs across the inner membrane to supply the highly polyspecific and powerful tripartite resistance-nodulation-cell division (RND) efflux pumps with compounds to be extruded across the outer membrane barrier. In Escherichia coli, the tripartite efflux system AcrAB-TolC is the archetype RND multiple drug efflux pump complex. The homotrimeric inner membrane component acriflavine resistance B (AcrB) is the drug specificity and energy transduction center for the drug/proton antiport process. Drugs are bound and expelled via a cycle of mainly three consecutive states in every protomer, constituting a flexible alternating access channel system. This review recapitulates the molecular basis of drug and inhibitor binding, including mechanistic insights into drug efflux by AcrB. It also summarizes 17 years of mutational analysis of the gene acrB, reporting the effect of every substitution on the ability of E. coli to confer resistance toward antibiotics (http://goethe.link/AcrBsubstitutions). We emphasize the functional robustness of AcrB toward single-site substitutions and highlight regions that are more sensitive to perturbation.

Máté Pálfy^*, Gunnar Schulze^*, Eivind Valen, Nadine Vastenhouw
Chromatin accessibility established by Pou5f3, Sox19b and Nanog primes genes for activity during zebrafish genome activation.
PLoS Genet, 16(1) Art. No. e1008546 (2020)
Open Access DOI

In many organisms, early embryonic development is driven by maternally provided factors until the controlled onset of transcription during zygotic genome activation. The regulation of chromatin accessibility and its relationship to gene activity during this transition remain poorly understood. Here, we generated chromatin accessibility maps with ATAC-seq from genome activation until the onset of lineage specification. During this period, chromatin accessibility increases at regulatory elements. This increase is independent of RNA polymerase II-mediated transcription, with the exception of the hypertranscribed miR-430 locus. Instead, accessibility often precedes the transcription of associated genes. Loss of the maternal transcription factors Pou5f3, Sox19b, and Nanog, which are known to be required for zebrafish genome activation, results in decreased accessibility at regulatory elements. Importantly, the accessibility of regulatory regions, especially when established by Pou5f3, Sox19b and Nanog, is predictive for future transcription. Our results show that the maternally provided transcription factors Pou5f3, Sox19b, and Nanog open up chromatin and prime genes for activity during zygotic genome activation in zebrafish.

Clemens Heissenberger, Lisa Liendl, Fabian Nagelreiter, Yulia Gonskikh, Guohuan Yang, Ernst H K Stelzer, Teresa L Krammer, Lucia Micutkova, Stefan Vogt, David P. Kreil, Gerhard Sekot, Emilio Siena, Ina Poser, Eva Harreither, Angela Linder, Viktoria Ehret, Thomas H Helbich, Regina Grillari-Voglauer, Pidder Jansen-Dürr, Martin Koš, Norbert Polacek, Johannes Grillari, Markus Schosserer
Loss of the ribosomal RNA methyltransferase NSUN5 impairs global protein synthesis and normal growth.
Nucleic Acids Res, 47(22) 11807-11825 (2019)
Open Access DOI

Modifications of ribosomal RNA expand the nucleotide repertoire and thereby contribute to ribosome heterogeneity and translational regulation of gene expression. One particular m5C modification of 25S ribosomal RNA, which is introduced by Rcm1p, was previously shown to modulate stress responses and lifespan in yeast and other small organisms. Here, we report that NSUN5 is the functional orthologue of Rcm1p, introducing m5C3782 into human and m5C3438 into mouse 28S ribosomal RNA. Haploinsufficiency of the NSUN5 gene in fibroblasts from William Beuren syndrome patients causes partial loss of this modification. The N-terminal domain of NSUN5 is required for targeting to nucleoli, while two evolutionary highly conserved cysteines mediate catalysis. Phenotypic consequences of NSUN5 deficiency in mammalian cells include decreased proliferation and size, which can be attributed to a reduction in total protein synthesis by altered ribosomes. Strikingly, Nsun5 knockout in mice causes decreased body weight and lean mass without alterations in food intake, as well as a trend towards reduced protein synthesis in several tissues. Together, our findings emphasize the importance of single RNA modifications for ribosome function and normal cellular and organismal physiology.

Georg Fritz, Noreen Walker, Ulrich Gerland
Heterogeneous Timing of Gene Induction as a Regulation Strategy.
J Mol Biol, 431(23) 4760-4774 (2019)
Open Access DOI

In response to environmental changes, cells often adapt by up-regulating genes to synthesize proteins that generate a benefit in the new environment. Several such cases of gene induction have been reported where the timing was heterogeneous, with some cells responding early and others responding late, although the microbial population was genetically homogeneous and the environment was well mixed. Here, we explore under which conditions heterogeneous timing of gene induction could be advantageous for the population as a whole. We base our study on a mathematical model that accounts for the cost of protein synthesis in terms of resources, which cells must provide immediately, whereas the associated benefit accumulates only slowly over the protein lifetime. Due to this delayed benefit, gene induction can be a risky investment, if resources are scarce and the environment fluctuates rapidly and unpredictably. Unprofitable gene induction then depletes the remaining limiting resource needed for maintenance of cell viability. We show that whenever gene induction is associated with a transient risk but beneficial in the long run, the stochastic timing of gene induction maximizes the reproductive success of a population. In particular, in an environment of stochastic periods of famine and feast, an optimum emerges from a trade-off between short-term growth, favoring rapid and homogeneous responses, and long-term survival, favoring a broadly heterogeneous response. Our analysis suggests that the optimal variability of induction times is just as large as the time required for the amortization of the initial investment into protein synthesis.

Bogdan Kirilenko, Lee R Hagey, Stephen Barnes, Charles N Falany, Michael Hiller
Evolutionary Analysis of Bile Acid-Conjugating Enzymes Reveals a Complex Duplication and Reciprocal Loss History.
Genome Biol Evol, 11(11) 3256-3268 (2019)
Open Access PDF DOI

To fulfill their physiological functions, bile acids are conjugated with amino acids. In humans, conjugation is catalyzed by bile acid coenzyme A: amino acid N-acyltransferase (BAAT), an enzyme with a highly conserved catalytic triad in its active site. Interestingly, the conjugated amino acids are highly variable among mammals, with some species conjugating bile acids with both glycine and taurine, whereas others conjugate only taurine. The genetic origin of these bile acid conjugation differences is unknown. Here, we tested whether mutations in BAAT's catalytic triad could explain bile acid conjugation differences. Our comparative analysis of 118 mammals first revealed that the ancestor of placental mammals and marsupials possessed two genes, BAAT and BAATP1, that arose by a tandem duplication. This duplication was followed by numerous gene losses, including BAATP1 in humans. Losses of either BAAT or BAATP1 largely happened in a reciprocal fashion, suggesting that a single conjugating enzyme is generally sufficient for mammals. In intact BAAT and BAATP1 genes, we observed multiple changes in the catalytic triad between Cys and Ser residues. Surprisingly, although mutagenesis experiments with the human enzyme have shown that replacing Cys for Ser greatly diminishes the glycine-conjugating ability, across mammals we found that this residue provides little power in predicting the experimentally measured amino acids that are conjugated with bile acids. This suggests that the mechanism of BAAT's enzymatic function is incompletely understood, despite relying on a classic catalytic triad. More generally, our evolutionary analysis indicates that results of mutagenesis experiments may not easily be extrapolatable to other species.

Kseniia Nikitina, Sandra Segeletz, Michael Kuhn, Yannis Kalaidzidis, Marino Zerial
Basic Phenotypes of Endocytic System Recognized by Independent Phenotypes Analysis of a High-throughput Genomic Screen.
In: Proceedings of the 2019 3rd International Conference on Computational Biology and Bioinformatics (2019), New York, ACM (2019), 69-75
DOI

High-content screens (HCS) using chemical and genomic interference based on light microscopy and quantitative image analysis yielded a large amount of multi-parametric (MP) phenotypic data. Such data-sets hold great promise for the understanding of cellular mechanisms by systems biology. However, extracting functional information from data-sets, such as links between cellular processes and the functions of unknown genes, remains challenging. The limitation of HCS analysis lies in the complexity of cellular organization. Here, we assumed that cellular processes have a modular structure, and deconvolved the MP data into separate signals from different cellular modules by Blind Source Separation. We applied a combination of quantitative MP image analysis (QMPIA) and Independent Component Analysis (ICA) to an image-based HCS of endocytosis, the process whereby cells uptake molecules from the outside and distribute them to different sub-cellular organelles. We named our approach Independent Phenotypes Analysis (IPA). Phenotypic traits revealed by IPA are interpretable in terms of perturbation of specific endosomal populations (e.g. specific cargo, specific molecular markers) and of specific functional modules (early stages of endocytosis, recycling, cell cycle, etc.). The profile of perturbation of each gene in such basic phenotypic coordinates intrinsically suggest its possible mode of action.

Juliana G. Roscito, Michael Hiller
Methods to Detect and Associate Divergence in Cis-Regulatory Elements to Phenotypic Divergence.
In: Evolution, Origin of Life, Concepts and Methods . (Eds.) Pierre Pontarotti,Cham,Springer International Publishing (2019),113-134 Ch. 6
PDF DOI

Understanding which genomic changes are responsible for morphological differences between species is a long-standing question in biology. While evolutionary theory predicts that morphology largely evolves by changing expression of important developmental genes, finding the underlying regulatory mutations is inherently difficult. Here, we discuss how the integration of comparative and functional genomics has provided valuable insights into the regulatory changes involved in morphological changes. By comparing genomes of species exhibiting differences in a morphological trait, comparative genomic methods enable the systematic detection of regulatory elements with divergence in sequence or transcription factor binding sites. To narrow this set of diverged elements down to those that likely contribute to differences in the trait of interest, one can leverage knowledge about gene function to assess which elements are associated with genes known to control the development of this trait. In addition, functional genomics can further prioritize diverged genomic regions based on overlap with experimentally determined regulatory elements that are active in tissues relevant for the trait. Further experiments can then evaluate whether sequence or binding site divergence translates into regulatory differences and affects the development of the trait. Thus, combining comparative and functional genomic approaches provide a widely applicable strategy to reveal regulatory changes contributing to morphological differences, which will enhance our understanding of how nature’s spectacular phenotypic diversity evolved.

Sabina Kanton, Michael James Boyle, Zhisong He, Malgorzata Santel, Anne Weigert, Fátima Sanchís-Calleja, Patricia Guijarro, Leila Sidow, Jonas Simon Fleck, Dingding Han, Zhengzong Qian, Michael Heide, Wieland Huttner, Philipp Khaitovich, Svante Pääbo, Barbara Treutlein, J Gray Camp
Organoid single-cell genomic atlas uncovers human-specific features of brain development.
Nature, 574(7778) 418-422 (2019)
DOI

The human brain has undergone substantial change since humans diverged from chimpanzees and the other great apes1,2. However, the genetic and developmental programs that underlie this divergence are not fully understood. Here we have analysed stem cell-derived cerebral organoids using single-cell transcriptomics and accessible chromatin profiling to investigate gene-regulatory changes that are specific to humans. We first analysed cell composition and reconstructed differentiation trajectories over the entire course of human cerebral organoid development from pluripotency, through neuroectoderm and neuroepithelial stages, followed by divergence into neuronal fates within the dorsal and ventral forebrain, midbrain and hindbrain regions. Brain-region composition varied in organoids from different iPSC lines, but regional gene-expression patterns remained largely reproducible across individuals. We analysed chimpanzee and macaque cerebral organoids and found that human neuronal development occurs at a slower pace relative to the other two primates. Using pseudotemporal alignment of differentiation paths, we found that human-specific gene expression resolved to distinct cell states along progenitor-to-neuron lineages in the cortex. Chromatin accessibility was dynamic during cortex development, and we identified divergence in accessibility between human and chimpanzee that correlated with human-specific gene expression and genetic change. Finally, we mapped human-specific expression in adult prefrontal cortex using single-nucleus RNA sequencing analysis and identified developmental differences that persist into adulthood, as well as cell-state-specific changes that occur exclusively in the adult brain. Our data provide a temporal cell atlas of great ape forebrain development, and illuminate dynamic gene-regulatory features that are unique to humans.

Yuko Sato, Lennart Hilbert, Haruka Oda, Yinan Wan, John M Heddleston, Teng-Leong Chew, Vasily Zaburdaev, Philipp Keller, Timothee Lionnet, Nadine Vastenhouw, Hiroshi Kimura
Histone H3K27 acetylation precedes active transcription during zebrafish zygotic genome activation as revealed by live-cell analysis.
Development, 146(19) Art. No. dev179127 (2019)
Open Access DOI

Histone post-translational modifications are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We applied a Fab-based live endogenous modification labeling technique to monitor the changes in histone modification levels during zygotic genome activation (ZGA) in living zebrafish embryos. Among various histone modifications, H3 Lys27 acetylation (H3K27ac) exhibited most drastic changes, accumulating in two nuclear foci in the 64- to 1k-cell-stage embryos. The elongating form of RNA polymerase II, which is phosphorylated at Ser2 in heptad repeats within the C-terminal domain (RNAP2 Ser2ph), and miR-430 transcripts were also concentrated in foci closely associated with H3K27ac. When treated with α-amanitin to inhibit transcription or JQ-1 to inhibit binding of acetyl-reader proteins, H3K27ac foci still appeared but RNAP2 Ser2ph and miR-430 morpholino were not concentrated in foci, suggesting that H3K27ac precedes active transcription during ZGA. We anticipate that the method presented here could be applied to a variety of developmental processes in any model and non-model organisms.

Kathrin Schmeisser, J Alex Parker
Pleiotropic Effects of mTOR and Autophagy During Development and Aging.
Front Cell Dev Biol, 7 Art. No. 192 (2019)
Open Access DOI

Autophagy as a ubiquitous catabolic process causes degradation of cytoplasmic components and is generally considered to have beneficial effects on health and lifespan. In contrast, inefficient autophagy has been linked with detrimental effects on the organism and various diseases, such as Parkinson's disease. Previous research, however, showed that this paradigm is far from being black and white. For instance, it has been reported that increased levels of autophagy during development can be harmful, but become advantageous in the aging cell or organism, causing enhanced healthspan and even longevity. The antagonistic pleiotropy hypothesis postulates that genes, which control various traits in an organism, can be fitness-promoting in early life, but subsequently trigger aging processes later. Autophagy is controlled by the mechanistic target of rapamycin (mTOR), a key player of nutrient sensing and signaling and classic example of a pleiotropic gene. mTOR acts upstream of transcription factors such as FOXO, NRF, and TFEB, controlling protein synthesis, degradation, and cellular growth, thereby regulating fertility as well as aging. Here, we review recent findings about the pleiotropic role of autophagy during development and aging, examine the upstream factors, and contemplate specific mechanisms leading to disease, especially neurodegeneration.

Matthias Huelsmann, Nikolai Hecker, Mark S Springer, John Gatesy, Virag Sharma, Michael Hiller
Genes lost during the transition from land to water in cetaceans highlight genomic changes associated with aquatic adaptations.
Sci Adv, 5(9) Art. No. eaaw6671 (2019)
Open Access PDF DOI

The transition from land to water in whales and dolphins (cetaceans) was accompanied by remarkable adaptations. To reveal genomic changes that occurred during this transition, we screened for protein-coding genes that were inactivated in the ancestral cetacean lineage. We found 85 gene losses. Some of these were likely beneficial for cetaceans, for example, by reducing the risk of thrombus formation during diving (F12 and KLKB1), erroneous DNA damage repair (POLM), and oxidative stress-induced lung inflammation (MAP3K19). Additional gene losses may reflect other diving-related adaptations, such as enhanced vasoconstriction during the diving response (mediated by SLC6A18) and altered pulmonary surfactant composition (SEC14L3), while loss of SLC4A9 relates to a reduced need for saliva. Last, loss of melatonin synthesis and receptor genes (AANAT, ASMT, and MTNR1A/B) may have been a precondition for adopting unihemispheric sleep. Our findings suggest that some genes lost in ancestral cetaceans were likely involved in adapting to a fully aquatic lifestyle.

Karin D Prummel, Christopher Hess, Susan Nieuwenhuize, Hugo J Parker, Katherine W Rogers, Iryna Kozmikova, Claudia Racioppi, Eline C Brombacher, Anna Czarkwiani, Dunja Knapp, Sibylle Burger, Elena Chiavacci, Gopi Shah, Alexa Burger, Jan Huisken, Maximina H Yun, Lionel Christiaen, Zbynek Kozmik, Patrick Müller, Marianne Bronner, Robb Krumlauf, Christian Mosimann
A conserved regulatory program initiates lateral plate mesoderm emergence across chordates.
Nat Commun, 10(1) Art. No. 3857 (2019)
Open Access DOI

Cardiovascular lineages develop together with kidney, smooth muscle, and limb connective tissue progenitors from the lateral plate mesoderm (LPM). How the LPM initially emerges and how its downstream fates are molecularly interconnected remain unknown. Here, we isolate a pan-LPM enhancer in the zebrafish-specific draculin (drl) gene that provides specific LPM reporter activity from early gastrulation. In toto live imaging and lineage tracing of drl-based reporters captures the dynamic LPM emergence as lineage-restricted mesendoderm field. The drl pan-LPM enhancer responds to the transcription factors EomesoderminA, FoxH1, and MixL1 that combined with Smad activity drive LPM emergence. We uncover specific activity of zebrafish-derived drl reporters in LPM-corresponding territories of several chordates including chicken, axolotl, lamprey, Ciona, and amphioxus, revealing a universal upstream LPM program. Altogether, our work provides a mechanistic framework for LPM emergence as defined progenitor field, possibly representing an ancient mesodermal cell state that predates the primordial vertebrate embryo.

Mauricio Rocha-Martins, Beatriz C de Toledo, Pedro Santos-França, Viviane Oliveira-Valença, Carlos Vieira-Vieira, Gabriel Matos-Rodrigues, Rafael Linden, Caren Norden, Rodrigo Martins^#, Mariana Silveira^#
De novo genesis of retinal ganglion cells by targeted expression of Klf4 in vivo.
Development, 146(16) Art. No. dev176586 (2019)
DOI

Retinal ganglion cell (RGC) degeneration is a hallmark of glaucoma, the most prevalent cause of irreversible blindness. Thus, therapeutic strategies are needed to protect and replace these projection neurons. One innovative approach is to promote de novo genesis of RGCs via manipulation of endogenous cell sources. Here, we demonstrate that the pluripotency regulator gene Krüppel-like factor 4 (Klf4) is sufficient to change the potency of lineage-restricted retinal progenitor cells to generate RGCs in vivo Transcriptome analysis disclosed that the overexpression of Klf4 induces crucial regulators of RGC competence and specification, including Atoh7 and Eya2 In contrast, loss-of-function studies in mice and zebrafish demonstrated that Klf4 is not essential for generation or differentiation of RGCs during retinogenesis. Nevertheless, induced RGCs (iRGCs) generated upon Klf4 overexpression migrate to the proper layer and project axons aligned with endogenous fascicles that reach the optic nerve head. Notably, iRGCs survive for up to 30 days after in vivo generation. We identified Klf4 as a promising candidate for reprogramming retinal cells and regenerating RGCs in the retina.This article has an associated 'The people behind the papers' interview.

Kassiani Skouloudaki^#, Ioannis Christodoulou, Dilan Khalili, Vasilios Tsarouhas, Christos Samakovlis, Pavel Tomancak, Elisabeth Knust^#, Dimitrios Papadopoulos^#
Yorkie controls tube length and apical barrier integrity during airway development.
J Cell Biol, 218(8) 2762-2781 (2019)
DOI

Epithelial organ size and shape depend on cell shape changes, cell-matrix communication, and apical membrane growth. The Drosophila melanogaster embryonic tracheal network is an excellent model to study these processes. Here, we show that the transcriptional coactivator of the Hippo pathway, Yorkie (YAP/TAZ in vertebrates), plays distinct roles in the developing Drosophila airways. Yorkie exerts a cytoplasmic function by binding Drosophila Twinstar, the orthologue of the vertebrate actin-severing protein Cofilin, to regulate F-actin levels and apical cell membrane size, which are required for proper tracheal tube elongation. Second, Yorkie controls water tightness of tracheal tubes by transcriptional regulation of the δ-aminolevulinate synthase gene (Alas). We conclude that Yorkie has a dual role in tracheal development to ensure proper tracheal growth and functionality.

Nikolai Hecker, Ulla Lächele, Heiko Stuckas, Peter Giere, Michael Hiller
Convergent vomeronasal system reduction in mammals coincides with convergent losses of calcium signalling and odorant-degrading genes.
Mol Ecol, 28(16) 3656-3668 (2019)
PDF DOI

The vomeronasal system (VNS) serves crucial functions for detecting olfactory clues often related to social and sexual behaviour. Intriguingly, two of the main components of the VNS, the vomeronasal organ (VNO) and the accessory olfactory bulb, are regressed in aquatic mammals, several bats and primates, likely due to adaptations to different ecological niches. To detect genomic changes that are associated with the convergent reduction of the VNS, we performed the first systematic screen for convergently inactivated protein-coding genes associated with convergent VNS reduction, considering 106 mammalian genomes. Extending previous studies, our results support that Trpc2, a cation channel that is important for calcium signalling in the VNO, is a predictive molecular marker for the presence of a VNS. Our screen also detected the convergent inactivation of the calcium-binding protein S100z, the aldehyde oxidase Aox2 that is involved in odorant degradation, and the uncharacterized Mslnl gene that is expressed in the VNO and olfactory epithelium. Furthermore, we found that Trpc2 and S100z or Aox2 are also inactivated in otters and Phocid seals for which no morphological data about the VNS are available yet. This predicts a VNS reduction in these semi-aquatic mammals. By examining the genomes of 115 species in total, our study provides a detailed picture of how the convergent reduction of the VNS coincides with gene inactivation in placental mammals. These inactivated genes provide experimental targets for studying the evolution and biological significance of the olfactory system under different environmental conditions.

Steven W Poser, Oliver Otto, Carina Arps-Forker, Yan Ge, Maik Herbig, Cordula Andree, Konrad Gruetzmann, Melissa F Adasme, Szymon Stodolak, Polyxeni Nikolakopoulou, Deric M Park, Alan Mcintyre, Mathias Lesche, Andreas Dahl, Petra Lennig, Stefan R. Bornstein, Evelin Schroeck, Barbara Klink, Ronen R Leker, Marc Bickle, George P Chrousos, Michael Schroeder, Carlo Vittorio Cannistraci, Jochen Guck, Andreas Androutsellis-Theotokis
Controlling distinct signaling states in cultured cancer cells provides a new platform for drug discovery.
FASEB J, 33(8) 9235-9249 (2019)
DOI

Cancer cells can switch between signaling pathways to regulate growth under different conditions. In the tumor microenvironment, this likely helps them evade therapies that target specific pathways. We must identify all possible states and utilize them in drug screening programs. One such state is characterized by expression of the transcription factor Hairy and Enhancer of Split 3 (HES3) and sensitivity to HES3 knockdown, and it can be modeled in vitro. Here, we cultured 3 primary human brain cancer cell lines under 3 different culture conditions that maintain low, medium, and high HES3 expression and characterized gene regulation and mechanical phenotype in these states. We assessed gene expression regulation following HES3 knockdown in the HES3-high conditions. We then employed a commonly used human brain tumor cell line to screen Food and Drug Administration (FDA)-approved compounds that specifically target the HES3-high state. We report that cells from multiple patients behave similarly when placed under distinct culture conditions. We identified 37 FDA-approved compounds that specifically kill cancer cells in the high-HES3-expression conditions. Our work reveals a novel signaling state in cancer, biomarkers, a strategy to identify treatments against it, and a set of putative drugs for potential repurposing.-Poser, S. W., Otto, O., Arps-Forker, C., Ge, Y., Herbig, M., Andree, C., Gruetzmann, K., Adasme, M. F., Stodolak, S., Nikolakopoulou, P., Park, D. M., Mcintyre, A., Lesche, M., Dahl, A., Lennig, P., Bornstein, S. R., Schroeck, E., Klink, B., Leker, R. R., Bickle, M., Chrousos, G. P., Schroeder, M., Cannistraci, C. V., Guck, J., Androutsellis-Theotokis, A. Controlling distinct signaling states in cultured cancer cells provides a new platform for drug discovery.

Ariel O Donayo, Radia M Johnson, Hsin-Wei Tseng, Said Izreig, Alexandra Gariepy, Vinay K Mayya, Edlyn Wu, Roshni Alam, Carine Lussier, Russell G Jones, Thomas F Duchaine
Oncogenic Biogenesis of pri-miR-17∼92 Reveals Hierarchy and Competition among Polycistronic MicroRNAs.
Mol Cell, 75(2) 340-356 (2019)
DOI

The microRNAs encoded by the miR-17∼92 polycistron are commonly overexpressed in cancer and orchestrate a wide range of oncogenic functions. Here, we identify a mechanism for miR-17∼92 oncogenic function through the disruption of endogenous microRNA (miRNA) processing. We show that, upon oncogenic overexpression of the miR-17∼92 primary transcript (pri-miR-17∼92), the microprocessor complex remains associated with partially processed intermediates that aberrantly accumulate. These intermediates reflect a series of hierarchical and conserved steps in the early processing of the pri-miR-17∼92 transcript. Encumbrance of the microprocessor by miR-17∼92 intermediates leads to the broad but selective downregulation of co-expressed polycistronic miRNAs, including miRNAs derived from tumor-suppressive miR-34b/c and from the Dlk1-Dio3 polycistrons. We propose that the identified steps of polycistronic miR-17∼92 biogenesis contribute to the oncogenic re-wiring of gene regulation networks. Our results reveal previously unappreciated functional paradigms for polycistronic miRNAs in cancer.

Aleksandra Spiegel, Mandy Bachmann, Gabriel Jurado Jiménez, Mihail Sarov
CRISPR/Cas9-based knockout pipeline for reverse genetics in mammalian cell culture.
Methods, 164/165 49-58 (2019)
Open Access DOI

We present a straightforward protocol for reverse genetics in cultured mammalian cells, using CRISPR/Cas9-mediated homology-dependent repair (HDR) based insertion of a protein trap cassette, resulting in a termination of the endogenous gene expression. Complete loss of function can be achieved with monoallelic trap cassette insertion, as the second allele is frequently disrupted by an error-prone non-homologous end joining (NHEJ) mechanism. The method should be applicable to any expressed gene in most cell lines, including those with low HDR efficiency, as the knockout alleles can be directly selected for.

Mattias Hogvall, Bruno C Vellutini, José M Martín-Durán, Andreas Hejnol, Graham E Budd, Ralf Janssen
Embryonic expression of priapulid Wnt genes.
Dev Genes Evol, 229(4) 125-135 (2019)
Open Access DOI

Posterior elongation of the developing embryo is a common feature of animal development. One group of genes that is involved in posterior elongation is represented by the Wnt genes, secreted glycoprotein ligands that signal to specific receptors on neighbouring cells and thereby establish cell-to-cell communication. In segmented animals such as annelids and arthropods, Wnt signalling is also likely involved in segment border formation and regionalisation of the segments. Priapulids represent unsegmented worms that are distantly related to arthropods. Despite their interesting phylogenetic position and their importance for the understanding of ecdysozoan evolution, priapulids still represent a highly underinvestigated group of animals. Here, we study the embryonic expression patterns of the complete sets of Wnt genes in the priapulids Priapulus caudatus and Halicryptus spinulosus. We find that both priapulids possess a complete set of 12 Wnt genes. At least in Priapulus, most of these genes are expressed in and around the posterior-located blastopore and thus likely play a role in posterior elongation. Together with previous work on the expression of other genetic factors such as caudal and even-skipped, this suggests that posterior elongation in priapulids is under control of the same (or very similar) conserved gene regulatory network as in arthropods.

Nadine Vastenhouw^#, Wen Xi Cao, Howard D Lipshitz^#
The maternal-to-zygotic transition revisited.
Development, 146(11) Art. No. dev161471 (2019)
DOI

The development of animal embryos is initially directed by maternal gene products. Then, during the maternal-to-zygotic transition (MZT), developmental control is handed to the zygotic genome. Extensive research in both vertebrate and invertebrate model organisms has revealed that the MZT can be subdivided into two phases, during which very different modes of gene regulation are implemented: initially, regulation is exclusively post-transcriptional and post-translational, following which gradual activation of the zygotic genome leads to predominance of transcriptional regulation. These changes in the gene expression program of embryos are precisely controlled and highly interconnected. Here, we review current understanding of the mechanisms that underlie handover of developmental control during the MZT.

Dimitrios Papadopoulos^#, Pavel Tomancak^#
Gene Regulation: Analog to Digital Conversion of Transcription Factor Gradients.
Curr Biol, 29(11) 422-424 (2019)
DOI

Transcription factor gradients trigger differential transcriptional responses based on concentration. But how, in some cases, do target genes maintain uniform transcription across portions of the gradient? Lessons from Drosophila demonstrate that organization of transcription into 'hubs' can lead to local increases in transcription factor concentration.

Falk Zakrzewski, Walter de Back, Martin Weigert, Torsten Wenke, Silke Zeugner, Robert Mantey, Christian Sperling, Katrin Friedrich, Ingo Roeder, Daniela Aust, Gustavo Baretton, Pia Hönscheid
Automated detection of the HER2 gene amplification status in Fluorescence in situ hybridization images for the diagnostics of cancer tissues.
Sci Rep, 9(1) Art. No. 8231 (2019)
Open Access DOI

The human epidermal growth factor receptor 2 (HER2) gene amplification status is a crucial marker for evaluating clinical therapies of breast or gastric cancer. We propose a deep learning-based pipeline for the detection, localization and classification of interphase nuclei depending on their HER2 gene amplification state in Fluorescence in situ hybridization (FISH) images. Our pipeline combines two RetinaNet-based object localization networks which are trained (1) to detect and classify interphase nuclei into distinct classes normal, low-grade and high-grade and (2) to detect and classify FISH signals into distinct classes HER2 or centromere of chromosome 17 (CEN17). By independently classifying each nucleus twice, the two-step pipeline provides both robustness and interpretability for the automated detection of the HER2 amplification status. The accuracy of our deep learning-based pipeline is on par with that of three pathologists and a set of 57 validation images containing several hundreds of nuclei are accurately classified. The automatic pipeline is a first step towards assisting pathologists in evaluating the HER2 status of tumors using FISH images, for analyzing FISH images in retrospective studies, and for optimizing the documentation of each tumor sample by automatically annotating and reporting of the HER2 gene amplification specificities.

Titus Franzmann, Simon Alberti
Protein Phase Separation as a Stress Survival Strategy.
Cold Spring Harb Perspect Biol, 11(6) Art. No. a034058 (2019)
DOI

Cells under stress must adjust their physiology, metabolism, and architecture to adapt to the new conditions. Most importantly, they must down-regulate general gene expression, but at the same time induce synthesis of stress-protective factors, such as molecular chaperones. Here, we investigate how the process of phase separation is used by cells to ensure adaptation to stress. We summarize recent findings and propose that the solubility of important translation factors is specifically affected by changes in physical-chemical parameters such temperature or pH and modulated by intrinsically disordered prion-like domains. These stress-triggered changes in protein solubility induce phase separation into condensates that regulate the activity of the translation factors and promote cellular fitness. Prion-like domains play important roles in this process as environmentally regulated stress sensors and modifier sequences that determine protein solubility and phase behavior. We propose that protein phase separation is an evolutionary conserved feature of proteins that cells harness to regulate adaptive stress responses and ensure survival in extreme environmental conditions.

Yaser Atlasi, Wout Megchelenbrink, Tianran Peng, Ehsan Habibi, Onkar Joshi, Shuang-Yin Wang, Cheng Wang, Colin Logie, Ina Poser, Hendrik Marks, Hendrik G Stunnenberg
Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency.
Nat Cell Biol, 21(5) 568-578 (2019)
DOI

The mechanisms underlying enhancer activation and the extent to which enhancer-promoter rewiring contributes to spatiotemporal gene expression are not well understood. Using integrative and time-resolved analyses we show that the extensive transcriptome and epigenome resetting during the conversion between 'serum' and '2i' states of mouse embryonic stem cells (ESCs) takes place with minimal enhancer-promoter rewiring that becomes more evident in primed-state pluripotency. Instead, differential gene expression is strongly linked to enhancer activation via H3K27ac. Conditional depletion of transcription factors and allele-specific enhancer analysis reveal an essential role for Esrrb in H3K27 acetylation and activation of 2i-specific enhancers. Restoration of a polymorphic ESRRB motif using CRISPR-Cas9 in a hybrid ESC line restores ESRRB binding and enhancer H3K27ac in an allele-specific manner but has no effect on chromatin interactions. Our study shows that enhancer activation in serum- and 2i-ESCs is largely driven by transcription factor binding and epigenetic marking in a hardwired network of chromatin interactions.

Ilaria Rossetti, Laura Zambusi, Paola Maccioni, Roberta Sau, Luciano Provini, M Paola Castelli, Krzysztof Gonciarz, Giancarlo Colombo, Stefano Morara
Predisposition to Alcohol Drinking and Alcohol Consumption Alter Expression of Calcitonin Gene-Related Peptide, Neuropeptide Y, and Microglia in Bed Nucleus of Stria Terminalis in a Subnucleus-Specific Manner.
Front Cell Neurosci, 13 Art. No. 158 (2019)
Open Access DOI

Excessive alcohol consumption is often linked to anxiety states and has a major relay center in the anterior part of bed nucleus of stria terminalis (BNST). We analyzed the impact of (i) genetic predisposition to high alcohol preference and consumption, and (ii) alcohol intake on anterior BNST, namely anterolateral (AL), anteromedial (AM), and anteroventral (lateral + medial subdivisions: AVl, AVm) subnuclei. We used two rat lines selectively bred for low- and high-alcohol preference and consumption, named Sardinian alcohol-non preferring (sNP) and -preferring (sP), respectively, the latter showing also inherent anxiety-related behaviors. We analyzed the modulation of calcitonin gene-related peptide (CGRP; exerting anxiogenic effects in BNST), neuropeptide Y (NPY; exerting mainly anxiolytic effects), and microglia activation (neuroinflammation marker, thought to increase anxiety). Calcitonin gene-related peptide-immunofluorescent fibers/terminals did not differ between alcohol-naive sP and sNP rats. Fiber/terminal NPY-immunofluorescent intensity was lower in BNST-AM and BNST-AVm of alcohol-naive sP rats. Activation of microglia (revealed by morphological analysis) was decreased in BNST-AM and increased in BNST-AVm of alcohol-naive sP rats. Prolonged (30 consecutive days), voluntary alcohol intake under the homecage 2-bottle "alcohol vs. water" regimen strongly increased CGRP intensity in BNST of sP rats in a subnucleus-specific manner: in BNST-AL, BNST-AVm, and BNST-AM. CGRP area sum, however, decreased in BNST-AM, without changes in other subnuclei. Alcohol consumption increased NPY expression, in a subnucleus-specific manner, in BNST-AL, BNST-AVl, and BNST-AVm. Alcohol consumption increased many size/shapes parameters in microglial cells, indicative of microglia de-activation. Finally, microglia density was increased in ventral anterior BNST (BNST-AVl, BNST-AVm) by alcohol consumption. In conclusion, genetic predisposition of sP rats to high alcohol intake could be in part mediated by anterior BNST subnuclei showing lower NPY expression and differential microglia activation. Alcohol intake in sP rats produced complex subnucleus-specific changes in BNST, affecting CGRP/NPY expression and microglia and leading to hypothesize that these changes might contribute to the anxiolytic effects of voluntarily consumed alcohol repeatedly observed in sP rats.

Mohamed A El-Brolosy, Zacharias Kontarakis, Andrea Rossi, Carsten Kuenne, Stefan Günther, Nana Fukuda, Khrievono Kikhi, Giulia L M Boezio, Carter M Takacs, Shih-Lei Lai, Ryuichi Fukuda, Claudia Gerri, Antonio J Giraldez, Didier Y.R. Stainier
Genetic compensation triggered by mutant mRNA degradation.
Nature, 568(7751) 193-197 (2019)
DOI

Genetic robustness, or the ability of an organism to maintain fitness in the presence of harmful mutations, can be achieved via protein feedback loops. Previous work has suggested that organisms may also respond to mutations by transcriptional adaptation, a process by which related gene(s) are upregulated independently of protein feedback loops. However, the prevalence of transcriptional adaptation and its underlying molecular mechanisms are unknown. Here, by analysing several models of transcriptional adaptation in zebrafish and mouse, we uncover a requirement for mutant mRNA degradation. Alleles that fail to transcribe the mutated gene do not exhibit transcriptional adaptation, and these alleles give rise to more severe phenotypes than alleles displaying mutant mRNA decay. Transcriptome analysis in alleles displaying mutant mRNA decay reveals the upregulation of a substantial proportion of the genes that exhibit sequence similarity with the mutated gene's mRNA, suggesting a sequence-dependent mechanism. These findings have implications for our understanding of disease-causing mutations, and will help in the design of mutant alleles with minimal transcriptional adaptation-derived compensation.

Lukas Huijbregts, Maja Borup Kjær Petersen, Claire Berthault, Mattias Hansson, Virginie Aiello, Latif Rachdi, Anne Grapin-Botton, Christian Honore, Raphael Scharfmann
Bromodomain and Extra Terminal Protein Inhibitors Promote Pancreatic Endocrine Cell Fate.
Diabetes, 68(4) 761-773 (2019)
DOI

Bromodomain and extraterminal (BET) proteins are epigenetic readers that interact with acetylated lysines of histone tails. Recent studies have demonstrated their role in cancer progression because they recruit key components of the transcriptional machinery to modulate gene expression. However, their role during embryonic development of the pancreas has never been studied. Using mouse embryonic pancreatic explants and human induced pluripotent stem cells (hiPSCs), we show that BET protein inhibition with I-BET151 or JQ1 enhances the number of neurogenin3 (NEUROG3) endocrine progenitors. In mouse explants, BET protein inhibition further led to increased expression of β-cell markers but in the meantime, strongly downregulated Ins1 expression. Similarly, although acinar markers, such as Cpa1 and CelA, were upregulated, Amy expression was repressed. In hiPSCs, BET inhibitors strongly repressed C-peptide and glucagon during endocrine differentiation. Explants and hiPSCs were then pulsed with BET inhibitors to increase NEUROG3 expression and further chased without inhibitors. Endocrine development was enhanced in explants with higher expression of insulin and maturation markers, such as UCN3 and MAFA. In hiPSCs, the outcome was different because C-peptide expression remained lower than in controls, but ghrelin expression was increased. Altogether, by using two independent models of pancreatic development, we show that BET proteins regulate multiple aspects of pancreatic development.

Shashank Rai^*, Maryam Arasteh^*, Matthew Jefferson^*, Timothy Pearson, Yingxue Wang, Weijiao Zhang, Bertalan Bicsak, Devina Divekar, Penny P Powell, Ronald Naumann, Naiara Beraza, Simon R Carding, Oliver Florey, Ulrike Mayer^#, Thomas Wileman^#
The ATG5-binding and coiled coil domains of ATG16L1 maintain autophagy and tissue homeostasis in mice independently of the WD domain required for LC3-associated phagocytosis.
Autophagy, 15(4) 599-612 (2019)
Open Access DOI

Macroautophagy/autophagy delivers damaged proteins and organelles to lysosomes for degradation, and plays important roles in maintaining tissue homeostasis by reducing tissue damage. The translocation of LC3 to the limiting membrane of the phagophore, the precursor to the autophagosome, during autophagy provides a binding site for autophagy cargoes, and facilitates fusion with lysosomes. An autophagy-related pathway called LC3-associated phagocytosis (LAP) targets LC3 to phagosome and endosome membranes during uptake of bacterial and fungal pathogens, and targets LC3 to swollen endosomes containing particulate material or apoptotic cells. We have investigated the roles played by autophagy and LAP in vivo by exploiting the observation that the WD domain of ATG16L1 is required for LAP, but not autophagy. Mice lacking the linker and WD domains, activate autophagy, but are deficient in LAP. The LAP-/- mice survive postnatal starvation, grow at the same rate as littermate controls, and are fertile. The liver, kidney, brain and muscle of these mice maintain levels of autophagy cargoes such as LC3 and SQSTM1/p62 similar to littermate controls, and prevent accumulation of SQSTM1 inclusions and tissue damage associated with loss of autophagy. The results suggest that autophagy maintains tissue homeostasis in mice independently of LC3-associated phagocytosis. Further deletion of glutamate E230 in the coiled-coil domain required for WIPI2 binding produced mice with defective autophagy that survived neonatal starvation. Analysis of brain lysates suggested that interactions between WIPI2 and ATG16L1 were less critical for autophagy in the brain, which may allow a low level of autophagy to overcome neonatal lethality. Abbreviations: CCD: coiled-coil domain; CYBB/NOX2: cytochrome b-245: beta polypeptide; GPT/ALT: glutamic pyruvic transaminase: soluble; LAP: LC3-associated phagocytosis; LC3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NOD: nucleotide-binding oligomerization domain; NADPH: nicotinamide adenine dinucleotide phosphate; RUBCN/Rubicon: RUN domain and cysteine-rich domain containing Beclin 1-interacting protein; SLE: systemic lupus erythematosus; SQSTM1/p62: sequestosome 1; TLR: toll-like receptor; TMEM: transmembrane protein; TRIM: tripartite motif-containing protein; UVRAG: UV radiation resistance associated gene; WD: tryptophan-aspartic acid; WIPI: WD 40 repeat domain: phosphoinositide interacting.

Naomi D Chrispijn, Dei M Elurbe, Michaela Mickoleit, Marco Aben, Dennis E M de Bakker, Karolina M Andralojc, Jan Huisken, Jeroen Bakkers, Leonie M Kamminga
Loss of the Polycomb group protein Rnf2 results in derepression of tbx-transcription factors and defects in embryonic and cardiac development.
Sci Rep, 9(1) Art. No. 4327 (2019)
Open Access DOI

The Polycomb group (PcG) protein family is a well-known group of epigenetic modifiers. We used zebrafish to investigate the role of Rnf2, the enzymatic subunit of PRC1. We found a positive correlation between loss of Rnf2 and upregulation of genes, especially of those whose promoter is normally bound by Rnf2. The heart of rnf2 mutants shows a tubular shaped morphology and to further understand the underlying mechanism, we studied gene expression of single wildtype and rnf2 mutant hearts. We detected the most pronounced differences at 3 dpf, including upregulation of heart transcription factors, such as tbx2a, tbx2b, and tbx3a. These tbx genes were decorated by broad PcG domains in wildtype whole embryo lysates. Chamber specific genes such as vmhc, myh6, and nppa showed downregulation in rnf2 mutant hearts. The marker of the working myocard, nppa, is negatively regulated by Tbx2 and Tbx3. Based on our findings and literature we postulate that loss of Rnf2-mediated repression results in upregulation and ectopic expression of tbx2/3, whose expression is normally restricted to the cardiac conductive system. This could lead to repression of chamber specific gene expression, a misbalance in cardiac cell types, and thereby to cardiac defects observed in rnf2 mutants.

Giorgio Fracasso, Yvonne Körner, David Thomas Gonzales, T-Y Dora Tang
In vitro gene expression and detergent-free reconstitution of active proteorhodopsin in lipid vesicles.
Exp Biol Med (Maywood), 244(4) 314-322 (2019)
DOI

Our results offer the potential for straightforward, additive-free, and molecularly simple routes to building complex bioreactors based on in vitro transcription-translation systems and lipid vesicles.

Björn Langer, Michael Hiller
TFforge utilizes large-scale binding site divergence to identify transcriptional regulators involved in phenotypic differences.
Nucleic Acids Res, 47(4) Art. No. e19 (2019)
Open Access PDF DOI

Changes in gene regulation are important for phenotypic and in particular morphological evolution. However, it remains challenging to identify the transcription factors (TFs) that contribute to differences in gene regulation and thus to phenotypic differences between species. Here, we present TFforge (Transcription Factor forward genomics), a computational method to identify TFs that are involved in the loss of phenotypic traits. TFforge screens an input set of regulatory genomic regions to detect TFs that exhibit a significant binding site divergence signature in species that lost a particular phenotypic trait. Using simulated data of modular and pleiotropic regulatory elements, we show that TFforge can identify the correct TFs for many different evolutionary scenarios. We applied TFforge to available eye regulatory elements to screen for TFs that exhibit a significant binding site decay signature in subterranean mammals. This screen identified interacting and co-binding eye-related TFs, and thus provides new insights into which TFs likely contribute to eye degeneration in these species. TFforge has broad applicability to identify the TFs that contribute to phenotypic changes between species, and thus can help to unravel the gene-regulatory differences that underlie phenotypic evolution.

Nikolai Hecker, Virag Sharma, Michael Hiller
Convergent gene losses illuminate metabolic and physiological changes in herbivores and carnivores.
Proc Natl Acad Sci U.S.A., 116(8) 3036-3041 (2019)
Open Access PDF DOI

The repeated evolution of dietary specialization represents a hallmark of mammalian ecology. To detect genomic changes that are associated with dietary adaptations, we performed a systematic screen for convergent gene losses associated with an obligate herbivorous or carnivorous diet in 31 placental mammals. For herbivores, our screen discovered the repeated loss of the triglyceride lipase inhibitor PNLIPRP1, suggesting enhanced triglyceride digestion efficiency. Furthermore, several herbivores lost the pancreatic exocytosis factor SYCN, providing an explanation for continuous pancreatic zymogen secretion in these species. For carnivores, we discovered the repeated loss of the hormone-receptor pair INSL5-RXFP4 that regulates appetite and glucose homeostasis, which likely relates to irregular feeding patterns and constant gluconeogenesis. Furthermore, reflecting the reduced need to metabolize plant-derived xenobiotics, several carnivores lost the xenobiotic receptors NR1I3 and NR1I2 Finally, the carnivore-associated loss of the gastrointestinal host defense gene NOX1 could be related to a reduced gut microbiome diversity. By revealing convergent gene losses associated with differences in dietary composition, feeding patterns, and gut microbiomes, our study contributes to understanding how similar dietary specializations evolved repeatedly in mammals.

Mark S Springer, Christopher A Emerling, John Gatesy, Jason Randall, Matthew A Collin, Nikolai Hecker, Michael Hiller, Frédéric Delsuc
Odontogenic ameloblast-associated (ODAM) is inactivated in toothless/enamelless placental mammals and toothed whales.
BMC Evol Biol, 19(1) Art. No. 31 (2019)
Open Access PDF DOI

The gene for odontogenic ameloblast-associated (ODAM) is a member of the secretory calcium-binding phosphoprotein gene family. ODAM is primarily expressed in dental tissues including the enamel organ and the junctional epithelium, and may also have pleiotropic functions that are unrelated to teeth. Here, we leverage the power of natural selection to test competing hypotheses that ODAM is tooth-specific versus pleiotropic. Specifically, we compiled and screened complete protein-coding sequences, plus sequences for flanking intronic regions, for ODAM in 165 placental mammals to determine if this gene contains inactivating mutations in lineages that either lack teeth (baleen whales, pangolins, anteaters) or lack enamel on their teeth (aardvarks, sloths, armadillos), as would be expected if the only essential functions of ODAM are related to tooth development and the adhesion of the gingival junctional epithelium to the enamel tooth surface.

Kassiani Skouloudaki^*^#, Dimitrios Papadopoulos^*, Pavel Tomancak, Elisabeth Knust^#
The apical protein Apnoia interacts with Crumbs to regulate tracheal growth and inflation.
PLoS Genet, 15(1) Art. No. e1007852 (2019)
Open Access DOI

Most organs of multicellular organisms are built from epithelial tubes. To exert their functions, tubes rely on apico-basal polarity, on junctions, which form a barrier to separate the inside from the outside, and on a proper lumen, required for gas or liquid transport. Here we identify apnoia (apn), a novel Drosophila gene required for tracheal tube elongation and lumen stability at larval stages. Larvae lacking Apn show abnormal tracheal inflation and twisted airway tubes, but no obvious defects in early steps of tracheal maturation. apn encodes a transmembrane protein, primarily expressed in the tracheae, which exerts its function by controlling the localization of Crumbs (Crb), an evolutionarily conserved apical determinant. Apn physically interacts with Crb to control its localization and maintenance at the apical membrane of developing airways. In apn mutant tracheal cells, Crb fails to localize apically and is trapped in retromer-positive vesicles. Consistent with the role of Crb in apical membrane growth, RNAi-mediated knockdown of Crb results in decreased apical surface growth of tracheal cells and impaired axial elongation of the dorsal trunk. We conclude that Apn is a novel regulator of tracheal tube expansion in larval tracheae, the function of which is mediated by Crb.

Andrei Rozanski, HongKee Moon, Holger Brandl, José M Martín-Durán, Markus Grohme, Katja Hüttner, Kerstin Bartscherer, Ian Henry, Jochen Rink
PlanMine 3.0-improvements to a mineable resource of flatworm biology and biodiversity.
Nucleic Acids Res, 47(D1) 812-820 (2019)
Open Access DOI

Flatworms (Platyhelminthes) are a basally branching phylum that harbours a wealth of fascinating biology, including planarians with their astonishing regenerative abilities and the parasitic tape worms and blood flukes that exert a massive impact on human health. PlanMine (http://planmine.mpi-cbg.de/) has the mission objective of providing both a mineable sequence repository for planarians and also a resource for the comparative analysis of flatworm biology. While the original PlanMine release was entirely based on transcriptomes, the current release transitions to a more genomic perspective. Building on the recent availability of a high quality genome assembly of the planarian model species Schmidtea mediterranea, we provide a gene prediction set that now assign existing transcripts to defined genomic coordinates. The addition of recent single cell and bulk RNA-seq datasets greatly expands the available gene expression information. Further, we add transcriptomes from a broad range of other flatworms and provide a phylogeny-aware interface that makes evolutionary species comparisons accessible to non-experts. At its core, PlanMine continues to utilize the powerful InterMine framework and consistent data annotations to enable meaningful inter-species comparisons. Overall, PlanMine 3.0 thus provides a host of new features that makes the fascinating biology of flatworms accessible to the wider research community.

Virag Sharma, Michael Hiller
Coding Exon-Structure Aware Realigner (CESAR): Utilizing Genome Alignments for Comparative Gene Annotation.
Methods Mol Biol, 1962 179-191 (2019)
PDF DOI

Alignment-based gene identification methods utilize sequence conservation between orthologous protein-coding genes to annotate genes in newly sequenced genomes. CESAR is an approach that makes use of existing genome alignments to transfer genes from one genome to other aligned genomes, and thus generates comparative gene annotations. To accurately detect conserved exons that exhibit an intact reading frame and consensus splice sites, CESAR produces a new alignment between orthologous exons, taking information about the exon's reading frame and splice site positions into account. Furthermore, CESAR is able to detect most evolutionary splice site shifts, which helps to annotate exon boundaries at high precision. Here, we describe how to apply CESAR to generate comparative gene annotations for one or many species, and discuss the strengths and limitations of this approach. CESAR is available at https://github.com/hillerlab/CESAR2.0 .

Malte Lehmann, Elisabeth Knust, Sarita Hebbar
Drosophila melanogaster: A Valuable Genetic Model Organism to Elucidate the Biology of Retinitis Pigmentosa.
Methods Mol Biol, 1834 221-249 (2019)
DOI

Retinitis pigmentosa (RP) is a complex inherited disease. It is associated with mutations in a wide variety of genes with many different functions. These mutations impact the integrity of rod photoreceptors and ultimately result in the progressive degeneration of rods and cone photoreceptors in the retina, leading to complete blindness. A hallmark of this disease is the variable degree to which symptoms are manifest in patients. This is indicative of the influence of the environment, and/or of the distinct genetic makeup of the individual.The fruit fly, Drosophila melanogaster, has effectively proven to be a great model system to better understand interconnected genetic networks. Unraveling genetic interactions and thereby different cellular processes is relatively easy because more than a century of research on flies has enabled the creation of sophisticated genetic tools to perturb gene function. A remarkable conservation of disease genes across evolution and the similarity of the general organization of the fly and vertebrate photoreceptor cell had prompted research on fly retinal degeneration. To date six fly models for RP, including RP4, RP11, RP12, RP14, RP25, and RP26, have been established, and have provided useful information on RP disease biology. In this chapter, an outline of approaches and experimental specifications are described to enable utilizing or developing new fly models of RP.

Rachel Kraut, Elisabeth Knust
Changes in endolysosomal organization define a pre-degenerative state in the crumbs mutant Drosophila retina.
PLoS ONE, 14(12) Art. No. e220220 (2019)
Open Access DOI

Mutations in the epithelial polarity gene crumbs (crb) lead to retinal degeneration in Drosophila and in humans. The overall morphology of the retina and its deterioration in Drosophila crb mutants has been well-characterized, but the cell biological origin of the degeneration is not well understood. Degenerative conditions in the retina and elsewhere in the nervous system often involve defects in degradative intracellular trafficking pathways. So far, however, effects of crb on the endolysosomal system, or on the spatial organization of these compartments in photoreceptor cells have not been described. We therefore asked whether photoreceptors in crb mutants exhibit alterations in endolysosomal compartments under pre-degenerative conditions, where the retina is still morphologically intact. Data presented here show that, already well before the onset of degeneration, Arl8, Rab7, and Atg8-carrying endolysosomal and autophagosomal compartments undergo changes in morphology and positioning with respect to each other in crb mutant retinas. We propose that these changes may be early signs of the degeneration-prone condition in crb retinas.

Monalisa Mishra, Elisabeth Knust
Analysis of the Drosophila Compound Eye with Light and Electron Microscopy.
Methods Mol Biol, 1834 345-364 (2019)
DOI

The Drosophila compound eye is composed of about 750 units, called ommatidia, which are arranged in a highly regular pattern. Eye development proceeds in a stereotypical fashion, where epithelial cells of the eye imaginal discs are specified, recruited, and differentiated in a sequential order that leads to the highly precise structure of an adult eye. Even small perturbations, for example in signaling pathways that control proliferation, cell death, or differentiation, can impair the regular structure of the eye, which can be easily detected and analyzed. In addition, the Drosophila eye has proven to be an ideal model for studying the genetic control of neurodegeneration, since the eye is not essential for viability. Several human neurodegeneration diseases have been modeled in the fly, leading to a better understanding of the function/misfunction of the respective gene. In many cases, the genes involved and their functions are conserved between flies and human. More strikingly, when ectopically expressed in the fly eye some human genes, even those without a Drosophila counterpart, can induce neurodegeneration, detectable by aberrant phototaxis, impaired electrophysiology, or defects in eye morphology and retinal histology. These defects are often rather subtle alteration in shape, size, or arrangement of the cells, and can be easily scored at the ultrastructural level. This chapter aims to provide an overview regarding the analysis of the retina by light and electron microscopy.

Anna Castells-Nobau, Ilse Eidhof, Michaela Fenckova, Dova B Brenman-Suttner, Jolanda M Scheffer-de Gooyert, Sheren Christine, Rosa L Schellevis, Kiran van der Laan, Christine Quentin, Lisa van Ninhuijs, Falko Hofmann, Radoslaw K Ejsmont, Simon E Fisher, Jamie M Kramer, Stephan J Sigrist, Anne F Simon, Annette Schenck
Conserved regulation of neurodevelopmental processes and behavior by FoxP in Drosophila.
PLoS ONE, 14(2) Art. No. e211652 (2019)
Open Access DOI

FOXP proteins form a subfamily of evolutionarily conserved transcription factors involved in the development and functioning of several tissues, including the central nervous system. In humans, mutations in FOXP1 and FOXP2 have been implicated in cognitive deficits including intellectual disability and speech disorders. Drosophila exhibits a single ortholog, called FoxP, but due to a lack of characterized mutants, our understanding of the gene remains poor. Here we show that the dimerization property required for mammalian FOXP function is conserved in Drosophila. In flies, FoxP is enriched in the adult brain, showing strong expression in ~1000 neurons of cholinergic, glutamatergic and GABAergic nature. We generate Drosophila loss-of-function mutants and UAS-FoxP transgenic lines for ectopic expression, and use them to characterize FoxP function in the nervous system. At the cellular level, we demonstrate that Drosophila FoxP is required in larvae for synaptic morphogenesis at axonal terminals of the neuromuscular junction and for dendrite development of dorsal multidendritic sensory neurons. In the developing brain, we find that FoxP plays important roles in α-lobe mushroom body formation. Finally, at a behavioral level, we show that Drosophila FoxP is important for locomotion, habituation learning and social space behavior of adult flies. Our work shows that Drosophila FoxP is important for regulating several neurodevelopmental processes and behaviors that are related to human disease or vertebrate disease model phenotypes. This suggests a high degree of functional conservation with vertebrate FOXP orthologues and established flies as a model system for understanding FOXP related pathologies.

Virag Sharma, Michael Hiller
Loss of Enzymes in the Bile Acid Synthesis Pathway Explains Differences in Bile Composition among Mammals.
Genome Biol Evol, 10(12) 3211-3217 (2018)
Open Access PDF DOI

Bile acids are important for absorbing nutrients. Most mammals produce cholic and chenodeoxycholic bile acids. Here, we investigated genes in the bile acid synthesis pathway in four mammals that deviate from the usual mammalian bile composition. First, we show that naked-mole rats, elephants, and manatees repeatedly inactivated CYP8B1, an enzyme uniquely required for cholic acid synthesis, which explains the absence of cholic acid in these species. Second, no gene-inactivating mutations were found in any pathway gene in the rhinoceros, a species that lacks bile acids, indicating an evolutionarily recent change in its bile composition. Third, elephants and/or manatees that also lack bile acids altogether have lost additional nonessential enzymes (SLC27A5, ACOX2). Apart from uncovering genomic differences explaining deviations in bile composition, our analysis of bile acid enzymes in bile acid-lacking species suggests that essentiality prevents gene loss, while loss of pleiotropic genes is permitted if their other functions are compensated by functionally related proteins.

Nereo Kalebic, Carlotta Gilardi, Mareike Albert, Takashi Namba, Katherine S. Long, Milos Kostic, Barbara Langen, Wieland Huttner
Human-specific ARHGAP11B induces hallmarks of neocortical expansion in developing ferret neocortex.
Elife, 7 Art. No. e41241 (2018)
Open Access DOI

The evolutionary increase in size and complexity of the primate neocortex is thought to underlie the higher cognitive abilities of humans. ARHGAP11B is a human-specific gene that, based on its expression pattern in fetal human neocortex and progenitor effects in embryonic mouse neocortex, has been proposed to have a key function in the evolutionary expansion of the neocortex. Here, we study the effects of ARHGAP11B expression in the developing neocortex of the gyrencephalic ferret. In contrast to its effects in mouse, ARHGAP11B markedly increases proliferative basal radial glia, a progenitor cell type thought to be instrumental for neocortical expansion, and results in extension of the neurogenic period and an increase in upper-layer neurons. Consequently, the postnatal ferret neocortex exhibits increased neuron density in the upper cortical layers and expands in both the radial and tangential dimensions. Thus, human-specific ARHGAP11B can elicit hallmarks of neocortical expansion in the developing ferret neocortex.

Juliana G. Roscito^*, Katrin Sameith^*, Genis Parra^*, Bjoern Langer, Andreas Petzold, Claudia Moebius, Marc Bickle, Miguel Trefaut Rodrigues, Michael Hiller
Phenotype loss is associated with widespread divergence of the gene regulatory landscape in evolution.
Nat Commun, 9(1) Art. No. 4737 (2018)
Open Access PDF DOI

Detecting the genomic changes underlying phenotypic changes between species is a main goal of evolutionary biology and genomics. Evolutionary theory predicts that changes in cis-regulatory elements are important for morphological changes. We combined genome sequencing, functional genomics and genome-wide comparative analyses to investigate regulatory elements in lineages that lost morphological traits. We first show that limb loss in snakes is associated with widespread divergence of limb regulatory elements. We next show that eye degeneration in subterranean mammals is associated with widespread divergence of eye regulatory elements. In both cases, sequence divergence results in an extensive loss of transcription factor binding sites. Importantly, diverged regulatory elements are associated with genes required for normal limb patterning or normal eye development and function, suggesting that regulatory divergence contributed to the loss of these phenotypes. Together, our results show that genome-wide decay of the phenotype-specific cis-regulatory landscape is a hallmark of lost morphological traits.

Samir Vaid, J Gray Camp, Lena Hersemann, Christina Eugster Oegema, Anne-Kristin Heninger, Sylke Winkler, Holger Brandl, Mihail Sarov, Barbara Treutlein, Wieland Huttner^#, Takashi Namba^#
A novel population of Hopx-dependent basal radial glial cells in the developing mouse neocortex.
Development, 145(20) Art. No. dev169276 (2018)
DOI

A specific subpopulation of neural progenitor cells, the basal radial glial cells (bRGCs) of the outer subventricular zone (OSVZ), are thought to have a key role in the evolutionary expansion of the mammalian neocortex. In the developing lissencephalic mouse neocortex, bRGCs exist at low abundance and show significant molecular differences from bRGCs in developing gyrencephalic species. Here, we demonstrate that the developing mouse medial neocortex (medNcx), in contrast to the canonically studied lateral neocortex (latNcx), exhibits an OSVZ and an abundance of bRGCs similar to that in developing gyrencephalic neocortex. Unlike bRGCs in developing mouse latNcx, the bRGCs in medNcx exhibit human bRGC-like gene expression, including expression of Hopx, a human bRGC marker. Disruption of Hopx expression in mouse embryonic medNcx and forced Hopx expression in mouse embryonic latNcx demonstrate that Hopx is required and sufficient, respectively, for bRGC abundance as found in the developing gyrencephalic neocortex. Taken together, our data identify a novel bRGC subpopulation in developing mouse medNcx that is highly related to bRGCs of developing gyrencephalic neocortex.

David Jebb, Michael Hiller
Recurrent loss of HMGCS2 shows that ketogenesis is not essential for the evolution of large mammalian brains.
Elife, 7 Art. No. e38906 (2018)
Open Access PDF DOI

Apart from glucose, fatty acid-derived ketone bodies provide metabolic energy for the brain during fasting and neonatal development. We investigated the evolution of HMGCS2, the key enzyme required for ketone body biosynthesis (ketogenesis). Unexpectedly, we found that three mammalian lineages, comprising cetaceans (dolphins and whales), elephants and mastodons, and Old World fruit bats have lost this gene. Remarkably, many of these species have exceptionally large brains and signs of intelligent behavior. While fruit bats are sensitive to starvation, cetaceans and elephants can still withstand periods of fasting. This suggests that alternative strategies to fuel large brains during fasting evolved repeatedly and reveals flexibility in mammalian energy metabolism. Furthermore, we show that HMGCS2 loss preceded brain size expansion in toothed whales and elephants. Thus, while ketogenesis was likely important for brain size expansion in modern humans, ketogenesis is not a universal precondition for the evolution of large mammalian brains.

Steven D. Briscoe, Clifton W. Ragsdale
Homology, neocortex, and the evolution of developmental mechanisms.
Science, 362(6411) 190-193 (2018)
DOI

The six-layered neocortex of the mammalian pallium has no clear homolog in birds or non-avian reptiles. Recent research indicates that although these extant amniotes possess a variety of divergent and nonhomologous pallial structures, they share a conserved set of neuronal cell types and circuitries. These findings suggest a principle of brain evolution: that natural selection preferentially preserves the integrity of information-processing pathways, whereas other levels of biological organization, such as the three-dimensional architectures of neuronal assemblies, are less constrained. We review the similarities of pallial neuronal cell types in amniotes, delineate candidate gene regulatory networks for their cellular identities, and propose a model of developmental evolution for the divergence of amniote pallial structures.

Maria Kotini, Elias H Barriga, Jonathan Leslie, Marc Gentzel, Verena Rauschenberger, Alexandra Schambon, Roberto Mayor
Gap junction protein Connexin-43 is a direct transcriptional regulator of N-cadherin in vivo.
Nat Commun, 9(1) Art. No. 3846 (2018)
Open Access DOI

Connexins are the primary components of gap junctions, providing direct links between cells under many physiological processes. Here, we demonstrate that in addition to this canonical role, Connexins act as transcriptional regulators. We show that Connexin 43 (Cx43) controls neural crest cell migration in vivo by directly regulating N-cadherin transcription. This activity requires interaction between Cx43 carboxy tail and the basic transcription factor-3, which drives the translocation of Cx43 tail to the nucleus. Once in the nucleus they form a complex with PolII which directly binds to the N-cadherin promoter. We found that this mechanism is conserved between amphibian and mammalian cells. Given the strong evolutionary conservation of connexins across vertebrates, this may reflect a common mechanism of gene regulation by a protein whose function was previously ascribed only to gap junctional communication.

Jordi Senserrich, Antoniana Batsivari, Stanislav Rybtsov, Sabrina Gordon-Keylock, Celine Souilhol, Frank Buchholz, David Hills, Suling Zhao, Alexander Medvinsky
Analysis of Runx1 Using Induced Gene Ablation Reveals Its Essential Role in Pre-liver HSC Development and Limitations of an In Vivo Approach.
Stem Cell Rep, 11(3) 784-794 (2018)
Open Access DOI

Hematopoietic stem cells (HSCs) develop in the embryonic aorta-gonad-mesonephros (AGM) region and subsequently relocate to fetal liver. Runx1 transcription factor is essential for HSC development, but is largely dispensable for adult HSCs. Here, we studied tamoxifen-inducible Runx1 inactivation in vivo. Induction at pre-liver stages (up to embryonic day 10.5) reduced erythromyeloid progenitor numbers, but surprisingly did not block the appearance of Runx1-null HSCs in liver. By contrast, ex vivo analysis showed an absolute Runx1 dependency of HSC development in the AGM region. We found that, contrary to current beliefs, significant Cre-inducing tamoxifen activity persists in mouse blood for at least 72 hr after injection. This deferred recombination can hit healthy HSCs, which escaped early Runx1 ablation and result in appearance of Runx1-null HSCs in liver. Such extended recombination activity in vivo is a potential source of misinterpretation, particularly in analysis of dynamic developmental processes during embryogenesis.

Christiane Rennert, Sebastian Vlaic, Eugenia Marbach-Breitrück, Carlo Thiel, Susanne Sales, Andrej Shevchenko, Rolf Gebhardt, Madlen Matz-Soja
The Diurnal Timing of Starvation Differently Impacts Murine Hepatic Gene Expression and Lipid Metabolism - A Systems Biology Analysis Using Self-Organizing Maps.
Front Physiol, 9 Art. No. 1180 (2018)
DOI

Organisms adapt their metabolism and draw on reserves as a consequence of food deprivation. The central role of the liver in starvation response is to coordinate a sufficient energy supply for the entire organism, which has frequently been investigated. However, knowledge of how circadian rhythms impact on and alter this response is scarce. Therefore, we investigated the influence of different timings of starvation on global hepatic gene expression. Mice (n = 3 each) were challenged with 24-h food deprivation started in the morning or evening, coupled with refeeding for different lengths and compared with ad libitum fed control groups. Alterations in hepatocyte gene expression were quantified using microarrays and confirmed or complemented with qPCR, especially for lowly detectable transcription factors. Analysis was performed using self-organizing maps (SOMs), which bases on clustering genes with similar expression profiles. This provides an intuitive overview of expression trends and allows easier global comparisons between complex conditions. Transcriptome analysis revealed a strong circadian-driven response to fasting based on the diurnal expression of transcription factors (e.g., Ppara, Pparg). Starvation initiated in the morning produced known metabolic adaptations in the liver; e.g., switching from glucose storage to consumption and gluconeogenesis. However, starvation initiated in the evening produced a different expression signature that was controlled by yet unknown regulatory mechanisms. For example, the expression of genes involved in gluconeogenesis decreased and fatty acid and cholesterol synthesis genes were induced. The differential regulation after morning and evening starvation were also reflected at the lipidome level. The accumulation of hepatocellular storage lipids (triacylglycerides, cholesteryl esters) was significantly higher after the initiation of starvation in the morning compared to the evening. Concerning refeeding, the gene expression pattern after a 12 h refeeding period largely resembled that of the corresponding starvation state but approached the ad libitum control state after refeeding for 21 h. Some components of these regulatory circuits are discussed. Collectively, these data illustrate a highly time-dependent starvation response in the liver and suggest that a circadian influence cannot be neglected when starvation is the focus of research or medicine, e.g., in the case of treating victims of sudden starvation events.

Cyrille Ramond, Belin Selcen Beydag-Tasöz, Ajuna Azad, Martijn van de Bunt, Maja Borup Kjær Petersen, Nicola L Beer, Nicolas Glaser, Claire Berthault, Anna L Gloyn, Mattias Hansson, Mark I McCarthy, Christian Honoré, Anne Grapin-Botton, Raphael Scharfmann
Understanding human fetal pancreas development using subpopulation sorting, RNA sequencing and single-cell profiling.
Development, 145(16) Art. No. dev165480 (2018)
DOI

To decipher the populations of cells present in the human fetal pancreas and their lineage relationships, we developed strategies to isolate pancreatic progenitors, endocrine progenitors and endocrine cells. Transcriptome analysis of the individual populations revealed a large degree of conservation among vertebrates in the drivers of gene expression changes that occur at different steps of differentiation, although notably, sometimes, different members of the same gene family are expressed. The transcriptome analysis establishes a resource to identify novel genes and pathways involved in human pancreas development. Single-cell profiling further captured intermediate stages of differentiation and enabled us to decipher the sequence of transcriptional events occurring during human endocrine differentiation. Furthermore, we evaluate how well individual pancreatic cells derived in vitro from human pluripotent stem cells mirror the natural process occurring in human fetuses. This comparison uncovers a few differences at the progenitor steps, a convergence at the steps of endocrine induction, and the current inability to fully resolve endocrine cell subtypes in vitro.

Marisa Karow, J Gray Camp, Sven Falk, Tobias Gerber, Abhijeet Pataskar, Malgorzata Gac-Santel, Jorge Kageyama, Agnieska Brazovskaja, Angela Garding, Wenqiang Fan, Therese Riedemann, Antonella Casamassa, Andrej Smiyakin, Christian Schichor, Magdalena Götz, Vijay K Tiwari, Barbara Treutlein, Benedikt Berninger
Direct pericyte-to-neuron reprogramming via unfolding of a neural stem cell-like program.
Nat Neurosci, 21(7) 932-940 (2018)
DOI

Ectopic expression of defined transcription factors can force direct cell-fate conversion from one lineage to another in the absence of cell division. Several transcription factor cocktails have enabled successful reprogramming of various somatic cell types into induced neurons (iNs) of distinct neurotransmitter phenotype. However, the nature of the intermediate states that drive the reprogramming trajectory toward distinct iN types is largely unknown. Here we show that successful direct reprogramming of adult human brain pericytes into functional iNs by Ascl1 and Sox2 encompasses transient activation of a neural stem cell-like gene expression program that precedes bifurcation into distinct neuronal lineages. During this transient state, key signaling components relevant for neural induction and neural stem cell maintenance are regulated by and functionally contribute to iN reprogramming and maturation. Thus, Ascl1- and Sox2-mediated reprogramming into a broad spectrum of iN types involves the unfolding of a developmental program via neural stem cell-like intermediates.

Virag Sharma, Thomas Lehmann, Heiko Stuckas, Liane Funke, Michael Hiller
Loss of RXFP2 and INSL3 genes in Afrotheria shows that testicular descent is the ancestral condition in placental mammals.
PLoS Biol, 16(6) Art. No. e2005293 (2018)
Open Access PDF DOI

Descent of testes from a position near the kidneys into the lower abdomen or into the scrotum is an important developmental process that occurs in all placental mammals, with the exception of five afrotherian lineages. Since soft-tissue structures like testes are not preserved in the fossil record and since key parts of the placental mammal phylogeny remain controversial, it has been debated whether testicular descent is the ancestral or derived condition in placental mammals. To resolve this debate, we used genomic data of 71 mammalian species and analyzed the evolution of two key genes (relaxin/insulin-like family peptide receptor 2 [RXFP2] and insulin-like 3 [INSL3]) that induce the development of the gubernaculum, the ligament that is crucial for testicular descent. We show that both RXFP2 and INSL3 are lost or nonfunctional exclusively in four afrotherians (tenrec, cape elephant shrew, cape golden mole, and manatee) that completely lack testicular descent. The presence of remnants of once functional orthologs of both genes in these afrotherian species shows that these gene losses happened after the split from the placental mammal ancestor. These "molecular vestiges" provide strong evidence that testicular descent is the ancestral condition, irrespective of persisting phylogenetic discrepancies. Furthermore, the absence of shared gene-inactivating mutations and our estimates that the loss of RXFP2 happened at different time points strongly suggest that testicular descent was lost independently in Afrotheria. Our results provide a molecular mechanism that explains the loss of testicular descent in afrotherians and, more generally, highlight how molecular vestiges can provide insights into the evolution of soft-tissue characters.

Peter Dobrowolski, Melina Fischer, Ronald Naumann
Novel insights into the genetic background of genetically modified mice.
Transgenic Res, 27(3) 265-275 (2018)
Open Access DOI

Unclear or misclassified genetic background of laboratory rodents or a lack of strain awareness causes a number of difficulties in performing or reproducing scientific experiments. Until now, genetic differentiation between strains and substrains of inbred mice has been a challenge. We have developed a screening method for analyzing inbred strains regarding their genetic background. It is based on 240 highly informative short tandem repeat (STR) markers covering the 19 autosomes as well as X and Y chromosomes. Combination of analysis results for presence of known C57BL/6 substrain-specific mutations together with autosomal STR markers and the Y-chromosomal STR-haplotype provides a comprehensive snapshot of the genetic background of mice. In this study, the genetic background of 72 mouse lines obtained from 18 scientific institutions in Germany and Austria was determined. By analyzing only 3 individuals per genetically modified line it was possible to detect mixed genetic backgrounds frequently. In several lines presence of a mispairing Y chromosome was detected. At least every second genetically modified line displayed a mixed genetic background which could lead to unexpected and non-reproducible results, irrespective of the investigated gene of interest.

Anastasia Felker, Karin D Prummel, Anne M Merks, Michaela Mickoleit, Eline C Brombacher, Jan Huisken, Daniela Panáková, Christian Mosimann
Continuous addition of progenitors forms the cardiac ventricle in zebrafish.
Nat Commun, 9(1) Art. No. 2001 (2018)
Open Access DOI

The vertebrate heart develops from several progenitor lineages. After early-differentiating first heart field (FHF) progenitors form the linear heart tube, late-differentiating second heart field (SHF) progenitors extend the atrium and ventricle, and form inflow and outflow tracts (IFT/OFT). However, the position and migration of late-differentiating progenitors during heart formation remains unclear. Here, we track zebrafish heart development using transgenics based on the cardiopharyngeal gene tbx1. Live imaging uncovers a tbx1 reporter-expressing cell sheath that continuously disseminates from the lateral plate mesoderm towards the forming heart tube. High-speed imaging and optogenetic lineage tracing corroborates that the zebrafish ventricle forms through continuous addition from the undifferentiated progenitor sheath followed by late-phase accrual of the bulbus arteriosus (BA). FGF inhibition during sheath migration reduces ventricle size and abolishes BA formation, refining the window of FGF action during OFT formation. Our findings consolidate previous end-point analyses and establish zebrafish ventricle formation as a continuous process.

Virag Sharma, Nikolai Hecker, Juliana G. Roscito, Leo Foerster, Björn Langer, Michael Hiller
A genomics approach reveals insights into the importance of gene losses for mammalian adaptations.
Nat Commun, 9(1) Art. No. 1215 (2018)
Open Access PDF DOI

Identifying the genomic changes that underlie phenotypic adaptations is a key challenge in evolutionary biology and genomics. Loss of protein-coding genes is one type of genomic change with the potential to affect phenotypic evolution. Here, we develop a genomics approach to accurately detect gene losses and investigate their importance for adaptive evolution in mammals. We discover a number of gene losses that likely contributed to morphological, physiological, and metabolic adaptations in aquatic and flying mammals. These gene losses shed light on possible molecular and cellular mechanisms that underlie these adaptive phenotypes. In addition, we show that gene loss events that occur as a consequence of relaxed selection following adaptation provide novel insights into species' biology. Our results suggest that gene loss is an evolutionary mechanism for adaptation that may be more widespread than previously anticipated. Hence, investigating gene losses has great potential to reveal the genomic basis underlying macroevolutionary changes.

Marta Florio^*, Michael Heide^*, Anneline Pinson, Holger Brandl, Mareike Albert, Sylke Winkler, Pauline Wimberger, Wieland B. Huttner^#, Michael Hiller^#
Evolution and cell-type specificity of human-specific genes preferentially expressed in progenitors of fetal neocortex.
Elife, 7 Art. No. e32332 (2018)
Open Access PDF DOI

Understanding the molecular basis that underlies the expansion of the neocortex during primate, and notably human, evolution requires the identification of genes that are particularly active in the neural stem and progenitor cells of the developing neocortex. Here, we have used existing transcriptome datasets to carry out a comprehensive screen for protein-coding genes preferentially expressed in progenitors of fetal human neocortex. We show that fifteen human-specific genes exhibit such expression, and many of them evolved distinct neural progenitor cell-type expression profiles and levels compared to their ancestral paralogs. Functional studies on one such gene,NOTCH2NL, demonstrate its ability to promote basal progenitor proliferation in mice. An additional 35 human genes with progenitor-enriched expression are shown to have orthologs only in primates. Our study provides a resource of genes that are promising candidates to exert specific, and novel, roles in neocortical development during primate, and notably human, evolution.

Pavel Vopalensky, Sabrina Pralow, Nadine Vastenhouw
Reduced expression of the Nodal co-receptor Oep causes loss of mesendodermal competence in zebrafish.
Development, 145(5) Art. No. dev158832 (2018)
DOI

The activation of specific gene expression programs depends on the presence of the appropriate signals and the competence of cells to respond to those signals. Although it is well established that cellular competence is regulated in space and time, the molecular mechanisms underlying the loss of competence remain largely unknown. Here, we determine the time window during which zebrafish prospective ectoderm loses its ability to respond to Nodal signals, and show that this coincides with a decrease in the levels of the Nodal co-receptor One-eyed pinhead (Oep). Bypassing Oep using a photoactivatable receptor, or an Oep-independent ligand, allows activation of Nodal target genes for an extended period of time. These results suggest that the reduced expression of Oep causes the loss of responsiveness to Nodal signals in the prospective ectoderm. Indeed, extending the presence of Oep prolongs the window of competence to respond to Nodal signals. Our findings suggest a simple mechanism in which the decreasing level of one component of the Nodal signaling pathway regulates the loss of mesendodermal competence in the prospective ectoderm.

Elisa Monzón-Casanova, Michael Screen, Manuel D Díaz-Muñoz, Richard M R Coulson, Sarah E Bell, Greta Lamers, Michele Solimena, Christopher W J Smith, Martin Turner
The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers.
Nat Immunol, 19(3) 267-278 (2018)
DOI

Antibody affinity maturation occurs in germinal centers (GCs), where B cells cycle between the light zone (LZ) and the dark zone. In the LZ, GC B cells bearing immunoglobulins with the highest affinity for antigen receive positive selection signals from helper T cells, which promotes their rapid proliferation. Here we found that the RNA-binding protein PTBP1 was needed for the progression of GC B cells through late S phase of the cell cycle and for affinity maturation. PTBP1 was required for proper expression of the c-MYC-dependent gene program induced in GC B cells receiving T cell help and directly regulated the alternative splicing and abundance of transcripts that are increased during positive selection to promote proliferation.

Claudia Gerri^*, Michele Marass^*, Andrea Rossi, Didier Y.R. Stainier
Hif-1α and Hif-2α regulate hemogenic endothelium and hematopoietic stem cell formation in zebrafish.
Blood, 131(9) 963-973 (2018)
DOI

During development, hematopoietic stem cells (HSCs) derive from specialized endothelial cells (ECs) called hemogenic endothelium (HE) via a process called endothelial-to-hematopoietic transition (EHT). Hypoxia-inducible factor-1α (HIF-1α) has been reported to positively modulate EHT in vivo, but current data indicate the existence of other regulators of this process. Here we show that in zebrafish, Hif-2α also positively modulates HSC formation. Specifically, HSC marker gene expression is strongly decreased in hif-1aa;hif-1ab (hif-1α) and in hif-2aa;hif-2ab (hif-2α) zebrafish mutants and morphants. Moreover, live imaging studies reveal a positive role for hif-1α and hif-2α in regulating HE specification. Knockdown of hif-2α in hif-1α mutants leads to a greater decrease in HSC formation, indicating that hif-1α and hif-2α have partially overlapping roles in EHT. Furthermore, hypoxic conditions, which strongly stimulate HSC formation in wild-type animals, have little effect in the combined absence of Hif-1α and Hif-2α function. In addition, we present evidence for Hif and Notch working in the same pathway upstream of EHT. Both notch1a and notch1b mutants display impaired EHT, which cannot be rescued by hypoxia. However, overexpression of the Notch intracellular domain in ECs is sufficient to rescue the hif-1α and hif-2α morphant EHT phenotype, suggesting that Notch signaling functions downstream of the Hif pathway during HSC formation. Altogether, our data provide genetic evidence that both Hif-1α and Hif-2α regulate EHT upstream of Notch signaling.

FoSheng Hsu, Stephanie Spannl, Charles Ferguson, Anthony Hyman, Robert G. Parton, Marino Zerial
Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria.
Elife, 7 Art. No. e32282 (2018)
Open Access DOI

Mitochondrial stress response is essential for cell survival, and damaged mitochondria are a hallmark of neurodegenerative diseases. Thus, it is fundamental to understand how mitochondria relay information within the cell. Here, by investigating mitochondrial-endosomal contact sites we made the surprising observation that the small GTPase Rab5 translocates from early endosomes to mitochondria upon oxidative stress. This process is reversible and accompanied by an increase in Rab5-positive endosomes in contact with mitochondria. Interestingly, activation of Rab5 on mitochondria depends on the Rab5-GEF ALS2/Alsin, encoded by a gene mutated in amyotrophic lateral sclerosis (ALS). Alsin-deficient human-induced pluripotent stem cell-derived spinal motor neurons are defective in relocating Rab5 to mitochondria and display increased susceptibility to oxidative stress. These findings define a novel pathway whereby Alsin catalyzes the assembly of the Rab5 endocytic machinery on mitochondria. Defects in stress-sensing by endosomes could be crucial for mitochondrial quality control during the onset of ALS.

T-Y Dora Tang, Dario Cecchi, Giorgio Fracasso, Davide Accardi, Angelique Coutable-Pennarun, Sheref S Mansy, Adam W Perriman, J L Ross Anderson, Stephen Mann
Gene-Mediated Chemical Communication in Synthetic Protocell Communities.
ACS Synth Biol, 7(2) 339-346 (2018)
DOI

A gene-directed chemical communication pathway between synthetic protocell signaling transmitters (lipid vesicles) and receivers (proteinosomes) was designed, built and tested using a bottom-up modular approach comprising small molecule transcriptional control, cell-free gene expression, porin-directed efflux, substrate signaling, and enzyme cascade-mediated processing.

J P Joos, A R Saadatmand, C Schnabel, Ivana Viktorinová, Thomas Brand, M Kramer, S Nattel, D Dobrev, Pavel Tomancak, J Backs, P Kleinbongard, G Heusch, K Lorenz, E Koch, S Weber, A El-Armouche
Ectopic expression of S28A-mutated Histone H3 modulates longevity, stress resistance and cardiac function in Drosophila.
Sci Rep, 8(1) Art. No. 2940 (2018)
Open Access DOI

Histone H3 serine 28 (H3S28) phosphorylation and de-repression of polycomb repressive complex (PRC)-mediated gene regulation is linked to stress conditions in mitotic and post-mitotic cells. To better understand the role of H3S28 phosphorylation in vivo, we studied a Drosophila strain with ectopic expression of constitutively-activated H3S28A, which prevents PRC2 binding at H3S28, thus mimicking H3S28 phosphorylation. H3S28A mutants showed prolonged life span and improved resistance against starvation and paraquat-induced oxidative stress. Morphological and functional analysis of heart tubes revealed smaller luminal areas and thicker walls accompanied by moderately improved cardiac function after acute stress induction. Whole-exome deep gene-sequencing from isolated heart tubes revealed phenotype-corresponding changes in longevity-promoting and myotropic genes. We also found changes in genes controlling mitochondrial biogenesis and respiration. Analysis of mitochondrial respiration from whole flies revealed improved efficacy of ATP production with reduced electron transport-chain activity. Finally, we analyzed posttranslational modification of H3S28 in an experimental heart failure model and observed increased H3S28 phosphorylation levels in HF hearts. Our data establish a critical role of H3S28 phosphorylation in vivo for life span, stress resistance, cardiac and mitochondrial function in Drosophila. These findings may pave the way for H3S28 phosphorylation as a putative target to treat stress-related disorders such as heart failure.

Masaya Oshima, Klaus-Peter Knoch, Marc Diedisheim, Antje Petzold, Pierre Cattan, Marco Bugliani, Piero Marchetti, Pratik Choudhary, Guo-Cai Huang, Stefan R. Bornstein, Michele Solimena, Olivier Albagli-Curiel, Raphael Scharfmann
Virus-like infection induces human β cell dedifferentiation.
JCI Insight, 3(3) Art. No. e97732 (2018)
DOI

Type 1 diabetes (T1D) is a chronic disease characterized by an autoimmune-mediated destruction of insulin-producing pancreatic β cells. Environmental factors such as viruses play an important role in the onset of T1D and interact with predisposing genes. Recent data suggest that viral infection of human islets leads to a decrease in insulin production rather than β cell death, suggesting loss of β cell identity. We undertook this study to examine whether viral infection could induce human β cell dedifferentiation. Using the functional human β cell line EndoC-βH1, we demonstrate that polyinosinic-polycytidylic acid (PolyI:C), a synthetic double-stranded RNA that mimics a byproduct of viral replication, induces a decrease in β cell-specific gene expression. In parallel with this loss, the expression of progenitor-like genes such as SOX9 was activated following PolyI:C treatment or enteroviral infection. SOX9 was induced by the NF-κB pathway and also in a paracrine non-cell-autonomous fashion through the secretion of IFN-α. Lastly, we identified SOX9 targets in human β cells as potentially new markers of dedifferentiation in T1D. These findings reveal that inflammatory signaling has clear implications in human β cell dedifferentiation.

Sergej Nowoshilow, Siegfried Schloissnig, Jifeng Fei, Andreas Dahl, Andy W C Pang, Martin Pippel, Sylke Winkler, Alex R Hastie, George Young, Juliana G. Roscito, Francisco Falcon, Dunja Knapp, Sean Powell, Alfredo Cruz, Han Cao, Bianca Habermann, Michael Hiller, Elly M. Tanaka, Eugene W Myers
The axolotl genome and the evolution of key tissue formation regulators.
Nature, 554(7690) 50-55 (2018)
Open Access PDF DOI

Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl (Ambystoma mexicanum) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3. However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3-/-and Pax7-/-mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies.

Matthias Kaiser, Florian Jug, Thomas Julou, Siddharth Deshpande, Thomas Pfohl, Olin Silander^#, Gene Myers^#, Erik van Nimwegen^#
Monitoring single-cell gene regulation under dynamically controllable conditions with integrated microfluidics and software.
Nat Commun, 9(1) Art. No. 212 (2018)
Open Access DOI

Much is still not understood about how gene regulatory interactions control cell fate decisions in single cells, in part due to the difficulty of directly observing gene regulatory processes in vivo. We introduce here a novel integrated setup consisting of a microfluidic chip and accompanying analysis software that enable long-term quantitative tracking of growth and gene expression in single cells. The dual-input Mother Machine (DIMM) chip enables controlled and continuous variation of external conditions, allowing direct observation of gene regulatory responses to changing conditions in single cells. The Mother Machine Analyzer (MoMA) software achieves unprecedented accuracy in segmenting and tracking cells, and streamlines high-throughput curation with a novel leveraged editing procedure. We demonstrate the power of the method by uncovering several novel features of an iconic gene regulatory program: the induction of Escherichia coli's lac operon in response to a switch from glucose to lactose.

Catarina Oliveira, Regis P. Lemaitre, Prayag Murawala, Akira Tazaki, David N. Drechsel, Elly M. Tanaka
Pseudotyped baculovirus is an effective gene expression tool for studying molecular function during axolotl limb regeneration.
Dev Biol, 433(2) 262-275 (2018)
DOI

Axolotls can regenerate complex structures through recruitment and remodeling of cells within mature tissues. Accessing the underlying mechanisms at a molecular resolution is crucial to understand how injury triggers regeneration and how it proceeds. However, gene transformation in adult tissues can be challenging. Here we characterize the use of pseudotyped baculovirus (BV) as an effective gene transfer method both for cells within mature limb tissue and within the blastema. These cells remain competent to participate in regeneration after transduction. We further characterize the effectiveness of BV for gene overexpression studies by overexpressing Shh in the blastema, which yields a high penetrance of classic polydactyly phenotypes. Overall, our work establishes BV as a powerful tool to access gene function in axolotl limb regeneration.

Alireza Haghighi, Joel B Krier, Agnes Toth-Petroczy, Christopher Cassa, Natasha Y Frank, Nikkola Carmichael, Elizabeth Fieg, Andrew Bjonnes, Anwoy Kumar Mohanty, Lauren C Briere, Sharyn Lincoln, Stephanie Lucia, Vandana A Gupta, Onuralp Söylemez, Sheila Sutti, Kameron Kooshesh, Haiyan Qiu, Christopher J Fay, Victoria Perroni, Jamie Valerius, Meredith Hanna, Alexander Frank, Jodie Ouahed, Scott B Snapper, Angeliki Pantazi, Sameer S Chopra, Ignaty Leshchiner, Nathan O Stitziel, Anna Feldweg, Michael Mannstadt, Joseph Loscalzo, David A Sweetser, Eric Liao, Joan M Stoler, Catherine B Nowak, Pedro A Sanchez-Lara, Ophir D. Klein, Hazel Perry, Nikolaos A Patsopoulos, Soumya Raychaudhuri, Wolfram Goessling, Robert C Green, Christine E Seidman, Calum A MacRae, Shamil Sunyaev, Richard L Maas, Dana Vuzman, Dana null
An integrated clinical program and crowdsourcing strategy for genomic sequencing and Mendelian disease gene discovery.
NPJ Genom Med, 3 21-21 (2018)
DOI

Despite major progress in defining the genetic basis of Mendelian disorders, the molecular etiology of many cases remains unknown. Patients with these undiagnosed disorders often have complex presentations and require treatment by multiple health care specialists. Here, we describe an integrated clinical diagnostic and research program using whole-exome and whole-genome sequencing (WES/WGS) for Mendelian disease gene discovery. This program employs specific case ascertainment parameters, a WES/WGS computational analysis pipeline that is optimized for Mendelian disease gene discovery with variant callers tuned to specific inheritance modes, an interdisciplinary crowdsourcing strategy for genomic sequence analysis, matchmaking for additional cases, and integration of the findings regarding gene causality with the clinical management plan. The interdisciplinary gene discovery team includes clinical, computational, and experimental biomedical specialists who interact to identify the genetic etiology of the disease, and when so warranted, to devise improved or novel treatments for affected patients. This program effectively integrates the clinical and research missions of an academic medical center and affords both diagnostic and therapeutic options for patients suffering from genetic disease. It may therefore be germane to other academic medical institutions engaged in implementing genomic medicine programs.

Mareike Albert^#, Wieland Huttner^#
Epigenetic and Transcriptional Pre-patterning-An Emerging Theme in Cortical Neurogenesis.
Front Neurosci, 12 Art. No. 359 (2018)
Open Access DOI

Neurogenesis is the process through which neural stem and progenitor cells generate neurons. During the development of the mouse neocortex, stem and progenitor cells sequentially give rise to neurons destined to different cortical layers and then switch to gliogenesis resulting in the generation of astrocytes and oligodendrocytes. Precise spatial and temporal regulation of neural progenitor differentiation is key for the proper formation of the complex structure of the neocortex. Dynamic changes in gene expression underlie the coordinated differentiation program, which enables the cells to generate the RNAs and proteins required at different stages of neurogenesis and across different cell types. Here, we review the contribution of epigenetic mechanisms, with a focus on Polycomb proteins, to the regulation of gene expression programs during mouse neocortical development. Moreover, we discuss the recent emerging concept of epigenetic and transcriptional pre-patterning in neocortical progenitor cells as well as post-transcriptional mechanisms for the fine-tuning of mRNA abundance.

Maja Borup Kjær Petersen, Carla A C Gonçalves, Yung Hae Kim, Anne Grapin-Botton
Recapitulating and Deciphering Human Pancreas Development From Human Pluripotent Stem Cells in a Dish.
Curr Top Dev Biol, 129 143-190 (2018)
DOI

Here, we review how human pluripotent stem cell models of pancreas development have emerged and became an important tool to study human development and disease. Initially developed toward the production of β cells for diabetes therapy, the protocols have been refined based on knowledge of pancreas development in model organisms. While the cells produced are closer and closer to the end goal of a functional β cell, these models have also been used to carry out functional experiments addressing gene function and expression as well as regulatory and epigenetic landscape changes during human pancreas development. They thereby complement model organisms and reports from human genetic variants predisposing to different forms of diabetes, as well as observations on human fetal tissue. In this review, we therefore compare these different sources of information and discuss how human stem cell models are evolving to inform us on pancreatic diseases and possible treatments.

Nicole Bäumer, Jan Rehkämper, Neele Appel, Lisa Terheyden, Wolfgang Hartmann, Eva Wardelmann, Frank Buchholz, Carsten Müller-Tidow, Wolfgang E Berdel, Sebastian Bäumer
Downregulation of PIK3CA via antibody-esiRNA-complexes suppresses human xenograft tumor growth.
PLoS ONE, 13(7) Art. No. e200163 (2018)
Open Access DOI

Precision cancer therapy requires on the one hand detailed knowledge about a tumor's driver oncogenes and on the other hand an effective targeted therapy that specifically inhibits these oncogenes. While the determination of genomic landscape of a tumor has reached a very precise level, the respective therapy options are scarce. The application of small inhibitory (si) RNAs is a promising field of investigation. Here, we present the effective in vivo-treatment of colorectal cancer (CRC) xenograft tumors with antibody-complexed, endoribonuclease-prepared small inhibitory (esi)RNAs. We chose heterogeneous endoribonuclease-prepared siRNA pools (esiRNAs) against the frequently mutated genes PIK3CA and KRAS and coupled them to the anti-EGFR antibody cetuximab, which was internalized specifically into the tumor cells. esiRNA pools have been shown to exhibit superior specificity in target gene knockdown compared to classic siRNAs. We identified a significant decrease in tumor growth upon this treatment due to decreased tumor cell proliferation. The ex vivo-analysis of the xenograft CRC tumors revealed the expected downregulation of the intended direct targets PIK3CA and KRAS on protein level. Moreover, known downstream targets for EGFR signaling such as p-ERK, p-AKT, and c-MYC were decreased as well. We therefore propose the use of antibody-esiRNA complexes as a novel experimental treatment option against key components of the EGFR signaling cascade.

Virag Sharma, Peter Schwede, Michael Hiller
CESAR 2.0 substantially improves speed and accuracy of comparative gene annotation.
Bioinformatics, 33(24) 3985-3987 (2017)
PDF DOI

Homology-based gene prediction is a powerful concept to annotate newly sequenced genomes. We have previously demonstrated that whole genome alignments can be utilized for accurate comparative coding gene annotation.

Debojyoti Chakraborty, Maciej Paszkowski-Rogacz, Nicolas Berger, Li Ding, Jovan Mircetic, Jun Fu, Vytautas Iesmantavicius, Chunaram Choudhary, Konstantinos Anastassiadis, A Francis Stewart, Frank Buchholz
lncRNA Panct1 Maintains Mouse Embryonic Stem Cell Identity by Regulating TOBF1 Recruitment to Oct-Sox Sequences in Early G1.
Cell Rep, 21(11) 3012-3021 (2017)
Open Access DOI

Long noncoding RNAs (lncRNAs) have been implicated in diverse biological processes, including embryonic stem cell (ESC) maintenance. However, their functional mechanisms remain largely undefined. Here, we show that the lncRNA Panct1 regulates the transient recruitment of a putative X-chromosome-encoded protein A830080D01Rik, hereafter referred to as transient octamer binding factor 1 (TOBF1), to genomic sites resembling the canonical Oct-Sox motif. TOBF1 physically interacts with Panct1 and exhibits a cell-cycle-specific punctate localization in ESCs. At the chromatin level, this correlates with its recruitment to promoters of pluripotency genes. Strikingly, mutating an octamer-like motif in Panct1 RNA abrogates the strength of TOBF1 localization and recruitment to its targets. Taken together, our data reveal a tightly controlled spatial and temporal pattern of lncRNA-mediated gene regulation in a cell-cycle-dependent manner and suggest that lncRNAs might function as barcodes for identifying genomic addresses for maintaining cellular states.

Michael Heide, Katherine Long, Wieland B. Huttner
Novel gene function and regulation in neocortex expansion.
Curr Opin Cell Biol, 49 22-30 (2017)
DOI

The expansion of the neocortex during human evolution is due to changes in our genome that result in increased and prolonged proliferation of neural stem and progenitor cells during neocortex development. Three principal types of such genomic changes can be distinguished, first, novel gene regulation in human, second, novel function in human of genes existing in both human and non-human species, and third, novel, human-specific genes. The latter comprise both, increases in the copy number of genes existing also in non-human species, and the emergence of genes giving rise to unique, human-specific gene products. Examples of all these types of changes in the human genome have been identified, with ARHGAP11B constituting a paradigmatic example of a unique, human-specific protein.

Sandra Richter, Ulrike Schulze, Pavel Tomançak, Andrew C. Oates
Small molecule screen in embryonic zebrafish using modular variations to target segmentation.
Nat Commun, 8(1) Art. No. 1901 (2017)
Open Access DOI

Small molecule in vivo phenotypic screening is used to identify drugs or biological activities by directly assessing effects in intact organisms. However, current screening designs may not exploit the full potential of chemical libraries due to false negatives. Here, we demonstrate a modular small molecule screen in embryonic zebrafish that varies concentration, genotype and timing to target segmentation disorders, birth defects that affect the spinal column. By testing each small molecule in multiple interrelated ways, this screen recovers compounds that a standard screening design would have missed, increasing the hit frequency from the chemical library three-fold. We identify molecular pathways and segmentation phenotypes, which we share in an open-access annotated database. These hits provide insight into human vertebral segmentation disorders and myopathies. This modular screening strategy is applicable to other developmental questions and disease models, highlighting the power of relatively small chemical libraries to accelerate gene discovery and disease study.

Laura Broutier, Gianmarco Mastrogiovanni, Monique Ma Verstegen, Hayley E Francies, Lena Morrill Gavarró, Charles R. Bradshaw, George E Allen, Robert Arnes-Benito, Olga Sidorova, Marcia P Gaspersz, Nikitas Georgakopoulos, Bon-Kyoung Koo, Sabine Dietmann, Susan E Davies, Raaj K Praseedom, Ruby Lieshout, Jan N M IJzermans, Stephen J Wigmore, Kourosh Saeb-Parsy, Mathew J Garnett, Luc J W van der Laan, Meritxell Huch
Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.
Nat Med, 23(12) 1424-1435 (2017)
DOI

Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.

Jifeng Fei, Maritta Schuez, Dunja Knapp, Yuka Taniguchi, David N. Drechsel, Elly M. Tanaka
Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration.
Proc Natl Acad Sci U.S.A., 114(47) 12501-12506 (2017)
DOI

Salamanders exhibit extensive regenerative capacities and serve as a unique model in regeneration research. However, due to the lack of targeted gene knockin approaches, it has been difficult to label and manipulate some of the cell populations that are crucial for understanding the mechanisms underlying regeneration. Here we have established highly efficient gene knockin approaches in the axolotl (Ambystoma mexicanum) based on the CRISPR/Cas9 technology. Using a homology-independent method, we successfully inserted both the Cherry reporter gene and a larger membrane-tagged Cherry-ERT2-Cre-ERT2 (∼5-kb) cassette into axolotl Sox2 and Pax7 genomic loci. Depending on the size of the DNA fragments for integration, 5-15% of the F0 transgenic axolotl are positive for the transgene. Using these techniques, we have labeled and traced the PAX7-positive satellite cells as a major source contributing to myogenesis during axolotl limb regeneration. Our work brings a key genetic tool to molecular and cellular studies of axolotl regeneration.

Jovan Mircetic, Antje Dietrich, Maciej Paszkowski-Rogacz, Mechthild Krause, Frank Buchholz
Development of a genetic sensor that eliminates p53 deficient cells.
Nat Commun, 8(1) Art. No. 1463 (2017)
Open Access DOI

The TP53 gene fulfills a central role in protecting cells from genetic insult. Given this crucial role it might be surprising that p53 itself is not essential for cell survival. Indeed, TP53 is the single most mutated gene across different cancer types. Thus, both a theoretical and a question of significant practical applicability arise: can cells be programmed to make TP53 an essential gene? Here we present a genetic p53 sensor, in which the loss of p53 is coupled to the rise of HSV-TK expression. We show that the sensor can distinguish both p53 knockout and cells expressing a common TP53 cancer mutation from otherwise isogenic TP53 wild-type cells. Importantly, the system is sensitive enough to specifically target TP53 loss-of-function cells with the HSV-TK pro-drug Ganciclovir both in vitro and in vivo. Our work opens new ways to programming cell intrinsic transformation protection systems that rely on endogenous components.

Yadira Boada, Alejandro Vignoni, Jesús Picó
Multi-objective optimization for gene expression noise reduction in a synthetic gene circuit
IFAC-PapersOnLine, 50(1) 4472-4477 (2017)
Open Access DOI

Stochasticity in biological systems often referred to as gene expression noise is ubiquitous. The main sources of this noise come about in two ways. The implicit randomness of the biochemical reactions generates intrinsic noise inside the cell. Other cellular processes are themselves products that vary over time and from each cell to another, producing the so-called extrinsic noise. Controlling the mean expression level of a gene while reducing its noise is a challenge in many applications of Synthetic Biology. In previous works, we proposed a gene synthetic circuit to reduce gene expression noise while achieving a desired mean expression level. The circuit combines a negative feedback loop and a cell-to-cell communication mechanism based on quorum sensing. In this work, we use a multi-objective optimization design approach to find the best values for the tunable-in-the-lab parameters that, for a given desired mean expression value, achieve minimization of gene expression noise caused by intrinsic and extrinsic fluctuations. Our approach allows tuning the circuit parameters required to minimize noise effects, providing results which prove in accordance with genome-wide experimental data reported in the literature. Exploring different scenarios, either considering only intrinsic noise or considering both extrinsic and intrinsic ones, we find that the design strategies obtained for both cases are not transferable. Thus, designing the circuit parameters taking into account only intrinsic noise yields a sub-optimal design with decreased performance when evaluated in a scenario where both extrinsic and intrinsic noise are present.

Yusuke Toyoda^*, Cedric J Cattin^*, Martin P Stewart^*, Ina Poser, Mirko Theis, Teymuras V. Kurzchalia, Frank Buchholz, Anthony Hyman^#, Daniel J. Müller^#
Genome-scale single-cell mechanical phenotyping reveals disease-related genes involved in mitotic rounding.
Nat Commun, 8(1) Art. No. 1266 (2017)
Open Access DOI

To divide, most animal cells drastically change shape and round up against extracellular confinement. Mitotic cells facilitate this process by generating intracellular pressure, which the contractile actomyosin cortex directs into shape. Here, we introduce a genome-scale microcantilever- and RNAi-based approach to phenotype the contribution of > 1000 genes to the rounding of single mitotic cells against confinement. Our screen analyzes the rounding force, pressure and volume of mitotic cells and localizes selected proteins. We identify 49 genes relevant for mitotic rounding, a large portion of which have not previously been linked to mitosis or cell mechanics. Among these, depleting the endoplasmic reticulum-localized protein FAM134A impairs mitotic progression by affecting metaphase plate alignment and pressure generation by delocalizing cortical myosin II. Furthermore, silencing the DJ-1 gene uncovers a link between mitochondria-associated Parkinson's disease and mitotic pressure. We conclude that mechanical phenotyping is a powerful approach to study the mechanisms governing cell shape.

Nikolai Hecker, Virag Sharma, Michael Hiller
Transition to an Aquatic Habitat Permitted the Repeated Loss of the Pleiotropic KLK8 Gene in Mammals.
Genome Biol Evol, 9(11) 3179-3188 (2017)
PDF DOI

Kallikrein related peptidase 8 (KLK8; also called neuropsin) is a serine protease that plays distinct roles in the skin and hippocampus. In the skin, KLK8 influences keratinocyte proliferation and desquamation, and activates antimicrobial peptides in sweat. In the hippocampus, KLK8 affects memory acquisition. Here, we examined the evolution of KLK8 in mammals and discovered that, out of 70 placental mammals, KLK8 is exclusively lost in three independent fully-aquatic lineages, comprising dolphin, killer whale, minke whale, and manatee. In addition, while the sperm whale has an intact KLK8 reading frame, the gene evolves neutrally in this species. We suggest that the distinct functions of KLK8 likely became obsolete in the aquatic environment, leading to the subsequent loss of KLK8 in several fully-aquatic mammalian lineages. First, the cetacean and manatee skin lacks sweat glands as an adaptation to the aquatic environment, which likely made the epidermal function of KLK8 obsolete. Second, cetaceans and manatees exhibit a proportionally small hippocampus, which may have rendered the hippocampal functions of KLK8 obsolete. Together, our results shed light on the genomic changes that correlate with skin and neuroanatomical differences of aquatic mammals, and show that even pleiotropic genes can be lost during evolution if an environmental change nullifies the need for the different functions of such genes.

Yadira Boada, Alejandro Vignoni, Jesús Picó
Engineered Control of Genetic Variability Reveals Interplay among Quorum Sensing, Feedback Regulation, and Biochemical Noise.
ACS Synth Biol, 6(10) 1903-1912 (2017)
DOI

Stochastic fluctuations in gene expression trigger both beneficial and harmful consequences for cell behavior. Therefore, achieving a desired mean protein expression level while minimizing noise is of interest in many applications, including robust protein production systems in industrial biotechnology. Here, we consider a synthetic gene circuit combining intracellular negative feedback and cell-to-cell communication based on quorum sensing. Accounting for both intrinsic and extrinsic noise, stochastic simulations allow us to analyze the capability of the circuit to reduce noise strength as a function of its parameters. We obtain mean expression levels and noise strengths for all species under different scenarios, showing good agreement with system-wide available experimental data of protein abundance and noise in Escherichia coli. Our in silico experiments, validated by preliminary in vivo results, reveal significant noise attenuation in gene expression through the interplay between quorum sensing and negative feedback and highlight the differential role that they play in regard to intrinsic and extrinsic noise.

Maja Borup Kjær Petersen, Ajuna Azad, Camilla Ingvorsen, Katja Hess, Mattias Hansson, Anne Grapin-Botton, Christian Honoré
Single-Cell Gene Expression Analysis of a Human ESC Model of Pancreatic Endocrine Development Reveals Different Paths to β-Cell Differentiation.
Stem Cell Rep, 9(4) 1246-1261 (2017)
Open Access DOI

The production of insulin-producing β cells from human embryonic stem cells (hESCs) in vitro represents a promising strategy for a cell-based therapy for type 1 diabetes mellitus. To explore the cellular heterogeneity and temporal progression of endocrine progenitors and their progeny, we performed single-cell qPCR on more than 500 cells across several stages of in vitro differentiation of hESCs and compared them with human islets. We reveal distinct subpopulations along the endocrine differentiation path and an early lineage bifurcation toward either polyhormonal cells or β-like cells. We uncover several similarities and differences with mouse development and reveal that cells can take multiple paths to the same differentiation state, a principle that could be relevant to other systems. Notably, activation of the key β-cell transcription factor NKX6.1 can be initiated before or after endocrine commitment. The single-cell temporal resolution we provide can be used to improve the production of functional β cells.

Kamran Rizzolo, Jennifer Huen, Ashwani Kumar, Sadhna Phanse, James Vlasblom, Yoshito Kakihara, Hussein A Zeineddine, Zoran Minic, Jamie Snider, Wen Wang, Carles Pons, Thiago V Seraphim, Edgar Boczek, Simon Alberti, Michael Costanzo, Chad L Myers, Igor Stagljar, Charles Boone, Mohan Babu, Walid A Houry
Features of the Chaperone Cellular Network Revealed through Systematic Interaction Mapping.
Cell Rep, 20(11) 2735-2748 (2017)
Open Access DOI

A comprehensive view of molecular chaperone function in the cell was obtained through a systematic global integrative network approach based on physical (protein-protein) and genetic (gene-gene or epistatic) interaction mapping. This allowed us to decipher interactions involving all core chaperones (67) and cochaperones (15) of Saccharomyces cerevisiae. Our analysis revealed the presence of a large chaperone functional supercomplex, which we named the naturally joined (NAJ) chaperone complex, encompassing Hsp40, Hsp70, Hsp90, AAA+, CCT, and small Hsps. We further found that many chaperones interact with proteins that form foci or condensates under stress conditions. Using an in vitro reconstitution approach, we demonstrate condensate formation for the highly conserved AAA+ ATPases Rvb1 and Rvb2, which are part of the R2TP complex that interacts with Hsp90. This expanded view of the chaperone network in the cell clearly demonstrates the distinction between chaperones having broad versus narrow substrate specificities in protein homeostasis.

L Carine Stapel, Christoph Zechner, Nadine Vastenhouw
Uniform gene expression in embryos is achieved by temporal averaging of transcription noise.
Genes Dev, 31(16) 1635-1640 (2017)
DOI

Transcription is often stochastic. This is seemingly incompatible with the importance of gene expression during development. Here we show that during zebrafish embryogenesis, transcription activation is stochastic due to (1) genes acquiring transcriptional competence at different times in different cells, (2) differences in cell cycle stage between cells, and (3) the stochastic nature of transcription. Initially, stochastic transcription causes large cell-to-cell differences in transcript levels. However, variability is reduced by lengthening cell cycles and the accumulation of transcription events in each cell. Temporal averaging might provide a general context in which to understand how embryos deal with stochastic transcription.

Koichiro Uriu, Rajasekaran Bhavna, Andrew C. Oates, Luis G Morelli
A framework for quantification and physical modeling of cell mixing applied to oscillator synchronization in vertebrate somitogenesis.
Biol Open, 6(8) 1235-1244 (2017)
Open Access DOI

In development and disease, cells move as they exchange signals. One example is found in vertebrate development, during which the timing of segment formation is set by a 'segmentation clock', in which oscillating gene expression is synchronized across a population of cells by Delta-Notch signaling. Delta-Notch signaling requires local cell-cell contact, but in the zebrafish embryonic tailbud, oscillating cells move rapidly, exchanging neighbors. Previous theoretical studies proposed that this relative movement or cell mixing might alter signaling and thereby enhance synchronization. However, it remains unclear whether the mixing timescale in the tissue is in the right range for this effect, because a framework to reliably measure the mixing timescale and compare it with signaling timescale is lacking. Here, we develop such a framework using a quantitative description of cell mixing without the need for an external reference frame and constructing a physical model of cell movement based on the data. Numerical simulations show that mixing with experimentally observed statistics enhances synchronization of coupled phase oscillators, suggesting that mixing in the tailbud is fast enough to affect the coherence of rhythmic gene expression. Our approach will find general application in analyzing the relative movements of communicating cells during development and disease.

Shambaditya Saha, Anthony Hyman
RNA gets in phase.
J Cell Biol, 216(8) 2235-2237 (2017)
DOI

Several neurological disorders are linked to tandem nucleotide repeat expansion in the mutated gene. Jain and Vale (2017. Nature. https://doi.org/10.1038/nature22386) show that, above a pathological threshold repeat number, base pairing interactions drive phase separation of RNA into membrane-less gels, suggesting that RNA can scaffold the assembly of phase-separated compartments that sequester proteins/RNAs causing toxicity.

V Albrecht, C Zweiniger, Vineeth Surendranath, Karl Lang, G Schöfl, Andreas Dahl, Sylke Winkler, V Lange, I Böhme, A H Schmidt
Dual redundant sequencing strategy: Full-length gene characterisation of 1056 novel and confirmatory HLA alleles.
HLA, 90(2) 79-87 (2017)
Open Access DOI

The high-throughput department of DKMS Life Science Lab encounters novel human leukocyte antigen (HLA) alleles on a daily basis. To characterise these alleles, we have developed a system to sequence the whole gene from 5'- to 3'-UTR for the HLA loci A, B, C, DQB1 and DPB1 for submission to the European Molecular Biology Laboratory - European Nucleotide Archive (EMBL-ENA) and the IPD-IMGT/HLA Database. Our workflow is based on a dual redundant sequencing strategy. Using shotgun sequencing on an Illumina MiSeq instrument and single molecule real-time (SMRT) sequencing on a PacBio RS II instrument, we are able to achieve highly accurate HLA full-length consensus sequences. Remaining conflicts are resolved using the R package DR2S (Dual Redundant Reference Sequencing). Given the relatively high throughput of this strategy, we have developed the semi-automated web service TypeLoader, to aid in the submission of sequences to the EMBL-ENA and the IPD-IMGT/HLA Database. In the IPD-IMGT/HLA Database release 3.24.0 (April 2016; prior to the submission of the sequences described here), only 5.2% of all known HLA alleles have been fully characterised together with intronic and UTR sequences. So far, we have applied our strategy to characterise and submit 1056 HLA alleles, thereby more than doubling the number of fully characterised alleles. Given the increasing application of next generation sequencing (NGS) for full gene characterisation in clinical practice, extending the HLA database concomitantly is highly desirable. Therefore, we propose this dual redundant sequencing strategy as a workflow for submission of novel full-length alleles and characterisation of sequences that are as yet incomplete. This would help to mitigate the predominance of partially known alleles in the database.

Frank Jülicher, Suzanne Eaton
Emergence of tissue shape changes from collective cell behaviours.
Semin Cell Dev Biol, 67 103-112 (2017)
DOI

Anyone watching a movie of embryonic development immediately appreciates the importance of morphogenetic movements and cell flows that reshape tissue. Dynamic tissue shape changes are genetically choreographed, but their execution is essentially a mechanical event. How the interplay between genetics and tissue mechanics controls tissue shape is a fundamental question. Key insights into this problem have emerged from studies in different model organisms as well as in cultured epithelia. These studies have revealed how gene expression patterns can generate patterns of planar cell polarity that orient cellular force generation and give rise to anisotropic mechanical properties of cells and tissues. These can autonomously bias the rate and orientation of cellular events such as cell divisions, extrusions, neighbor exchanges and shape changes that drive morphogenesis. However recent studies also highlight how autonomously controlled cell dynamics lead to tissue-wide stress patterns framed by mechanical constraints such as cellular connections to extracellular matrices. These stress patterns themselves can orient the cell behaviours underlying morphogenesis. As a result of this interplay, tissue shape emerges in a mechanical process that tightly couples mechanics and genetics.

Virag Sharma, Michael Hiller
Increased alignment sensitivity improves the usage of genome alignments for comparative gene annotation.
Nucleic Acids Res, 45(14) 8369-8377 (2017)
Open Access PDF DOI

Genome alignments provide a powerful basis to transfer gene annotations from a well-annotated reference genome to many other aligned genomes. The completeness of these annotations crucially depends on the sensitivity of the underlying genome alignment. Here, we investigated the impact of the genome alignment parameters and found that parameters with a higher sensitivity allow the detection of thousands of novel alignments between orthologous exons that have been missed before. In particular, comparisons between species separated by an evolutionary distance of >0.75 substitutions per neutral site, like human and other non-placental vertebrates, benefit from increased sensitivity. To systematically test if increased sensitivity improves comparative gene annotations, we built a multiple alignment of 144 vertebrate genomes and used this alignment to map human genes to the other 143 vertebrates with CESAR. We found that higher alignment sensitivity substantially improves the completeness of comparative gene annotations by adding on average 2382 and 7440 novel exons and 117 and 317 novel genes for mammalian and non-mammalian species, respectively. Our results suggest a more sensitive alignment strategy that should generally be used for genome alignments between distantly-related species. Our 144-vertebrate genome alignment and the comparative gene annotations (https://bds.mpi-cbg.de/hillerlab/144VertebrateAlignment_CESAR/) are a valuable resource for comparative genomics.

Patricia Heyn, Hanna Salmonowicz, Jonathan Rodenfels, Karla M. Neugebauer
Activation of transcription enforces the formation of distinct nuclear bodies in zebrafish embryos.
RNA Biol, 14(6) 752-760 (2017)
DOI

Nuclear bodies are cellular compartments that lack lipid bilayers and harbor specific RNAs and proteins. Recent proposals that nuclear bodies form through liquid-liquid phase separation leave the question of how different nuclear bodies maintain their distinct identities unanswered. Here we investigate Cajal bodies (CBs), histone locus bodies (HLBs) and nucleoli - involved in assembly of the splicing machinery, histone mRNA 3' end processing, and rRNA processing, respectively - in the embryos of the zebrafish, Danio rerio. We take advantage of the transcriptional silence of the 1-cell embryo and follow nuclear body appearance as zygotic transcription becomes activated. CBs are present from fertilization onwards, while HLB and nucleolar components formed foci several hours later when histone genes and rDNA became active. HLB formation was blocked by transcription inhibition, suggesting nascent histone transcripts recruit HLB components like U7 snRNP. Surprisingly, we found that U7 base-pairing with nascent histone transcripts was not required for localization to HLBs. Rather, the type of Sm ring assembled on U7 determined its targeting to HLBs or CBs; the spliceosomal Sm ring targeted snRNAs to CBs while the specialized U7 Sm-ring localized to HLBs, demonstrating the contribution of protein constituents to the distinction among nuclear bodies. Thus, nucleolar, HLB, and CB components can mix in early embryogenesis when transcription is naturally or artificially silenced. These data support a model in which transcription of specific gene loci nucleates nuclear body components with high specificity and fidelity to perform distinct regulatory functions.

Jovan Mircetic, Iris Steinebrunner, Li Ding, Jifeng Fei, Aliona Bogdanova, David N. Drechsel, Frank Buchholz
Purified Cas9 Fusion Proteins for Advanced Genome Manipulation.
Small Methods, 1(4) Art. No. 1600052 (2017)
DOI

The CRISPR/Cas9 system (CRISPR = clustered regularly interspaced short palindromic repeats) has rapidly become one of the most versatile genome manipulation technologies, and different methods to introduce the Cas9 nuclease activity into cells have been developed. The direct delivery of purified Cas9 protein complexed with a guide RNA as a ribonucleoprotein (RNP) has emerged as an advantageous approach, as it provides instant, but limited activity of the enzyme, thereby reducing off-target cleavage. The usefulness of the CRISPR/Cas9 system has recently been extended by the generation of Cas9 or dead (d) Cas9 fusion genes. However, these systems have so far been mainly explored when delivered by expression plasmids. Here, a variety of purified Cas9 fusion proteins are generated, and their utility is tested in a number of assays. This work illustrates that Cas9 fused to green-or redfluorescent proteins can be usefully employed to increase the frequency of targeted cells when transfected as RNPs. Furthermore, it is demonstrated that purified dCas9 fused to a dual transactivation domain can potently activate gene expression when transfected as an RNP into embryonic stem cells. The results show that purified Cas9 fusion proteins are versatile and efficient reagents that facilitate advanced genome manipulation.

Máté Pálfy, Shai Joseph, Nadine Vastenhouw
The timing of zygotic genome activation.
Curr Opin Genet Dev, 43 53-60 (2017)
DOI

After fertilization, the embryonic genome is inactive until transcription is initiated during the maternal-to-zygotic transition. How the onset of transcription is regulated in a precisely timed manner, however, is a long standing question in biology. Several mechanisms have been shown to contribute to the temporal regulation of genome activation but none of them can fully explain the general absence of transcription as well the gene specific onset that follows. Here we review the work that has been done toward elucidating the mechanisms underlying the temporal regulation of transcription in embryos.

R Urrego, Sandra Milena Bernal-Ulloa, N A Chavarría, E Herrera-Puerta, Andrea Lucas-Hahn, D Herrmann, Sylke Winkler, Dorit Pache, H Niemann, N Rodriguez-Osorio
Satellite DNA methylation status and expression of selected genes in Bos indicus blastocysts produced in vivo and in vitro.
Zygote, 25(2) 131-140 (2017)
DOI

Bovine embryos produced in vivo and in vitro differ with respect to molecular profiles, including epigenetic marks and gene expression profiles. This study investigated the CpG methylation status in bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) DNA sequences, and concomitantly the relative abundance of transcripts, critically involved in DNA methylation (DNMT1 and DNMT3A), growth and development (IGF2R) and pluripotency (POU5F1) in Bos indicus embryos produced in vitro or in vivo. Results revealed that methylation of BTS were higher (P < 0.05) in embryos produced in vitro compared with their in vivo produced counterparts, while the methylation status of BTαS was similar in both groups. There were no significant differences in transcript abundance for DNMT3A, IGF2R and POU5F1 between blastocysts produced in vivo and in vitro. However, a significantly lower amount of DNMT1 transcripts was found in the in vitro cultured embryos (P < 0.05) compared with their in vivo derived counterparts. In conclusion, this study reported only minor changes in the expression of developmentally important genes and satellite DNA methylation related to the in vitro embryo production system.

Stephanie Spannl^*, Alexandra Kumichel^*, Sarita Hebbar, Katja Kapp, Marcos Gonzalez-Gaitan, Sylke Winkler, Rosana Blawid, Gregor Jessberger, Elisabeth Knust
The Crumbs_C isoform of Drosophila shows tissue- and stage-specific expression and prevents light-dependent retinal degeneration.
Biol Open, 6(2) 165-175 (2017)
Open Access PDF DOI

Drosophila Crumbs (Crb) is a key regulator of epithelial polarity and fulfils a plethora of other functions, such as growth regulation, morphogenesis of photoreceptor cells and prevention of retinal degeneration. This raises the question how a single gene regulates such diverse functions, which in mammals are controlled by three different paralogs. Here, we show that in Drosophila different Crb protein isoforms are differentially expressed as a result of alternative splicing. All isoforms are transmembrane proteins that differ by just one EGF-like repeat in their extracellular portion. Unlike Crb_A, which is expressed in most embryonic epithelia from early stages onward, Crb_C is expressed later and only in a subset of embryonic epithelia. Flies specifically lacking Crb_C are homozygous viable and fertile. Strikingly, these flies undergo light-dependent photoreceptor degeneration despite the fact that the other isoforms are expressed and properly localised at the stalk membrane. This allele now provides an ideal possibility to further unravel the molecular mechanisms by which Drosophila crb protects photoreceptor cells from the detrimental consequences of light-induced cell stress.

Tom Stückemann^*, James Cleland^*, Steffen Werner, Hanh Thi-Kim Vu, Robert Bayersdorf, Shang-Yun Liu, Benjamin Friedrich, Frank Jülicher, Jochen Rink
Antagonistic Self-Organizing Patterning Systems Control Maintenance and Regeneration of the Anteroposterior Axis in Planarians.
Dev Cell, 40(3) 248-263 (2017)
DOI

Planarian flatworms maintain their body plan in the face of constant internal turnover and can regenerate from arbitrary tissue fragments. Both phenomena require self-maintaining and self-organizing patterning mechanisms, the molecular mechanisms of which remain poorly understood. We show that a morphogenic gradient of canonical Wnt signaling patterns gene expression along the planarian anteroposterior (A/P) axis. Our results demonstrate that gradient formation likely occurs autonomously in the tail and that an autoregulatory module of Wnt-mediated Wnt expression both shapes the gradient at steady state and governs its re-establishment during regeneration. Functional antagonism between the tail Wnt gradient and an unknown head patterning system further determines the spatial proportions of the planarian A/P axis and mediates mutually exclusive molecular fate choices during regeneration. Overall, our results suggest that the planarian A/P axis is patterned by self-organizing patterning systems deployed from either end that are functionally coupled by mutual antagonism.

Gaelle R Carrat, Ming Hu, Marie-Sophie Nguyen-Tu, Pauline Chabosseau, Kyle J Gaulton, Martijn van de Bunt, Afshan Siddiq, Mario Falchi, Matthias Thurner, Mickaël Canouil, Francois Pattou, Isabelle Leclerc, Timothy J Pullen, Matthew C Cane, Priyanka Prabhala, William Greenwald, Anke Schulte, Piero Marchetti, Mark Ibberson, Patrick E MacDonald, Jocelyn E Manning Fox, Anna L Gloyn, Philippe Froguel, Michele Solimena, Mark I McCarthy, Guy A Rutter
Decreased STARD10 Expression Is Associated with Defective Insulin Secretion in Humans and Mice.
Am J Hum Genet, 100(2) 238-256 (2017)
Open Access PDF DOI

Genetic variants near ARAP1 (CENTD2) and STARD10 influence type 2 diabetes (T2D) risk. The risk alleles impair glucose-induced insulin secretion and, paradoxically but characteristically, are associated with decreased proinsulin:insulin ratios, indicating improved proinsulin conversion. Neither the identity of the causal variants nor the gene(s) through which risk is conferred have been firmly established. Whereas ARAP1 encodes a GTPase activating protein, STARD10 is a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer protein family. By integrating genetic fine-mapping and epigenomic annotation data and performing promoter-reporter and chromatin conformational capture (3C) studies in β cell lines, we localize the causal variant(s) at this locus to a 5 kb region that overlaps a stretch-enhancer active in islets. This region contains several highly correlated T2D-risk variants, including the rs140130268 indel. Expression QTL analysis of islet transcriptomes from three independent subject groups demonstrated that T2D-risk allele carriers displayed reduced levels of STARD10 mRNA, with no concomitant change in ARAP1 mRNA levels. Correspondingly, β-cell-selective deletion of StarD10 in mice led to impaired glucose-stimulated Ca(2+) dynamics and insulin secretion and recapitulated the pattern of improved proinsulin processing observed at the human GWAS signal. Conversely, overexpression of StarD10 in the adult β cell improved glucose tolerance in high fat-fed animals. In contrast, manipulation of Arap1 in β cells had no impact on insulin secretion or proinsulin conversion in mice. This convergence of human and murine data provides compelling evidence that the T2D risk associated with variation at this locus is mediated through reduction in STARD10 expression in the β cell.

Marta Florio, Víctor Borrell, Wieland B. Huttner
Human-specific genomic signatures of neocortical expansion.
Curr Opin Neurobiol, 42 33-44 (2017)
DOI

Neocortex evolutionary expansion is primarily due to increased proliferative capacity of neural progenitor cells during cortical development. Exploiting insights into the cell biology of cortical progenitors gained during the past two decades, recent studies uncovered a variety of gene expression differences that underlie differential cortical progenitor behavior. These comprise both, differences between cortical areas that likely provide a molecular basis for cortical folding, and differences across species thought to be responsible for increases in neocortex size. Human-specific signatures have been identified for gene regulatory elements, non-coding gene products, and protein-encoding genes, and have been functionally examined in in vivo as well as novel in vitro model systems.

Takashi Namba, Wieland B. Huttner
Neural progenitor cells and their role in the development and evolutionary expansion of the neocortex.
Wiley Interdiscip Rev Dev Biol, 6(1) Art. No. e256 (2017)
DOI

The evolutionary expansion of the mammalian brain, notably the neocortex, provides a platform for the higher cognitive abilities that characterize humans. Cortical expansion is accompanied by increased folding of the pial surface, which gives rise to a gyrencephalic (folded) rather than lissencephalic (unfolded) neocortex. This expansion reflects the prolonged and increased proliferation of neural stem and progenitor cells (NPCs). Distinct classes of NPCs can be distinguished based on either cell biological criteria (apical progenitors [APs], basal progenitors [BPs]) or lineage (primary progenitors and secondary progenitors). Cortical expansion in development and evolution is linked to an increased abundance and proliferative capacity of BPs, notably basal radial glial cells, a recently characterized type of secondary progenitor derived from apical radial glial cells, the primary progenitors. To gain insight into the molecular basis underlying the prolonged and increased proliferation of NPCs and in particular BPs, comparative genomic and transcriptomic approaches, mostly for human versus mouse, have been employed and applied to specific NPC types and subpopulations. These have revealed two principal sets of molecular changes. One concerns differences in the expression of common genes between species with different degrees of cortical expansion. The other comprises human-specific genes or genomic regulatory sequences. Various systems that allow functional testing of these genomic and gene expression differences between species have emerged, including transient and stable transgenesis, genome editing, cerebral organoids, and organotypic slice cultures. These provide future avenues for uncovering the molecular basis of cortical expansion. For further resources related to this article, please visit the WIREs website.

Marie Skogs, Charlotte Stadler, Rutger Schutten, Martin Hjelmare, Christian Gnann, Lars Björk, Ina Poser, Anthony Hyman, Mathias Uhlén, Emma Lundberg
Antibody Validation in Bioimaging Applications Based on Endogenous Expression of Tagged Proteins.
J Proteome Res, 16(1) 147-155 (2017)
DOI

Antibodies are indispensible research tools, yet the scientific community has not adopted standardized procedures to validate their specificity. Here we present a strategy to systematically validate antibodies for immunofluorescence (IF) applications using gene tagging. We have assessed the on- and off-target binding capabilities of 197 antibodies using 108 cell lines expressing EGFP-tagged target proteins at endogenous levels. Furthermore, we assessed batch-to-batch effects for 35 target proteins, showing that both the on- and off-target binding patterns vary significantly between antibody batches and that the proposed strategy serves as a reliable procedure for ensuring reproducibility upon production of new antibody batches. In summary, we present a systematic scheme for antibody validation in IF applications using endogenous expression of tagged proteins. This is an important step toward a reproducible approach for context- and application-specific antibody validation and improved reliability of antibody-based experiments and research data.

Nereo Kalebic, Katherine S. Long, Wieland B. Huttner
Neocortex expansion in development and evolution: the cell biology of neural stem and progenitor cells and the impact of human-specific gene expression.
In: Evolution of Nervous Systems . Vol. 3 : The nervous systems of non-human primates. (Eds.) Jon H Kaas,Amsterdam, Netherlands,Elsevier (2017),73-89

Peter Horvath, Nathalie Aulner, Marc Bickle, Anthony M Davies, Elaine Del Nery, Daniel Ebner, Maria Montoya, Päivi Östling, Vilja Pietiäinen, Leo S Price, Spencer L Shorte, Gerardo Turcatti, Carina von Schantz, Neil O Carragher
Screening out irrelevant cell-based models of disease.
Nat Rev Drug Discov, 15(11) 751-769 (2016)
DOI

The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell- and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates.

Veronika Fitz, Jaeoh Shin, Christoph Ehrlich, Lucas Farnung, Patrick Cramer, Vasily Zaburdaev, Stephan W. Grill
Nucleosomal arrangement affects single-molecule transcription dynamics.
Proc Natl Acad Sci U.S.A., 113(45) 12733-12738 (2016)
DOI

In eukaryotes, gene expression depends on chromatin organization. However, how chromatin affects the transcription dynamics of individual RNA polymerases has remained elusive. Here, we use dual trap optical tweezers to study single yeast RNA polymerase II (Pol II) molecules transcribing along a DNA template with two nucleosomes. The slowdown and the changes in pausing behavior within the nucleosomal region allow us to determine a drift coefficient, χ, which characterizes the ability of the enzyme to recover from a nucleosomal backtrack. Notably, χ can be used to predict the probability to pass the first nucleosome. Importantly, the presence of a second nucleosome changes χ in a manner that depends on the spacing between the two nucleosomes, as well as on their rotational arrangement on the helical DNA molecule. Our results indicate that the ability of Pol II to pass the first nucleosome is increased when the next nucleosome is turned away from the first one to face the opposite side of the DNA template. These findings help to rationalize how chromatin arrangement affects Pol II transcription dynamics.

Felipe Mora-Bermúdez, Farhath Badsha, Sabina Kanton, J Gray Camp, Benjamin Vernot, Kathrin Köhler, Birger Voigt, Keisuke Okita, Tomislav Maricic, Zhisong He, Robert Lachmann, Svante Pääbo, Barbara Treutlein, Wieland B. Huttner
Differences and similarities between human and chimpanzee neural progenitors during cerebral cortex development.
Elife, 5 Art. No. e18683 (2016)
Open Access PDF DOI

Human neocortex expansion likely contributed to the remarkable cognitive abilities of humans. This expansion is thought to primarily reflect differences in proliferation versus differentiation of neural progenitors during cortical development. Here, we have searched for such differences by analysing cerebral organoids from human and chimpanzees using immunohistofluorescence, live imaging, and single-cell transcriptomics. We find that the cytoarchitecture, cell type composition, and neurogenic gene expression programs of humans and chimpanzees are remarkably similar. Notably, however, live imaging of apical progenitor mitosis uncovered a lengthening of prometaphase-metaphase in humans compared to chimpanzees that is specific to proliferating progenitors and not observed in non-neural cells. Consistent with this, the small set of genes more highly expressed in human apical progenitors points to increased proliferative capacity, and the proportion of neurogenic basal progenitors is lower in humans. These subtle differences in cortical progenitors between humans and chimpanzees may have consequences for human neocortex evolution.

L Carine Stapel
The road to uniform gene expression
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2016)

Kaiser Matthias, Florian Jug, Olin Silander, Siddharth Deshpande, Thomas Pfohl, Thomas Julou^#, Gene Myers^#, Erik van Nimwegen^#
Tracking single-cell gene regulation in dynamically controlled environments using an integrated microfluidic and computational setup
bioRxiv, Art. No. https://doi.org/10.1101/076224 (2016)
Open Access DOI

Jacqueline Tabler, Christopher P Rice, Karen J Liu, John Wallingford
A novel ciliopathic skull defect arising from excess neural crest.
Dev Biol, 417(1) 4-10 (2016)
DOI

The skull is essential for protecting the brain from damage, and birth defects involving disorganization of skull bones are common. However, the developmental trajectories and molecular etiologies by which many craniofacial phenotypes arise remain poorly understood. Here, we report a novel skull defect in ciliopathic Fuz mutant mice in which only a single bone pair encases the forebrain, instead of the usual paired frontal and parietal bones. Through genetic lineage analysis, we show that this defect stems from a massive expansion of the neural crest-derived frontal bone. This expansion occurs at the expense of the mesodermally-derived parietal bones, which are either severely reduced or absent. A similar, though less severe, phenotype was observed in Gli3 mutant mice, consistent with a role for Gli3 in cilia-mediated signaling. Excess crest has also been shown to drive defective palate morphogenesis in ciliopathic mice, and that defect is ameliorated by reduction of Fgf8 gene dosage. Strikingly, skull defects in Fuz mutant mice are also rescued by loss of one allele of fgf8, suggesting a potential route to therapy. In sum, this work is significant for revealing a novel skull defect with a previously un-described developmental etiology and for suggesting a common developmental origin for skull and palate defects in ciliopathies.

Laura Broutier, Amanda Andersson-Rolf, Christopher J Hindley, Sylvia F Boj, Hans Clevers, Bon-Kyoung Koo, Meritxell Huch
Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.
Nat Protoc, 11(9) 1724-1743 (2016)
DOI

Adult somatic tissues have proven difficult to expand in vitro, largely because of the complexity of recreating appropriate environmental signals in culture. We have overcome this problem recently and developed culture conditions for adult stem cells that allow the long-term expansion of adult primary tissues from small intestine, stomach, liver and pancreas into self-assembling 3D structures that we have termed 'organoids'. We provide a detailed protocol that describes how to grow adult mouse and human liver and pancreas organoids, from cell isolation and long-term expansion to genetic manipulation in vitro. Liver and pancreas cells grow in a gel-based extracellular matrix (ECM) and a defined medium. The cells can self-organize into organoids that self-renew in vitro while retaining their tissue-of-origin commitment, genetic stability and potential to differentiate into functional cells in vitro (hepatocytes) and in vivo (hepatocytes and endocrine cells). Genetic modification of these organoids opens up avenues for the manipulation of adult stem cells in vitro, which could facilitate the study of human biology and allow gene correction for regenerative medicine purposes. The complete protocol takes 1-4 weeks to generate self-renewing 3D organoids and to perform genetic manipulation experiments. Personnel with basic scientific training can conduct this protocol.

M Gai, Federico Bianchi, Cristiana Vagnoni, Fiammetta Vernì, Silvia Bonaccorsi, Selina Pasquero, G E Berto, F Sgrò, A M A Chiotto, Laura Annaratone, Anna Sapino, Anna Bergo, Nicoletta Landsberger, Jacqueline Bond, Wieland B. Huttner, F Di Cunto
ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules.
EMBO Rep, 17(10) 1396-1409 (2016)
DOI

Correct orientation of cell division is considered an important factor for the achievement of normal brain size, as mutations in genes that affect this process are among the leading causes of microcephaly. Abnormal spindle orientation is associated with reduction of the neuronal progenitor symmetric divisions, premature cell cycle exit, and reduced neurogenesis. This mechanism has been involved in microcephaly resulting from mutation of ASPM, the most frequently affected gene in autosomal recessive human primary microcephaly (MCPH), but it is presently unknown how ASPM regulates spindle orientation. In this report, we show that ASPM may control spindle positioning by interacting with citron kinase (CITK), a protein whose loss is also responsible for severe microcephaly in mammals. We show that the absence of CITK leads to abnormal spindle orientation in mammals and insects. In mouse cortical development, this phenotype correlates with increased production of basal progenitors. ASPM is required to recruit CITK at the spindle, and CITK overexpression rescues ASPM phenotype. ASPM and CITK affect the organization of astral microtubules (MT), and low doses of MT-stabilizing drug revert the spindle orientation phenotype produced by their knockdown. Finally, CITK regulates both astral-MT nucleation and stability. Our results provide a functional link between two established microcephaly proteins.

Sara Cuylen, Claudia Blaukopf, Antonio Z Politi, Thomas Müller-Reichert, Beate Neumann, Ina Poser, Jan Ellenberg, Anthony Hyman, Daniel W Gerlich
Ki-67 acts as a biological surfactant to disperse mitotic chromosomes.
Nature, 535(7611) 308-312 (2016)
DOI

Eukaryotic genomes are partitioned into chromosomes that form compact and spatially well-separated mechanical bodies during mitosis. This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes and the discovery of proteins at the chromosome surface, the molecular and biophysical bases of mitotic chromosome structural individuality have remained unclear. Here we report that the proliferation marker protein Ki-67 (encoded by the MKI67 gene), a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of human Ki-67 is not confined within a specific protein domain, but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrostatic charge barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-colour labelling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic of polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery in mammalian cells and suggests that natural proteins can function as surfactants in intracellular compartmentalization.

Michael Heide, Marta Florio, Wieland B. Huttner
Die Rolle humanspezifischer Gene in der Gehirnentwicklung und -evolution
BIOspektrum, 22(4) 352-355 (2016)
DOI

Virag Sharma, Anas Elghafari, Michael Hiller
Coding exon-structure aware realigner (CESAR) utilizes genome alignments for accurate comparative gene annotation.
Nucleic Acids Res, 44(11) Art. No. e103 (2016)
Open Access PDF DOI

Identifying coding genes is an essential step in genome annotation. Here, we utilize existing whole genome alignments to detect conserved coding exons and then map gene annotations from one genome to many aligned genomes. We show that genome alignments contain thousands of spurious frameshifts and splice site mutations in exons that are truly conserved. To overcome these limitations, we have developed CESAR (Coding Exon-Structure Aware Realigner) that realigns coding exons, while considering reading frame and splice sites of each exon. CESAR effectively avoids spurious frameshifts in conserved genes and detects 91% of shifted splice sites. This results in the identification of thousands of additional conserved exons and 99% of the exons that lack inactivating mutations match real exons. Finally, to demonstrate the potential of using CESAR for comparative gene annotation, we applied it to 188 788 exons of 19 865 human genes to annotate human genes in 99 other vertebrates. These comparative gene annotations are available as a resource (http://bds.mpi-cbg.de/hillerlab/CESAR/). CESAR (https://github.com/hillerlab/CESAR/) can readily be applied to other alignments to accurately annotate coding genes in many other vertebrate and invertebrate genomes.

Yin Ning Chiang, Kah Junn Tan, Henry Chung, Oksana Lavrynenko, Andrej Shevchenko, Joanne Y Yew
Steroid Hormone Signaling Is Essential for Pheromone Production and Oenocyte Survival.
PLoS Genet, 12(6) Art. No. e1006126 (2016)
Open Access PDF DOI

Many of the lipids found on the cuticles of insects function as pheromones and communicate information about age, sex, and reproductive status. In Drosophila, the composition of the information-rich lipid profile is dynamic and changes over the lifetime of an individual. However, the molecular basis of this change is not well understood. To identify genes that control cuticular lipid production in Drosophila, we performed a RNA interference screen and used Direct Analysis in Real Time and gas chromatography mass spectrometry to quantify changes in the chemical profiles. Twelve putative genes were identified whereby transcriptional silencing led to significant differences in cuticular lipid production. Amongst them, we characterized a gene which we name spidey, and which encodes a putative steroid dehydrogenase that has sex- and age-dependent effects on viability, pheromone production, and oenocyte survival. Transcriptional silencing or overexpression of spidey during embryonic development results in pupal lethality and significant changes in levels of the ecdysone metabolite 20-hydroxyecdysonic acid and 20-hydroxyecdysone. In contrast, inhibiting gene expression only during adulthood resulted in a striking loss of oenocyte cells and a concomitant reduction of cuticular hydrocarbons, desiccation resistance, and lifespan. Oenocyte loss and cuticular lipid levels were partially rescued by 20-hydroxyecdysone supplementation. Taken together, these results identify a novel regulator of pheromone synthesis and reveal that ecdysteroid signaling is essential for the maintenance of cuticular lipids and oenocytes throughout adulthood.

Édgar Roldán, Ana Lisica, Daniel Sánchez-Taltavull, Stephan W. Grill
Stochastic resetting in backtrack recovery by RNA polymerases.
Phys Rev E, 93(6-1) Art. No. 062411 (2016)
DOI

Transcription is a key process in gene expression, in which RNA polymerases produce a complementary RNA copy from a DNA template. RNA polymerization is frequently interrupted by backtracking, a process in which polymerases perform a random walk along the DNA template. Recovery of polymerases from the transcriptionally inactive backtracked state is determined by a kinetic competition between one-dimensional diffusion and RNA cleavage. Here we describe backtrack recovery as a continuous-time random walk, where the time for a polymerase to recover from a backtrack of a given depth is described as a first-passage time of a random walker to reach an absorbing state. We represent RNA cleavage as a stochastic resetting process and derive exact expressions for the recovery time distributions and mean recovery times from a given initial backtrack depth for both continuous and discrete-lattice descriptions of the random walk. We show that recovery time statistics do not depend on the discreteness of the DNA lattice when the rate of one-dimensional diffusion is large compared to the rate of cleavage.

Qian Li, Song Guo, Xi Jiang, Jaroslaw Bryk, Ronald Naumann, Wolfgang Enard, Masaru Tomita, Masahiro Sugimoto, Philipp Khaitovich, Svante Pääbo
Mice carrying a human GLUD2 gene recapitulate aspects of human transcriptome and metabolome development.
Proc Natl Acad Sci U.S.A., 113(19) 5358-5363 (2016)
Open Access PDF DOI

Whereas all mammals have one glutamate dehydrogenase gene (GLUD1), humans and apes carry an additional gene (GLUD2), which encodes an enzyme with distinct biochemical properties. We inserted a bacterial artificial chromosome containing the human GLUD2 gene into mice and analyzed the resulting changes in the transcriptome and metabolome during postnatal brain development. Effects were most pronounced early postnatally, and predominantly genes involved in neuronal development were affected. Remarkably, the effects in the transgenic mice partially parallel the transcriptome and metabolome differences seen between humans and macaques analyzed. Notably, the introduction of GLUD2 did not affect glutamate levels in mice, consistent with observations in the primates. Instead, the metabolic effects of GLUD2 center on the tricarboxylic acid cycle, suggesting that GLUD2 affects carbon flux during early brain development, possibly supporting lipid biosynthesis.

Berta Terré, Gabriele Piergiovanni, Sandra Segura-Bayona, Gabriel Gil-Gómez, Sameh A Youssef, Camille Stephan-Otto Attolini, Michaela Wilsch-Bräuninger, Carole Jung, Ana M Rojas, Marko Marjanović, Philip A Knobel, Lluís Palenzuela, Teresa López-Rovira, Stephen Forrow, Wieland B. Huttner, Miguel A Valverde, Alain de Bruin, Vincenzo Costanzo, Travis H Stracker
GEMC1 is a critical regulator of multiciliated cell differentiation.
EMBO J, 35(9) 942-960 (2016)
DOI

The generation of multiciliated cells (MCCs) is required for the proper function of many tissues, including the respiratory tract, brain, and germline. Defects in MCC development have been demonstrated to cause a subclass of mucociliary clearance disorders termed reduced generation of multiple motile cilia (RGMC). To date, only two genes, Multicilin (MCIDAS) and cyclin O (CCNO) have been identified in this disorder in humans. Here, we describe mice lacking GEMC1 (GMNC), a protein with a similar domain organization as Multicilin that has been implicated in DNA replication control. We have found that GEMC1-deficient mice are growth impaired, develop hydrocephaly with a high penetrance, and are infertile, due to defects in the formation of MCCs in the brain, respiratory tract, and germline. Our data demonstrate that GEMC1 is a critical regulator of MCC differentiation and a candidate gene for human RGMC or related disorders.

Fernando Carrillo Oesterreich, Lydia Herzel, Korinna Straube, Katja Hujer, Jonathon Howard, Karla M. Neugebauer
Splicing of Nascent RNA Coincides with Intron Exit from RNA Polymerase II.
Cell, 165(2) 372-381 (2016)
DOI

Protein-coding genes in eukaryotes are transcribed by RNA polymerase II (Pol II) and introns are removed from pre-mRNA by the spliceosome. Understanding the time lag between Pol II progression and splicing could provide mechanistic insights into the regulation of gene expression. Here, we present two single-molecule nascent RNA sequencing methods that directly determine the progress of splicing catalysis as a function of Pol II position. Endogenous genes were analyzed on a global scale in budding yeast. We show that splicing is 50% complete when Pol II is only 45 nt downstream of introns, with the first spliced products observed as introns emerge from Pol II. Perturbations that slow the rate of spliceosome assembly or speed up the rate of transcription caused splicing delays, showing that regulation of both processes determines in vivo splicing profiles. We propose that matched rates streamline the gene expression pathway, while allowing regulation through kinetic competition.

Hao Yin, Roman L Bogorad, Carmen Barnes, Stephen Walsh, Iris Zhuang, Hidenori Nonaka, Vera M Ruda, Satya Kuchimanchi, Lubomir Nechev, Akin Akinc, Wen Xue, Marino Zerial, Robert Langer, Daniel G Anderson, Victor Koteliansky
RNAi-nanoparticulate manipulation of gene expression as a new functional genomics tool in the liver.
J Hepatol, 64(4) 899-907 (2016)
PDF DOI

The Hippo pathway controls organ size through a negative regulation of the transcription co-activator Yap1. The overexpression of hyperactive mutant Yap1 or deletion of key components in the Hippo pathway leads to increased organ size in different species. Analysis of interactions of this pathway with other cellular signals corroborating organ size control is limited in part due to the difficulties associated with development of rodent models.

Michal Levin, Leon Anavy, Alison G Cole, Eitan Winter, Natalia Mostov, Sally Khair, Naftalie Senderovich, Ekaterina Kovalev, David H Silver, Martin Feder, Selene L Fernandez-Valverde, Nagayasu Nakanishi, David Simmons, Oleg Simakov, Tomas Larsson, Shang-Yun Liu, Ayelet Jerafi-Vider, Karina Yaniv, Joseph F Ryan, Mark Q Martindale, Jochen Rink, Detlev Arendt, Sandie M Degnan, Bernard M Degnan, Tamar Hashimshony, Itai Yanai
The mid-developmental transition and the evolution of animal body plans.
Nature, 531(7596) 637-641 (2016)
DOI

Animals are grouped into ~35 'phyla' based upon the notion of distinct body plans. Morphological and molecular analyses have revealed that a stage in the middle of development--known as the phylotypic period--is conserved among species within some phyla. Although these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals. Here we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that in all ten species, development comprises the coupling of early and late phases of conserved gene expression. These phases are linked by a divergent 'mid-developmental transition' that uses species-specific suites of signalling pathways and transcription factors. This mid-developmental transition overlaps with the phylotypic period that has been defined previously for three of the ten phyla, suggesting that transcriptional circuits and signalling mechanisms active during this transition are crucial for defining the phyletic body plan and that the mid-developmental transition may be used to define phylotypic periods in other phyla. Placing these observations alongside the reported conservation of mid-development within phyla, we propose that a phylum may be defined as a collection of species whose gene expression at the mid-developmental transition is both highly conserved among them, yet divergent relative to other species.

Nereo Kalebic, Elena Taverna, Stefania Tavano, Fong Kuan Wong, Dana Suchold, Sylke Winkler, Wieland B. Huttner, Mihail Sarov
CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo.
EMBO Rep, 17(3) 338-348 (2016)
Open Access PDF DOI

We have applied the CRISPR/Cas9 system in vivo to disrupt gene expression in neural stem cells in the developing mammalian brain. Two days after in utero electroporation of a single plasmid encoding Cas9 and an appropriate guide RNA (gRNA) into the embryonic neocortex of Tis21::GFP knock-in mice, expression of GFP, which occurs specifically in neural stem cells committed to neurogenesis, was found to be nearly completely (≈90%) abolished in the progeny of the targeted cells. Importantly, upon in utero electroporation directly of recombinant Cas9/gRNA complex, near-maximal efficiency of disruption of GFP expression was achieved already after 24 h. Furthermore, by using microinjection of the Cas9 protein/gRNA complex into neural stem cells in organotypic slice culture, we obtained disruption of GFP expression within a single cell cycle. Finally, we used either Cas9 plasmid in utero electroporation or Cas9 protein complex microinjection to disrupt the expression of Eomes/Tbr2, a gene fundamental for neocortical neurogenesis. This resulted in a reduction in basal progenitors and an increase in neuronal differentiation. Thus, the present in vivo application of the CRISPR/Cas9 system in neural stem cells provides a rapid, efficient and enduring disruption of expression of specific genes to dissect their role in mammalian brain development.

Ibrahim Ömer Çiçek, Samir Karaca, Marko Brankatschk, Suzanne Eaton, Henning Urlaub, Halyna R Shcherbata
Hedgehog Signaling Strength Is Orchestrated by the mir-310 Cluster of MicroRNAs in Response to Diet.
Genetics, 202(3) 1167-1183 (2016)
DOI

Since the discovery of microRNAs (miRNAs) only two decades ago, they have emerged as an essential component of the gene regulatory machinery. miRNAs have seemingly paradoxical features: a single miRNA is able to simultaneously target hundreds of genes, while its presence is mostly dispensable for animal viability under normal conditions. It is known that miRNAs act as stress response factors; however, it remains challenging to determine their relevant targets and the conditions under which they function. To address this challenge, we propose a new workflow for miRNA function analysis, by which we found that the evolutionarily young miRNA family, the mir-310s (mir-310/mir-311/mir-312/mir-313), are important regulators of Drosophila metabolic status. mir-310s-deficient animals have an abnormal diet-dependent expression profile for numerous diet-sensitive components, accumulate fats, and show various physiological defects. We found that the mir-310s simultaneously repress the production of several regulatory factors (Rab23, DHR96, and Ttk) of the evolutionarily conserved Hedgehog (Hh) pathway to sharpen dietary response. As the mir-310s expression is highly dynamic and nutrition sensitive, this signal relay model helps to explain the molecular mechanism governing quick and robust Hh signaling responses to nutritional changes. Additionally, we discovered a new component of the Hh signaling pathway in Drosophila, Rab23, which cell autonomously regulates Hh ligand trafficking in the germline stem cell niche. How organisms adjust to dietary fluctuations to sustain healthy homeostasis is an intriguing research topic. These data are the first to report that miRNAs can act as executives that transduce nutritional signals to an essential signaling pathway. This suggests miRNAs as plausible therapeutic agents that can be used in combination with low calorie and cholesterol diets to manage quick and precise tissue-specific responses to nutritional changes.

L Carine Stapel, Benoit Lombardot, Coleman Broaddus, Dagmar Kainmueller, Florian Jug, Eugene W Myers, Nadine Vastenhouw
Automated detection and quantification of single RNAs at cellular resolution in zebrafish embryos.
Development, 143(3) 540-546 (2016)
Open Access PDF DOI

Analysis of differential gene expression is crucial for the study of cell fate and behavior during embryonic development. However, automated methods for the sensitive detection and quantification of RNAs at cellular resolution in embryos are lacking. With the advent of single-molecule fluorescence in situ hybridization (smFISH), gene expression can be analyzed at single-molecule resolution. However, the limited availability of protocols for smFISH in embryos and the lack of efficient image analysis pipelines have hampered quantification at the (sub)cellular level in complex samples such as tissues and embryos. Here, we present a protocol for smFISH on zebrafish embryo sections in combination with an image analysis pipeline for automated transcript detection and cell segmentation. We use this strategy to quantify gene expression differences between different cell types and identify differences in subcellular transcript localization between genes. The combination of our smFISH protocol and custom-made, freely available, analysis pipeline will enable researchers to fully exploit the benefits of quantitative transcript analysis at cellular and subcellular resolution in tissues and embryos.

Holger Brandl^*, HongKee Moon^*, Miquel Vila-Farré, Shang-Yun Liu, Ian Henry^#, Jochen Rink^#
PlanMine - a mineable resource of planarian biology and biodiversity.
Nucleic Acids Res, 44 Art. No. D764–D773 (2016)
Open Access PDF DOI

Planarian flatworms are in the midst of a renaissance as a model system for regeneration and stem cells. Besides two well-studied model species, hundreds of species exist worldwide that present a fascinating diversity of regenerative abilities, tissue turnover rates, reproductive strategies and other life history traits. PlanMine (http://planmine.mpi-cbg.de/) aims to accomplish two primary missions: First, to provide an easily accessible platform for sharing, comparing and value-added mining of planarian sequence data. Second, to catalyze the comparative analysis of the phenotypic diversity amongst planarian species. Currently, PlanMine houses transcriptomes independently assembled by our lab and community contributors. Detailed assembly/annotation statistics, a custom-developed BLAST viewer and easy export options enable comparisons at the contig and assembly level. Consistent annotation of all transcriptomes by an automated pipeline, the integration of published gene expression information and inter-relational query tools provide opportunities for mining planarian gene sequences and functions. For inter-species comparisons, we include transcriptomes of, so far, six planarian species, along with images, expert-curated information on their biology and pre-calculated cross-species sequence homologies. PlanMine is based on the popular InterMine system in order to make the rich biology of planarians accessible to the general life sciences research community.

Jaime Huerta-Cepas, Damian Szklarczyk, Kristoffer Forslund, Helen Cook, Davide Heller, Mathias C Walter, Thomas Rattei, Daniel R Mende, Shinichi Sunagawa, Michael Kuhn, Lars Juhl Jensen, Christian von Mering, Peer Bork
eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences.
Nucleic Acids Res, 44(D1) 286-293 (2016)
Open Access DOI

eggNOG is a public resource that provides Orthologous Groups (OGs) of proteins at different taxonomic levels, each with integrated and summarized functional annotations. Developments since the latest public release include changes to the algorithm for creating OGs across taxonomic levels, making nested groups hierarchically consistent. This allows for a better propagation of functional terms across nested OGs and led to the novel annotation of 95 890 previously uncharacterized OGs, increasing overall annotation coverage from 67% to 72%. The functional annotations of OGs have been expanded to also provide Gene Ontology terms, KEGG pathways and SMART/Pfam domains for each group. Moreover, eggNOG now provides pairwise orthology relationships within OGs based on analysis of phylogenetic trees. We have also incorporated a framework for quickly mapping novel sequences to OGs based on precomputed HMM profiles. Finally, eggNOG version 4.5 incorporates a novel data set spanning 2605 viral OGs, covering 5228 proteins from 352 viral proteomes. All data are accessible for bulk downloading, as a web-service, and through a completely redesigned web interface. The new access points provide faster searches and a number of new browsing and visualization capabilities, facilitating the needs of both experts and less experienced users. eggNOG v4.5 is available at http://eggnog.embl.de.

Sandra Milena Bernal-Ulloa, Julia Heinzmann, Doris Herrmann, Klaus-Gerd Hadeler, Patrick Aldag, Sylke Winkler, Dorit Pache, Ulrich Baulain, Andrea Lucas-Hahn, Heiner Niemann
Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle.
PLoS ONE, 11(2) Art. No. e0150264 (2016)
Open Access PDF DOI

High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24h IVM/control), cAMP30 (2h pre-IVM (forskolin-IBMX), 30h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency.

Nicole Bieberstein, Eva Kozáková, Martina Huranová, Prasoon K Thakur, Zuzana Krchňáková, Michaela Krausová, Fernando Carrillo Oesterreich, David Staněk
TALE-directed local modulation of H3K9 methylation shapes exon recognition.
Sci Rep, 6 Art. No. 29961 (2016)
Open Access PDF DOI

In search for the function of local chromatin environment on pre-mRNA processing we established a new tool, which allows for the modification of chromatin using a targeted approach. Using Transcription Activator-Like Effector domains fused to histone modifying enzymes (TALE-HME), we show locally restricted alteration of histone methylation modulates the splicing of target exons. We provide evidence that a local increase in H3K9 di- and trimethylation promotes inclusion of the target alternative exon, while demethylation by JMJD2D leads to exon skipping. We further demonstrate that H3K9me3 is localized on internal exons genome-wide suggesting a general role in splicing. Consistently, targeting of the H3K9 demethylase to a weak constitutive exon reduced co-transcriptional splicing. Together our data show H3K9 methylation within the gene body is a factor influencing recognition of both constitutive and alternative exons.

Bo-Kai Liao, David J. Jörg, Andrew C. Oates
Faster embryonic segmentation through elevated Delta-Notch signalling.
Nat Commun, 7 Art. No. 11861 (2016)
Open Access PDF DOI

An important step in understanding biological rhythms is the control of period. A multicellular, rhythmic patterning system termed the segmentation clock is thought to govern the sequential production of the vertebrate embryo's body segments, the somites. Several genetic loss-of-function conditions, including the Delta-Notch intercellular signalling mutants, result in slower segmentation. Here, we generate DeltaD transgenic zebrafish lines with a range of copy numbers and correspondingly increased signalling levels, and observe faster segmentation. The highest-expressing line shows an altered oscillating gene expression wave pattern and shortened segmentation period, producing embryos with more, shorter body segments. Our results reveal surprising differences in how Notch signalling strength is quantitatively interpreted in different organ systems, and suggest a role for intercellular communication in regulating the output period of the segmentation clock by altering its spatial pattern.

Alexis Webb, Iván M Lengyel, David J. Jörg, Guillaume Valentin, Frank Jülicher, Luis G. Morelli, Andrew C. Oates
Persistence, period and precision of autonomous cellular oscillators from the zebrafish segmentation clock.
Elife, 5 Art. No. e08438 (2016)
Open Access PDF DOI

In vertebrate development, the sequential and rhythmic segmentation of the body axis is regulated by a "segmentation clock". This clock is comprised of a population of coordinated oscillating cells that together produce rhythmic gene expression patterns in the embryo. Whether individual cells autonomously maintain oscillations, or whether oscillations depend on signals from neighboring cells is unknown. Using a transgenic zebrafish reporter line for the cyclic transcription factor Her1, we recorded single tailbud cells in vitro. We demonstrate that individual cells can behave as autonomous cellular oscillators. We described the observed variability in cell behavior using a theory of generic oscillators with correlated noise. Single cells have longer periods and lower precision than the tissue, highlighting the role of collective processes in the segmentation clock. Our work reveals a population of cells from the zebrafish segmentation clock that behave as self-sustained, autonomous oscillators with distinctive noisy dynamics.

Artur Yakimovich, Yauhen Yakimovich, Michael Schmid, Jason Mercer, Ivo F. Sbalzarini, Urs F Greber
Infectio: a Generic Framework for Computational Simulation of Virus Transmission between Cells.
mSphere, 1(1) Art. No. e00078-15 (2016)
Open Access PDF DOI

Viruses spread between cells, tissues, and organisms by cell-free and cell-cell mechanisms, depending on the cell type, the nature of the virus, or the phase of the infection cycle. The mode of viral transmission has a large impact on disease development, the outcome of antiviral therapies or the efficacy of gene therapy protocols. The transmission mode of viruses can be addressed in tissue culture systems using live-cell imaging. Yet even in relatively simple cell cultures, the mechanisms of viral transmission are difficult to distinguish. Here we present a cross-platform software framework called "Infectio," which is capable of simulating transmission phenotypes in tissue culture of virtually any virus. Infectio can estimate interdependent biological parameters, for example for vaccinia virus infection, and differentiate between cell-cell and cell-free virus spreading. Infectio assists in elucidating virus transmission mechanisms, a feature useful for designing strategies of perturbing or enhancing viral transmission. The complexity of the Infectio software is low compared to that of other software commonly used to quantitate features of cell biological images, which yields stable and relatively error-free output from Infectio. The software is open source (GPLv3 license), and operates on the major platforms (Windows, Mac, and Linux). The complete source code can be downloaded from http://infectio.github.io/index.html. IMPORTANCE Infectio presents a generalized platform to analyze virus infection spread between cells. It allows the simulation of plaque phenotypes from image-based assays. Viral plaques are the result of virus spreading from primary infected cells to neighboring cells. This is a complex process and involves neighborhood effects at cell-cell contact sites or fluid dynamics in the extracellular medium. Infectio differentiates between two major modes of virus transmission between cells, allowing in silico testing of hypotheses about spreading mechanisms of any virus which can be grown in cell cultures, based on experimentally measured parameters, such as infection intensity or cell killing. The results of these tests can be compared with experimental data and allow interpretations with regard to biophysical mechanisms. Infectio also facilitates characterizations of the mode of action of therapeutic agents, such as oncolytic viruses or other infectious or cytotoxic agents.

J Gray Camp, Farhath Badsha, Marta Florio, Sabina Kanton, Tobias Gerber, Michaela Wilsch-Bräuninger, Eric Lewitus, Alex Sykes, Wulf Hevers, Madeline Lancaster, Jürgen A. Knoblich, Robert Lachmann, Svante Pääbo, Wieland B. Huttner, Barbara Treutlein
Human cerebral organoids recapitulate gene expression programs of fetal neocortex development.
Proc Natl Acad Sci U.S.A., 112(51) 15672-15677 (2015)
Open Access PDF DOI

Cerebral organoids-3D cultures of human cerebral tissue derived from pluripotent stem cells-have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.

Kathrin Reichwald, Andreas Petzold, Philipp Koch, Bryan R Downie, Nils Hartmann, Stefan Pietsch, Mario Baumgart, Domitille Chalopin, Marius Felder, Martin Bens, Arne Sahm, Karol Szafranski, Stefan Taudien, Marco Groth, Ivan Arisi, Anja Weise, Samarth S Bhatt, Virag Sharma, Johann M Kraus, Florian Schmid, Steffen Priebe, Thomas Liehr, Matthias Görlach, Manuel E Than, Michael Hiller, Hans A Kestler, Jean-Nicolas Volff, Manfred Schartl, Alessandro Cellerino, Christoph Englert, Matthias Platzer
Insights into Sex Chromosome Evolution and Aging from the Genome of a Short-Lived Fish.
Cell, 163(6) 1527-1538 (2015)
PDF DOI

The killifish Nothobranchius furzeri is the shortest-lived vertebrate that can be bred in the laboratory. Its rapid growth, early sexual maturation, fast aging, and arrested embryonic development (diapause) make it an attractive model organism in biomedical research. Here, we report a draft sequence of its genome that allowed us to uncover an intra-species Y chromosome polymorphism representing-in real time-different stages of sex chromosome formation that display features of early mammalian XY evolution "in action." Our data suggest that gdf6Y, encoding a TGF-β family growth factor, is the master sex-determining gene in N. furzeri. Moreover, we observed genomic clustering of aging-related genes, identified genes under positive selection, and revealed significant similarities of gene expression profiles between diapause and aging, particularly for genes controlling cell cycle and translation. The annotated genome sequence is provided as an online resource (http://www.nothobranchius.info/NFINgb).

Marta Florio
Transcriptional Signatures of Neocortical Expansion - Evolutionary Role of the Human-Specific Gene ARHGAP11B
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2015)

Gesa I Albert, Christoph Schell, Karin M Kirschner, Sebastian Schäfer, Ronald Naumann, Alexandra Müller, Oliver Kretz, Benno Kuropka, Mathias Girbig, Norbert Hübner, Eberhard Krause, Holger Scholz, Tobias B Huber, Klaus-Peter Knobeloch, Christian Freund
The GYF domain protein CD2BP2 is critical for embryogenesis and podocyte function.
J Mol Cell Biol, 7(5) 402-414 (2015)
DOI

Scaffolding proteins play pivotal roles in the assembly of macromolecular machines such as the spliceosome. The adaptor protein CD2BP2, originally identified as a binding partner of the adhesion molecule CD2, is a pre-spliceosomal assembly factor that utilizes its glycine-tyrosine-phenylalanine (GYF) domain to co-localize with spliceosomal proteins. So far, its function in vertebrates is unknown. Using conditional gene targeting in mice, we show that CD2BP2 is crucial for embryogenesis, leading to growth retardation, defects in vascularization, and premature death at embryonic day 10.5 when absent. Ablation of the protein in bone marrow-derived macrophages indicates that CD2BP2 is involved in the alternative splicing of mRNA transcripts from diverse origins. At the molecular level, we identified the phosphatase PP1 to be recruited to the spliceosome via the N-terminus of CD2BP2. Given the strong expression of CD2BP2 in podocytes of the kidney, we use selective depletion of CD2BP2, in combination with next-generation sequencing, to monitor changes in exon usage of genes critical for podocyte functions, including VEGF and actin regulators. CD2BP2-depleted podocytes display foot process effacement, and cause proteinuria and ultimately lethal kidney failure in mice. Collectively, our study defines CD2BP2 as a non-redundant splicing factor essential for embryonic development and podocyte integrity.

Jerome Gilleron, Prasath Paramasivam, Anja Zeigerer, William Querbes, Giovanni Marsico, Cordula Andree, Sarah Seifert, Pablo Amaya, Martin Stöter, Victor Koteliansky, Herbert Waldmann, Kevin Fitzgerald, Yannis Kalaidzidis, Akin Akinc, Martin A Maier, Muthiah Manoharan, Marc Bickle, Marino Zerial
Identification of siRNA delivery enhancers by a chemical library screen.
Nucleic Acids Res, 43(16) 7984-8001 (2015)
Open Access PDF DOI

Most delivery systems for small interfering RNA therapeutics depend on endocytosis and release from endo-lysosomal compartments. One approach to improve delivery is to identify small molecules enhancing these steps. It is unclear to what extent such enhancers can be universally applied to different delivery systems and cell types. Here, we performed a compound library screen on two well-established siRNA delivery systems, lipid nanoparticles and cholesterol conjugated-siRNAs. We identified fifty-one enhancers improving gene silencing 2-5 fold. Strikingly, most enhancers displayed specificity for one delivery system only. By a combination of quantitative fluorescence and electron microscopy we found that the enhancers substantially differed in their mechanism of action, increasing either endocytic uptake or release of siRNAs from endosomes. Furthermore, they acted either on the delivery system itself or the cell, by modulating the endocytic system via distinct mechanisms. Interestingly, several compounds displayed activity on different cell types. As proof of principle, we showed that one compound enhanced siRNA delivery in primary endothelial cells in vitro and in the endocardium in the mouse heart. This study suggests that a pharmacological approach can improve the delivery of siRNAs in a system-specific fashion, by exploiting distinct mechanisms and acting upon multiple cell types.

Meritxell Huch, Bon-Kyoung Koo
Modeling mouse and human development using organoid cultures.
Development, 142(18) 3113-3125 (2015)
DOI

In vitro three-dimensional (3D) cultures are emerging as novel systems with which to study tissue development, organogenesis and stem cell behavior ex vivo. When grown in a 3D environment, embryonic stem cells (ESCs) self-organize into organoids and acquire the right tissue patterning to develop into several endoderm- and ectoderm-derived tissues, mimicking their in vivo counterparts. Tissue-resident adult stem cells (AdSCs) also form organoids when grown in 3D and can be propagated in vitro for long periods of time. In this Review, we discuss recent advances in the generation of pluripotent stem cell- and AdSC-derived organoids, highlighting their potential for enhancing our understanding of human development. We will also explore how this new culture system allows disease modeling and gene repair for a personalized regenerative medicine approach.

David J. Jörg, Luis G. Morelli, Daniele Soroldoni, Andrew C. Oates, Frank Jülicher
Continuum theory of gene expression waves during vertebrate segmentation
New J Phys, 17 Art. No. 093042 (2015)
Open Access PDF DOI

Jan Böttger, Katrin Arnold, Carlo Thiel, Christiane Rennert, Susanne Aleithe, Ute Hofmann, Sebastian Vlaic, Susanne Sales, Andrej Shevchenko, Madlen Matz-Soja
RNAi in murine hepatocytes: the agony of choice-a study of the influence of lipid-based transfection reagents on hepatocyte metabolism.
Arch Toxicol, 89(9) 1579-1588 (2015)
DOI

Primary hepatocyte cell cultures are widely used for studying hepatic diseases with alterations in hepatic glucose and lipid metabolism, such as diabetes and non-alcoholic fatty liver disease. Therefore, small interfering RNAs (siRNAs) provide a potent and specific tool to elucidate the signaling pathways and gene functions involved in these pathologies. Although RNA interference (RNAi) in vitro is frequently used in these investigations, the metabolic alterations elucidated by different siRNA delivery strategies have hardly been investigated in transfected hepatocytes. To elucidate the influence of the most commonly used lipid-based transfection reagents on cultured primary hepatocytes, we studied the cytotoxic effects and transfection efficiencies of INTERFERin(®), Lipofectamine(®)RNAiMAX, and HiPerFect(®). All of these transfection agents displayed low cytotoxicity (5.6-9.0 ± 1.3-3.4 %), normal cell viability, and high transfection efficiency (fold change 0.08-0.13 ± 0.03-0.05), and they also favored the satisfactory down-regulation of target gene expression. However, when effects on the metabolome and lipidome were studied, considerable differences were observed among the transfection reagents. Cellular triacylglycerides levels were either up- or down-regulated [maximum fold change: INTERFERin(®) (48 h) 2.55 ± 0.34, HiPerFect(®) (24 h) 0.79 ± 0.08, Lipofectamine(®)RNAiMAX (48 h) 1.48 ± 0.21], and mRNA levels of genes associated with lipid metabolism were differentially affected. Likewise, metabolic functions such as amino acid utilization from were perturbed (alanine, arginine, glycine, ornithine, and pyruvate). In conclusion, these findings demonstrate that the choice of non-viral siRNA delivery agent is critical in hepatocytes. This should be remembered, especially if RNA silencing is used for studying hepatic lipid homeostasis and its regulation.

Lydia Herzel, Karla M. Neugebauer
Quantification of co-transcriptional splicing from RNA-Seq data.
Methods, 85 36-43 (2015)
DOI

During gene expression, protein-coding transcripts are shaped by multiple processing events: 5' end capping, pre-mRNA splicing, RNA editing, and 3' end cleavage and polyadenylation. These events are required to produce mature mRNA, which can be subsequently translated. Nearly all of these RNA processing steps occur during transcription, while the nascent RNA is still attached to the DNA template by RNA polymerase II (i.e. co-transcriptionally). Polyadenylation occurs after 3' end cleavage or post-transcriptionally. Pre-mRNA splicing - the removal of introns and ligation of exons - can be initiated and concluded co-transcriptionally, although this is not strictly required. Recently, a number of studies using global methods have shown that the majority of splicing is co-transcriptional, yet not all published studies agree in their conclusions. Short read sequencing of RNA (RNA-Seq) is the prevailing approach to measuring splicing levels in nascent RNA, mRNA or total RNA. Here, we compare four different strategies for analyzing and quantifying co-transcriptional splicing. To do so, we reanalyze two nascent RNA-Seq datasets of the same species, but different cell type and RNA isolation procedure. Average co-transcriptional splicing values calculated on a per intron basis are similar, independent of the strategy used. We emphasize the technical requirements for identifying co-transcriptional splicing events with high confidence, e.g. how to calculate co-transcriptional splicing from nascent RNA- versus mRNA-Seq data, the number of biological replicates needed, depletion of polyA+RNA, and appropriate normalization. Finally, we present guidelines for planning a nascent RNA-Seq experiment.

Liat Rockah-Shmuel, Ágnes Tóth-Petróczy, Dan S Tawfik
Systematic Mapping of Protein Mutational Space by Prolonged Drift Reveals the Deleterious Effects of Seemingly Neutral Mutations.
PLoS Comput Biol, 11(8) Art. No. e1004421 (2015)
Open Access DOI

Systematic mappings of the effects of protein mutations are becoming increasingly popular. Unexpectedly, these experiments often find that proteins are tolerant to most amino acid substitutions, including substitutions in positions that are highly conserved in nature. To obtain a more realistic distribution of the effects of protein mutations, we applied a laboratory drift comprising 17 rounds of random mutagenesis and selection of M.HaeIII, a DNA methyltransferase. During this drift, multiple mutations gradually accumulated. Deep sequencing of the drifted gene ensembles allowed determination of the relative effects of all possible single nucleotide mutations. Despite being averaged across many different genetic backgrounds, about 67% of all nonsynonymous, missense mutations were evidently deleterious, and an additional 16% were likely to be deleterious. In the early generations, the frequency of most deleterious mutations remained high. However, by the 17th generation, their frequency was consistently reduced, and those remaining were accepted alongside compensatory mutations. The tolerance to mutations measured in this laboratory drift correlated with sequence exchanges seen in M.HaeIII's natural orthologs. The biophysical constraints dictating purging in nature and in this laboratory drift also seemed to overlap. Our experiment therefore provides an improved method for measuring the effects of protein mutations that more closely replicates the natural evolutionary forces, and thereby a more realistic view of the mutational space of proteins.

Andrea Rossi^*, Zacharias Kontarakis^*, Claudia Gerri, Hendrik Nolte, Soraya Hölper, Marcus Krüger, Didier Y.R. Stainier
Genetic compensation induced by deleterious mutations but not gene knockdowns.
Nature, 524(7564) 230-233 (2015)
DOI

Cells sense their environment and adapt to it by fine-tuning their transcriptome. Wired into this network of gene expression control are mechanisms to compensate for gene dosage. The increasing use of reverse genetics in zebrafish, and other model systems, has revealed profound differences between the phenotypes caused by genetic mutations and those caused by gene knockdowns at many loci, an observation previously reported in mouse and Arabidopsis. To identify the reasons underlying the phenotypic differences between mutants and knockdowns, we generated mutations in zebrafish egfl7, an endothelial extracellular matrix gene of therapeutic interest, as well as in vegfaa. Here we show that egfl7 mutants do not show any obvious phenotypes while animals injected with egfl7 morpholino (morphants) exhibit severe vascular defects. We further observe that egfl7 mutants are less sensitive than their wild-type siblings to Egfl7 knockdown, arguing against residual protein function in the mutants or significant off-target effects of the morpholinos when used at a moderate dose. Comparing egfl7 mutant and morphant proteomes and transcriptomes, we identify a set of proteins and genes that are upregulated in mutants but not in morphants. Among them are extracellular matrix genes that can rescue egfl7 morphants, indicating that they could be compensating for the loss of Egfl7 function in the phenotypically wild-type egfl7 mutants. Moreover, egfl7 CRISPR interference, which obstructs transcript elongation and causes severe vascular defects, does not cause the upregulation of these genes. Similarly, vegfaa mutants but not morphants show an upregulation of vegfab. Taken together, these data reveal the activation of a compensatory network to buffer against deleterious mutations, which was not observed after translational or transcriptional knockdown.

T-Y Dora Tang, Dirk van Swaay, Andrew deMello, J L Ross Anderson, Stephen Mann
In vitro gene expression within membrane-free coacervate protocells.
Chem Commun (Camb), 51(57) 11429-11432 (2015)
DOI

Cell-free gene expression of a fluorescent protein (mCherry) is demonstrated within the molecularly crowded matrix of a polysaccharide/polypeptide coacervate.

Marco Nousch, Christian R. Eckmann
Translational activation maintains germline tissue homeostasis during adulthood.
Worm, 4(3) Art. No. e1042644 (2015)
DOI

Adult tissue maintenance is achieved through a tightly controlled equilibrium of 2 opposing cell fates: stem cell proliferation and differentiation. In recent years, the germ line emerged as a powerful in vivo model tissue to investigate the underlying gene expression mechanisms regulating this balance. Studies in numerous organisms highlighted the prevalence of post-transcriptional mRNA regulation, which relies on RNA-targeting factors that influence mRNA fates (e.g. decay or translational efficiency). Conserved translational repressors were identified that build negative feedback loops to ensure one or the other cell fate. However, to facilitate a fast and efficient transition between 2 opposing cell fates, translational repression per se appears not to be sufficient, suggesting the involvement of additional modes of gene expression regulation. Cytoplasmic poly(A) polymerases (cytoPAPs) represent a unique class of post-transcriptional mRNA regulators that modify mRNA 3' ends and positively influence cytoplasmic mRNA fates. We recently discovered that the 2 main cytoPAPs, GLD-2 and GLD-4, use distinct mechanisms to promote gene expression and that cytoPAP-mediated mRNA activation is important for regulating the size of the proliferative germ cell pool in the adult Caenorhabditis elegans gonad. Here, we comment on the different mechanisms of the 2 cytoPAPs as translational activators in germ cell development and focus on their biological roles in maintaining the balance between germline stem cell proliferation and differentiation in the Caenorhabditis elegans gonad.

Anja Zeigerer, Roman L Bogorad, Kirti Sharma, Jerome Gilleron, Sarah Seifert, Susanne Sales, Nikolaus Berndt, Sascha Bulik, Giovanni Marsico, Rochelle C J D'Souza, Naharajan Lakshmanaperumal, Kesavan Meganathan, Karthick Natarajan, Agapios Sachinidis, Andreas Dahl, Hermann-Georg Holzhütter, Andrej Shevchenko, Matthias Mann, Victor Koteliansky, Marino Zerial
Regulation of Liver Metabolism by the Endosomal GTPase Rab5.
Cell Rep, 11(6) 884-892 (2015)
Open Access DOI

The liver maintains glucose and lipid homeostasis by adapting its metabolic activity to the energy needs of the organism. Communication between hepatocytes and extracellular environment via endocytosis is key to such homeostasis. Here, we addressed the question of whether endosomes are required for gluconeogenic gene expression. We took advantage of the loss of endosomes in the mouse liver upon Rab5 silencing. Strikingly, we found hepatomegaly and severe metabolic defects such as hypoglycemia, hypercholesterolemia, hyperlipidemia, and glycogen accumulation that phenocopied those found in von Gierke's disease, a glucose-6-phosphatase (G6Pase) deficiency. G6Pase deficiency alone can account for the reduction in hepatic glucose output and glycogen accumulation as determined by mathematical modeling. Interestingly, we uncovered functional alterations in the transcription factors, which regulate G6Pase expression. Our data highlight a requirement of Rab5 and the endosomal system for the regulation of gluconeogenic gene expression that has important implications for metabolic diseases.

Ana Tomasovic, Nina Kurrle, Duran Sürün, Juliana Heidler, Koraljka Husnjak, Ina Poser, Frank Schnütgen, Susan Scheibe, Michael Seimetz, Peter Jaksch, Anthony Hyman, Norbert Weissmann, Harald von Melchner
Sestrin 2 Protein Regulates Platelet-derived Growth Factor Receptor β (Pdgfrβ) Expression by Modulating Proteasomal and Nrf2 Transcription Factor Functions.
J Biol Chem, 290(15) 9738-9752 (2015)
DOI

We recently identified the antioxidant protein Sestrin 2 (Sesn2) as a suppressor of platelet-derived growth factor receptor β (Pdgfrβ) signaling and Pdgfrβ signaling as an inducer of lung regeneration and injury repair. Here, we identified Sesn2 and the antioxidant gene inducer nuclear factor erythroid 2-related factor 2 (Nrf2) as positive regulators of proteasomal function. Inactivation of Sesn2 or Nrf2 induced reactive oxygen species-mediated proteasomal inhibition and Pdgfrβ accumulation. Using bacterial artificial chromosome (BAC) transgenic HeLa and mouse embryonic stem cells stably expressing enhanced green fluorescent protein-tagged Sesn2 at nearly endogenous levels, we also showed that Sesn2 physically interacts with 2-Cys peroxiredoxins and Nrf2 albeit under different reductive conditions. Overall, we characterized a novel, redox-sensitive Sesn2/Pdgfrβ suppressor pathway that negatively interferes with lung regeneration and is up-regulated in the emphysematous lungs of patients with chronic obstructive pulmonary disease (COPD).

Marta Florio, Mareike Albert, Elena Taverna, Takashi Namba, Holger Brandl, Eric Lewitus, Christiane Haffner, Alex Sykes, Fong Kuan Wong, Jula Peters, E. Guhr, Sylvia Klemroth, Kay Prüfer, Janet Kelso, Ronald Naumann, Ina Nüsslein, Andreas Dahl, Robert Lachmann, Svante Pääbo, Wieland B. Huttner
Human-specific gene ARHGAP11B promotes basal progenitor amplification and neocortex expansion.
Science, 347(6229) 1465-1470 (2015)
DOI

Evolutionary expansion of the human neocortex reflects increased amplification of basal progenitors in the subventricular zone, producing more neurons during fetal corticogenesis. In this work, we analyze the transcriptomes of distinct progenitor subpopulations isolated by a cell polarity-based approach from developing mouse and human neocortex. We identify 56 genes preferentially expressed in human apical and basal radial glia that lack mouse orthologs. Among these, ARHGAP11B has the highest degree of radial glia-specific expression. ARHGAP11B arose from partial duplication of ARHGAP11A (which encodes a Rho guanosine triphosphatase-activating protein) on the human lineage after separation from the chimpanzee lineage. Expression of ARHGAP11B in embryonic mouse neocortex promotes basal progenitor generation and self-renewal and can increase cortical plate area and induce gyrification. Hence, ARHGAP11B may have contributed to evolutionary expansion of human neocortex.

Anne Eugster, Aaron Lindner, Mara Catani, Anne-Kristin Heninger, Andreas Dahl, Sylvia Klemroth, Denise Kühn, Susanne M Dietz, Marc Bickle, Anette-Gabrielle Ziegler, Enzio Bonifacio
High Diversity in the TCR Repertoire of GAD65 Autoantigen-Specific Human CD4+ T Cells.
J Immunol, 194(6) 2531-2538 (2015)
DOI

Autoreactive CD4(+) T cells are an essential feature of type 1 diabetes mellitus. We applied single-cell TCR α- and β-chain sequencing to peripheral blood GAD65-specific CD4(+) T cells, and TCR α-chain next-generation sequencing to bulk memory CD4(+) T cells to provide insight into TCR diversity in autoimmune diabetes mellitus. TCRs obtained for 1650 GAD65-specific CD4(+) T cells isolated from GAD65 proliferation assays and/or GAD65 557I tetramer staining in 6 patients and 10 islet autoantibody-positive children showed large diversity with 1003 different TCRs identified. TRAV and TRBV gene usage was broad, and the TRBV5.1 gene was most prominent within the GAD65 557I tetramer(+) cells. Limited overlap (<5%) was observed between TCRs of GAD65-proliferating and GAD65 557I tetramer(+) CD4(+) T cells. Few TCRs were repeatedly found in GAD65-specific cells at different time points from individual patients, and none was seen in more than one subject. However, single chains were often shared between patients and used in combination with different second chains. Next-generation sequencing revealed a wide frequency range (<0.00001-1.62%) of TCR α-chains corresponding to GAD65-specific T cells. The findings support minor selection of genes and TCRs for GAD65-specific T cells, but fail to provide strong support for TCR-targeted therapies.

Laurence A Lemaire, Joan Goulley, Yung Hae Kim, Solenne Carat, Patrick Jacquemin, Jacques Rougemont, Daniel B Constam, Anne Grapin-Botton
Bicaudal C1 promotes pancreatic NEUROG3+ endocrine progenitor differentiation and ductal morphogenesis.
Development, 142(5) 858-870 (2015)
DOI

In human, mutations in bicaudal C1 (BICC1), an RNA binding protein, have been identified in patients with kidney dysplasia. Deletion of Bicc1 in mouse leads to left-right asymmetry randomization and renal cysts. Here, we show that BICC1 is also expressed in both the pancreatic progenitor cells that line the ducts during development, and in the ducts after birth, but not in differentiated endocrine or acinar cells. Genetic inactivation of Bicc1 leads to ductal cell over-proliferation and cyst formation. Transcriptome comparison between WT and Bicc1 KO pancreata, before the phenotype onset, reveals that PKD2 functions downstream of BICC1 in preventing cyst formation in the pancreas. Moreover, the analysis highlights immune cell infiltration and stromal reaction developing early in the pancreas of Bicc1 knockout mice. In addition to these functions in duct morphogenesis, BICC1 regulates NEUROG3(+) endocrine progenitor production. Its deletion leads to a late but sustained endocrine progenitor decrease, resulting in a 50% reduction of endocrine cells. We show that BICC1 functions downstream of ONECUT1 in the pathway controlling both NEUROG3(+) endocrine cell production and ductal morphogenesis, and suggest a new candidate gene for syndromes associating kidney dysplasia with pancreatic disorders, including diabetes.

Patricia Heyn^#, Alex T. Kalinka, Pavel Tomancak^#, Karla M. Neugebauer
Introns and gene expression: Cellular constraints, transcriptional regulation, and evolutionary consequences.
Bioessays, 37(2) 148-154 (2015)
DOI

A gene's "expression profile" denotes the number of transcripts present relative to all other transcripts. The overall rate of transcript production is determined by transcription and RNA processing rates. While the speed of elongating RNA polymerase II has been characterized for many different genes and organisms, gene-architectural features - primarily the number and length of exons and introns - have recently emerged as important regulatory players. Several new studies indicate that rapidly cycling cells constrain gene-architecture toward short genes with a few introns, allowing efficient expression during short cell cycles. In contrast, longer genes with long introns exhibit delayed expression, which can serve as timing mechanisms for patterning processes. These findings indicate that cell cycle constraints drive the evolution of gene-architecture and shape the transcriptome of a given cell type. Furthermore, a tendency for short genes to be evolutionarily young hints at links between cellular constraints and the evolution of animal ontogeny.

Hanchuan Peng, Eugene W Myers
Constructing 5D developing gene expression patterns without live animal imaging
Biomed Eng Lett, 4(4) 338-346 (2015)
DOI

Meritxell Huch, Helmuth Gehart, Ruben van Boxtel, Karien Hamer, Francis Blokzijl, Monique Ma Verstegen, Ewa Ellis, Martien van Wenum, Sabine A Fuchs, Joep de Ligt, Marc van de Wetering, Nobuo Sasaki, Susanne J Boers, Hans Kemperman, Jeroen de Jonge, Jan N M Ijzermans, Edward E S Nieuwenhuis, Ruurdtje Hoekstra, Stephen Strom, Robert R G Vries, Luc J W van der Laan, Edwin Cuppen, Hans Clevers
Long-term culture of genome-stable bipotent stem cells from adult human liver.
Cell, 160(1-2) 299-312 (2015)
DOI

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.

Helena Jambor^#, Vineeth Surendranath, Alex T. Kalinka, Pavel Mejstrik, Stephan Saalfeld, Pavel Tomancak^#
Systematic imaging reveals features and changing localization of mRNAs in Drosophila development.
Elife, 4 Art. No. e05003 (2015)
Open Access DOI

mRNA localization is critical for eukaryotic cells and affects numerous transcripts, yet how cells regulate distribution of many mRNAs to their subcellular destinations is still unknown. We combined transcriptomics and systematic imaging to determine the tissue-specific expression and subcellular distribution of 5862 mRNAs during Drosophila oogenesis. mRNA localization is widespread in the ovary and detectable in all of its cell types-the somatic epithelial, the nurse cells, and the oocyte. Genes defined by a common RNA localization share distinct gene features and differ in expression level, 3'UTR length and sequence conservation from unlocalized mRNAs. Comparison of mRNA localizations in different contexts revealed that localization of individual mRNAs changes over time in the oocyte and between ovarian and embryonic cell types. This genome scale image-based resource (Dresden Ovary Table, DOT.

Eric Lewitus, Wieland B. Huttner
Neurodevelopmental LincRNA Microsyteny Conservation and Mammalian Brain Size Evolution.
PLoS ONE, 10(7) Art. No. e0131818 (2015)
Open Access PDF DOI

The mammalian neocortex has undergone repeated selection for increases and decreases in size and complexity, often over relatively short evolutionary time. But because probing developmental mechanisms across many species is experimentally unfeasible, it is unknown whether convergent morphologies in distantly related species are regulated by conserved developmental programs. In this work, we have taken advantage of the abundance of available mammalian genomes to find evidence of selection on genomic regions putatively regulating neurogenesis in large- versus small-brained species. Using published fetal human RNA-seq data, we show that the gene-neighborhood (i.e., microsynteny) of long intergenic non-coding RNAs (lincRNAs) implicated in cortical development is differentially conserved in large-brained species, lending support to the hypothesis that lincRNAs regulating neurogenesis are selectively lost in small-brained species. We provide evidence that this is not a phenomenon attributable to lincRNA expressed in all tissue types and is therefore likely to represent an adaptive function in the evolution of neurogenesis. A strong correlation between transcription factor-adjacency and lincRNA sequence conservation reinforces this conclusion.

Arne Schwelm, Johan Fogelqvist, Andrea Knaust, Sabine Jülke, Tua Lilja, German Bonilla-Rosso, Magnus Karlsson, Andrej Shevchenko, Vignesh Dhandapani, Su Ryun Choi, Hong Gi Kim, Ju Young Park, Yong Pyo Lim, Jutta Ludwig-Müller, Christina Dixelius
The Plasmodiophora brassicae genome reveals insights in its life cycle and ancestry of chitin synthases.
Sci Rep, 5 Art. No. 11153 (2015)
Open Access DOI

Plasmodiophora brassicae causes clubroot, a major disease of Brassica oil and vegetable crops worldwide. P. brassicae is a Plasmodiophorid, obligate biotrophic protist in the eukaryotic kingdom of Rhizaria. Here we present the 25.5 Mb genome draft of P. brassicae, developmental stage-specific transcriptomes and a transcriptome of Spongospora subterranea, the Plasmodiophorid causing powdery scab on potato. Like other biotrophic pathogens both Plasmodiophorids are reduced in metabolic pathways. Phytohormones contribute to the gall phenotypes of infected roots. We report a protein (PbGH3) that can modify auxin and jasmonic acid. Plasmodiophorids contain chitin in cell walls of the resilient resting spores. If recognized, chitin can trigger defense responses in plants. Interestingly, chitin-related enzymes of Plasmodiophorids built specific families and the carbohydrate/chitin binding (CBM18) domain is enriched in the Plasmodiophorid secretome. Plasmodiophorids chitin synthases belong to two families, which were present before the split of the eukaryotic Stramenopiles/Alveolates/Rhizaria/Plantae and Metazoa/Fungi/Amoebozoa megagroups, suggesting chitin synthesis to be an ancient feature of eukaryotes. This exemplifies the importance of genomic data from unexplored eukaryotic groups, such as the Plasmodiophorids, to decipher evolutionary relationships and gene diversification of early eukaryotes.

Christoph Zechner, Heinz Koeppl
Uncoupled analysis of stochastic reaction networks in fluctuating environments.
PLoS Comput Biol, 10(12) Art. No. e1003942 (2014)
Open Access PDF DOI

The dynamics of stochastic reaction networks within cells are inevitably modulated by factors considered extrinsic to the network such as, for instance, the fluctuations in ribosome copy numbers for a gene regulatory network. While several recent studies demonstrate the importance of accounting for such extrinsic components, the resulting models are typically hard to analyze. In this work we develop a general mathematical framework that allows to uncouple the network from its dynamic environment by incorporating only the environment's effect onto the network into a new model. More technically, we show how such fluctuating extrinsic components (e.g., chemical species) can be marginalized in order to obtain this decoupled model. We derive its corresponding process- and master equations and show how stochastic simulations can be performed. Using several case studies, we demonstrate the significance of the approach.

Spencer G Willet, Michael A Hale, Anne Grapin-Botton, Mark A Magnuson, Raymond J MacDonald, Christopher V E Wright
Dominant and context-specific control of endodermal organ allocation by Ptf1a.
Development, 141(22) 4385-4394 (2014)
DOI

The timing and gene regulatory logic of organ-fate commitment from within the posterior foregut of the mammalian endoderm is largely unexplored. Transient misexpression of a presumed pancreatic-commitment transcription factor, Ptf1a, in embryonic mouse endoderm (Ptf1a(EDD)) dramatically expanded the pancreatic gene regulatory network within the foregut. Ptf1a(EDD) temporarily suppressed Sox2 broadly over the anterior endoderm. Pancreas-proximal organ territories underwent full tissue conversion. Early-stage Ptf1a(EDD) rapidly expanded the endogenous endodermal Pdx1-positive domain and recruited other pancreas-fate-instructive genes, thereby spatially enlarging the potential for pancreatic multipotency. Early Ptf1a(EDD) converted essentially the entire glandular stomach, rostral duodenum and extrahepatic biliary system to pancreas, with formation of many endocrine cell clusters of the type found in normal islets of Langerhans. Sliding the Ptf1a(EDD) expression window through embryogenesis revealed differential temporal competencies for stomach-pancreas respecification. The response to later-stage Ptf1a(EDD) changed radically towards unipotent, acinar-restricted conversion. We provide strong evidence, beyond previous Ptf1a inactivation or misexpression experiments in frog embryos, for spatiotemporally context-dependent activity of Ptf1a as a potent gain-of-function trigger of pro-pancreatic commitment.

Jan Philipp Junker, Emily S. Noël, Victor Guryev, Kevin A Peterson, Gopi Shah, Jan Huisken, Andrew P. McMahon, Eugene Berezikov, Jeroen Bakkers, Alexander van Oudenaarden
Genome-wide RNA Tomography in the Zebrafish Embryo.
Cell, 159(3) 662-675 (2014)
DOI

Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. Although genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using in situ hybridization, for example, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. Importantly, our technique enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs.

Marco Nousch, Assa Yeroslaviz, Bianca Habermann, Christian R. Eckmann
The cytoplasmic poly(A) polymerases GLD-2 and GLD-4 promote general gene expression via distinct mechanisms.
Nucleic Acids Res, 42(18) 11622-11633 (2014)
PDF DOI

Post-transcriptional gene regulation mechanisms decide on cellular mRNA activities. Essential gatekeepers of post-transcriptional mRNA regulation are broadly conserved mRNA-modifying enzymes, such as cytoplasmic poly(A) polymerases (cytoPAPs). Although these non-canonical nucleotidyltransferases efficiently elongate mRNA poly(A) tails in artificial tethering assays, we still know little about their global impact on poly(A) metabolism and their individual molecular roles in promoting protein production in organisms. Here, we use the animal model Caenorhabditis elegans to investigate the global mechanisms of two germline-enriched cytoPAPs, GLD-2 and GLD-4, by combining polysome profiling with RNA sequencing. Our analyses suggest that GLD-2 activity mediates mRNA stability of many translationally repressed mRNAs. This correlates with a general shortening of long poly(A) tails in gld-2-compromised animals, suggesting that most if not all targets are stabilized via robust GLD-2-mediated polyadenylation. By contrast, only mild polyadenylation defects are found in gld-4-compromised animals and few mRNAs change in abundance. Interestingly, we detect a reduced number of polysomes in gld-4 mutants and GLD-4 protein co-sediments with polysomes, which together suggest that GLD-4 might stimulate or maintain translation directly. Our combined data show that distinct cytoPAPs employ different RNA-regulatory mechanisms to promote gene expression, offering new insights into translational activation of mRNAs.

Angela Simeone, Giovanni Marsico, Claudio Collinet, Thierry Galvez, Yannis Kalaidzidis, Marino Zerial, Andreas Beyer
Revealing molecular mechanisms by integrating high-dimensional functional screens with protein interaction data.
PLoS Comput Biol, 10(9) Art. No. e1003801 (2014)
DOI

Functional genomics screens using multi-parametric assays are powerful approaches for identifying genes involved in particular cellular processes. However, they suffer from problems like noise, and often provide little insight into molecular mechanisms. A bottleneck for addressing these issues is the lack of computational methods for the systematic integration of multi-parametric phenotypic datasets with molecular interactions. Here, we present Integrative Multi Profile Analysis of Cellular Traits (IMPACT). The main goal of IMPACT is to identify the most consistent phenotypic profile among interacting genes. This approach utilizes two types of external information: sets of related genes (IMPACT-sets) and network information (IMPACT-modules). Based on the notion that interacting genes are more likely to be involved in similar functions than non-interacting genes, this data is used as a prior to inform the filtering of phenotypic profiles that are similar among interacting genes. IMPACT-sets selects the most frequent profile among a set of related genes. IMPACT-modules identifies sub-networks containing genes with similar phenotype profiles. The statistical significance of these selections is subsequently quantified via permutations of the data. IMPACT (1) handles multiple profiles per gene, (2) rescues genes with weak phenotypes and (3) accounts for multiple biases e.g. caused by the network topology. Application to a genome-wide RNAi screen on endocytosis showed that IMPACT improved the recovery of known endocytosis-related genes, decreased off-target effects, and detected consistent phenotypes. Those findings were confirmed by rescreening 468 genes. Additionally we validated an unexpected influence of the IGF-receptor on EGF-endocytosis. IMPACT facilitates the selection of high-quality phenotypic profiles using different types of independent information, thereby supporting the molecular interpretation of functional screens.

Sophia Millonigg, Ryuji Minasaki, Marco Nousch, Christian R. Eckmann
GLD-4-Mediated Translational Activation Regulates the Size of the Proliferative Germ Cell Pool in the Adult C. elegans Germ Line.
PLoS Genet, 10(9) Art. No. e1004647 (2014)
DOI

To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult Caenorhabditis elegans reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A) polymerase (cytoPAP) GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that gld-4 activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with glp-1 mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of glp-1 mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant translational circuit with GLD-2 cytoPAP and the translational repressor GLD-1 to restrict proliferation. Together with previous findings, our combined data reveals two interconnected translational activation/repression circuitries of broadly conserved RNA regulators that maintain the balance between adult germ cell proliferation and differentiation.

Yusuke Toyoda, Cihan Erkut, Francisco Pan-Montojo, Sebastian Boland, Martin P Stewart, Daniel J. Müller, Wolfgang Wurst, Anthony Hyman, Teymuras V. Kurzchalia
Products of the Parkinson's disease-related glyoxalase DJ-1, D-lactate and glycolate, support mitochondrial membrane potential and neuronal survival.
Biol Open, 3(8) 777-784 (2014)
PDF DOI

Parkinson's disease is associated with mitochondrial decline in dopaminergic neurons of the substantia nigra. One of the genes linked with the onset of Parkinson's disease, DJ-1/PARK7, belongs to a novel glyoxalase family and influences mitochondrial activity. It has been assumed that glyoxalases fulfill this task by detoxifying aggressive aldehyde by-products of metabolism. Here we show that supplying either D-lactate or glycolate, products of DJ-1, rescues the requirement for the enzyme in maintenance of mitochondrial potential. We further show that glycolic acid and D-lactic acid can elevate lowered mitochondrial membrane potential caused by silencing PINK-1, another Parkinson's related gene, as well as by paraquat, an environmental toxin known to be linked with Parkinson's disease. We propose that DJ-1 and consequently its products are components of a novel pathway that stabilizes mitochondria during cellular stress. We go on to show that survival of cultured mesencephalic dopaminergic neurons, defective in Parkinson's disease, is enhanced by glycolate and D-lactate. Because glycolic and D-lactic acids occur naturally, they are therefore a potential therapeutic route for treatment or prevention of Parkinson's disease.

Ellie S Heckscher, Fuhui Long, Michael J Layden, Chein-Hui Chuang, Laurina Manning, Jourdain Richart, Joseph C Pearson, Stephen T Crews, Hanchuan Peng, Gene Myers, Chris Q Doe
Atlas-builder software and the eNeuro atlas: resources for developmental biology and neuroscience.
Development, 141(12) 2524-2532 (2014)
PDF DOI

A major limitation in understanding embryonic development is the lack of cell type-specific markers. Existing gene expression and marker atlases provide valuable tools, but they typically have one or more limitations: a lack of single-cell resolution; an inability to register multiple expression patterns to determine their precise relationship; an inability to be upgraded by users; an inability to compare novel patterns with the database patterns; and a lack of three-dimensional images. Here, we develop new 'atlas-builder' software that overcomes each of these limitations. A newly generated atlas is three-dimensional, allows the precise registration of an infinite number of cell type-specific markers, is searchable and is open-ended. Our software can be used to create an atlas of any tissue in any organism that contains stereotyped cell positions. We used the software to generate an 'eNeuro' atlas of the Drosophila embryonic CNS containing eight transcription factors that mark the major CNS cell types (motor neurons, glia, neurosecretory cells and interneurons). We found neuronal, but not glial, nuclei occupied stereotyped locations. We added 75 new Gal4 markers to the atlas to identify over 50% of all interneurons in the ventral CNS, and these lines allowed functional access to those interneurons for the first time. We expect the atlas-builder software to benefit a large proportion of the developmental biology community, and the eNeuro atlas to serve as a publicly accessible hub for integrating neuronal attributes - cell lineage, gene expression patterns, axon/dendrite projections, neurotransmitters--and linking them to individual neurons.

Alicia Tapias, Zhong-Wei Zhou, Yue Shi, Zechen Chong, Pei Wang, Marco Groth, Matthias Platzer, Wieland Huttner, Zdenko Herceg, Yun-Gui Yang, Zhao-Qi Wang
Trrap-dependent histone acetylation specifically regulates cell-cycle gene transcription to control neural progenitor fate decisions.
Cell Stem Cell, 14(5) 632-643 (2014)
DOI

Fate decisions in neural progenitor cells are orchestrated via multiple pathways, and the role of histone acetylation in these decisions has been ascribed to a general function promoting gene activation. Here, we show that the histone acetyltransferase (HAT) cofactor transformation/transcription domain-associated protein (Trrap) specifically regulates activation of cell-cycle genes, thereby integrating discrete cell-intrinsic programs of cell-cycle progression and epigenetic regulation of gene transcription in order to control neurogenesis. Deletion of Trrap impairs recruitment of HATs and transcriptional machinery specifically to E2F cell-cycle target genes, disrupting their transcription with consequent cell-cycle lengthening specifically within cortical apical neural progenitors (APs). Consistently, Trrap conditional mutants exhibit microcephaly because of premature differentiation of APs into intermediate basal progenitors and neurons, and overexpressing cell-cycle regulators in vivo can rescue these premature differentiation defects. These results demonstrate an essential and highly specific role for Trrap-mediated histone regulation in controlling cell-cycle progression and neurogenesis.

Reinhard Roessler, Sebastien A Smallwood, Jesse V Veenvliet, Petros Pechlivanoglou, Su-Ping Peng, Koushik Chakrabarty, Marian J A Groot-Koerkamp, R Jeroen Pasterkamp, Evelyn Wesseling, Gavin Kelsey, Erik Boddeke, Marten P Smidt, Sjef Copray
Detailed analysis of the genetic and epigenetic signatures of iPSC-derived mesodiencephalic dopaminergic neurons.
Stem Cell Rep, 2(4) 520-533 (2014)
Open Access DOI

Induced pluripotent stem cells (iPSCs) hold great promise for in vitro generation of disease-relevant cell types, such as mesodiencephalic dopaminergic (mdDA) neurons involved in Parkinson's disease. Although iPSC-derived midbrain DA neurons have been generated, detailed genetic and epigenetic characterizations of such neurons are lacking. The goal of this study was to examine the authenticity of iPSC-derived DA neurons obtained by established protocols. We FACS purified mdDA (Pitx3 (Gfp/+) ) neurons derived from mouse iPSCs and primary mdDA (Pitx3 (Gfp/+) ) neurons to analyze and compare their genetic and epigenetic features. Although iPSC-derived DA neurons largely adopted characteristics of their in vivo counterparts, relevant deviations in global gene expression and DNA methylation were found. Hypermethylated genes, mainly involved in neurodevelopment and basic neuronal functions, consequently showed reduced expression levels. Such abnormalities should be addressed because they might affect unambiguous long-term functionality and hamper the potential of iPSC-derived DA neurons for in vitro disease modeling or cell-based therapy.

Jifeng Fei, Christiane Haffner, Wieland B. Huttner
3' UTR-Dependent, miR-92-Mediated Restriction of Tis21 Expression Maintains Asymmetric Neural Stem Cell Division to Ensure Proper Neocortex Size.
Cell Rep, 7(2) 398-411 (2014)
DOI

Mammalian neocortex size primarily reflects the number and mode of divisions of neural stem and progenitor cells. Cortical stem cells (apical progenitors) switching from symmetric divisions, which expand their population, to asymmetric divisions, which generate downstream neuronal progenitors (basal progenitors), start expressing Tis21, a so-called antiproliferative/prodifferentiative gene. Tis21 encodes a small (17.5 kDa), functionally poorly characterized protein and a relatively large (2 kb), highly conserved 3' UTR. Here, we show that mice lacking the Tis21 3' UTR develop a microcephalic neocortex with fewer neurons, notably in the upper layers. This reflects a progressive decrease in basal progenitors, which in turn is due to a fraction of apical progenitors prematurely switching from asymmetric self-renewing to symmetric self-consuming divisions. This switch is caused by the markedly increased Tis21 protein level resulting from lack of microRNA-, notably miR-92-, dependent restriction of Tis21 expression. Our data show that a premature onset of consumptive neural stem cell divisions can lead to microcephaly.

L Carine Stapel, Nadine Vastenhouw
Message control in developmental transitions; deciphering chromatin's role using zebrafish genomics.
Brief Funct Genomics, 13(2) 106-120 (2014)
DOI

Now that the sequencing of genomes has become routine, understanding how a given genome is used in different ways to obtain cell type diversity in an organism is the next frontier. How specific transcription programs are established during vertebrate embryogenesis, however, remains poorly understood. Transcription is influenced by chromatin structure, which determines the accessibility of DNA-binding proteins to the genome. Although large-scale genomics approaches have uncovered specific features of chromatin structure that are diagnostic for different cell types and developmental stages, our functional understanding of chromatin in transcriptional regulation during development is very limited. In recent years, zebrafish embryogenesis has emerged as an excellent vertebrate model system to investigate the functional relationship between chromatin organization, gene regulation and development in a dynamic environment. Here, we review how studies in zebrafish have started to improve our understanding of the role of chromatin structure in genome activation and pluripotency and in the potential inheritance of transcriptional states from parent to progeny.

Evan Heller, K Vijay Kumar, Stephan W. Grill, Elaine Fuchs
Forces generated by cell intercalation tow epidermal sheets in Mammalian tissue morphogenesis.
Dev Cell, 28(6) 617-632 (2014)
DOI

While gastrulation movements offer mechanistic paradigms for how collective cellular movements shape developing embryos, far less is known about coordinated cellular movements that occur later in development. Studying eyelid closure, we explore a case where an epithelium locally reshapes, expands, and moves over another epithelium. Live imaging, gene targeting, and cell-cycle inhibitors reveal that closure does not require overlying periderm, proliferation, or supracellular actin cable assembly. Laser ablation and quantitative analyses of tissue deformations further distinguish the mechanism from wound repair and dorsal closure. Rather, cell intercalations parallel to the tissue front locally compress it perpendicularly, pulling the surrounding epidermis along the closure axis. Functional analyses in vivo show that the mechanism requires localized myosin-IIA- and α5β1 integrin/fibronectin-mediated migration and E-cadherin downregulation likely stimulated by Wnt signaling. These studies uncover a mode of epithelial closure in which forces generated by cell intercalation are leveraged to tow the surrounding tissue.

Julie Hanotel, Nathalie Bessodes, Aurore Thélie, Marie Hedderich, Karine Parain, Benoit Van Driessche, Karina De Oliveira Brandão, Sadia Kricha, Mette C Jorgensen, Anne Grapin-Botton, Palle Serup, Carine Van Lint, Muriel Perron, Tomas Pieler, Kristine A Henningfeld, Eric J Bellefroid
The Prdm13 histone methyltransferase encoding gene is a Ptf1a-Rbpj downstream target that suppresses glutamatergic and promotes GABAergic neuronal fate in the dorsal neural tube.
Dev Biol, 386(2) 340-357 (2014)
DOI

The basic helix-loop-helix (bHLH) transcriptional activator Ptf1a determines inhibitory GABAergic over excitatory glutamatergic neuronal cell fate in progenitors of the vertebrate dorsal spinal cord, cerebellum and retina. In an in situ hybridization expression survey of PR domain containing genes encoding putative chromatin-remodeling zinc finger transcription factors in Xenopus embryos, we identified Prdm13 as a histone methyltransferase belonging to the Ptf1a synexpression group. Gain and loss of Ptf1a function analyses in both frog and mice indicates that Prdm13 is positively regulated by Ptf1a and likely constitutes a direct transcriptional target. We also showed that this regulation requires the formation of the Ptf1a-Rbp-j complex. Prdm13 knockdown in Xenopus embryos and in Ptf1a overexpressing ectodermal explants lead to an upregulation of Tlx3/Hox11L2, which specifies a glutamatergic lineage and a reduction of the GABAergic neuronal marker Pax2. It also leads to an upregulation of Prdm13 transcription, suggesting an autonegative regulation. Conversely, in animal caps, Prdm13 blocks the ability of the bHLH factor Neurog2 to activate Tlx3. Additional gain of function experiments in the chick neural tube confirm that Prdm13 suppresses Tlx3(+)/glutamatergic and induces Pax2(+)/GABAergic neuronal fate. Thus, Prdm13 is a novel crucial component of the Ptf1a regulatory pathway that, by modulating the transcriptional activity of bHLH factors such as Neurog2, controls the balance between GABAergic and glutamatergic neuronal fate in the dorsal and caudal part of the vertebrate neural tube.

Andreas Begitt, Mathias Droescher, Thomas Meyer, Christoph D Schmid, Michelle Baker, Filipa Antunes, Markus R Owen, Ronald Naumann, Thomas Decker, Uwe Vinkemeier
STAT1-cooperative DNA binding distinguishes type 1 from type 2 interferon signaling.
Nat Immunol, 15(2) 168-176 (2014)
DOI

STAT1 is an indispensable component of a heterotrimer (ISGF3) and a STAT1 homodimer (GAF) that function as transcription regulators in type 1 and type 2 interferon signaling, respectively. To investigate the importance of STAT1-cooperative DNA binding, we generated gene-targeted mice expressing cooperativity-deficient STAT1 with alanine substituted for Phe77. Neither ISGF3 nor GAF bound DNA cooperatively in the STAT1F77A mouse strain, but type 1 and type 2 interferon responses were affected differently. Type 2 interferon-mediated transcription and antibacterial immunity essentially disappeared owing to defective promoter recruitment of GAF. In contrast, STAT1 recruitment to ISGF3 binding sites and type 1 interferon-dependent responses, including antiviral protection, remained intact. We conclude that STAT1 cooperativity is essential for its biological activity and underlies the cellular responses to type 2, but not type 1 interferon.

Yong Zhang^*^#, Nadine Vastenhouw^*^#, Jianxing Feng, Kai Fu, Chenfei Wang, Ying Ge, Paul van Hummelen, Alexander F Schier^#, Xiaole Shirley Liu
Canonical nucleosome organization at promoters forms during genome activation.
Genome Res, 24(2) 260-266 (2014)
PDF DOI

The organization of nucleosomes influences transcriptional activity by controlling accessibility of DNA binding proteins to the genome. Genome-wide nucleosome binding profiles have identified a canonical nucleosome organization at gene promoters, where arrays of well-positioned nucleosomes emanate from nucleosome-depleted regions. The mechanisms of formation and the function of canonical promoter nucleosome organization remain unclear. Here we analyze the genome-wide location of nucleosomes during zebrafish embryogenesis and show that well-positioned nucleosome arrays appear on thousands of promoters during the activation of the zygotic genome. The formation of canonical promoter nucleosome organization is independent of DNA sequence preference, transcriptional elongation, and robust RNA polymerase II (Pol II) binding. Instead, canonical promoter nucleosome organization correlates with the presence of Histone H3 Lysine 4 trimethylation (H3K4me3) and affects future transcriptional activation. These findings reveal that genome activation is central to the organization of nucleosome arrays during early embryogenesis.

Christoph Zechner, Maria F Unger, Serge Pelet, Matthias Peter, Heinz Koeppl
Scalable inference of heterogeneous reaction kinetics from pooled single-cell recordings.
Nat Methods, 11(2) 197-202 (2014)
DOI

Mathematical methods combined with measurements of single-cell dynamics provide a means to reconstruct intracellular processes that are only partly or indirectly accessible experimentally. To obtain reliable reconstructions, the pooling of measurements from several cells of a clonal population is mandatory. However, cell-to-cell variability originating from diverse sources poses computational challenges for such process reconstruction. We introduce a scalable Bayesian inference framework that properly accounts for population heterogeneity. The method allows inference of inaccessible molecular states and kinetic parameters; computation of Bayes factors for model selection; and dissection of intrinsic, extrinsic and technical noise. We show how additional single-cell readouts such as morphological features can be included in the analysis. We use the method to reconstruct the expression dynamics of a gene under an inducible promoter in yeast from time-lapse microscopy data.

Nicole Bieberstein, Korinna Straube, Karla M. Neugebauer
Chromatin immunoprecipitation approaches to determine co-transcriptional nature of splicing.
Methods Mol Biol, 1126 315-323 (2014)
DOI

Chromatin immunoprecipitation (ChIP) is a common method used to determine the position along DNA where an antigen is found. The method was initially devised for protein antigens that come in direct contact with genomic DNA, such as components of the transcriptional machinery and histones. However, ChIP can also be extended to antigens that bind RNA, as demonstrated by the specific localization of spliceosomal components to particular gene regions that correlate with when and where introns and exons are transcribed. The activities of any RNA binding protein can in principle be monitored using ChIP, and RNA dependency of binding can also be assessed through RNase treatment. Combined with qPCR or high-throughput sequencing, this method allows the detection of RNA bound proteins at individual genes or genome-wide. Here, we present a detailed protocol for "splicing factor ChIP" in tissue culture cells.

Daiki Umetsu, Sebastian Dunst, Christian Dahmann
An RNA interference screen for genes required to shape the anteroposterior compartment boundary in Drosophila identifies the eph receptor.
PLoS ONE, 9(12) Art. No. e114340 (2014)
Open Access PDF DOI

The formation of straight compartment boundaries separating groups of cells with distinct fates and functions is an evolutionarily conserved strategy during animal development. The physical mechanisms that shape compartment boundaries have recently been further elucidated, however, the molecular mechanisms that underlie compartment boundary formation and maintenance remain poorly understood. Here, we report on the outcome of an RNA interference screen aimed at identifying novel genes involved in maintaining the straight shape of the anteroposterior compartment boundary in Drosophila wing imaginal discs. Out of screening 3114 transgenic RNA interference lines targeting a total of 2863 genes, we identified a single novel candidate that interfered with the formation of a straight anteroposterior compartment boundary. Interestingly, the targeted gene encodes for the Eph receptor tyrosine kinase, an evolutionarily conserved family of signal transducers that has previously been shown to be important for maintaining straight compartment boundaries in vertebrate embryos. Our results identify a hitherto unknown role of the Eph receptor tyrosine kinase in Drosophila and suggest that Eph receptors have important functions in shaping compartment boundaries in both vertebrate and insect development.

Alexis Webb, Daniele Soroldoni, Annelie Oswald, Johannes Schindelin, Andrew C. Oates
Generation of dispersed presomitic mesoderm cell cultures for imaging of the zebrafish segmentation clock in single cells.
J Vis Exp, (89) Art. No. e50307 (2014)
DOI

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Alexandra Kumichel, Elisabeth Knust
Apical localisation of crumbs in the boundary cells of the Drosophila hindgut is independent of its canonical interaction partner stardust.
PLoS ONE, 9(4) Art. No. e94038 (2014)
PDF DOI

The transmembrane protein Crumbs/Crb is a key regulator of apico-basal epithelial cell polarity, both in Drosophila and in vertebrates. In most cases studied so far, the apical localisation of Drosophila Crumbs depends on the interaction of its C-terminal amino acids with the scaffolding protein Stardust. Consequently, embryos lacking either Crumbs or Stardust develop a very similar phenotype, characterised by the loss of epithelial tissue integrity and cell polarity in many epithelia. An exception is the hindgut, which is not affected by the loss of either gene. The hindgut is a single layered epithelial tube composed of two cell populations, the boundary cells and the principal cells. Here we show that Crumbs localisation in the principal cells depends on Stardust, similarly to other embryonic epithelia. In contrast, localisation of Crumbs in the boundary cells does not require Stardust and is independent of its PDZ domain- and FERM-domain binding motifs. In line with this, the considerable upregulation of Crumbs in boundary cells is not followed by a corresponding upregulation of its canonical binding partners. Our data are the first to suggest a mechanism controlling apical Crumbs localisation, which is independent of its conserved FERM- and PDZ-domain binding motifs.

Eric R Geertsma
FX cloning: a simple and robust high-throughput cloning method for protein expression.
Methods Mol Biol, 1116 153-164 (2014)
DOI

The immense amount of gene sequences available nowadays allows scientist to screen broadly for extraordinary proteins. Reliable cloning tools that allow the parallel processing of many targets are vital for the success of this strategy. The FX cloning procedure detailed here is such a straightforward and efficient tool. It is dedicated to the cloning of open reading frames (ORFs) with the final aim of expressing the corresponding proteins. FX cloning combines attractive features of established high-throughput cloning methods that were thus far not unified in one single method. It facilitates the subcloning of a sequence-verified ORF to a variety of expression vectors, but is sufficiently versatile to accept PCR products as well. Moreover, the common, but undesirable feature of extending target ORFs with long cloning-related sequences is avoided. It leads to the addition of only one amino acid to each side of the protein. As a consequence, only one primer pair or PCR product suffices to generate expression vectors for both N- and C-terminal translational fusions. FX cloning is highly efficient and economical in its use. The method is suited for high-throughput cloning projects and also for everyday cloning of single targets. FX cloning is based on the use of type IIS restriction enzymes and negative selection markers. The full procedure takes place in one pot in less than 3 h and does not require intermediate purification steps nor extensive handling. The method has proven to be very robust and suitable for all common expression systems.

Jarmila Hnilicová, Samira Hozeifi, Eva Stejskalová, Eva Dušková, Ina Poser, Jana Humpolíčková, Martin Hof, David Staněk
The C-terminal domain of Brd2 is important for chromatin interaction and regulation of transcription and alternative splicing.
Mol Biol Cell, 24(22) 3557-3568 (2013)
DOI

Brd2 is a member of the bromodomain extra terminal (BET) protein family, which consists of four chromatin-interacting proteins that regulate gene expression. Each BET protein contains two N-terminal bromodomains, which recognize acetylated histones, and the C-terminal protein-protein interaction domain. Using a genome-wide screen, we identify 1450 genes whose transcription is regulated by Brd2. In addition, almost 290 genes change their alternative splicing pattern upon Brd2 depletion. Brd2 is specifically localized at promoters of target genes, and our data show that Brd2 interaction with chromatin cannot be explained solely by histone acetylation. Using coimmunoprecipitation and live-cell imaging, we show that the C-terminal part is crucial for Brd2 association with chromatin. Live-cell microscopy also allows us to map the average binding time of Brd2 to chromatin and quantify the contributions of individual Brd2 domains to the interaction with chromatin. Finally, we show that bromodomains and the C-terminal domain are equally important for transcription and splicing regulation, which correlates with the role of these domains in Brd2 binding to chromatin.

Liat Rockah-Shmuel, Ágnes Tóth-Petróczy, Asaf Sela, Omri Wurtzel, Rotem Sorek, Dan S Tawfik
Correlated occurrence and bypass of frame-shifting insertion-deletions (InDels) to give functional proteins.
PLoS Genet, 9(10) Art. No. e1003882 (2013)
Open Access DOI

Short insertions and deletions (InDels) comprise an important part of the natural mutational repertoire. InDels are, however, highly deleterious, primarily because two-thirds result in frame-shifts. Bypass through slippage over homonucleotide repeats by transcriptional and/or translational infidelity is known to occur sporadically. However, the overall frequency of bypass and its relation to sequence composition remain unclear. Intriguingly, the occurrence of InDels and the bypass of frame-shifts are mechanistically related - occurring through slippage over repeats by DNA or RNA polymerases, or by the ribosome, respectively. Here, we show that the frequency of frame-shifting InDels, and the frequency by which they are bypassed to give full-length, functional proteins, are indeed highly correlated. Using a laboratory genetic drift, we have exhaustively mapped all InDels that occurred within a single gene. We thus compared the naive InDel repertoire that results from DNA polymerase slippage to the frame-shifting InDels tolerated following selection to maintain protein function. We found that InDels repeatedly occurred, and were bypassed, within homonucleotide repeats of 3-8 bases. The longer the repeat, the higher was the frequency of InDels formation, and the more frequent was their bypass. Besides an expected 8A repeat, other types of repeats, including short ones, and G and C repeats, were bypassed. Although obtained in vitro, our results indicate a direct link between the genetic occurrence of InDels and their phenotypic rescue, thus suggesting a potential role for frame-shifting InDels as bridging evolutionary intermediates.

Marco Nousch, Nora Techritz, Daniel Hampel, Sophia Millonigg, Christian R. Eckmann
The Ccr4-Not deadenylase complex constitutes the main poly(A) removal activity in C. elegans.
J Cell Sci, 126(18) 4274-4285 (2013)
DOI

Post-transcriptional regulatory mechanisms are widely used to control gene expression programs of tissue development and physiology. Controlled 3' poly(A) tail-length changes of mRNAs provide a mechanistic basis of such regulation, affecting mRNA stability and translational competence. Deadenylases are a conserved class of enzymes that facilitate poly(A) tail removal, and their biochemical activities have been mainly studied in the context of single-cell systems. Little is known about the different deadenylases and their biological role in multicellular organisms. In this study, we identify and characterize all known deadenylases of Caenorhabditis elegans, and identify the germ line as tissue that depends strongly on deadenylase activity. Most deadenylases are required for hermaphrodite fertility, albeit to different degrees. Whereas ccr-4 and ccf-1 deadenylases promote germline function under physiological conditions, panl-2 and parn-1 deadenylases are only required under heat-stress conditions. We also show that the Ccr4-Not core complex in nematodes is composed of the two catalytic subunits CCR-4 and CCF-1 and the structural subunit NTL-1, which we find to regulate the stability of CCF-1. Using bulk poly(A) tail measurements with nucleotide resolution, we detect strong deadenylation defects of mRNAs at the global level only in the absence of ccr-4, ccf-1 and ntl-1, but not of panl-2, parn-1 and parn-2. Taken together, this study suggests that the Ccr4-Not complex is the main deadenylase complex in C. elegans germ cells. On the basis of this and as a result of evidence in flies, we propose that the conserved Ccr4-Not complex is an essential component in post-transcriptional regulatory networks promoting animal reproduction.

Matthew A Benton, Michael Akam, Anastasios Pavlopoulos
Cell and tissue dynamics during Tribolium embryogenesis revealed by versatile fluorescence labeling approaches.
Development, 140(15) 3210-3220 (2013)
DOI

Studies on new arthropod models such as the beetle Tribolium castaneum are shifting our knowledge of embryonic patterning and morphogenesis beyond the Drosophila paradigm. In contrast to Drosophila, Tribolium embryos exhibit the short-germ type of development and become enveloped by extensive extra-embryonic membranes, the amnion and serosa. The genetic basis of these processes has been the focus of active research. Here, we complement genetic approaches with live fluorescence imaging of Tribolium embryos to make the link between gene function and morphogenetic cell behaviors during blastoderm formation and differentiation, germband condensation and elongation, and extra-embryonic development. We first show that transient labeling methods result in strong, homogeneous and persistent expression of fluorescent markers in Tribolium embryos, labeling the chromatin, membrane, cytoskeleton or combinations thereof. We then use co-injection of fluorescent markers with dsRNA for live imaging of embryos with disrupted caudal gene function caused by RNA interference. Using these approaches, we describe and compare cell and tissue dynamics in Tribolium embryos with wild-type and altered fate maps. We find that Tribolium germband condensation is effected by cell contraction and intercalation, with the latter being dependent on the anterior-posterior patterning system. We propose that germband condensation drives initiation of amnion folding, whereas expansion of the amniotic fold and closure of the amniotic cavity are likely driven by contraction of an actomyosin cable at the boundary between the amnion and serosa. Our methodology provides a comprehensive framework for testing quantitative models of patterning, growth and morphogenetic mechanisms in Tribolium and other arthropod species.

Jesse V Veenvliet^*, Maria T M Alves Dos Santos^*, Willemieke M Kouwenhoven, Lars von Oerthel, Jamie L Lim, Annemarie J A van der Linden, Marian J A Groot Koerkamp, Frank C P Holstege, Marten P Smidt
Specification of dopaminergic subsets involves interplay of En1 and Pitx3.
Development, 140(16) 3373-3384 (2013)
DOI

Mesodiencephalic dopaminergic (mdDA) neurons control locomotion and emotion and are affected in multiple psychiatric and neurodegenerative diseases, including Parkinson's disease (PD). The homeodomain transcription factor Pitx3 is pivotal in mdDA neuron development and loss of Pitx3 results in programming deficits in a rostrolateral subpopulation of mdDA neurons destined to form the substantia nigra pars compacta (SNc), reminiscent of the specific cell loss observed in PD. We show here that in adult mice in which the gene encoding a second homeoprotein, engrailed 1 (En1), has been deleted, dramatic loss of mdDA neurons and striatal innervation defects were observed, partially reminiscent of defects observed in Pitx3(-/-) mice. We then continue to reveal developmental crosstalk between En1 and Pitx3 through genome-wide expression analysis. During development, both En1 and Pitx3 are required to induce expression of mdDA genes in the rostrolateral subset destined to form the SNc. By contrast, Pitx3 and En1 reciprocally regulate a separate gene cluster, which includes Cck, demarcating a caudal mdDA subset in wild-type embryos. Whereas En1 is crucial for induction of this caudal phenotype, Pitx3 antagonizes it rostrolaterally. The combinatorial action of En1 and Pitx3 is potentially realized through at least three levels of molecular interaction: (1) influencing each other's expression level, (2) releasing histone deacetylase-mediated repression of Nurr1 target genes and (3) modulating En1 activity through Pitx3-driven activation of En1 modulatory proteins. These findings show how two crucial mediators of mdDA neuronal development, En1 and Pitx3, interact in dopaminergic subset specification, the importance of which is exemplified by the specific vulnerability of the SNc found in PD.

Raymond Behrendt, Tina Schumann, Alexander Gerbaulet, Laura A Nguyen, Nadja Schubert, Dimitra Alexopoulou, Ursula Berka, Stefan Lienenklaus, Katrin Peschke, Kathrin Gibbert, Sabine Wittmann, Dirk Lindemann, Siegfried Weiss, Andreas Dahl, Ronald Naumann, Ulf Dittmer, Baek Kim, Werner Mueller, Thomas Gramberg, Axel Roers
Mouse SAMHD1 has antiretroviral activity and suppresses a spontaneous cell-intrinsic antiviral response.
Cell Rep, 4(4) 689-696 (2013)
DOI

Aicardi-Goutières syndrome (AGS), a hereditary autoimmune disease, clinically and biochemically overlaps with systemic lupus erythematosus (SLE) and, like SLE, is characterized by spontaneous type I interferon (IFN) production. The finding that defects of intracellular nucleases cause AGS led to the concept that intracellular accumulation of nucleic acids triggers inappropriate production of type I IFN and autoimmunity. AGS can also be caused by defects of SAMHD1, a 3' exonuclease and deoxynucleotide (dNTP) triphosphohydrolase. Human SAMHD1 is an HIV-1 restriction factor that hydrolyzes dNTPs and decreases their concentration below the levels required for retroviral reverse transcription. We show in gene-targeted mice that also mouse SAMHD1 reduces cellular dNTP concentrations and restricts retroviral replication in lymphocytes, macrophages, and dendritic cells. Importantly, the absence of SAMHD1 triggered IFN-β-dependent transcriptional upregulation of type I IFN-inducible genes in various cell types indicative of spontaneous IFN production. SAMHD1-deficient mice may be instrumental for elucidating the mechanisms that trigger pathogenic type I IFN responses in AGS and SLE.

Michael Hiller, Saatvik Agarwal, James H Notwell, Ravi Parikh, Harendra Guturu, Aaron M Wenger, Gill Bejerano
Computational methods to detect conserved non-genic elements in phylogenetically isolated genomes: application to zebrafish.
Nucleic Acids Res, 41(15) Art. No. e151 (2013)
PDF DOI

Many important model organisms for biomedical and evolutionary research have sequenced genomes, but occupy a phylogenetically isolated position, evolutionarily distant from other sequenced genomes. This phylogenetic isolation is exemplified for zebrafish, a vertebrate model for cis-regulation, development and human disease, whose evolutionary distance to all other currently sequenced fish exceeds the distance between human and chicken. Such large distances make it difficult to align genomes and use them for comparative analysis beyond gene-focused questions. In particular, detecting conserved non-genic elements (CNEs) as promising cis-regulatory elements with biological importance is challenging. Here, we develop a general comparative genomics framework to align isolated genomes and to comprehensively detect CNEs. Our approach integrates highly sensitive and quality-controlled local alignments and uses alignment transitivity and ancestral reconstruction to bridge large evolutionary distances. We apply our framework to zebrafish and demonstrate substantially improved CNE detection and quality compared with previous sets. Our zebrafish CNE set comprises 54 533 CNEs, of which 11 792 (22%) are conserved to human or mouse. Our zebrafish CNEs (http://zebrafish.stanford.edu) are highly enriched in known enhancers and extend existing experimental (ChIP-Seq) sets. The same framework can now be applied to the isolated genomes of frog, amphioxus, Caenorhabditis elegans and many others.

Denise Stenzel, Wieland B. Huttner
Role of maternal thyroid hormones in the developing neocortex and during human evolution
Front Neuroanat, 7 Art. No. 19 (2013)
DOI

The importance of thyroid hormones during brain development has been appreciated for many decades. In humans, low levels of circulating maternal thyroid hormones, e.g., caused by maternal hypothyroidism or lack of iodine in diet, results in a wide spectrum of severe neurological defects, including neurological cretinism characterized by profound neurologic impairment and mental retardation, underlining the importance of the maternal thyroid hormone contribution. In fact, iodine intake, which is essential for thyroid hormone production in the thyroid gland, has been related to the expansion of the brain, associated with the increased cognitive capacities during human evolution. Because thyroid hormones regulate transcriptional activity of target genes via their nuclear thyroid hormone receptors (THRs), even mild and transient changes in maternal thyroid hormone levels can directly affect and alter the gene expression profile, and thus disturb fetal brain development. Here we summarize how thyroid hormones may have influenced human brain evolution through the adaptation to new habitats, concomitant with changes in diet and, therefore, iodine intake. Further, we review the current picture we gained from experimental studies in rodents on the function of maternal thyroid hormones during developmental neurogenesis. We aim to evaluate the effects of maternal thyroid hormone deficiency as well as lack of THRs and transporters on brain development and function, shedding light on the cellular behavior conducted by thyroid hormones.

Jacqueline Tabler, William B Barrell, Heather L Szabo-Rogers, Christopher Healy, Yvonne Yeung, Elisa Gomez Perdiguero, Christian Schulz, Basil Z Yannakoudakis, Aida Mesbahi, Bogdan Wlodarczyk, Frederic Geissmann, Richard H Finnell, John Wallingford, Karen J Liu
Fuz mutant mice reveal shared mechanisms between ciliopathies and FGF-related syndromes.
Dev Cell, 25(6) 623-635 (2013)
Open Access PDF DOI

Ciliopathies are a broad class of human disorders with craniofacial dysmorphology as a common feature. Among these is high arched palate, a condition that affects speech and quality of life. Using the ciliopathic Fuz mutant mouse, we find that high arched palate does not, as commonly suggested, arise from midface hypoplasia. Rather, increased neural crest expands the maxillary primordia. In Fuz mutants, this phenotype stems from dysregulated Gli processing, which in turn results in excessive craniofacial Fgf8 gene expression. Accordingly, genetic reduction of Fgf8 ameliorates the maxillary phenotypes. Similar phenotypes result from mutation of oral-facial-digital syndrome 1 (Ofd1), suggesting that aberrant transcription of Fgf8 is a common feature of ciliopathies. High arched palate is also a prevalent feature of fibroblast growth factor (FGF) hyperactivation syndromes. Thus, our findings elucidate the etiology for a common craniofacial anomaly and identify links between two classes of human disease: FGF-hyperactivation syndromes and ciliopathies.

Patricia Heyn
Nuclear architecture and gene expression during early zebrafish development
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2013)

Jörg Schönheit, Christiane Kuhl, Marie Luise Gebhardt, Francisco Fernández Klett, Pia Riemke, Marina Scheller, Gang Huang, Ronald Naumann, Achim Leutz, Carol Stocking, Josef Priller, Miguel A Andrade-Navarro, Frank Rosenbauer
PU.1 level-directed chromatin structure remodeling at the Irf8 gene drives dendritic cell commitment.
Cell Rep, 3(5) 1617-1628 (2013)
DOI

Dendritic cells (DCs) are essential regulators of immune responses; however, transcriptional mechanisms that establish DC lineage commitment are poorly defined. Here, we report that the PU.1 transcription factor induces specific remodeling of the higher-order chromatin structure at the interferon regulatory factor 8 (Irf8) gene to initiate DC fate choice. An Irf8 reporter mouse enabled us to pinpoint an initial progenitor stage at which DCs separate from other myeloid lineages in the bone marrow. In the absence of Irf8, this progenitor undergoes DC-to-neutrophil reprogramming, indicating that DC commitment requires an active, Irf8-dependent escape from alternative myeloid lineage potential. Mechanistically, myeloid Irf8 expression depends on high PU.1 levels, resulting in local chromosomal looping and activation of a lineage- and developmental-stage-specific cis-enhancer. These data delineate PU.1 as a concentration-dependent rheostat of myeloid lineage selection by controlling long-distance contacts between regulatory elements and suggest that specific higher-order chromatin remodeling at the Irf8 gene determines DC differentiation.

Eric Lewitus, Alex T. Kalinka
Neocortical development as an evolutionary platform for intragenomic conflict.
Front Neuroanat, 7 Art. No. 2 (2013)
DOI

Embryonic development in mammals has evolved a platform for genomic conflict between mothers and embryos and, by extension, between maternal and paternal genomes. The evolutionary interests of the mother and embryo may be maximized through the promotion of sex-chromosome genes and imprinted alleles, resulting in the rapid evolution of postzygotic phenotypes preferential to either the maternal or paternal genome. In eutherian mammals, extraordinary in utero maternal investment in the brain, and neocortex especially, suggests that convergent evolution of an expanded mammalian neocortex along divergent lineages may be explained, in part, by parent-of-origin-linked gene expression arising from parent-offspring conflict. The influence of this conflict on neocortical development and evolution, however, has not been investigated at the genomic level. In this hypothesis and theory article, we provide preliminary evidence for positive selection in humans in the regions of two platforms of intragenomic conflict-chromosomes 15q11-q13 and X-and explore the potential relevance of cis-regulated imprinted domains to neocortical expansion in mammalian evolution. We present the hypothesis that maternal- and paternal-specific pressures on the developing neocortex compete intragenomically to influence neocortical expansion in mammalian evolution.

Tomislav Maricic, Viola Günther, Oleg Georgiev, Sabine Gehre, Marija Curlin, Christiane Schreiweis, Ronald Naumann, Hernán A Burbano, Matthias Meyer, Carles Lalueza-Fox, Marco de la Rasilla, Antonio Rosas, Srecko Gajovic, Janet Kelso, Wolfgang Enard, Walter Schaffner, Svante Pääbo
A Recent Evolutionary Change Affects a Regulatory Element in the Human FOXP2 Gene.
Mol Biol Evol, 30(4) 844-852 (2013)
DOI

The FOXP2 gene is required for normal development of speech and language. By isolating and sequencing FOXP2 genomic DNA fragments from a 49,000-year-old Iberian Neandertal and 50 present-day humans, we have identified substitutions in the gene shared by all or nearly all present-day humans but absent or polymorphic in Neandertals. One such substitution is localized in intron 8 and affects a binding site for the transcription factor POU3F2, which is highly conserved among vertebrates. We find that the derived allele of this site is less efficient than the ancestral allele in activating transcription from a reporter construct. The derived allele also binds less POU3F2 dimers than POU3F2 monomers compared with the ancestral allele. Because the substitution in the POU3F2 binding site is likely to alter the regulation of FOXP2 expression, and because it is localized in a region of the gene associated with a previously described signal of positive selection, it is a plausible candidate for having caused a recent selective sweep in the FOXP2 gene.

Xicotencatly Gracida, Christian R. Eckmann
Fertility and germline stem cell maintenance under different diets requires nhr-114/HNF4 in C. elegans.
Curr Biol, 23(7) 607-613 (2013)
PDF DOI

Animals can thrive on variable food resources as a result of autonomous processes and beneficial relationships with their gut microbes [1]. Food intake elicits major physiological changes, which are counteracted by transient systemic responses that maintain homeostasis in the organism. This integration of external information occurs through cellular sensory elements, such as nuclear receptors, which modulate gene expression in response to specific cues [2]. Given the importance of germline stem cells (GSCs) for the development of the germline and the continuity of species, it is reasonable to assume that GSCs might be shielded from the negative influence of environmental perturbations. To our knowledge, however, there are no mechanisms reported that protect GSCs from harmful dietary metabolites. Using Caenorhabditis elegans as a model, we report that the somatic activity of the conserved nuclear receptor nhr-114/HNF4 protects GSC integrity from dietary metabolites. In the absence of nhr-114 and on certain bacterial diets, otherwise somatically normal animals accumulate germ cell division defects during development and become sterile. We found that, in nhr-114(-) animals, the induction of germline defects and sterility depend on bacterial metabolic status, with respect to the essential amino acid tryptophan. This illustrates an animal-microbe interaction in which somatic nuclear receptor activity preserves the germline by buffering against dietary metabolites, most likely through a somatic detoxifying response. Overall, our findings uncover an unprecedented, and presumably evolutionarily conserved, soma-to-germline axis of communication that maintains reproductive robustness on variable food resources.

Germana Rappa, Javier Mercapide, Fabio Anzanello, Thuc T Le, Mary G Johlfs, Ronald R Fiscus, Michaela Wilsch-Bräuninger, Denis Corbeil, Aurelio Lorico
Wnt interaction and extracellular release of prominin-1/CD133 in human malignant melanoma cells.
Exp Cell Res, 319(6) 810-819 (2013)
DOI

Prominin-1 (CD133) is the first identified gene of a novel class of pentaspan membrane glycoproteins. It is expressed by various epithelial and non-epithelial cells, and notably by stem and cancer stem cells. In non-cancerous cells such as neuro-epithelial and hematopoietic stem cells, prominin-1 is selectively concentrated in plasma membrane protrusions, and released into the extracellular milieu in association with small vesicles. Previously, we demonstrated that prominin-1 contributes to melanoma cells pro-metastatic properties and suggested that it may constitute a molecular target to prevent prominin-1-expressing melanomas from colonizing and growing in lymph nodes and distant organs. Here, we report that three distinct pools of prominin-1 co-exist in cultures of human FEMX-I metastatic melanoma. Morphologically, in addition to the plasma membrane localization, prominin-1 is found within the intracellular compartments, (e.g., Golgi apparatus) and in association with extracellular membrane vesicles. The latter prominin-1-positive structures appeared in three sizes (small, ≤40 nm; intermediates ~40-80 nm, and large, >80 nm). Functionally, the down-regulation of prominin-1 in FEMX-I cells resulted in a significant reduction of number of lipid droplets as observed by coherent anti-Stokes Raman scattering image analysis and Oil red O staining, and surprisingly in a decrease in the nuclear localization of beta-catenin, a surrogate marker of Wnt activation. Moreover, the T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity was 2 to 4 times higher in parental than in prominin-1-knockdown cells. Collectively, our results point to Wnt signaling and/or release of prominin-1-containing membrane vesicles as mediators of the pro-metastatic activity of prominin-1 in FEMX-I melanoma.

Xiaoling Qu, Solomon Afelik, Jan Nygaard Jensen, Michael A Bukys, Sune Kobberup, Martin Schmerr, Fan Xiao, Pia Nyeng, Maria Veronica Albertoni, Anne Grapin-Botton, Jan Nygaard Jensen
Notch-mediated post-translational control of Ngn3 protein stability regulates pancreatic patterning and cell fate commitment.
Dev Biol, 376(1) 1-12 (2013)
DOI

Ngn3 is recognized as a regulator of pancreatic endocrine formation, and Notch signaling as an important negative regulator Ngn3 gene expression. By conditionally controlling expression of Ngn3 in the pancreas, we find that these two signaling components are dynamically linked. This connection involves transcriptional repression as previously shown, but also incorporates a novel post-translational mechanism. In addition to its ability to promote endocrine fate, we provide evidence of a competing ability of Ngn3 in the patterning of multipotent progenitor cells in turn controlling the formation of ducts. On one hand, Ngn3 cell-intrinsically activates endocrine target genes; on the other, Ngn3 cell-extrinsically promotes lateral signaling via the Dll1>Notch>Hes1 pathway which substantially limits its ability to sustain endocrine formation. Prior to endocrine commitment, the Ngn3-mediated activation of the Notch>Hes1 pathway impacts formation of the trunk domain in the pancreas causing multipotent progenitors to lose acinar, while gaining endocrine and ductal, competence. The subsequent selection of fate from such bipotential progenitors is then governed by lateral inhibition, where Notch>Hes1-mediated Ngn3 protein destabilization serves to limit endocrine differentiation by reducing cellular levels of Ngn3. This system thus allows for rapid dynamic changes between opposing bHLH proteins in cells approaching a terminal differentiation event. Inhibition of Notch signaling leads to Ngn3 protein stabilization in the normal mouse pancreas explants. We conclude that the mutually exclusive expression pattern of Ngn3/Hes1 proteins in the mammalian pancreas is partially controlled through Notch-mediated post-translational regulation and we demonstrate that the formation of insulin-producing beta-cells can be significantly enhanced upon induction of a pro-endocrine drive combined with the inhibition of Notch processing.

Wilhelm Palm, Marta Maria Swierczynska, Veena Kumari, Monika Ehrhart-Bornstein, Stefan R. Bornstein, Suzanne Eaton
Secretion and signaling activities of lipoprotein-associated hedgehog and non-sterol-modified hedgehog in flies and mammals
PLoS Biol, 11(3) Art. No. e1001505 (2013)
Open Access DOI

Hedgehog (Hh) proteins control animal development and tissue homeostasis. They activate gene expression by regulating processing, stability, and activation of Gli/Cubitus interruptus (Ci) transcription factors. Hh proteins are secreted and spread through tissue, despite becoming covalently linked to sterol during processing. Multiple mechanisms have been proposed to release Hh proteins in distinct forms; in Drosophila, lipoproteins facilitate long-range Hh mobilization but also contain lipids that repress the pathway. Here, we show that mammalian lipoproteins have conserved roles in Sonic Hedgehog (Shh) release and pathway repression. We demonstrate that lipoprotein-associated forms of Hh and Shh specifically block lipoprotein-mediated pathway inhibition. We also identify a second conserved release form that is not sterol-modified and can be released independently of lipoproteins (Hh-N*/Shh-N*). Lipoprotein-associated Hh/Shh and Hh-N*/Shh-N* have complementary and synergistic functions. In Drosophila wing imaginal discs, lipoprotein-associated Hh increases the amount of full-length Ci, but is insufficient for target gene activation. However, small amounts of non-sterol-modified Hh synergize with lipoprotein-associated Hh to fully activate the pathway and allow target gene expression. The existence of Hh secretion forms with distinct signaling activities suggests a novel mechanism for generating a diversity of Hh responses.

Meritxell Huch, Craig Dorrell, Sylvia F Boj, Johan H van Es, Vivian S W Li, Marc van de Wetering, Toshiro Sato, Karien Hamer, Nobuo Sasaki, Milton J Finegold, Annelise Haft, Robert R G Vries, Markus Grompe, Hans Clevers
In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.
Nature, 494(7436) 247-250 (2013)
DOI

The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single Lgr5(+) stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ(+) cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5(+) cells generate hepatocytes and bile ducts in vivo. Single Lgr5(+) cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah(-/-) mice. These findings indicate that previous observations concerning Lgr5(+) stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.

Bo-Kai Liao
Quantitative study of deltaD gene dosage in zebrafish somitogenesis
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2013)

Michael C Bassik, Martin Kampmann, Robert Jan Lebbink, Shuyi Wang, Marco Y Hein, Ina Poser, Jimena Weibezahn, Max A Horlbeck, Siyuan Chen, Matthias Mann, Anthony A. Hyman, Emily M Leproust, Michael T McManus, Jonathan S. Weissman
A systematic Mammalian genetic interaction map reveals pathways underlying ricin susceptibility.
Cell, 152(4) 909-922 (2013)
PDF DOI

Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultracomplex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a noncanonical role for COPI, a previously uncharacterized protein complex affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs.

Mariya Rostovskaya, Ronald Naumann, Jun Fu, Mandy Obst, Doris Mueller, A Francis Stewart, Konstantinos Anastassiadis
Transposon mediated BAC transgenesis via pronuclear injection of mouse zygotes.
Genesis, 51(2) 135-141 (2013)
DOI

Pronuclear microinjection of bacterial artificial chromosomes (BACs) is the preferred way to generate transgenic mice because the transgene accurately recapitulates expression of the endogenous gene. However, the method is demanding and the integrity and copy number of the BAC transgene is difficult to control. Here, we describe a simpler pronuclear injection method that relies on transposition to introduce full-length BACs into the mouse genome. The bacterial backbone of a hPAX6-GFP reporter BAC was retrofitted with PiggyBac transposon inverted terminal repeats and co-injected with PiggyBac transposase mRNA. Both the frequency of transgenic founders as well as intact, full-length, single copy integrations were increased. Transposition was determined by a rapid PCR screen for a transpositional signature and confirmation by splinkerette sequencing to show that theBACs were integrated as a single copy either in one or two different genomic sites. BAC transposons displayed improved functional accuracy over random integrants as evaluated by expression of the hPAX6-GFP reporter in embryonic neural tube and absence of ectopic expression. This method involves less work to achieve increased frequencies of both transgenesis and single copy, full-length integrations. These advantages are not only relevant to rodents but also for transgenesis in all systems. genesis 51:135-141, 2013. © 2012 Wiley Periodicals, Inc.

Shahryar Khattak, Tatiana Sandoval-Guzmán, Nicole Stanke, Stephanie Protze, Elly M. Tanaka, Dirk Lindemann
Foamy virus for efficient gene transfer in regeneration studies.
BMC Dev Biol, 13 Art. No. 17 (2013)
DOI

Molecular studies of appendage regeneration have been hindered by the lack of a stable and efficient means of transferring exogenous genes. We therefore sought an efficient integrating virus system that could be used to study limb and tail regeneration in salamanders.

Dunja Knapp, Herbert Schulz, Cynthia Alexander Rascon, Michael Volkmer, Juliane Scholz, Eugeniu Nacu, Mu Le, Sergey Novozhilov, Akira Tazaki, Stephanie Protze, Tina Jacob, Norbert Hubner, Bianca Habermann, Elly M. Tanaka
Comparative transcriptional profiling of the axolotl limb identifies a tripartite regeneration-specific gene program.
PLoS ONE, 8(5) Art. No. e61352 (2013)
DOI

Understanding how the limb blastema is established after the initial wound healing response is an important aspect of regeneration research. Here we performed parallel expression profile time courses of healing lateral wounds versus amputated limbs in axolotl. This comparison between wound healing and regeneration allowed us to identify amputation-specific genes. By clustering the expression profiles of these samples, we could detect three distinguishable phases of gene expression - early wound healing followed by a transition-phase leading to establishment of the limb development program, which correspond to the three phases of limb regeneration that had been defined by morphological criteria. By focusing on the transition-phase, we identified 93 strictly amputation-associated genes many of which are implicated in oxidative-stress response, chromatin modification, epithelial development or limb development. We further classified the genes based on whether they were or were not significantly expressed in the developing limb bud. The specific localization of 53 selected candidates within the blastema was investigated by in situ hybridization. In summary, we identified a set of genes that are expressed specifically during regeneration and are therefore, likely candidates for the regulation of blastema formation.

Céline Fernandez, Marianne Sandin, Julio Sampaio, Peter Almgren, Krzysztof Narkiewicz, Michaela Hoffmann, Thomas Hedner, Björn Wahlstrand, Kai Simons, Andrej Shevchenko, Peter James, Olle Melander
Plasma lipid composition and risk of developing cardiovascular disease.
PLoS ONE, 8(8) Art. No. e71846 (2013)
DOI

We tested whether characteristic changes of the plasma lipidome in individuals with comparable total lipids level associate with future cardiovascular disease (CVD) outcome and whether 23 validated gene variants associated with coronary artery disease (CAD) affect CVD associated lipid species.

Claes Andréasson, Anna J Schick, Susanne M Pfeiffer, Mihail Sarov, Francis Stewart, Wolfgang Wurst, Joel A Schick
Direct cloning of isogenic murine DNA in yeast and relevance of isogenicity for targeting in embryonic stem cells
PLoS ONE, 8(9) Art. No. e74207 (2013)
Open Access DOI

Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors.

Marco Nousch, Christian R. Eckmann
Translational Control in the Caenorhabditis elegans Germ Line.
Adv Exp Med Biol, 757 205-247 (2013)
PDF DOI

Translational control is a prevalent form of gene expression regulation in the Caenorhabditis elegans germ line. Linking the amount of protein synthesis to mRNA quantity and translational accessibility in the cell cytoplasm provides unique advantages over DNA-based controls for developing germ cells. This mode of gene expression is especially exploited in germ cell fate decisions and during oogenesis, when the developing oocytes stockpile hundreds of different mRNAs required for early embryogenesis. Consequently, a dense web of RNA regulators, consisting of diverse RNA-binding proteins and RNA-modifying enzymes, control the translatability of entire mRNA expression programs. These RNA regulatory networks are tightly coupled to germ cell developmental progression and are themselves under translational control. The underlying molecular mechanisms and RNA codes embedded in the mRNA molecules are beginning to be understood. Hence, the C. elegans germ line offers fertile grounds for discovering post-transcriptional mRNA regulatory mechanisms and emerges as great model for a systems level understanding of translational control during development.

Bruno Thomas Fievet, Josana Rodriguez, Sundar Naganathan, Christine Lee, Eva Zeiser, Takao Ishidate, Masaki Shirayama, Stephan W. Grill, Julie Ahringer
Systematic genetic interaction screens uncover cell polarity regulators and functional redundancy.
Nat Cell Biol, 15(1) 103-112 (2013)
DOI

Although single-gene loss-of-function analyses can identify components of particular processes, important molecules are missed owing to the robustness of biological systems. Here we show that large-scale RNAi screening for suppression interactions with functionally related mutants greatly expands the repertoire of genes known to act in a shared process and reveals a new layer of functional relationships. We performed RNAi screens for 17 Caenorhabditis elegans cell polarity mutants, generating the most comprehensive polarity network in a metazoan, connecting 184 genes. Of these, 72% were not previously linked to cell polarity and 80% have human homologues. We experimentally confirmed functional roles predicted by the network and characterized through biophysical analyses eight myosin regulators. In addition, we discovered functional redundancy between two unknown polarity genes. Similar systematic genetic interaction screens for other biological processes will help uncover the inventory of relevant genes and their patterns of interactions.

Vineeth Surendranath, Mirko Theis, Bianca Habermann, Frank Buchholz
Designing efficient and specific endoribonuclease-prepared siRNAs.
Methods Mol Biol, 942 193-204 (2013)
PDF DOI

RNA interference (RNAi) has grown to be one of the main techniques for loss-of-function studies, leading to the elucidation of biological function of genes in various cellular systems and model organisms. While for many invertebrates such as Drosophila melanogaster (D. melanogaster) and Caenorhabditis elegans (C. elegans) long double-stranded RNA (dsRNA) can directly be used to induce a RNAi response, chemically synthesized small interfering RNAs (siRNAs) are typically employed in mammalian cells to avoid an interferon-like response triggered by long dsRNA (Reynolds et al., RNA 12:988-993, 2006). However, siRNAs are expensive and beset with unintentional gene targeting effects (off-targets) confounding the analysis of results from such studies. We, and others, have developed an alternative technology for RNAi in mammalian cells, termed endoribonuclease-prepared siRNA (esiRNA), which is based on the enzymatic generation of siRNA pools by digestion of long dsRNAs with recombinant RNase III in vitro (Yang et al., Proc Natl Acad Sci USA 99: 9942-9947, 2002; Myers et al., Nat Biotechnol 21:324-328; 2003). This technology has proven to be cost-efficient and reliable. Furthermore, several studies have demonstrated that complex pools of siRNAs, as inherent in esiRNAs, which target one transcript reduce off-target effects (Myers et al., J RNAi Gene Silencing 2:181, 2006; Kittler et al., Nat Methods 4:337-344, 2007). Within this chapter we describe design criteria for the generation of target-optimized esiRNAs.

Monalisa Mishra, Elisabeth Knust
Analysis of the Drosophila compound eye with light and electron microscopy.
Methods Mol Biol, 935 161-182 (2013)
PDF DOI

The Drosophila compound eye is a regular structure, in which about 750 units, called ommatidia, are arranged in a highly regular pattern. Eye development proceeds in a stereotypical fashion, where epithelial cells of the eye imaginal discs are specified, recruited, and differentiated in a sequential order that leads to the highly precise structure of an adult eye. Even small perturbations, for example in signaling pathways that control proliferation, cell death, or differentiation, can impair the regular structure of the eye, which can be easily detected and analyzed. In addition, the Drosophila eye has proven to be an ideal model for studying the genetic control of neurodegeneration, since the eye is not essential for viability. Several human neurodegeneration diseases have been modeled in the fly, leading to a better understanding of the function/misfunction of the respective gene. In many cases, the genes involved and their function are conserved between flies and human. More strikingly, when ectopically expressed in the fly eye some human genes without a Drosophila counterpart can induce neurodegeneration, detectable by aberrant phototaxis, impaired electrophysiology, or defects in eye morphology. These defects are often rather subtle alteration in shape, size, or arrangement of the cells, and can be easily scored at the ultrastructural level. This chapter aims to provide an overview regarding the analysis of the retina by various means.

Martin Machyna, Patricia Heyn, Karla M. Neugebauer
Cajal bodies: where form meets function.
Wiley Interdiscip Rev RNA, 4(1) 17-34 (2013)
PDF DOI

The cell nucleus contains dozens of subcompartments that separate biochemical processes into confined spaces. Cajal bodies (CBs) were discovered more than 100 years ago, but only extensive research in the past decades revealed the surprising complexity of molecular and cellular functions taking place in these structures. Many protein and RNA species are modified and assembled within CBs, which have emerged as a meeting place and factory for ribonucleoprotein (RNP) particles involved in splicing, ribosome biogenesis and telomere maintenance. Recently, a distinct structure near histone gene clusters--the Histone locus body (HLB)--was discovered. Involved in histone mRNA 3'-end formation, HLBs can share several components with CBs. Whether the appearance of distinct HLBs is simply a matter of altered affinity between these structures or of an alternate mode of CB assembly is unknown. However, both structures share basic assembly properties, in which transcription plays a decisive role in initiation. After this seeding event, additional components associate in random order. This appears to be a widespread mechanism for body assembly. CB assembly encompasses an additional layer of complexity, whereby a set of pre-existing substructures can be integrated into mature CBs. We propose this as a multi-seeding model of CB assembly.

Melek Asli Kayserili^*, Dave T. Gerrard^*, Pavel Tomancak^#, Alex T. Kalinka^#
An excess of gene expression divergence on the x chromosome in Drosophila embryos: implications for the faster-x hypothesis.
PLoS Genet, 8(12) Art. No. e1003200 (2012)
DOI

The X chromosome is present as a single copy in the heterogametic sex, and this hemizygosity is expected to drive unusual patterns of evolution on the X relative to the autosomes. For example, the hemizgosity of the X may lead to a lower chromosomal effective population size compared to the autosomes, suggesting that the X might be more strongly affected by genetic drift. However, the X may also experience stronger positive selection than the autosomes, because recessive beneficial mutations will be more visible to selection on the X where they will spend less time being masked by the dominant, less beneficial allele-a proposal known as the faster-X hypothesis. Thus, empirical studies demonstrating increased genetic divergence on the X chromosome could be indicative of either adaptive or non-adaptive evolution. We measured gene expression in Drosophila species and in D. melanogaster inbred strains for both embryos and adults. In the embryos we found that expression divergence is on average more than 20% higher for genes on the X chromosome relative to the autosomes; but in contrast, in the inbred strains, gene expression variation is significantly lower on the X chromosome. Furthermore, expression divergence of genes on Muller's D element is significantly greater along the branch leading to the obscura sub-group, in which this element segregates as a neo-X chromosome. In the adults, divergence is greatest on the X chromosome for males, but not for females, yet in both sexes inbred strains harbour the lowest level of gene expression variation on the X chromosome. We consider different explanations for our results and conclude that they are most consistent within the framework of the faster-X hypothesis.

Kim Schneider, Thomas Köcher, Teemu Andersin, Teymuras V. Kurzchalia, Ueli Schibler, David Gatfield
CAVIN-3 regulates circadian period length and PER:CRY protein abundance and interactions.
EMBO Rep, 13(12) 1138-1144 (2012)
DOI

In mammals, transcriptional autorepression by Period (PER) and Cryptochrome (CRY) protein complexes is essential for the generation of circadian rhythms. We have identified CAVIN-3 as a new, cytoplasmic PER2-interacting protein influencing circadian clock properties. Thus, CAVIN-3 loss- and gain-of-function shortened and lengthened, respectively, the circadian period in fibroblasts and affected PER:CRY protein abundance and interaction. While depletion of protein kinase Cδ (PKCδ), a known partner of CAVIN-3, had little effect on circadian gene expression, CAVIN-3 required the PKCδ-binding site to exert its effect on period length. This suggests the involvement of yet uncharacterized protein kinases. Finally, CAVIN-3 activity in circadian gene expression was independent of caveolae.

Andrew Dauber, Stephen H Lafranchi, Zoltan Maliga, Julian C Lui, Jennifer E Moon, Cailin McDeed, Katrin Henke, Jonathan Zonana, Garrett A Kingman, Tune H Pers, Jeffrey Baron, Ron G Rosenfeld, Joel N Hirschhorn, Matthew P Harris, Vivian Hwa
Novel microcephalic primordial dwarfism disorder associated with variants in the centrosomal protein ninein.
J Clin Endocrinol Metab, 97(11) 2140-2151 (2012)
DOI

Microcephalic primordial dwarfism (MPD) is a rare, severe form of human growth failure in which growth restriction is evident in utero and continues into postnatal life. Single causative gene defects have been identified in a number of patients with MPD, and all involve genes fundamental to cellular processes including centrosome functions.

Eunyoung Choi, Marine R-C Kraus, Laurence A Lemaire, Momoko Yoshimoto, Sasidhar Vemula, Leah A Potter, Elisabetta Manduchi, Christian J Stoeckert, Anne Grapin-Botton, Mark A Magnuson
Dual lineage-specific expression of Sox17 during mouse embryogenesis.
Stem Cells, 30(10) 2297-2308 (2012)
DOI

Sox17 is essential for both endoderm development and fetal hematopoietic stem cell (HSC) maintenance. While endoderm-derived organs are well known to originate from Sox17-expressing cells, it is less certain whether fetal HSCs also originate from Sox17-expressing cells. By generating a Sox17(GFPCre) allele and using it to assess the fate of Sox17-expressing cells during embryogenesis, we confirmed that both endodermal and a part of definitive hematopoietic cells are derived from Sox17-positive cells. Prior to E9.5, the expression of Sox17 is restricted to the endoderm lineage. However, at E9.5 Sox17 is expressed in the endothelial cells (ECs) at the para-aortic splanchnopleural region that contribute to the formation of HSCs at a later stage. The identification of two distinct progenitor cell populations that express Sox17 at E9.5 was confirmed using fluorescence-activated cell sorting together with RNA-Seq to determine the gene expression profiles of the two cell populations. Interestingly, this analysis revealed differences in the RNA processing of the Sox17 mRNA during embryogenesis. Taken together, these results indicate that Sox17 is expressed in progenitor cells derived from two different germ layers, further demonstrating the complex expression pattern of this gene and suggesting caution when using Sox17 as a lineage-specific marker.

Michael Hiller^#, Bruce T Schaar, Vahan B Indjeian, David M Kingsley, Lee R Hagey, Gill Bejerano^#
A "Forward Genomics" Approach Links Genotype to Phenotype using Independent Phenotypic Losses among Related Species.
Cell Rep, 2(4) 817-823 (2012)
PDF DOI

Genotype-phenotype mapping is hampered by countless genomic changes between species. We introduce a computational "forward genomics" strategy that-given only an independently lost phenotype and whole genomes-matches genomic and phenotypic loss patterns to associate specific genomic regions with this phenotype. We conducted genome-wide screens for two metabolic phenotypes. First, our approach correctly matches the inactivated Gulo gene exactly with the species that lost the ability to synthesize vitamin C. Second, we attribute naturally low biliary phospholipid levels in guinea pigs and horses to the inactivated phospholipid transporter Abcb4. Human ABCB4 mutations also result in low phospholipid levels but lead to severe liver disease, suggesting compensatory mechanisms in guinea pig and horse. Our simulation studies, counts of independent changes in existing phenotype surveys, and the forthcoming availability of many new genomes all suggest that forward genomics can be applied to many phenotypes, including those relevant for human evolution and disease.

Yoanne M Clovis, Wolfgang Enard, Federica Marinaro, Wieland B. Huttner, Davide De Pietri Tonelli
Convergent repression of Foxp2 3'UTR by miR-9 and miR-132 in embryonic mouse neocortex: implications for radial migration of neurons.
Development, 139(18) 3332-3342 (2012)
DOI

MicroRNAs (miRNAs) are rapidly emerging as a new layer of regulation of mammalian brain development. However, most of the miRNA target genes remain unidentified. Here, we explore gene expression profiling upon miRNA depletion and in vivo target validation as a strategy to identify novel miRNA targets in embryonic mouse neocortex. By this means, we find that Foxp2, a transcription factor associated with speech and language development and evolution, is a novel miRNA target. In particular, we find that miR-9 and miR-132 are able to repress ectopic expression of Foxp2 protein by targeting its 3' untranslated region (3'UTR) in vivo. Interestingly, ectopic expression of Foxp2 in cortical projection neurons (a scenario that mimics the absence of miRNA-mediated silencing of Foxp2 expression) delays neurite outgrowth in vitro and impairs their radial migration in embryonic mouse neocortex in vivo. Our results uncover a new layer of control of Foxp2 expression that may be required for proper neuronal maturation.

Liliana Malinovska^*, Sonja Kroschwald^*, Matthias Munder^*, Doris Richter, Simon Alberti
Molecular chaperones and stress-inducible protein-sorting factors coordinate the spatiotemporal distribution of protein aggregates
Mol Biol Cell, 23(16) 3041-3056 (2012)
PDF DOI

Acute stress causes a rapid redistribution of protein quality control components and aggregation-prone proteins to diverse subcellular compartments. How these remarkable changes come about is currently not well understood. Using a phenotypic reporter for a synthetic yeast prion, we identified two protein sorting factors of the Hook family termed Btn2 and Cur1 as key regulators of spatial protein quality control in Saccharomyces cerevisiae. Btn2 and Cur1 are undetectable under normal growth conditions, but accumulate in stressed cells due to increased gene expression and reduced proteasomal turnover. Newly synthesized Btn2 can associate with the small heat shock protein Hsp42 to promote the sorting of misfolded proteins to a peripheral protein deposition site. Alternatively, Btn2 can bind to the chaperone Sis1 to facilitate the targeting of misfolded proteins to a juxtanuclear compartment. Protein redistribution by Btn2 is accompanied by a gradual depletion of Sis1 from the cytosol, which is mediated by the sorting factor Cur1. Based on these findings, we propose a dynamic model that explains the subcellular distribution of misfolded proteins as a function of the cytosolic concentrations of molecular chaperones and protein sorting factors. Our model suggests that protein aggregation is not a haphazard process, but rather an orchestrated cellular response that adjusts the flux of misfolded proteins to the capacities of the protein quality control system.

Nicole Bieberstein^*, Fernando Carrillo Oesterreich^*, Korinna Straube, Karla M. Neugebauer
First exon length controls active chromatin signatures and transcription.
Cell Rep, 2(1) 62-68 (2012)
PDF DOI

Here, we explore the role of splicing in transcription, employing both genome-wide analysis of human ChIP-seq data and experimental manipulation of exon-intron organization in transgenic cell lines. We show that the activating histone modifications H3K4me3 and H3K9ac map specifically to first exon-intron boundaries. This is surprising, because these marks help recruit general transcription factors (GTFs) to promoters. In genes with long first exons, promoter-proximal levels of H3K4me3 and H3K9ac are greatly reduced; consequently, GTFs and RNA polymerase II are low at transcription start sites (TSSs) and exhibit a second, promoter-distal peak from which transcription also initiates. In contrast, short first exons lead to increased H3K4me3 and H3K9ac at promoters, higher expression levels, accuracy in TSS usage, and a lower frequency of antisense transcription. Therefore, first exon length is predictive for gene activity. Finally, splicing inhibition and intron deletion reduce H3K4me3 levels and transcriptional output. Thus, gene architecture and splicing determines transcription quantity and quality as well as chromatin signatures.

Hung Ping Shih, Janel L Kopp, Manbir Sandhu, Claire L Dubois, Philip Allan Seymour, Anne Grapin-Botton, Maike Sander
A Notch-dependent molecular circuitry initiates pancreatic endocrine and ductal cell differentiation.
Development, 139(14) 2488-2499 (2012)
DOI

In the pancreas, Notch signaling is thought to prevent cell differentiation, thereby maintaining progenitors in an undifferentiated state. Here, we show that Notch renders progenitors competent to differentiate into ductal and endocrine cells by inducing activators of cell differentiation. Notch signaling promotes the expression of Sox9, which cell-autonomously activates the pro-endocrine gene Ngn3. However, at high Notch activity endocrine differentiation is blocked, as Notch also induces expression of the Ngn3 repressor Hes1. At the transition from high to intermediate Notch activity, only Sox9, but not Hes1, is maintained, thus de-repressing Ngn3 and initiating endocrine differentiation. In the absence of Sox9 activity, endocrine and ductal cells fail to differentiate, resulting in polycystic ducts devoid of primary cilia. Although Sox9 is required for Ngn3 induction, endocrine differentiation necessitates subsequent Sox9 downregulation and evasion from Notch activity via cell-autonomous repression of Sox9 by Ngn3. If high Notch levels are maintained, endocrine progenitors retain Sox9 and undergo ductal fate conversion. Taken together, our findings establish a novel role for Notch in initiating both ductal and endocrine development and reveal that Notch does not function in an on-off mode, but that a gradient of Notch activity produces distinct cellular states during pancreas development.

Asifa Akhtar, Karla M. Neugebauer
The nucleus and gene expression: the center of the cyclone
Curr Opin Cell Biol, 24(3) 293-295 (2012)
PDF DOI

Christoph Zechner, Jakob Ruess, Peter Krenn, Serge Pelet, Matthias Peter, John Lygeros, Heinz Koeppl
Moment-based inference predicts bimodality in transient gene expression.
Proc Natl Acad Sci U.S.A., 109(21) 8340-8345 (2012)
DOI

Recent computational studies indicate that the molecular noise of a cellular process may be a rich source of information about process dynamics and parameters. However, accessing this source requires stochastic models that are usually difficult to analyze. Therefore, parameter estimation for stochastic systems using distribution measurements, as provided for instance by flow cytometry, currently remains limited to very small and simple systems. Here we propose a new method that makes use of low-order moments of the measured distribution and thereby keeps the essential parts of the provided information, while still staying applicable to systems of realistic size. We demonstrate how cell-to-cell variability can be incorporated into the analysis obviating the need for the ubiquitous assumption that the measurements stem from a homogeneous cell population. We demonstrate the method for a simple example of gene expression using synthetic data generated by stochastic simulation. Subsequently, we use time-lapsed flow cytometry data for the osmo-stress induced transcriptional response in budding yeast to calibrate a stochastic model, which is then used as a basis for predictions. Our results show that measurements of the mean and the variance can be enough to determine the model parameters, even if the measured distributions are not well-characterized by low-order moments only--e.g., if they are bimodal.

Bettina C Kirchmaier, Kar Lai Poon, Thorsten Schwerte, Jan Huisken, Christoph Winkler, Benno Jungblut, Didier Y.R. Stainier, Thomas Brand
The Popeye domain containing 2 (popdc2) gene in zebrafish is required for heart and skeletal muscle development.
Dev Biol, 363(2) 438-450 (2012)
DOI

The Popeye domain containing (Popdc) genes encode a family of transmembrane proteins with an evolutionary conserved Popeye domain. These genes are abundantly expressed in striated muscle tissue, however their function is not well understood. In this study we have investigated the role of the popdc2 gene in zebrafish. Popdc2 transcripts were detected in the embryonic myocardium and transiently in the craniofacial and tail musculature. Morpholino oligonucleotide-mediated knockdown of popdc2 resulted in aberrant development of skeletal muscle and heart. Muscle segments in the trunk were irregularly shaped and craniofacial muscles were severely reduced or even missing. In the heart, pericardial edema was prevalent in the morphants and heart chambers were elongated and looping was abnormal. These pathologies in muscle and heart were alleviated after reducing the morpholino concentration. However the heart still was abnormal displaying cardiac arrhythmia at later stages of development. Optical recordings of cardiac contractility revealed irregular ventricular contractions with a 2:1, or 3:1 atrial/ventricular conduction ratio, which caused a significant reduction in heart frequency. Recordings of calcium transients with high spatiotemporal resolution using a transgenic calcium indicator line (Tg(cmlc2:gCaMP)(s878)) and SPIM microscopy confirmed the presence of a severe arrhythmia phenotype. Our results identify popdc2 as a gene important for striated muscle differentiation and cardiac morphogenesis. In addition it is required for the development of the cardiac conduction system.

Li Ding, Ina Poser, Maciej Paszkowski-Rogacz, Frank Buchholz
From RNAi screens to molecular function in embryonic stem cells
Stem cell rev, 8(1) 32-42 (2012)
DOI

The ability of embryonic stem (ES) cells to generate any of the around 220 cell types of the adult body has fascinated scientists ever since their discovery. The capacity to re-program fully differentiated cells into induced pluripotent stem (iPS) cells has further stimulated the interest in ES cell research. Fueled by this interest, intense research has provided new insights into the biology of ES cells in the recent past. The development of large-scale and high throughput RNAi technologies has made it possible to sample the role of every gene in maintaining ES cell identity. Here, we review the RNAi screens performed in ES cells to date and discuss the challenges associated with these large-scale experiments. Furthermore, we provide a perspective on how to streamline the molecular characterization following the initial phenotypic description utilizing bacterial artificial chromosome (BAC) transgenesis.

Cristina Aguirre-Portolés, Alexander W. Bird, Anthony A. Hyman, Marta Cañamero, Ignacio Pérez de Castro, Marcos Malumbres
Tpx2 controls spindle integrity, genome stability, and tumor development.
Cancer Res, 72(6) 1518-1528 (2012)
PDF DOI

Tpx2 is a microtubule-associated protein that activates the cell-cycle kinase Aurora A and regulates the mitotic spindle. Overexpression of Tpx2 is associated with the development of different human tumors and strongly correlates with chromosomal instability. By analyzing a conditional null mutation in the mouse Tpx2 gene, we show here that Tpx2 expression is essential for spindle function and chromosome segregation in the mouse embryo. Conditional genetic ablation of Tpx2 in primary cultures resulted in deficient microtubule nucleation from DNA and aberrant spindles during prometaphase. These cells eventually exited from mitosis without chromosome segregation. In addition, Tpx2 haploinsufficiency led to the accumulation of aneuploidies in vivo and increased susceptibility to spontaneous lymphomas and lung tumors. Together, our findings indicate that Tpx2 is essential for maintaining genomic stability through its role in spindle regulation. Subtle changes in Tpx2 expression may favor tumor development in vivo.

Nancy Thompson, Emilie Gésina, Peter Scheinert, Philipp Bucher, Anne Grapin-Botton
RNA profiling and chromatin immunoprecipitation-sequencing reveal that PTF1a stabilizes pancreas progenitor identity via the control of MNX1/HLXB9 and a network of other transcription factors.
Mol Cell Biol, 32(6) 1189-1199 (2012)
DOI

Pancreas development is initiated by the specification and expansion of a small group of endodermal cells. Several transcription factors are crucial for progenitor maintenance and expansion, but their interactions and the downstream targets mediating their activity are poorly understood. Among those factors, PTF1a, a basic helix-loop-helix (bHLH) transcription factor which controls pancreas exocrine cell differentiation, maintenance, and functionality, is also needed for the early specification of pancreas progenitors. We used RNA profiling and chromatin immunoprecipitation (ChIP) sequencing to identify a set of targets in pancreas progenitors. We demonstrate that Mnx1, a gene that is absolutely required in pancreas progenitors, is a major direct target of PTF1a and is regulated by a distant enhancer element. Pdx1, Nkx6.1, and Onecut1 are also direct PTF1a targets whose expression is promoted by PTF1a. These proteins, most of which were previously shown to be necessary for pancreas bud maintenance or formation, form a transcription factor network that allows the maintenance of pancreas progenitors. In addition, we identify Bmp7, Nr5a2, RhoV, and P2rx1 as new targets of PTF1a in pancreas progenitors.

Elena Taverna, Christiane Haffner, Rainer Pepperkok, Wieland B. Huttner
A new approach to manipulate the fate of single neural stem cells in tissue.
Nat Neurosci, 15(2) 329-337 (2012)
DOI

A challenge in the field of neural stem cell biology is the mechanistic dissection of single stem cell behavior in tissue. Although such behavior can be tracked by sophisticated imaging techniques, current methods of genetic manipulation do not allow researchers to change the level of a defined gene product on a truly acute time scale and are limited to very few genes at a time. To overcome these limitations, we established microinjection of neuroepithelial/radial glial cells (apical progenitors) in organotypic slice culture of embryonic mouse brain. Microinjected apical progenitors showed cell cycle parameters that were indistinguishable to apical progenitors in utero, underwent self-renewing divisions and generated neurons. Microinjection of single genes, recombinant proteins or complex mixtures of RNA was found to elicit acute and defined changes in apical progenitor behavior and progeny fate. Thus, apical progenitor microinjection provides a new approach to acutely manipulating single neural stem and progenitor cells in tissue.

Natalia Tapia, Peter Reinhardt, Annett Duemmler, Guangming Wu, Marcos J Araúzo-Bravo, Daniel Esch, Boris Greber, Vlad Cojocaru, Cynthia Alexander Rascon, Akira Tazaki, Kevin Kump, Randal Voss, Elly M. Tanaka, Hans R Schöler
Reprogramming to pluripotency is an ancient trait of vertebrate Oct4 and Pou2 proteins.
Nat Commun, 3 Art. No. 1279 (2012)
DOI

The evolutionary origins of the gene network underlying cellular pluripotency, a central theme in developmental biology, have yet to be elucidated. In mammals, Oct4 is a factor crucial in the reprogramming of differentiated cells into induced pluripotent stem cells. The Oct4 and Pou2 genes evolved from a POU class V gene ancestor, but it is unknown whether pluripotency induced by Oct4 gene activity is a feature specific to mammals or was already present in ancestral vertebrates. Here we report that different vertebrate Pou2 and Oct4 homologues can induce pluripotency in mouse and human fibroblasts and that the inability of zebrafish Pou2 to establish pluripotency is not representative of all Pou2 genes, as medaka Pou2 and axolotl Pou2 are able to reprogram somatic cells into pluripotent cells. Therefore, our results indicate that induction of pluripotency is not a feature specific to mammals, but existed in the Oct4/Pou2 common ancestral vertebrate.

Alexander W. Bird, Axel Erler, Jun Fu, Jean-Karim Hériché, Marcello Maresca, Youming Zhang, Anthony A. Hyman, A Francis Stewart
High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes.
Nat Methods, 9(1) 103-109 (2012)
PDF DOI

Whereas bacterial artificial chromosomes (BACs) offer many advantages in studies of gene and protein function, generation of seamless, precisely mutated BACs has been difficult. Here we describe a counterselection-based recombineering method and its accompanying reagents. After identifying intramolecular recombination as the major problem in counterselection, we built a strategy to reduce these unwanted events by expressing Redβ alone at the crucial step. We enhanced this method by using phosphothioated oligonucleotides, using a sequence-altered rpsL counterselection gene and developing online software for oligonucleotide design. We illustrated this method by generating transgenic mammalian cell lines carrying small interfering RNA-resistant and point-mutated BAC transgenes. Using this approach, we generated mutated TACC3 transgenes to identify phosphorylation-specific spindle defects after knockdown of endogenous TACC3 expression. Our results highlight the complementary use of precisely mutated BAC transgenes and RNA interference in the study of cell biology at physiological expression levels and regulation.

Shubha Vij^*, Jochen Rink^*, Hao Kee Ho, Deepak Babu, Michael Eitel, Vijayashankaranarayanan Narasimhan, Varnesh Tiku, Jody Westbrook, Bernd Schierwater, Sudipto Roy
Evolutionarily ancient association of the FoxJ1 transcription factor with the motile ciliogenic program.
PLoS Genet, 8(11) Art. No. e1003019 (2012)
DOI

It is generally believed that the last eukaryotic common ancestor (LECA) was a unicellular organism with motile cilia. In the vertebrates, the winged-helix transcription factor FoxJ1 functions as the master regulator of motile cilia biogenesis. Despite the antiquity of cilia, their highly conserved structure, and their mechanism of motility, the evolution of the transcriptional program controlling ciliogenesis has remained incompletely understood. In particular, it is presently not known how the generation of motile cilia is programmed outside of the vertebrates, and whether and to what extent the FoxJ1-dependent regulation is conserved. We have performed a survey of numerous eukaryotic genomes and discovered that genes homologous to foxJ1 are restricted only to organisms belonging to the unikont lineage. Using a mis-expression assay, we then obtained evidence of a conserved ability of FoxJ1 proteins from a number of diverse phyletic groups to activate the expression of a host of motile ciliary genes in zebrafish embryos. Conversely, we found that inactivation of a foxJ1 gene in Schmidtea mediterranea, a platyhelminth (flatworm) that utilizes motile cilia for locomotion, led to a profound disruption in the differentiation of motile cilia. Together, all of these findings provide the first evolutionary perspective into the transcriptional control of motile ciliogenesis and allow us to propose a conserved FoxJ1-regulated mechanism for motile cilia biogenesis back to the origin of the metazoans.

Thomas Kurth, Susanne Weiche, Daniela Vorkel, Susanne Kretschmar, Anja Menge
Histology of plastic embedded amphibian embryos and larvae.
Genesis, 50(3) 235-250 (2012)
DOI

Amphibians including the South African clawed frog Xenopus laevis, its close relative Xenopus tropicalis, and the Mexican axolotl (Ambystoma mexicanum) are important vertebrate models for cell biology, development and regeneration. For the analysis of embryos and larva with altered gene expression in gain-of-function or loss-of-function studies histology is increasingly important. Here, we discuss plastic or resin embedding of embryos as valuable alternatives to conventional paraffin embedding. For example, microwave-assisted tissue processing, combined with embedding in the glycol methacrylate Technovit 7100, is a fast, simple and reliable method to obtain state-of-the-art histology with high resolution of cellular details in less than a day. Microwave-processed samples embedded in Epon 812 are also useful for transmission electron microscopy. Finally, Technovit-embedded samples are well suited for serial section analysis of embryos labeled either by whole-mount immunofluorescence, or with tracers such as GFP or fluorescent dextrans. Therefore, plastic embedding offers a versatile alternative to paraffin embedding for routine histology and immunocytochemistry of amphibian embryos. © 2011 Wiley-Liss, Inc.

Lakshmikanth Mariyanna^*, Poornima Priyadarshini^*, Helga Hofmann-Sieber, Marcel Krepstakies, Nicole Walz, Adam Grundhoff, Frank Buchholz, Eberhard Hildt, Joachim Hauber
Excision of HIV-1 proviral DNA by recombinant cell permeable tre-recombinase.
PLoS ONE, 7(2) Art. No. e31576 (2012)
DOI

Over the previous years, comprehensive studies on antiretroviral drugs resulted in the successful introduction of highly active antiretroviral therapy (HAART) into clinical practice for treatment of HIV/AIDS. However, there is still need for new therapeutic approaches, since HAART cannot eradicate HIV-1 from the infected organism and, unfortunately, can be associated with long-term toxicity and the development of drug resistance. In contrast, novel gene therapy strategies may have the potential to reverse the infection by eradicating HIV-1. For example, expression of long terminal repeat (LTR)-specific recombinase (Tre-recombinase) has been shown to result in chromosomal excision of proviral DNA and, in consequence, in the eradication of HIV-1 from infected cell cultures. However, the delivery of Tre-recombinase currently depends on the genetic manipulation of target cells, a process that is complicating such therapeutic approaches and, thus, might be undesirable in a clinical setting. In this report we demonstrate that E.coli expressed Tre-recombinases, tagged either with the protein transduction domain (PTD) from the HIV-1 Tat trans-activator or the translocation motif (TLM) of the Hepatitis B virus PreS2 protein, were able to translocate efficiently into cells and showed significant recombination activity on HIV-1 LTR sequences. Tre activity was observed using episomal and stable integrated reporter constructs in transfected HeLa cells. Furthermore, the TLM-tagged enzyme was able to excise the full-length proviral DNA from chromosomal integration sites of HIV-1-infected HeLa and CEM-SS cells. The presented data confirm Tre-recombinase activity on integrated HIV-1 and provide the basis for the non-genetic transient application of engineered recombinases, which may be a valuable component of future HIV eradication strategies.

Ursula Ehmer, Sandra Kalthoff, Bastian Fakundiny, Brigitte Pabst, Nicole Freiberg, Ronald Naumann, Michael P Manns, Christian P Strassburg
Gilbert syndrome redefined: A complex genetic haplotype influences the regulation of glucuronidation.
Hepatology, 55(6) 1912-1921 (2012)
DOI

Gilbert syndrome (GS) is characterized by intermittent unconjugated hyperbilirubinemia without structural liver damage affecting about 10% of the white population. In GS the UGT1A1*28 variant reduces bilirubin conjugation by 70% and is associated with irinotecan and protease inhibitor side effects. Aim of this study was to characterize potential in vivo consequences of UGT1A gene variability in GS. Three hundred GS patients (UGT1A1*28 homozygous) and 249 healthy blood donors (HBD) were genotyped for UGT1A (UGT1A1*28, UGT1A3-66 T>C, UGT1A6*3a, UGT1A7*3) and transporter SNPs (SCLO1B1 p.V174A, SCLO1B1 p.N130D, ABCC2 p.I1324I, ABCC2 -24 UTR) using TaqMan-5'-nuclease-assays. A humanized transgenic UGT1A-SNP and corresponding wild-type mouse model were established carrying the GS-associated UGT1A variant haplotype. UGT1A transcript and protein expression, and transcriptional activation were studied in vivo. Homozygous UGT1A1*28 GS individuals were simultaneously homozygous for UGT1A3-66 T>C (91%), UGT1A6*2a (77%), and UGT1A7*3 (77%). Seventy-six percent of GS and only 9% of HBD were homozygous for the variant haplotype spanning 4 UGT1A genes. SCLO1B1 and ABCC2 single nucleotide polymorphisms (SNPs) showed no differences. In transgenic humanized UGT1A SNP and wild-type mice this UGT1A haplotype led to lower UGT1A mRNA expression and UGT1A protein synthesis. UGT1A transcriptional activation by dioxin, phenobarbital and endotoxin was significantly reduced in SNP mice. CONCLUSION: Our data redefine the genetic basis behind GS. In vivo data studying the genotype present in 76% of GS individuals suggest that transcription and transcriptional activation of glucuronidation genes responsible for conjugation and detoxification is directly affected leading to lower responsiveness. This study suggests that GS should be considered as a potential risk factor for drug toxicity. (HEPATOLOGY 2011.).

Bon-Kyoung Koo^*, Daniel E Stange^*, Toshiro Sato, Wouter Karthaus, Henner F Farin, Meritxell Huch, Johan H van Es, Hans Clevers
Controlled gene expression in primary Lgr5 organoid cultures.
Nat Methods, 9(1) 81-83 (2011)
DOI

The study of gene function in endodermal epithelia such as of stomach, small intestine and colon relies heavily on transgenic approaches. Establishing such animal models is laborious, expensive and time-consuming. We present here a method based on Cre recombinase-inducible retrovirus vectors that allows the conditional manipulation of gene expression in primary mouse organoid culture systems.

Frank M J Jacobs^*, Jesse V Veenvliet^*, Wadia H Almirza, Elisa J Hoekstra, Lars von Oerthel, Annemarie J A van der Linden, Roel Neijts, Marian J A Groot Koerkamp, Dik van Leenen, Frank C P Holstege, J Peter H Burbach, Marten P Smidt
Retinoic acid-dependent and -independent gene-regulatory pathways of Pitx3 in meso-diencephalic dopaminergic neurons.
Development, 138(23) 5213-5222 (2011)
DOI

Development of meso-diencephalic dopamine (mdDA) neurons requires the combined actions of the orphan nuclear receptor Nurr1 and the paired-like homeobox transcription factor Pitx3. Whereas all mdDA neurons require Nurr1 for expression of Th and survival, dependence on Pitx3 is displayed only by the mdDA subpopulation that will form the substantia nigra (SNc). Previously, we have demonstrated that Pitx3(-/-) embryos lack the expression of the retinoic acid (RA)-generating enzyme Ahd2, which is normally selectively expressed in the Pitx3-dependent DA neurons of the SNc. Restoring RA signaling in Pitx3(-/-) embryos revealed a selective dependence of SNc neurons on the presence of RA for differentiation into Th-positive neurons and maintenance throughout embryonic development. Whereas these data are suggestive of an important developmental role for RA in neurons of the SNc, it remained unclear whether other Nurr1 and Pitx3 target genes depend on RA signaling in a manner similar to Th. In the search for genes that were affected in Pitx3-deficient mdDA neurons and restored upon embryonic RA treatment, we provide evidence that Delta-like 1, D2R (Drd2) and Th are regulated by Pitx3 and RA signaling, which influences the mdDA terminal differentiated phenotype. Furthermore, we show that regulation of Ahd2-mediated RA signaling represents only one aspect of the Pitx3 downstream cascade, as Vmat2, Dat, Ahd2 (Aldh1a1), En1, En2 and Cck were unaffected by RA treatment and are (subset) specifically modulated by Pitx3. In conclusion, our data reveal several RA-dependent and -independent aspects of the Pitx3-regulated gene cascade, suggesting that Pitx3 acts on multiple levels in the molecular subset-specification of mdDA neurons.

Daniele Soroldoni, Andrew C. Oates
Live transgenic reporters of the vertebrate embryo's Segmentation Clock
Curr Opin Genet Dev, 21(5) 600-605 (2011)
DOI

Imaging rapidly changing gene expression during embryogenesis is a challenge for the development of probes and imaging techniques. The vertebrate Segmentation Clock is a genetic network that controls the subdivision of the elongating embryonic body axis into somites, the precursors of adult segmented structures, such as vertebrae. Because of its rapid oscillations, direct observation of gene expression in this system has proven difficult, and so is a benchmark for transgene design and imaging in vivo. Transgenic approaches using destabilized reporter cassettes in the mouse embryo have provided the first glimpses of this dynamic expression system. Nevertheless, improvements in temporal and spatial resolution, paired with the ability to make precise quantifications, will be necessary to connect observations and theory.

Lei Qu, Fuhui Long, Xiao Liu, Stuart Kim, Eugene Myers, Hanchuan Peng
Simultaneous recognition and segmentation of cells: application in C.elegans.
Bioinformatics, 27(20) 2895-2902 (2011)
PDF DOI

Automatic recognition of cell identities is critical for quantitative measurement, targeting and manipulation of cells of model animals at single-cell resolution. It has been shown to be a powerful tool for studying gene expression and regulation, cell lineages and cell fates. Existing methods first segment cells, before applying a recognition algorithm in the second step. As a result, the segmentation errors in the first step directly affect and complicate the subsequent cell recognition step. Moreover, in new experimental settings, some of the image features that have been previously relied upon to recognize cells may not be easy to reproduce, due to limitations on the number of color channels available for fluorescent imaging or to the cost of building transgenic animals. An approach that is more accurate and relies on only a single signal channel is clearly desirable.

Ivana Viktorinova, Lucie Kucerova, Marta Bohmova, Ian Henry, Marek Jindra, Petr Dolezal, Martina Zurovcova, Michal Zurovec
Characterization of two closely related α-amylase paralogs in the bark beetle, Ips typographus (L.).
Arch Insect Biochem Physiol, 77(4) 179-198 (2011)
DOI

Ips typographus (L.), the eight-spined spruce bark beetle, causes severe damage throughout Eurasian spruce forests and suitable nuclear markers are needed in order to study its population structure on a genetic level. Two closely related genes encoding α-amylase in I. typographus were characterized and named AmyA and AmyB. Both α-amylase paralogs consisted of six exons and five introns. AmyA encodes a polypeptide of 483 amino acids, whereas AmyB has two alternative transcripts encoding polypeptides of 483 and 370 amino acids. The expression levels of both genes were high during larval stage and adulthood. The AmyB transcripts were absent in the pupal stage. A modification of the allozyme staining method allowed us to detect two clusters of bands on the electrophoretic gel that may correspond to the two α-amylase genes. There was a correlation between the lack of AmyB expression in pupa and the absence of the fast migrating isozyme cluster at this stage, suggesting that the faster migrating isoforms are products of the AmyB gene, whereas the slowly migrating bands are derived from the AmyA.

Dragomir Krastev, Mikolaj Slabicki, Maciej Paszkowski-Rogacz, Nina C Hubner, Magno Junqueira, Andrej Shevchenko, Matthias Mann, Karla M. Neugebauer, Frank Buchholz
A systematic RNAi synthetic interaction screen reveals a link between p53 and snoRNP assembly.
Nat Cell Biol, 13(7) 809-818 (2011)
DOI

TP53 (tumour protein 53) is one of the most frequently mutated genes in human cancer and its role during cellular transformation has been studied extensively. However, the homeostatic functions of p53 are less well understood. Here, we explore the molecular dependency network of TP53 through an RNAi-mediated synthetic interaction screen employing two HCT116 isogenic cell lines and a genome-scale endoribonuclease-prepared short interfering RNA library. We identify a variety of TP53 synthetic interactions unmasking the complex connections of p53 to cellular physiology and growth control. Molecular dissection of the TP53 synthetic interaction with UNRIP indicates an enhanced dependency of TP53-negative cells on small nucleolar ribonucleoprotein (snoRNP) assembly. This dependency is mediated by the snoRNP chaperone gene NOLC1 (also known as NOPP140), which we identify as a physiological p53 target gene. This unanticipated function of TP53 in snoRNP assembly highlights the potential of RNAi-mediated synthetic interaction screens to dissect molecular pathways of tumour suppressor genes.

Dominic Rose, Michael Hiller, Katharina Schutt, Jörg Hackermüller, Rolf Backofen, Peter F Stadler
Computational discovery of human coding and non-coding transcripts with conserved splice sites.
Bioinformatics, 27(14) 1894-1900 (2011)
PDF DOI

MOTIVATION: Long non-coding RNAs (lncRNAs) resemble protein-coding mRNAs but do not encode proteins. Most lncRNAs are under lower sequence constraints than protein-coding genes and lack conserved secondary structures, making it hard to predict them computationally. RESULTS: We introduce an approach to predict spliced lncRNAs in vertebrate genomes combining comparative genomics and machine learning. It is based on detecting signatures of characteristic splice site evolution in vertebrate whole genome alignments. First, we predict individual splice sites, then assemble compatible sites into exon candidates, and finally predict multi-exon transcripts. Using a novel method to evaluate typical splice site substitution patterns that explicitly takes the species phylogeny into account, we show that individual splice sites can be accurately predicted. Since our approach relies only on predicted splice sites, it can uncover both coding and non-coding exons. We show that our predicted exons and partial transcripts are mostly non-coding and lack conserved secondary structures. These exons are of particular interest, since existing computational approaches cannot detect them. Transcriptome sequencing data indicate tissue-specific expression patterns of predicted exons and there is evidence that increasing sequencing depth and breadth will validate additional predictions. We also found a significant enrichment of predicted exons that form multi-exon transcript parts, and we experimentally validate such a novel multi-exon gene. Overall, we obtain 336 novel multi-exon transcript predictions from human intergenic regions. Our results indicate the existence of novel human transcripts that are conserved in evolution and our approach contributes to the completion of the human transcript catalog. AVAILABILITY AND IMPLEMENTATION: Predicted human splice sites, exons and gene structures together with a Perl implementation of the tree-based log-odds scoring and a supplementary PDF file containing additional figures and tables are available at: http://www.bioinf.uni-leipzig.de/publications/supplements/10-010. The five experimentally confirmed partial transcript isoforms have been deposited in GenBank under accession numbers HM587422-HM587426.

Rebecca A Green, Huey-Ling Kao, Anjon Audhya, Swathi Arur, Jonathan R Mayers, Heidi N Fridolfsson, Monty Schulman, Siegfried Schloissnig, Sherry Niessen, Kimberley Laband, Shaohe Wang, Daniel A Starr, Anthony A. Hyman, Tim Schedl, Arshad Desai, Fabio Piano, Kristin C. Gunsalus^#, Karen Oegema^#
A high-resolution C. elegans essential gene network based on phenotypic profiling of a complex tissue.
Cell, 145(3) 470-482 (2011)
PDF DOI

High-content screening for gene profiling has generally been limited to single cells. Here, we explore an alternative approach-profiling gene function by analyzing effects of gene knockdowns on the architecture of a complex tissue in a multicellular organism. We profile 554 essential C. elegans genes by imaging gonad architecture and scoring 94 phenotypic features. To generate a reference for evaluating methods for network construction, genes were manually partitioned into 102 phenotypic classes, predicting functions for uncharacterized genes across diverse cellular processes. Using this classification as a benchmark, we developed a robust computational method for constructing gene networks from high-content profiles based on a network context-dependent measure that ranks the significance of links between genes. Our analysis reveals that multi-parametric profiling in a complex tissue yields functional maps with a resolution similar to genetic interaction-based profiling in unicellular eukaryotes-pinpointing subunits of macromolecular complexes and components functioning in common cellular processes.

Helmut Hofemeister, Giovanni Ciotta, Jun Fu, Philipp Martin Seibert, Alexander Schulz, Marcello Maresca, Mihail Sarov, Konstantinos Anastassiadis, A. Francis Stewart
Recombineering, transfection, Western, IP and ChIP methods for protein tagging via gene targeting or BAC transgenesis.
Methods, 53(4) 437-452 (2011)
PDF DOI

Protein tagging offers many advantages for proteomic and regulomic research. Ideally, protein tagging is equivalent to having a high affinity antibody for every chosen protein. However, these advantages are compromised if the tagged protein is overexpressed, which is usually the case from cDNA expression vectors. Physiological expression of tagged proteins can be achieved by gene targeting to knock-in the protein tag or by BAC transgenesis. BAC transgenes usually retain the native gene architecture including all cis-regulatory elements as well as the exon-intron configurations. Consequently most BAC transgenes are authentically regulated (e.g. by transcription factors, cell cycle, miRNA) and can be alternatively spliced. Recombineering has become the method of choice for generating targeting constructs or modifying BACs. Here we present methods with detailed protocols for protein tagging by recombineering for BAC transgenesis and/or gene targeting, including the evaluation of tagged protein expression, the retrieval of associated protein complexes for mass spectrometry and the use of the tags in ChIP (chromatin immunoprecipitation).

Mirko Theis, Frank Buchholz
High-throughput RNAi screening in mammalian cells with esiRNAs
Methods, 53(4) 424-429 (2011)
DOI

The development of advanced functional genomic tools has paved the way for systematic investigations of biological processes in health and disease. In particular, the implementation of RNA interference (RNAi) as a genome-wide, loss-of-function screening tool has enabled scientists to probe the role for every gene in cellular assays and many new factors for various processes have been discovered employing RNAi screens in recent years. However, the results also demonstrate the complexity of biological systems and indicate that we are still a long way from understanding functional networks in depth. Nevertheless, RNAi screens present a powerful method to interrogate gene function in high-throughput and different methods to elicit RNAi in mammalian cells have been developed. Here, we describe steps that should be considered when planning an RNAi screen employing endoribonuclease prepared (e)siRNAs. We provide useful information on how to implement the screen and analyze the results. Furthermore, we discuss strategies for hit validation and present an outline on how to follow-up on verified hits to gain a molecular understanding of the underlying phenotypes.

Fernando Carrillo Oesterreich, Nicole Bieberstein, Karla M. Neugebauer
Pause locally, splice globally.
Trends Cell Biol, 21(6, SI) 328-335 (2011)
PDF DOI

Most eukaryotic protein-coding transcripts contain introns, which vary in number and position along the transcript body. Intron removal through pre-mRNA splicing is tightly linked to transcription by RNA polymerase II as it translocates along each gene. Here, we review recent evidence that transcription and splicing are functionally coupled. We focus on how RNA polymerase II elongation rates impact splicing through local regulation and transcriptional pausing within genes. Emerging concepts of how splicing-related changes in elongation might be achieved are highlighted. We place the interplay between transcription and splicing in the context of chromatin where nucleosome positioning influences elongation, and histone modifications participate directly in the recruitment of splicing regulators to nascent transcripts.

Stefanie Redemann, Siegfried Schloissnig, Susanne Ernst, Andrei I. Pozniakovsky, Swathi Ayloo, Anthony A. Hyman, Henrik Bringmann
Codon adaptation-based control of protein expression in C. elegans.
Nat Methods, 8(3) 250-252 (2011)
PDF DOI

We present a method to control protein levels under native genetic regulation in Caenorhabditis elegans by using synthetic genes with adapted codons. We found that the force acting on the spindle in C. elegans embryos was related to the amount of the G-protein regulator GPR-1/2. Codon-adapted versions of any C. elegans gene can be designed using our web tool, C. elegans codon adapter.

Alexander Gerbaulet, Claudia Wickenhauser, Julia Scholten, Katrin Peschke, Sebastian Drube, Hans-Peter Horny, Thomas Kamradt, Ronald Naumann, Werner Müller, Thomas Krieg, Claudia Waskow, Karin Hartmann, Axel Roers
Mast cell hyperplasia, B cell malignancy, and intestinal inflammation in mice with conditional expression of a constitutively active kit.
Blood, 117(6) 2012-2021 (2011)
PDF DOI

Signaling through the receptor tyrosine kinase kit controls proliferation and differentiation of hematopoietic precursor cells and mast cells. Somatic point mutations of the receptor that constitutively activate kit signaling are associated with mastocytosis and various hematopoietic malignancies. We generated a Cre/loxP-based bacterial artificial chromosome (BAC) transgenic mouse model that allows conditional expression of a kit gene carrying the kitD814V mutation (the murine homolog of the most common mutation in human mastocytosis, kitD816V) driven by the kit promoter. Expression of the mutant kit in adult mice caused severe mastocytosis with 100 % penetrance at young age frequently associated with additional hematopoietic (mostly B lineage-derived) neoplasms and focal colitis. Restriction of transgene expression to mature mast cells resulted in a similar mast cell disease developing with slower kinetics. Embryonic expression led to a hyperproliferative dysregulation of the erythroid lineage with a high rate of perinatal lethality. In addition, most adult animals developed colitis associated with mucosal mast cell accumulation. Our findings demonstrate that the effects of constitutive kit signaling critically depend on the developmental stage and the state of differentiation of the cell hit by the gain of function mutation.

Wei Niu, Zhi John Lu, Mei Zhong, Mihail Sarov, James T Murray, Cathleen M Brdlik, Judith Janette, Chao Chen, Pedro Alves, Elicia A. Preston, Cindie Slightam, Lixia Jiang, Anthony A. Hyman, Stuart K Kim, Robert H Waterston, Mark Gerstein, Michael Snyder, Valerie Reinke
Diverse transcription factor binding features revealed by genome-wide ChIP-seq in C. elegans.
Genome Res, 21(2) 245-254 (2011)
PDF DOI

Regulation of gene expression by sequence-specific transcription factors is central to developmental programs and depends on the binding of transcription factors with target sites in the genome. To date, most such analyses in Caenorhabditis elegans have focused on the interactions between a single transcription factor with one or a few select target genes. As part of the modENCODE Consortium, we have used chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) to determine the genome-wide binding sites of 22 transcription factors (ALR-1, BLMP-1, CEH-14, CEH-30, EGL-27, EGL-5, ELT-3, EOR-1, GEI-11, HLH-1, LIN-11, LIN-13, LIN-15B, LIN-39, MAB-5, MDL-1, MEP-1, PES-1, PHA-4, PQM-1, SKN-1, and UNC-130) at diverse developmental stages. For each factor we determined candidate gene targets, both coding and non-coding. The typical binding sites of almost all factors are within a few hundred nucleotides of the transcript start site. Most factors target a mixture of coding and non-coding target genes, although one factor preferentially binds to non-coding RNA genes. We built a regulatory network among the 22 factors to determine their functional relationships to each other and found that some factors appear to act preferentially as regulators and others as target genes. Examination of the binding targets of three related HOX factors-LIN-39, MAB-5, and EGL-5-indicates that these factors regulate genes involved in cellular migration, neuronal function, and vulval differentiation, consistent with their known roles in these developmental processes. Ultimately, the comprehensive mapping of transcription factor binding sites will identify features of transcriptional networks that regulate C. elegans developmental processes.

Sven Danckwardt, Anne-Susan Gantzert, Stephan Macher-Goeppinger, Hans Christian Probst, Marc Gentzel, Matthias Wilm, Hermann-Josef Gröne, Peter Schirmacher, Matthias W. Hentze, Andreas E Kulozik
p38 MAPK controls prothrombin expression by regulated RNA 3' end processing.
Mol Cell, 41(3) 298-310 (2011)
DOI

Thrombin is a key protease involved in blood coagulation, complement activation, inflammation, angiogenesis, and tumor invasion. Although induced in many (patho-)physiological conditions, the underlying mechanisms controlling prothrombin expression remained enigmatic. We have now discovered that prothrombin expression is regulated by a posttranscriptional regulatory mechanism responding to stress and inflammation. This mechanism is triggered by external stimuli that activate p38 MAPK. In turn, p38 MAPK upmodulates canonical 3' end processing components and phosphorylates the RNA-binding proteins FBP2 and FBP3, which inhibit 3' end processing of mRNAs, such as prothrombin mRNA, that bear a defined upstream sequence element (USE) in their 3'UTRs. Upon phosphorylation, FBP2 and FBP3 dissociate from the USE, making it accessible to proteins that stimulate 3' end processing. We provide in vivo evidence suggesting the importance of this mechanism in inflammatory hypercoagulation and tumor invasion. Regulated 3' end processing thus emerges as a key mechanism of gene regulation with broad biological and medical implications.

Annelie Oswald, Andrew C. Oates
Control of endogenous gene expression timing by introns
Genome Biol, 12(3) Art. No. 107 (2011)
DOI

Comparison of gene expression from transgenes and endogenous genes with or without introns reveals a time-regulating role of introns in natural biological systems.

Radoslaw K Ejsmont
A toolkit for visualization of patterns of gene expression in live Drosophila embryos
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2011)

Radoslaw K Ejsmont, Maria Bogdanzaliewa, Kamil A Lipinski, Pavel Tomancak
Production of fosmid genomic libraries optimized for liquid culture recombineering and cross-species transgenesis.
Methods Mol Biol, 772 423-443 (2011)
PDF DOI

Genomic DNA libraries are a valuable source of large constructs that can contain all the regulatory elements necessary for recapitulating wild-type gene expression when introduced into animal genomes as a transgene. Such clones can be directly used in complementation studies. In combination with recombineering manipulation, the tagged genomic clones can serve as faithful in vivo gene activity reporters that enable studies of tissue specificity of gene expression, subcellular protein localization, and affinity purification of complexes. We present a detailed protocol for generating an unbiased genomic library in a custom pFlyFos vector that is optimized for liquid culture recombineering manipulation and site-specific transgenesis of fosmid-size loci across different Drosophila species. The cross-species properties of the library can be used, for example, to establish the specificity of RNAi phenotypes or to selectively introgress specific genomic loci among different Drosophila species making it an ideal tool for experimental evolutionary studies. The FlyFos system can be easily adapted to other organisms.

Radoslaw K Ejsmont, Peter Ahlfeld, Andrei I. Pozniakovsky, A Francis Stewart, Pavel Tomancak^#, Mihail Sarov^#
Recombination-mediated genetic engineering of large genomic DNA transgenes.
Methods Mol Biol, 772 445-458 (2011)
PDF DOI

Faithful gene activity reporters are a useful tool for evo-devo studies enabling selective introduction of specific loci between species and assaying the activity of large gene regulatory sequences. The use of large genomic constructs such as BACs and fosmids provides an efficient platform for exploration of gene function under endogenous regulatory control. Despite their large size they can be easily engineered using in vivo homologous recombination in Escherichia coli (recombineering). We have previously demonstrated that the efficiency and fidelity of recombineering are sufficient to allow high-throughput transgene engineering in liquid culture, and have successfully applied this approach in several model systems. Here, we present a detailed protocol for recombineering of BAC/fosmid transgenes for expression of fluorescent or affinity tagged proteins in Drosophila under endogenous in vivo regulatory control. The tag coding sequence is seamlessly recombineered into the genomic region contained in the BAC/fosmid clone, which is then integrated into the fly genome using ?C31 recombination. This protocol can be easily adapted to other recombineering projects.

Yehuda Brody, Noa Neufeld, Nicole Bieberstein, Sebastien Z Causse, Eva-Maria Böhnlein, Karla M. Neugebauer, Xavier Darzacq, Yaron Shav-Tal
The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing.
PLoS Biol, 9(1) Art. No. e1000573 (2011)
Open Access PDF DOI

RNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3' processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. How Pol II elongation rates are influenced by splicing is not well understood. We generated a family of inducible gene constructs containing increasing numbers of introns and exons, which were stably integrated in human cells to serve as actively transcribing gene loci. By monitoring the association of the transcription and splicing machineries on these genes in vivo, we showed that only U1 snRNP localized to the intronless gene, consistent with a splicing-independent role for U1 snRNP in transcription. In contrast, all snRNPs accumulated on intron-containing genes, and increasing the number of introns increased the amount of spliceosome components recruited. This indicates that nascent RNA can assemble multiple spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo, Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors showed that polymerase elongation rates were uncoupled from ongoing splicing. This study shows that transcription elongation kinetics proceed independently of splicing at the model genes studied here. Surprisingly, retention of polyadenylated mRNA was detected at the transcription site after transcription termination. This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end.

Sylke Winkler, Nicola Gscheidel, Michael Brand
Mutant generation in vertebrate model organisms by TILLING.
Methods Mol Biol, 770 475-504 (2011)
PDF DOI

TILLING (Targeting Induced Local Lesions IN Genomes) is a popular reverse genetic approach that has been successfully applied in several genetic model organisms such as zebrafish, rat, Drosophila, Arabidopsis, or medaka. In contrast to classical targeted knockout technologies that work in mice by directly targeting a gene of interest, TILLING follows an indirect strategy. The first step of the TILLING pipeline is the generation of a TILLING library that consists of large numbers of mutagenized individuals. In a second step, these individuals are screened for mutations in any gene of interest. Screening is performed by PCR amplification of specific exons from each individual of a library followed by mutation detection. This could be done, for example, by direct re-sequencing of PCR fragments or alternatively, by CEL1 endonuclease-mediated mutation discovery. Individuals carrying potentially deleterious point mutations are isolated from the library and mutant lines are established. TILLING allows the identification of a whole range of point mutations, covering nonsense, splice site, and missense mutations in only one screening round, because the generation of mutations by mutagenesis as well as the screening tools is not biased. Potential knockout mutations are initially the mutations of choice, but TILLING screens can also be used to isolate allelic series of point mutations ranging from complete null phenotypes to hypomorphic or even dominant-negative or conditional alleles. These allelic series can be helpful for a comprehensive functional analysis of a gene of interest. TILLING is applicable to any kind of genetically tractable model organism, as long as this model organism is amenable to chemical mutagenesis, and genomic sequence information for a gene of interest is available. This chapter describes the design and pipeline of a TILLING facility as we are currently operating it for zebrafish in Dresden. Protocols for mutation detection by direct re-sequencing are described in detail. However, alternatives to this pipeline do exist and will be mentioned briefly.

Andreas Ettinger, Michaela Wilsch-Bräuninger, Anne-Marie Marzesco, Marc Bickle, Annett Lohmann, Zoltan Maliga, Jana Karbanová, Denis Corbeil, Anthony A. Hyman, Wieland B. Huttner
Proliferating versus differentiating stem and cancer cells exhibit distinct midbody-release behaviour.
Nat Commun, 2 Art. No. 503 (2011)
PDF DOI

The central portion of the midbody, a cytoplasmic bridge between nascent daughter cells at the end of cell division, has generally been thought to be retained by one of the daughter cells, but has, recently, also been shown to be released into the extracellular space. The significance of midbody-retention versus -release is unknown. Here we show, by quantitatively analysing midbody-fate in various cell lines under different growth conditions, that the extent of midbody-release is significantly greater in stem cells than cancer-derived cells. Induction of cell differentiation is accompanied by an increase in midbody-release. Knockdown of the endosomal sorting complex required for transport family members, Alix and tumour-suppressor gene 101, or of their interaction partner, centrosomal protein 55, impairs midbody-release, suggesting mechanistic similarities to abscission. Cells with such impaired midbody-release exhibit enhanced responsiveness to a differentiation stimulus. Taken together, midbody-release emerges as a characteristic feature of cells capable of differentiation.

Rajesh Ramaswamy, Ivo F. Sbalzarini
A partial-propensity formulation of the stochastic simulation algorithm for chemical reaction networks with delays
J Chem Phys, 134(1) Art. No. 014106 (2011)
PDF DOI

Several real-world systems, such as gene expression networks in biological cells, contain coupled chemical reactions with a time delay between reaction initiation and completion. The non-Markovian kinetics of such reaction networks can be exactly simulated using the delay stochastic simulation algorithm (dSSA). The computational cost of dSSA scales with the total number of reactions in the network. We reduce this cost to scale at most with the smaller number of species by using the concept of partial reaction propensities. The resulting delay partial-propensity direct method (dPDM) is an exact dSSA formulation for well-stirred systems of coupled chemical reactions with delays. We detail dPDM and present a theoretical analysis of its computational cost. Furthermore, we demonstrate the implications of the theoretical cost analysis in two prototypical benchmark applications. The dPDM formulation is shown to be particularly efficient for strongly coupled reaction networks, where the number of reactions is much larger than the number of species.

József Jászai, Christine A. Fargeas, Sylvi Graupner, Elly M. Tanaka, Michael Brand, Wieland B. Huttner, Denis Corbeil
Distinct and conserved prominin-1/CD133-positive retinal cell populations identified across species.
PLoS ONE, 6(3) Art. No. e17590 (2011)
DOI

Besides being a marker of various somatic stem cells in mammals, prominin-1 (CD133) plays a role in maintaining the photoreceptor integrity since mutations in the PROM1 gene are linked with retinal degeneration. In spite of that, little information is available regarding its distribution in eyes of non-mammalian vertebrates endowed with high regenerative abilities. To address this subject, prominin-1 cognates were isolated from axolotl, zebrafish and chicken, and their retinal compartmentalization was investigated and compared to that of their mammalian orthologue. Interestingly, prominin-1 transcripts--except for the axolotl--were not strictly restricted to the outer nuclear layer (i.e., photoreceptor cells), but they also marked distinct subdivisions of the inner nuclear layer (INL). In zebrafish, where the prominin-1 gene is duplicated (i.e., prominin-1a and prominin-1b), a differential expression was noted for both paralogues within the INL being localized either to its vitreal or scleral subdivision, respectively. Interestingly, expression of prominin-1a within the former domain coincided with Pax-6-positive cells that are known to act as progenitors upon injury-induced retino-neurogenesis. A similar, but minute population of prominin-1-positive cells located at the vitreal side of the INL was also detected in developing and adult mice. In chicken, however, prominin-1-positive cells appeared to be aligned along the scleral side of the INL reminiscent of zebrafish prominin-1b. Taken together our data indicate that in addition to conserved expression of prominin-1 in photoreceptors, significant prominin-1-expressing non-photoreceptor retinal cell populations are present in the vertebrate eye that might represent potential sources of stem/progenitor cells for regenerative therapies.

María E Primo, Jean Jakoncic, Martín E Noguera, Valeria A Risso, Laura Sosa, Mauricio P Sica, Michele Solimena, Edgardo Poskus, Mario R Ermácora
Protein-protein interactions in crystals of the human receptor-type protein tyrosine phosphatase ICA512 ectodomain
PLoS ONE, 6(9) Art. No. e24191 (2011)
DOI

ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.

Marc Leushacke, Ralf Spörle, Christof Bernemann, Antje Brouwer-Lehmitz, Johannes Fritzmann, Mirko Theis, Frank Buchholz, Bernhard G Herrmann, Markus Morkel
An RNA interference phenotypic screen identifies a role for FGF signals in colon cancer progression
PLoS ONE, 6(8) Art. No. e23381 (2011)
DOI

In tumor cells, stepwise oncogenic deregulation of signaling cascades induces alterations of cellular morphology and promotes the acquisition of malignant traits. Here, we identified a set of 21 genes, including FGF9, as determinants of tumor cell morphology by an RNA interference phenotypic screen in SW480 colon cancer cells. Using a panel of small molecular inhibitors, we subsequently established phenotypic effects, downstream signaling cascades, and associated gene expression signatures of FGF receptor signals. We found that inhibition of FGF signals induces epithelial cell adhesion and loss of motility in colon cancer cells. These effects are mediated via the mitogen-activated protein kinase (MAPK) and Rho GTPase cascades. In agreement with these findings, inhibition of the MEK1/2 or JNK cascades, but not of the PI3K-AKT signaling axis also induced epithelial cell morphology. Finally, we found that expression of FGF9 was strong in a subset of advanced colon cancers, and overexpression negatively correlated with patients' survival. Our functional and expression analyses suggest that FGF receptor signals can contribute to colon cancer progression.

Yoko Arai, Jeremy N. Pulvers, Christiane Haffner, Britta Schilling, Ina Nüsslein, Federico Calegari, Wieland B. Huttner
Neural stem and progenitor cells shorten S-phase on commitment to neuron production.
Nat Commun, 2 Art. No. 154 (2011)
DOI

During mammalian cerebral cortex development, the G1-phase of the cell cycle is known to lengthen, but it has been unclear which neural stem and progenitor cells are affected. In this paper, we develop a novel approach to determine cell-cycle parameters in specific classes of neural stem and progenitor cells, identified by molecular markers rather than location. We found that G1 lengthening was associated with the transition from stem cell-like apical progenitors to fate-restricted basal (intermediate) progenitors. Unexpectedly, expanding apical and basal progenitors exhibit a substantially longer S-phase than apical and basal progenitors committed to neuron production. Comparative genome-wide gene expression analysis of expanding versus committed progenitor cells revealed changes in key factors of cell-cycle regulation, DNA replication and repair and chromatin remodelling. Our findings suggest that expanding neural stem and progenitor cells invest more time during S-phase into quality control of replicated DNA than those committed to neuron production.

Mark Gerstein^*, Zhi John Lu^*, Eric L. Van Nostrand^*, Chao Cheng^*, Bradley I. Arshinoff^*, Tao Liu^*, Kevin Y. Yip^*, Rebecca Robilotto^*, Andreas Rechtsteiner^*, Kohta Ikegami^*, Pedro Alves^*, Aurelien Chateigner^*, Marc Perry^*, Mitzi Morris^*, Raymond Auerbach^*, Xin Feng^*, Jing Leng^*, Anne Vielle^*, Wei Niu^*, Kahn Rhrissorrakrai^*, Ashish Agarwal, Roger P. Alexander, Galt Barber, Cathleen M Brdlik, Jennifer Brennan, Jeremy Jean Brouillet, Adrian Carr, Ming-Sin Cheung, Hiram Clawson, Sergio Contrino, Luke O. Dannenberg, Abby F. Dernburg, Arshad Desai, Lindsay Dick, Andréa Dosé, Jiang Du, Thea Egelhofer, Sevinc Ercan, Ghia Euskirchen, Brent Ewing, Elise A. Feingold, Reto Gassmann, Peter J. Good, Phil Green, Francois Gullier, Michelle Gutwein, Mark S. Guyer, Lukas Habegger, Ting Han, Jorja G. Henikoff, Stefan R. Henz, Angie Hinrichs, Heather Holster, Anthony A. Hyman, A. Leo Iniguez, Judith Janette, Morten Jensen, Masaomi Kato, W. James Kent, Ellen Kephart, Vishal Khivansara, Ekta Khurana, John K. Kim, Paulina Kolasinska-Zwierz, Eric C. Lai, Isabel Latorre, Amber Leahey, Suzanna E Lewis, Paul Lloyd, Lucas Lochovsky, Rebecca F. Lowdon, Yaniv Lubling, Rachel Lyne, Michael MacCoss, Sebastian D. Mackowiak, Marco Mangone, Sheldon McKay, Desirea Mecenas, Gennifer Merrihew, David M. Miller, Andrew Muroyama, John I. Murray, Siew-Loon Ooi, Hoang Pham, Taryn Phippen, Elicia A. Preston, Nikolaus Rajewski, Gunnar Rätsch, Heidi Rosenbaum, Joel Rozowksy, Kim Rutherford, Peter Ruzanov, Mihail Sarov, Rajkumar Sasidharan, Andrea Sboner, Paul Scheid, Eran Segal, Hyunjin Shin, Chong Shou, Frank J. Slack, Cindie Slightam, Richard Smith, William C. Spencer, E. O. Stinson, Scott Taing, Teruaki Takasaki, Dionne Vafeados, Ksenia Voronina, Guilin Wang, Nicole L. Washington, Christina M. Whittle, Beijing Wu, Koon-Kiu Yan, Georg Zeller, Zheng Zha, Mei Zhong, Xingliang Zhou, Julie Ahringer, Susan Strome, Kristin C. Gunsalus, Gos Micklem, X. Shirley Liu, Valerie Reinke, Stuart K Kim, LaDeana W Hillier, Steven Henikoff, Fabio Piano, Michael Snyder, Lincoln Stein, Jason D. Lieb, Robert H Waterston
Integrative analysis of the Caenorhabditis elegans genome by the modENCODE project
Science, 330(6012) 1775-1787 (2010)
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We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor–binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor–binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.

Alex T. Kalinka^*, Karolina M Varga^*, Dave T. Gerrard, Stephan Preibisch, David L. Corcoran, Julia Jarrells, Uwe Ohler, Casey M. Bergman, Pavel Tomancak
Gene expression divergence recapitulates the developmental hourglass model.
Nature, 468(7325) 811-814 (2010)
PDF DOI

The observation that animal morphology tends to be conserved during the embryonic phylotypic period (a period of maximal similarity between the species within each animal phylum) led to the proposition that embryogenesis diverges more extensively early and late than in the middle, known as the hourglass model. This pattern of conservation is thought to reflect a major constraint on the evolution of animal body plans. Despite a wealth of morphological data confirming that there is often remarkable divergence in the early and late embryos of species from the same phylum, it is not yet known to what extent gene expression evolution, which has a central role in the elaboration of different animal forms, underpins the morphological hourglass pattern. Here we address this question using species-specific microarrays designed from six sequenced Drosophila species separated by up to 40 million years. We quantify divergence at different times during embryogenesis, and show that expression is maximally conserved during the arthropod phylotypic period. By fitting different evolutionary models to each gene, we show that at each time point more than 80% of genes fit best to models incorporating stabilizing selection, and that for genes whose evolutionarily optimal expression level is the same across all species, selective constraint is maximized during the phylotypic period. The genes that conform most to the hourglass pattern are involved in key developmental processes. These results indicate that natural selection acts to conserve patterns of gene expression during mid-embryogenesis, and provide a genome-wide insight into the molecular basis of the hourglass pattern of developmental evolution.

Haiyan Lei, Tetsunari Fukushige, Wei Niu, Mihail Sarov, Valerie Reinke, Michael Krause
A widespread distribution of genomic CeMyoD binding sites revealed and cross validated by ChIP-Chip and ChIP-Seq techniques.
PLoS ONE, 5(12) Art. No. e15898 (2010)
PDF DOI

Identifying transcription factor binding sites genome-wide using chromatin immunoprecipitation (ChIP)-based technology is becoming an increasingly important tool in addressing developmental questions. However, technical problems associated with factor abundance and suitable ChIP reagents are common obstacles to these studies in many biological systems. We have used two completely different, widely applicable methods to determine by ChIP the genome-wide binding sites of the master myogenic regulatory transcription factor HLH-1 (CeMyoD) in C. elegans embryos. The two approaches, ChIP-seq and ChIP-chip, yield strongly overlapping results revealing that HLH-1 preferentially binds to promoter regions of genes enriched for E-box sequences (CANNTG), known binding sites for this well-studied class of transcription factors. HLH-1 binding sites were enriched upstream of genes known to be expressed in muscle, consistent with its role as a direct transcriptional regulator. HLH-1 binding was also detected at numerous sites unassociated with muscle gene expression, as has been previously described for its mouse homolog MyoD. These binding sites may reflect several additional functions for HLH-1, including its interactions with one or more co-factors to activate (or repress) gene expression or a role in chromatin organization distinct from direct transcriptional regulation of target genes. Our results also provide a comparison of ChIP methodologies that can overcome limitations commonly encountered in these types of studies while highlighting the complications of assigning in vivo functions to identified target sites.

Fernando Carrillo Oesterreich, Stephan Preibisch, Karla M. Neugebauer
Global analysis of nascent RNA reveals transcriptional pausing in terminal exons.
Mol Cell, 40(4) 571-581 (2010)
PDF DOI

Pre-mRNA splicing is catalyzed by the spliceosome, which can assemble on pre-mRNA cotranscriptionally. However, whether splicing generally occurs during transcription has not been addressed. Indeed, splicing catalysis is expected to occur posttranscriptionally in yeast, where the shortness of terminal exons should leave insufficient time for splicing. Here, we isolate endogenous S. cerevisiae nascent RNA and determine gene-specific splicing efficiencies and transcription profiles, using high-density tiling microarrays. Surprisingly, we find that splicing occurs cotranscriptionally for the majority of intron-containing genes. Analysis of transcription profiles reveals Pol II pausing within the terminal exons of these genes. Intronless and inefficiently spliced genes lack this pause. In silico simulations of transcription and splicing kinetics confirm that this pausing event provides sufficient time for splicing before termination. The discovery of terminal exon pausing demonstrates functional coupling of transcription and splicing near gene ends.

Natalia Bulgakova, Michaela Rentsch, Elisabeth Knust
Antagonistic functions of two stardust isoforms in Drosophila photoreceptor cells.
Mol Biol Cell, 21(22) 3915-3925 (2010)
PDF DOI

Membrane-associated guanylate kinases (MAGUKs) are scaffolding proteins that organize supramolecular protein complexes, thereby partitioning the plasma membrane into spatially and functionally distinct subdomains. Their modular organization is ideally suited to organize protein complexes with cell type- or stage-specific composition, or both. Often more than one MAGUK isoform is expressed by one gene in the same cell, yet very little is known about their individual in vivo functions. Here, we show that two isoforms of Drosophila stardust, Sdt-H (formerly called Sdt-B2) and Sdt-D, which differ in their N terminus, are expressed in adult photoreceptors. Both isoforms associate with Crumbs and PATJ, constituents of the conserved Crumbs-Stardust complex. However, they form distinct complexes, localized at the stalk, a restricted region of the apical plasma membrane. Strikingly, Sdt-H and Sdt-D have antagonistic functions. While Sdt-H overexpression increases stalk membrane length and prevents light-dependent retinal degeneration, Sdt-D overexpression reduces stalk length and enhances light-dependent retinal degeneration. These results suggest that a fine-tuned balance of different Crumbs complexes regulates photoreceptor homeostasis.

Yanmei Liu, Jakob Suckale, Jimmy Masjkur, Maria Grazia Magro, Anja Steffen, Konstantinos Anastassiadis, Michele Solimena
Tamoxifen-independent recombination in the RIP-CreER mouse.
PLoS ONE, 5(10) Art. No. e13533 (2010)
PDF DOI

The inducible Cre-lox system is a valuable tool to study gene function in a spatial and time restricted fashion in mouse models. This strategy relies on the limited background activity of the modified Cre recombinase (CreER) in the absence of its inducer, the competitive estrogen receptor ligand, tamoxifen. The RIP-CreER mouse (Tg (Ins2-cre/Esr1) 1Dam) is among the few available β-cell specific CreER mouse lines and thus it has been often used to manipulate gene expression in the insulin-producing cells of the endocrine pancreas.

Michel Bagnat, Adam Navis, Sara Herbstreith, Koroboshka Brand-Arzamendi, Silvia Curado, Sherif Gabriel, Keith Mostov, Jan Huisken, Didier Y.R. Stainier
Cse1l Is a Negative Regulator of CFTR-Dependent Fluid Secretion.
Curr Biol, 20(20) 1840-1845 (2010)
DOI

Transport of chloride through the cystic fibrosis transmembrane conductance regulator (CFTR) channel is a key step in regulating fluid secretion in vertebrates [1, 2]. Loss of CFTR function leads to cystic fibrosis [1, 3, 4], a disease that affects the lungs, pancreas, liver, intestine, and vas deferens. Conversely, uncontrolled activation of the channel leads to increased fluid secretion and plays a major role in several diseases and conditions including cholera [5, 6] and other secretory diarrheas [7] as well as polycystic kidney disease [8-10]. Understanding how CFTR activity is regulated in vivo has been limited by the lack of a genetic model. Here, we used a forward genetic approach in zebrafish to uncover CFTR regulators. We report the identification, isolation, and characterization of a mutation in the zebrafish cse1l gene that leads to the sudden and dramatic expansion of the gut tube. We show that this phenotype results from a rapid accumulation of fluid due to the uncontrolled activation of the CFTR channel. Analyses in zebrafish larvae and mammalian cells indicate that Cse1l is a negative regulator of CFTR-dependent fluid secretion. This work demonstrates the importance of fluid homeostasis in development and establishes the zebrafish as a much-needed model system to study CFTR regulation in vivo.

Michiel Vermeulen^*^#, H Christian Eberl^*, Filomena Matarese^*, Hendrik Marks, Sergei Denissov, Falk Butter, Kenneth K Lee, Jesper V Olsen, Anthony A. Hyman, Hendrik G Stunnenberg^#, Matthias Mann^#
Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers.
Cell, 142(6) 967-980 (2010)
PDF DOI

Trimethyl-lysine (me3) modifications on histones are the most stable epigenetic marks and they control chromatin-mediated regulation of gene expression. Here, we determine proteins that bind these marks by high-accuracy, quantitative mass spectrometry. These chromatin "readers" are assigned to complexes by interaction proteomics of full-length BAC-GFP-tagged proteins. ChIP-Seq profiling identifies their genomic binding sites, revealing functional properties. Among the main findings, the human SAGA complex binds to H3K4me3 via a double Tudor-domain in the C terminus of Sgf29, and the PWWP domain is identified as a putative H3K36me3 binding motif. The ORC complex, including LRWD1, binds to the three most prominent transcriptional repressive lysine methylation sites. Our data reveal a highly adapted interplay between chromatin marks and their associated protein complexes. Reading specific trimethyl-lysine sites by specialized complexes appears to be a widespread mechanism to mediate gene expression.

Julia Elisabeth Pulverer, Ulfert Rand, Stefan Lienenklaus, Daniela Kugel, Natalia Zietara, Georg Kochs, Ronald Naumann, Siegfried Weiss, Peter Staeheli, Hansjörg Hauser^#, Mario Köster^#
Temporal and spatial resolution of type I and III interferon responses in vivo.
J Virol, 84(17) 8626-8638 (2010)
PDF DOI

Although the action of interferons (IFNs) has been extensively studied in vitro, limited information is available on the spatial and temporal activation pattern of IFN-induced genes in vivo. We created BAC transgenic mice expressing firefly luciferase under transcriptional control of the Mx2 gene promoter. Expression of the reporter with regard to onset and kinetics of induction parallels that of Mx2 and is thus a hallmark for the host response. Substantial constitutive expression of the reporter gene was observed in the liver and most other tissues of transgenic mice, whereas this expression was strongly reduced in animals lacking functional type I IFN receptors. As expected, the reporter gene was induced not only in response to type I (alpha and beta) and type III (lambda) IFNs but also in response to a variety of IFN inducers such as double-stranded RNA, lipopolysaccharide (LPS), and viruses. In vivo IFN subtypes show clear differences with respect to their kinetics of action and to their spatial activation pattern: while the type I IFN response was strong in liver, spleen, and kidney, type III IFN reactivity was most prominent in organs with mucosal surfaces. Infection of reporter mice with virus strains that differ in their pathogenicity shows that the IFN response is significantly altered in the strength of IFN action at sites which are not primarily infected as well as by the onset and duration of gene induction.

Daniel M Linares, Eric R Geertsma, B Poolman
Evolved Lactococcus lactis strains for enhanced expression of recombinant membrane proteins.
J Mol Biol, 401(1) 45-55 (2010)
DOI

The production of complex multidomain (membrane) proteins is a major hurdle in structural genomics and a generic approach for optimizing membrane protein expression is still lacking. We have devised a selection method to isolate mutant strains with improved functional expression of recombinant membrane proteins. By fusing green fluorescent protein and an erythromycin resistance marker (ErmC) to the C-terminus of a target protein, one simultaneously selects for variants with enhanced expression (increased erythromycin resistance) and correct folding (green fluorescent protein fluorescence). Three evolved hosts, displaying 2- to 8-fold increased expression of a plethora of proteins, were fully sequenced and shown to carry single-site mutations in the nisK gene. NisK is the sensor protein of a two-component regulatory system that directs nisin-A-mediated expression. The levels of recombinant membrane proteins were increased in the evolved strains, and in some cases their folding states were improved. The generality and simplicity of our approach allow rapid improvements of protein production yields by directed evolution in a high-throughput way.

Minna-Liisa Änkö, Lucia Morales, Ian Henry, Andreas Beyer, Karla M. Neugebauer
Global analysis reveals SRp20- and SRp75-specific mRNPs in cycling and neural cells.
Nat Struct Mol Biol, 17(8) 962-970 (2010)
PDF DOI

Members of the SR protein family of RNA-binding proteins have numerous roles in mRNA metabolism, from transcription to translation. To understand how SR proteins coordinate gene regulation, comprehensive knowledge of endogenous mRNA targets is needed. Here we establish physiological expression of GFP-tagged SR proteins from stable transgenes. Using the GFP tag for immunopurification of mRNPs, mRNA targets of SRp20 and SRp75 were identified in cycling and neurally induced P19 cells. Genome-wide analysis showed that SRp20 and SRp75 associate with hundreds of distinct, functionally related groups of transcripts that change in response to neural differentiation. Knockdown of either SRp20 or SRp75 led to up- or downregulation of specific transcripts, including identified targets, and rescue by the GFP-tagged SR proteins proved their functionality. Thus, SR proteins contribute to the execution of gene-expression programs through their association with distinct endogenous mRNAs.

Adam P. Kupinski, Thomas Müller-Reichert, Christian R. Eckmann
The Caenorhabditis elegans Ste20 kinase, GCK-3, is essential for postembryonic developmental timing and regulates meiotic chromosome segregation.
Dev Biol, 344(2) 758-771 (2010)
PDF DOI

Ste20 kinases constitute a large family of serine/threonine kinases with a plethora of biological functions. Members of the GCK-VI subfamily have been identified as important regulators of osmohomeostasis across species functioning upstream of ion channels. Although the expression of the two highly similar mammalian GCK-VI kinases is eminent in a wide variety of tissues, which includes also the testis, their potential roles in development remain elusive. Caenorhabditis elegans contains a single ancestral ortholog termed GCK-3. Here, we report a comprehensive analysis of gck-3 function and demonstrate its requirement for several developmental processes independent of ion homeostasis, i.e., larval progression, vulva, and germ line formation. Consistent with a wide range of gck-3 function we find that endogenous GCK-3 is expressed ubiquitously. The serine/threonine kinase activity of GCK-3, but not its presumed C-terminal substrate interaction domain, is essential for gck-3 gene function. Although expressed in female germ cells, we find GCK-3 progressively accumulating during spermatogenesis where it promotes the first meiotic cell division and facilitates faithful chromosome segregation. In particular, we find that different levels of gck-3 activity appear to be important for various aspects of germ line development. Taken together, our findings suggest that members of the GCK-VI kinase subfamily may act as key regulators of many developmental processes and that this newly described role in meiotic progression might be conserved and an important part of sexual reproduction.

Manonmani Arunachalam, Karthik Jayasurya, Pavel Tomancák, Uwe Ohler
An alignment-free method to identify candidate orthologous enhancers in multiple Drosophila genomes.
Bioinformatics, 26(17) 2109-2115 (2010)
PDF DOI

MOTIVATION: Evolutionarily conserved non-coding genomic sequences represent a potentially rich source for the discovery of gene regulatory region such as transcriptional enhancers. However, detecting orthologous enhancers using alignment-based methods in higher eukaryotic genomes is particularly challenging, as regulatory regions can undergo considerable sequence changes while maintaining their functionality. RESULTS: We have developed an alignment-free method which identifies conserved enhancers in multiple diverged species. Our method is based on similarity metrics between two sequences based on the co-occurrence of sequence patterns regardless of their order and orientation, thus tolerating sequence changes observed in non-coding evolution. We show that our method is highly successful in detecting orthologous enhancers in distantly related species without requiring additional information such as knowledge about transcription factors involved, or predicted binding sites. By estimating the significance of similarity scores, we are able to discriminate experimentally validated functional enhancers from seemingly equally conserved candidates without function. We demonstrate the effectiveness of this approach on a wide range of enhancers in Drosophila, and also present encouraging results to detect conserved functional regions across large evolutionary distances. Our work provides encouraging steps on the way to ab initio unbiased enhancer prediction to complement ongoing experimental efforts. AVAILABILITY: The software, data and the results used in this paper are available at http://www.genome.duke.edu/labs/ohler/research/transcription/fly_enhancer/ CONTACT: uwe.ohler@duke.edu, tomancak@mpi-cbg.de.

Christian Schröter
Segmentation clock dynamics in zebrafish her gene mutants
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2010)

Christian Schröter, Andrew C. Oates
Segment number and axial identity in a segmentation clock period mutant.
Curr Biol, 20(14) 1254-1258 (2010)
PDF DOI

A species-specific number of segments is a hallmark of the vertebrate body plan. The first segmental structures in the vertebrate embryo are the somites, which bud sequentially from the growing presomitic mesoderm (PSM). The Clock and Wavefront model for somitogenesis proposes that the total number of somites is determined by the period of an oscillator or clock operating in the PSM and the total duration of PSM growth. Furthermore, the number of oscillations of the segmentation clock has been suggested to regulate the regional identity of segments along the body axis. Here we test these two ideas in a zebrafish mutant in which the segmentation clock is specifically slowed. This reduces segment number as predicted, but hox gene expression and posterior anatomical markers align with lower segmental counts in mutants compared to the wild-type, arguing against an instructive role of the segmentation clock in determining axial identities. Our data therefore suggest that precise control of segmentation clock period in relation to axial growth ensures a species-specific segment number and that during evolution modulating the clock's period through genetic mutations may have been a relevant way to vary segment number independently of axial regionalization.

Mikolaj Slabicki, Mirko Theis, Dragomir Krastev, Sergey Samsonov, Emeline Mundwiller, Magno Junqueira, Maciej Paszkowski-Rogacz, Joan Teyra, Anne-Kristin Heninger, Ina Poser, Fabienne Prieur, Jérémy Truchetto, Christian Confavreux, Cécilia Marelli, Alexandra Durr, Jean Philippe Camdessanche, Alexis Brice, Andrej Shevchenko, Maria Teresa Pisabarro, Giovanni Stevanin, Frank Buchholz
A genome-scale DNA repair RNAi screen identifies SPG48 as a novel gene associated with hereditary spastic paraplegia.
PLoS Biol, 8(6) Art. No. e1000408 (2010)
Open Access PDF DOI

DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair.

Indranil Sinha^*, Luke Buchanan^*, Michelle Rönnerblad, Carolina Bonilla, Mickaël Durand-Dubief, Andrej Shevchenko, Michael Grunstein, A. Francis Stewart, Karl Ekwall
Genome-wide mapping of histone modifications and mass spectrometry reveal H4 acetylation bias and H3K36 methylation at gene promoters in fission yeast
Epigenomics, 2(3) 377-393 (2010)

Aims: To map histone modifications with unprecedented resolution both globally and locus-specifically, and to link modification patterns to gene expression. Materials & methods: Using correlations between quantitative mass spectrometry and chromatin immunoprecipitation/microarray analyses, we have mapped histone post-translational modifications in fission yeast (Schizosaccharomyces pombe). Results: Acetylations at lysine 9, 18 and 27 of histone H3 give the best positive correlations with gene expression in this organism. Using clustering analysis and gene ontology search tools, we identified promoter histone modification patterns that characterize several classes of gene function. For example, gene promoters of genes involved in cytokinesis have high H3K36me2 and low H3K4me2, whereas the converse pattern is found ar promoters of gene involved in positive regulation of the cell cycle. We detected acetylation of H4 preferentially at lysine 16 followed by lysine 12, 8 and 5. Our analysis shows that this H4 acetylation bias in the coding regions is dependent upon gene length and linked to gene expression. Our analysis also reveals a role for H3K36 methylation at gene promoters where it functions in a crosstalk between the histone methyltransferase Set2KMT3 and the histone deacetylase Clr6, which removes H3K27ac leading to repression of transcription. Conclusion: Histone modification patterns could be linked to gene expression in fission yeast.

Yuansheng Yang, - Mariati, Janet Chusainow, Miranda G S Yap
DNA methylation contributes to loss in productivity of monoclonal antibody-producing CHO cell lines.
J Biotechnol, 147(3-4) 180-185 (2010)
PDF DOI

Production instability currently limits the use of mammalian cells for industrial production of therapeutic proteins. We have previously reported that the loss of productivity in recombinant monoclonal antibody producing Chinese Hamster Ovary (CHO-mAb) cell lines is mainly due to a decrease in heavy chain (HC) and light chain (LC) transcripts. Molecular analysis indicates that the decreased mRNA levels are not due to a loss in gene copies and change of integration sites. In this work, we further demonstrate that impaired trans-acting factors and spontaneous mutations to the DNA are not responsible for the reduced HC and LC transcription. Examination of two CpG sites by methyl-assisted quantitative real-time PCR assay revealed an increase in methylation of the human cytomegalovirus major immediate-early enhancer and promoter (hCMV-MIE) controlling the expression of LC and HC in cells which exhibited loss in productivity. Treatment of these cells with a DNA methylation inhibitor, 5-aza-2'-deoxycytidine, partially restored the lost specific mAb productivity. The increase in productivity correlated to the increase in mRNA levels of HC and LC and the demethylation of hCMV-MIE promoter. This finding, which indicates that DNA methylation contributes to production instability, will be beneficial for generation of high-producing cell lines with stable productivity.

Maciej Paszkowski-Rogacz, Mikolaj Slabicki, Maria Teresa Pisabarro, Frank Buchholz
PhenoFam-gene set enrichment analysis through protein structural information.
BMC Bioinformatics, 11 Art. No. 254 (2010)
PDF DOI

With the current technological advances in high-throughput biology, the necessity to develop tools that help to analyse the massive amount of data being generated is evident. A powerful method of inspecting large-scale data sets is gene set enrichment analysis (GSEA) and investigation of protein structural features can guide determining the function of individual genes. However, a convenient tool that combines these two features to aid in high-throughput data analysis has not been developed yet. In order to fill this niche, we developed the user-friendly, web-based application, PhenoFam.

Sergey Lekomtsev, Julien Guizetti, Andrei I. Pozniakovsky, Daniel W Gerlich, Mark Petronczki
Evidence that the tumor-suppressor protein BRCA2 does not regulate cytokinesis in human cells.
J Cell Sci, 123(Pt 9) 1395-1400 (2010)
PDF DOI

Germline mutations in the tumor-suppressor gene BRCA2 predispose to breast and ovarian cancer. BRCA2 plays a well-established role in maintaining genome stability by regulating homologous recombination. BRCA2 has more recently been implicated in cytokinesis, the final step of cell division, but the molecular basis for this remains unknown. We have used time-lapse microscopy, recently developed cytokinesis assays and BAC recombineering (bacterial artificial chromosome recombinogenic engineering) to investigate the function and localization of BRCA2 during cell division. Our analysis suggests that BRCA2 does not regulate cytokinesis in human cells. Thus, cytokinesis defects are unlikely to contribute to chromosomal instability and tumorigenesis in BRCA2-related cancers.

Cory Y McLean, Dave Bristor, Michael Hiller, Shoa L Clarke, Bruce T Schaar, Craig B Lowe, Aaron M Wenger, Gill Bejerano
GREAT improves functional interpretation of cis-regulatory regions.
Nat Biotechnol, 28(5) 495-501 (2010)
PDF DOI

We developed the Genomic Regions Enrichment of Annotations Tool (GREAT) to analyze the functional significance of cis-regulatory regions identified by localized measurements of DNA binding events across an entire genome. Whereas previous methods took into account only binding proximal to genes, GREAT is able to properly incorporate distal binding sites and control for false positives using a binomial test over the input genomic regions. GREAT incorporates annotations from 20 ontologies and is available as a web application. Applying GREAT to data sets from chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-seq) of multiple transcription-associated factors, including SRF, NRSF, GABP, Stat3 and p300 in different developmental contexts, we recover many functions of these factors that are missed by existing gene-based tools, and we generate testable hypotheses. The utility of GREAT is not limited to ChIP-seq, as it could also be applied to open chromatin, localized epigenomic markers and similar functional data sets, as well as comparative genomics sets.

Daniela Röllig
The role of the hes5 gene family in the evolution of the somitogenesis clock
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2010)

Susanne Bechstedt, Jörg Albert, David P. Kreil, Thomas Müller-Reichert, Martin Göpfert, Jonathon Howard
A doublecortin containing microtubule-associated protein is implicated in mechanotransduction in Drosophila sensory cilia.
Nat Commun, 1(1) Art. No. 11 (2010)
PDF DOI

Mechanoreceptors are sensory cells that transduce mechanical stimuli into electrical signals and mediate the perception of sound, touch and acceleration. Ciliated mechanoreceptors possess an elaborate microtubule cytoskeleton that facilitates the coupling of external forces to the transduction apparatus. In a screen for genes preferentially expressed in Drosophila campaniform mechanoreceptors, we identified DCX-EMAP, a unique member of the EMAP family (echinoderm-microtubule-associated proteins) that contains two doublecortin domains. DCX-EMAP localizes to the tubular body in campaniform receptors and to the ciliary dilation in chordotonal mechanoreceptors in Johnston's organ, the fly's auditory organ. Adult flies carrying a piggyBac insertion in the DCX-EMAP gene are uncoordinated and deaf and display loss of mechanosensory transduction and amplification. Electron microscopy of mutant sensilla reveals loss of electron-dense materials within the microtubule cytoskeleton in the tubular body and ciliary dilation. Our results establish a catalogue of candidate genes for Drosophila mechanosensation and show that one candidate, DCX-EMAP, is likely to be required for mechanosensory transduction and amplification.

Beate Neumann^*, Thomas Walter^*, Jean-Karim Hériché, Jutta Bulkescher, Holger Erfle, Christian Conrad, Phill Rogers, Ina Poser, Michael Held, Urban Liebel, Cihan Cetin, Frank Sieckmann, Gregoire Pau, Rolf Kabbe, Annelie Wünsche, Venkata Satagopam, Michael H A Schmitz, Catherine Chapuis, Daniel W Gerlich, Reinhard Schneider, Roland Eils, Wolfgang Huber, Jan-Michael Peters, Anthony A. Hyman, Richard Durbin, Rainer Pepperkok, Jan Ellenberg
Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes.
Nature, 464(7289) 721-727 (2010)
PDF DOI

Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the approximately 21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community.

James R A Hutchins^*, Yusuke Toyoda^*, Björn Hegemann^*, Ina Poser^*, Jean-Karim Hériché, Martina M Sykora, Martina Augsburg, Otto Hudecz, Bettina A Buschhorn, Jutta Bulkescher, Christian Conrad, David Comartin, Alexander Schleiffer, Mihail Sarov, Andrei I. Pozniakovsky, Mikolaj Slabicki, Siegfried Schloissnig, Ines Steinmacher, Marit Leuschner, Andrea Ssykor, Steffen Lawo, Laurence Pelletier, Holger Stark, Kim Nasmyth, Jan Ellenberg, Richard Durbin, Frank Buchholz, Karl Mechtler, Anthony A. Hyman^#, Jan-Michael Peters^#
Systematic analysis of human protein complexes identifies chromosome segregation proteins.
Science, 328(5978) 593-599 (2010)
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Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells.

Falk Butter, Dennis Kappei, Frank Buchholz^#, Michiel Vermeulen^#, Matthias Mann
A domesticated transposon mediates the effects of a single-nucleotide polymorphism responsible for enhanced muscle growth.
EMBO Rep, 11(4) 305-311 (2010)
PDF DOI

Single-nucleotide polymorphisms (SNPs) in the regulatory regions of the genome can have a profound impact on phenotype. The G3072A polymorphism in intron 3 of insulin-like growth factor 2 (IGF2) is implicated in higher muscle content and reduced fat in European pigs and is bound by a putative repressor. Here, we identify this repressor--which we call muscle growth regulator (MGR)--by using a DNA protein interaction screen based on quantitative mass spectrometry. MGR has a bipartite nuclear localization signal, two BED-type zinc fingers and is highly conserved between placental mammals. Surprisingly, the gene is located in an intron and belongs to the hobo-Ac-Tam3 transposase superfamily, suggesting regulatory use of a formerly parasitic element. In transactivation assays, MGR differentially represses the expression of the two SNP variants. Knockdown of MGR in C2C12 myoblast cells upregulates Igf2 expression and mild overexpression retards growth. Thus, MGR is the repressor responsible for enhanced muscle growth in the IGF2 G3072A polymorphism in commercially bred pigs.

David K. Breslow, Sean R. Collins, Bernd Bodenmiller, Ruedi Aebersold, Kai Simons, Andrej Shevchenko, Christer S. Ejsing, Jonathan S. Weissman
Orm family proteins mediate sphingolipid homeostasis
Nature, 463(7284) 1048-1053 (2010)
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Despite the essential roles of sphingolipids both as structural components of membranes and critical signalling molecules, we have a limited understanding of how cells sense and regulate their levels. Here we reveal the function in sphingolipid metabolism of the ORM genes (known as ORMDL genes in humans)—a conserved gene family that includes ORMDL3, which has recently been identified as a potential risk factor for childhood asthma. Starting from an unbiased functional genomic approach in Saccharomyces cerevisiae, we identify Orm proteins as negative regulators of sphingolipid synthesis that form a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production. We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted. Changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism. Our work identifies the Orm proteins as critical mediators of sphingolipid homeostasis and raises the possibility that sphingolipid misregulation contributes to the development of childhood asthma.

Marcus Gentsch^*, Aneta Kaczmarczyk^*, Karin van Leeuwen, Martin de Boer, Magdalena Kaus-Drobek, Marie-Claire Dagher, Petra Kaiser, Peter D Arkwright, Manfred Gahr, Angela Rösen-Wolff, Matthias Bochtler, Elizabeth Secord, Pamela Britto-Williams, Gulam Mustafa Saifi, Anne Maddalena, Ghassan Dbaibo, Jacinta Bustamante, Jean-Laurent Casanova, Dirk Roos^*, Joachim Roesler^*
Alu-repeat-induced deletions within the NCF2 gene causing p67-phox-deficient chronic granulomatous disease (CGD).
Hum Mutat, 31(2) 151-158 (2010)
PDF DOI

Mutations that impair expression or function of the components of the phagocyte NADPH oxidase complex cause chronic granulomatous disease (CGD), which is associated with life-threatening infections and dysregulated granulomatous inflammation. In five CGD patients from four consanguineous families of two different ethnic backgrounds, we found similar genomic homozygous deletions of 1,380 bp comprising exon 5 of NCF2, which could be traced to Alu-mediated recombination events. cDNA sequencing showed in-frame deletions of phase zero exon 5, which encodes one of the tandem repeat motifs in the tetratricopeptide (TPR4) domain of p67-phox. The resulting shortened protein (p67Delta5) had a 10-fold reduced intracellular half-life and was unable to form a functional NADPH oxidase complex. No dominant negative inhibition of oxidase activity by p67Delta5 was observed. We conclude that Alu-induced deletion of the TPR4 domain of p67-phox leads to loss of function and accelerated degradation of the protein, and thus represents a new mechanism causing p67-phox-deficient CGD.

Michael Weber
Interspecimen registration of long-term, time-lapse selective plane illumination microscopy recordings of Drosophila melanogaster embryogenesis
Diploma Thesis,Technische Universität Dresden, Dresden, Germany (2010)

The investigation of gene expression patterns plays a key role in Drosophila developmental research. Previous techniques such as in situ hybridization and microarray time course analysis are nowadays supplemented by advanced 4d imaging techniques, offering unprecedented possibilities to visualize organisms and allow the collection of systematic image data characterizing gene expression on a large scale. A prominent representative of those techniques is Selective Plane Illumination Microscopy (SPIM), which combines the advantages of fluorescence stereomicroscopy and high-resolution imaging of optical sections in a unique in vivo imaging system. This work utilizes SPIM to collect in toto time-lapse datasets of the formation of prominent gene expression patterns during D. melanogaster embryogenesis. Several parameters of acquisition and sample preparation were optimized to improve the microscope performance as well as the viability of the imaged flies. An approach for the registration of individual SPIM datasets in space and time based on Pearson’s correlation will be presented. This interspecimen registration allows to align time-lapse data in a fast and reliable way. The approach may be used to investigate gene regulatory networks which facilitate the maturation of a single cell to a complex organism.

Jie Shen, Christian Dahmann, Gert Pflugfelder
Spatial discontinuity of optomotor-blind expression in the Drosophila wing imaginal disc disrupts epithelial architecture and promotes cell sorting.
BMC Dev Biol, 10 Art. No. 23 (2010)
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BACKGROUND: Decapentaplegic (Dpp) is one of the best characterized morphogens, required for dorso-ventral patterning of the Drosophila embryo and for anterior-posterior (A/P) patterning of the wing imaginal disc. In the larval wing pouch, the Dpp target gene optomotor-blind (omb) is generally assumed to be expressed in a step function above a certain threshold of Dpp signaling activity. RESULTS: We show that the transcription factor Omb forms, in fact, a symmetrical gradient on both sides of the A/P compartment boundary. Disruptions of the Omb gradient lead to a re-organization of the epithelial cytoskeleton and to a retraction of cells toward the basal membrane suggesting that the Omb gradient is required for correct epithelial morphology. Moreover, by analysing the shape of omb gain- and loss-of-function clones, we find that Omb promotes cell sorting along the A/P axis in a concentration-dependent manner. CONCLUSIONS: Our findings show that Omb distribution in the wing imaginal disc is described by a gradient rather than a step function. Graded Omb expression is necessary for normal cell morphogenesis and cell affinity and sharp spatial discontinuities must be avoided to allow normal wing development.

Nick Barker, Meritxell Huch, Pekka Kujala, Marc van de Wetering, Hugo J Snippert, Johan H van Es, Toshiro Sato, Daniel E Stange, Harry Begthel, Maaike van den Born, Esther Danenberg, Stieneke van den Brink, Jeroen Korving, Arie Abo, Peter J Peters, Nick Wright, Richard Poulsom, Hans Clevers
Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.
Cell Stem Cell, 6(1) 25-36 (2010)
DOI

The study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene Lgr5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated Lgr5 expression in the stomach and assessed the stem cell potential of the Lgr5(+ve) cells by using in vivo lineage tracing. In neonatal stomach, Lgr5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these Lgr5(+ve) cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single Lgr5(+ve) cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The Lgr5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer.

Jésica Díaz-Vera, Yézer G Morales, Juan R Hernández-Fernaud, Marcial Camacho, Mónica S Montesinos, Federico Calegari, Wieland B. Huttner, Ricardo Borges, José D Machado
Chromogranin B gene ablation reduces the catecholamine cargo and decelerates exocytosis in chromaffin secretory vesicles.
J Neurosci, 30(3) 950-957 (2010)
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Chromogranins/secretogranins (Cgs) are the major soluble proteins of large dense-core secretory vesicles (LDCVs). We have recently reported that the absence of chromogranin A (CgA) caused important changes in the accumulation and in the exocytosis of catecholamines (CAs) using a CgA-knock-out (CgA-KO) mouse. Here, we have analyzed a CgB-KO mouse strain that can be maintained in homozygosis. These mice have 36% less adrenomedullary epinephrine when compared to Chgb(+/+) [wild type (WT)], whereas the norepinephrine content was similar. The total evoked release of CA was 33% lower than WT mice. This decrease was not due to a lower frequency of exocytotic events but to less secretion per quantum (approximately 30%) measured by amperometry; amperometric spikes exhibited a slower ascending but a normal decaying phase. Cell incubation with L-DOPA increased the vesicle CA content of WT but not of the CgB-KO cells. Intracellular electrochemistry, using patch amperometry, showed that L-DOPA overload produced a significantly larger increase in cytosolic CAs in cells from the KO animals than chromaffin cells from the WT. These data indicate that the mechanisms for vesicular accumulation of CAs in the CgB-KO cells were saturated, while there was ample capacity for further accumulation in WT cells. Protein analysis of LDCVs showed the overexpression of CgA as well as other proteins apparently unrelated to the secretory process. We conclude that CgB, like CgA, is a highly efficient system directly involved in monoamine accumulation and in the kinetics of exocytosis from LDCVs.

Karolina Jastrzebowska
Evolution of gene expression regulation during embryonic development of Drosophila species
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2010)

Magdalena Strzelecka, Andrew C. Oates, Karla M. Neugebauer
Dynamic control of Cajal body number during zebrafish embryogenesis
Nucleus, 1(1) 96-108 (2010)
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The Cajal body (CB) is an evolutionarily conserved nuclear subcompartment, enriched in components of the RNA processing machinery. The composition and dynamics of CBs in cells of living organisms is not well understood. Here we establish the zebrafish embryo as a model system to investigate the properties of CBs during rapid growth and cell division, taking advantage of the ease of live-cell imaging. We show that zebrafish embryo CBs contain coilin and multiple components of the pre-mRNA splicing machinery. Histone mRNA 3' end processing factors, present in CBs in some systems, were instead concentrated in a distinct nuclear body. CBs were present in embryos before and after activation of zygotic gene expression, indicating a maternal contribution of CB components. During the first 24 hours of development, embryonic cells displayed up to 30 CBs per nucleus; these dispersed prior to mitosis and reassembled within minutes upon daughter cell nucleus formation. Following zygotic genome activation, snRNP biogenesis was required for CB assembly and maintenance, suggesting a self-assembly process that determines CB numbers in embryos. Differentiation into muscle, neurons and epidermis was associated with the achievement of a steady state number of 2 CBs per nucleus. We propose that CB number is regulated during development to respond to the demands of gene expression in a rapidly growing embryo.

Xiao Liu, Fuhui Long, Hanchuan Peng, Sarah J Aerni, Min Jiang, Adolfo Sánchez-Blanco, John I Murray, Elicia A. Preston, Barbara Mericle, Serafim Batzoglou, Eugene W Myers, Stuart K Kim
Analysis of cell fate from single-cell gene expression profiles in C. elegans.
Cell, 139(3) 623-633 (2009)
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The C. elegans cell lineage provides a unique opportunity to look at how cell lineage affects patterns of gene expression. We developed an automatic cell lineage analyzer that converts high-resolution images of worms into a data table showing fluorescence expression with single-cell resolution. We generated expression profiles of 93 genes in 363 specific cells from L1 stage larvae and found that cells with identical fates can be formed by different gene regulatory pathways. Molecular signatures identified repeating cell fate modules within the cell lineage and enabled the generation of a molecular differentiation map that reveals points in the cell lineage when developmental fates of daughter cells begin to diverge. These results demonstrate insights that become possible using computational approaches to analyze quantitative expression from many genes in parallel using a digital gene expression atlas.

Fuhui Long, Hanchuan Peng, Xiao Liu, Stuart K Kim, Eugene Myers
A 3D digital atlas of C. elegans and its application to single-cell analyses.
Nat Methods, 6(9) 667-672 (2009)
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We built a digital nuclear atlas of the newly hatched, first larval stage (L1) of the wild-type hermaphrodite of Caenorhabditis elegans at single-cell resolution from confocal image stacks of 15 individual worms. The atlas quantifies the stereotypy of nuclear locations and provides other statistics on the spatial patterns of the 357 nuclei that could be faithfully segmented and annotated out of the 558 present at this developmental stage. We then developed an automated approach to assign cell names to each nucleus in a three-dimensional image of an L1 worm. We achieved 86% accuracy in identifying the 357 nuclei automatically. This computational method will allow high-throughput single-cell analyses of the post-embryonic worm, such as gene expression analysis, or ablation or stimulation of cells under computer control in a high-throughput functional screen.

Sidney B Cambridge, Daniel Geissler, Federico Calegari, Konstantinos Anastassiadis, Mazahir T Hasan, A. Francis Stewart, Wieland B. Huttner, Volker Hagen, Tobias Bonhoeffer
Doxycycline-dependent photoactivated gene expression in eukaryotic systems.
Nat Methods, 6(7) 527-531 (2009)
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High spatial and temporal resolution of conditional gene expression is typically difficult to achieve in whole tissues or organisms. We synthesized two reversibly inhibited, photoactivatable ('caged') doxycycline derivatives with different membrane permeabilities for precise spatial and temporal light-controlled activation of transgenes based on the 'Tet-on' system. After incubation with caged doxycycline or caged cyanodoxycycline, we induced gene expression by local irradiation with UV light or by two-photon uncaging in diverse biological systems, including mouse organotypic brain cultures, developing mouse embryos and Xenopus laevis tadpoles. The amount of UV light needed for induction was harmless as we detected no signs of toxicity. This method allows high-resolution conditional transgene expression at different spatial scales, ranging from single cells to entire complex organisms.

Wiebke Herzog, Katja Müller, Jan Huisken, Didier Y.R. Stainier
Genetic evidence for a noncanonical function of seryl-tRNA synthetase in vascular development.
Circ Res, 104(11) 1260-1266 (2009)
DOI

In a recent genetic screen, we identified mutations in genes important for vascular development and maintenance in zebrafish (Jin et al. Dev Biol. 2007;307:29-42). Mutations [corrected] at the adrasteia (adr) locus cause a pronounced dilatation of the aortic arch vessels as well as aberrant patterning of the hindbrain capillaries and, to a lesser extent, intersomitic vessels. This dilatation of the aortic arch vessels does not appear to be caused by increased cell proliferation but is dependent on vascular endothelial growth factor (Vegf) signaling. By positional cloning, we isolated seryl-tRNA synthetase (sars) as the gene affected by the adr mutations. Small interfering RNA knockdown experiments in human umbilical vein endothelial cell cultures indicate that SARS also regulates endothelial sprouting. These analyses of zebrafish and human endothelial cells reveal a new noncanonical function of Sars in endothelial development.

Jeremy N. Pulvers, Wieland B. Huttner
Brca1 is required for embryonic development of the mouse cerebral cortex to normal size by preventing apoptosis of early neural progenitors.
Development, 136(11) 1859-1868 (2009)
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The extent of apoptosis of neural progenitors is known to influence the size of the cerebral cortex. Mouse embryos lacking Brca1, the ortholog of the human breast cancer susceptibility gene BRCA1, show apoptosis in the neural tube, but the consequences of this for brain development have not been studied. Here we investigated the role of Brca1 during mouse embryonic cortical development by deleting floxed Brca1 using Emx1-Cre, which leads to conditional gene ablation specifically in the dorsal telencephalon after embryonic day (E) 9.5. The postnatal Brca1-ablated cerebral cortex was substantially reduced in size with regard to both cortical thickness and surface area. Remarkably, although the thickness of the cortical layers (except for the upper-most layer) was decreased, cortical layering as such was essentially unperturbed. High levels of apoptosis were found at E11.5 and E13.5, but dropped to near-control levels by E16.5. The apoptosis at the early stage of neurogenesis occurred in both BrdU pulse-labeled neural progenitors and the neurons derived therefrom. No changes were observed in the mitotic index of apical (neuroepithelial, radial glial) progenitors and basal (intermediate) progenitors, indicating that Brca1 ablation did not affect cell cycle progression. Brca1 ablation did, however, result in the nuclear translocation of p53 in neural progenitors, suggesting that their apoptosis involved activation of the p53 pathway. Our results show that Brca1 is required for the cerebral cortex to develop to normal size by preventing the apoptosis of early cortical progenitors and their immediate progeny.

Radoslaw K Ejsmont, Mihail Sarov, Sylke Winkler, Kamil A Lipinski, Pavel Tomancák
A toolkit for high-throughput, cross-species gene engineering in Drosophila.
Nat Methods, 6(6) 435-437 (2009)
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We generated two complementary genomic fosmid libraries for Drosophila melanogaster and Drosophila pseudoobscura that permit seamless modification of large genomic clones by high-throughput recombineering and direct transgenesis. The fosmid transgenes recapitulated endogenous gene expression patterns. These libraries, in combination with recombineering technology, will be useful to rescue mutant phenotypes, allow imaging of gene products in living flies and enable systematic analysis and manipulation of gene activity across species.

Jesper B Bramsen, Maria B Laursen, Anne F Nielsen, Thomas B Hansen, Claus Bus, Niels Langkjaer, B Ravindra Babu, Torben Højland, Mikhail Abramov, Arthur Van Aerschot, Dalibor Odadzic, Romualdas Smicius, Jens Haas, Cordula Andree, Jharna Barman, Malgorzata Wenska, Puneet Srivastava, Chuanzheng Zhou, Dmytro Honcharenko, Simone Hess, Elke Müller, Georgii V Bobkov, Sergey N Mikhailov, Eugenio Fava, Thomas F Meyer, Jyoti Chattopadhyaya, Marino Zerial, Joachim W Engels, Piet Herdewijn, Jesper Wengel, Jørgen Kjems
A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity.
Nucleic Acids Res, 37(9) 2867-2881 (2009)
PDF DOI

The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3'-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.

Bettina Heinrich, Zhaiyi Zhang, Oleg Raitskin, Michael Hiller, Natalya Benderska, Annette M Hartmann, Laurent Bracco, David Elliott, Shani Ben-Ari, Hermona Soreq, Joseph Sperling, Ruth Sperling, Stefan Stamm
Heterogeneous nuclear ribonucleoprotein G regulates splice site selection by binding to CC(A/C)-rich regions in pre-mRNA.
J Biol Chem, 284(21) 14303-14315 (2009)
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Almost every protein-coding gene undergoes pre-mRNA splicing, and the majority of these pre-mRNAs are alternatively spliced. Alternative exon usage is regulated by the transient formation of protein complexes on the pre-mRNA that typically contain heterogeneous nuclear ribonucleoproteins (hnRNPs). Here we characterize hnRNP G, a member of the hnRNP class of proteins. We show that hnRNP G is a nuclear protein that is expressed in different concentrations in various tissues and that interacts with other splicing regulatory proteins. hnRNP G is part of the supraspliceosome, where it regulates alternative splice site selection in a concentration-dependent manner. Its action on alternative exons can occur without a functional RNA-recognition motif by binding to other splicing regulatory proteins. The RNA-recognition motif of hnRNP G binds to a loose consensus sequence containing a CC(A/C) motif, and hnRNP G preferentially regulates alternative exons where this motif is clustered in close proximity. The X-chromosomally encoded hnRNP G regulates different RNAs than its Y-chromosomal paralogue RNA-binding motif protein, Y-linked (RBMY), suggesting that differences in alternative splicing, evoked by the sex-specific expression of hnRNP G and RBMY, could contribute to molecular sex differences in mammals.

Mirko Theis, Mikolaj Slabicki, Magno Junqueira, Maciej Paszkowski-Rogacz, Jana Sontheimer, Ralf Kittler, Anne-Kristin Heninger, Timo Glatter, Kristi Kruusmaa, Ina Poser, Anthony A. Hyman, Maria Teresa Pisabarro, Matthias Gstaiger, Rudolf Aebersold, Andrej Shevchenko, Frank Buchholz
Comparative profiling identifies C13orf3 as a component of the Ska complex required for mammalian cell division.
EMBO J, 28(10) 1453-1465 (2009)
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Proliferation of mammalian cells requires the coordinated function of many proteins to accurately divide a cell into two daughter cells. Several RNAi screens have identified previously uncharacterised genes that are implicated in mammalian cell division. The molecular function for these genes needs to be investigated to place them into pathways. Phenotypic profiling is a useful method to assign putative functions to uncharacterised genes. Here, we show that the analysis of protein localisation is useful to refine a phenotypic profile. We show the utility of this approach by defining a function of the previously uncharacterised gene C13orf3 during cell division. C13orf3 localises to centrosomes, the mitotic spindle, kinetochores, spindle midzone, and the cleavage furrow during cell division and is specifically phosphorylated during mitosis. Furthermore, C13orf3 is required for centrosome integrity and anaphase onset. Depletion by RNAi leads to mitotic arrest in metaphase with an activation of the spindle assembly checkpoint and loss of sister chromatid cohesion. Proteomic analyses identify C13orf3 (Ska3) as a new component of the Ska complex and show a direct interaction with a regulatory subunit of the protein phosphatase PP2A. All together, these data identify C13orf3 as an important factor for metaphase to anaphase progression and highlight the potential of combined RNAi screening and protein localisation analyses.

Aparna K Sapra, Minna-Liisa Änkö, Inna Grishina, Mike Lorenz, Marta Pabis, Ina Poser, Jarod Rollins, Eva-Marie Weiland, Karla M. Neugebauer
SR protein family members display diverse activities in the formation of nascent and mature mRNPs in vivo.
Mol Cell, 34(2) 179-190 (2009)
PDF DOI

The SR proteins are a family of pre-mRNA splicing factors with additional roles in gene regulation. To investigate individual family members in vivo, we generated a comprehensive panel of stable cell lines expressing GFP-tagged SR proteins under endogenous promoter control. Recruitment of SR proteins to nascent FOS RNA was transcription dependent and RNase sensitive, with unique patterns of accumulation along the gene specified by the RNA recognition motifs (RRMs). In addition, all SR protein interactions with Pol II were RNA dependent, indicating that SR proteins are not preassembled with Pol II. SR protein interactions with RNA were confirmed in situ by FRET/FLIM. Interestingly, SC35-GFP also exhibited FRET with DNA and failed to associate with cytoplasmic mRNAs, whereas all other SR proteins underwent nucleocytoplasmic shuttling and associated with specific nuclear and cytoplasmic mRNAs. Because different constellations of SR proteins bound nascent, nuclear, and cytoplasmic mRNAs, mRNP remodeling must occur throughout an mRNA's lifetime.

Mark Schmid, Beate Küchler, Christian R. Eckmann
Two conserved regulatory cytoplasmic poly(A) polymerases, GLD-4 and GLD-2, regulate meiotic progression in C. elegans.
Genes Dev, 23(7) 824-836 (2009)
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Translational regulation is heavily employed during developmental processes to control the timely accumulation of proteins independently of gene transcription. In particular, mRNA poly(A) tail metabolism in the cytoplasm is a key determinant for balancing an mRNA's translational output and its decay rate. Noncanonical poly(A) polymerases (PAPs), such as germline development defective-2 (GLD-2), can mediate poly(A) tail extension. Little is known about the regulation and functional complexity of cytoplasmic PAPs. Here we report the discovery of Caenorhabditis elegans GLD-4, a cytoplasmic PAP present in P granules that is orthologous to Trf4/5p from budding yeast. GLD-4 enzymatic activity is enhanced by its interaction with GLS-1, a protein associated with the RNA-binding protein GLD-3. GLD-4 is predominantly expressed in germ cells, and its activity is essential for early meiotic progression of male and female gametes in the absence of GLD-2. For commitment into female meiosis, both PAPs converge on at least one common target mRNA-i.e., gld-1 mRNA-and, as a consequence, counteract the repressive action of two PUF proteins and the putative deadenylase CCR-4. Together our findings suggest that two different cytoplasmic PAPs stabilize and translationally activate several meiotic mRNAs to provide a strong fail-safe mechanism for early meiotic progression.

Luis G. Morelli, Saul Ares, Leah Herrgen, Christian Schröter, Frank Jülicher, Andrew C. Oates
Delayed coupling theory of vertebrate segmentation.
HFSP J, 3(1) 55-66 (2009)
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Rhythmic and sequential subdivision of the elongating vertebrate embryonic body axis into morphological somites is controlled by an oscillating multicellular genetic network termed the segmentation clock. This clock operates in the presomitic mesoderm (PSM), generating dynamic stripe patterns of oscillatory gene-expression across the field of PSM cells. How these spatial patterns, the clock's collective period, and the underlying cellular-level interactions are related is not understood. A theory encompassing temporal and spatial domains of local and collective aspects of the system is essential to tackle these questions. Our delayed coupling theory achieves this by representing the PSM as an array of phase oscillators, combining four key elements: a frequency profile of oscillators slowing across the PSM; coupling between neighboring oscillators; delay in coupling; and a moving boundary describing embryonic axis elongation. This theory predicts that the segmentation clock's collective period depends on delayed coupling. We derive an expression for pattern wavelength across the PSM and show how this can be used to fit dynamic wildtype gene-expression patterns, revealing the quantitative values of parameters controlling spatial and temporal organization of the oscillators in the system. Our theory can be used to analyze experimental perturbations, thereby identifying roles of genes involved in segmentation.

Bret J Pearson^*, George T Eisenhoffer^*, Kyle A Gurley^*, Jochen Rink^*, Diane E Miller, Alejandro Sánchez Alvarado
Formaldehyde-based whole-mount in situ hybridization method for planarians.
Dev Dyn, 238(2) 443-450 (2009)
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Whole-mount in situ hybridization (WISH) is a powerful tool for visualizing gene expression patterns in specific cell and tissue types. Each model organism presents its own unique set of challenges for achieving robust and reproducible staining with cellular resolution. Here, we describe a formaldehyde-based WISH method for the freshwater planarian Schmidtea mediterranea developed by systematically comparing and optimizing techniques for fixation, permeabilization, hybridization, and postprocessing. The new method gives robust, high-resolution labeling in fine anatomical detail, allows co-labeling with fluorescent probes, and is sufficiently sensitive to resolve the expression pattern of a microRNA in planarians. Our WISH methodology not only provides significant advancements over current protocols that make it a valuable asset for the planarian community, but should also find wide applicability in WISH methods used in other systems.

Marina Trouillas, Claire Saucourt, Bertrand Guillotin, Xavier Gauthereau, Li Ding, Frank Buchholz, Michael Xavier Doss, Agapios Sachinidis, Juergen Hescheler, Oliver Hummel, Norbert Huebner, Raivo Kolde, Jaak Vilo, Herbert Schulz, Hélène Boeuf
Three LIF-dependent signatures and gene clusters with atypical expression profiles, identified by transcriptome studies in mouse ES cells and early derivatives.
BMC Genomics, 10(73) 1-20 (2009)
PDF DOI

BACKGROUND: Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of the cytokine Leukaemia Inhibitory Factor (LIF). Identification of LIF targets and of genes regulating the transition between pluripotent and early differentiated cells is a critical step for understanding the control of ES cell pluripotency. RESULTS: By gene profiling studies carried out with mRNAs from ES cells and their early derivatives treated or not with LIF, we have identified i) LIF-dependent genes, highly expressed in pluripotent cells, whose expression level decreases sharply upon LIF withdrawal [Pluri genes], ii) LIF induced genes [Lifind genes] whose expression is differentially regulated depending upon cell context and iii) genes specific to the reversible or irreversible committed states. In addition, by hierarchical gene clustering, we have identified, among eight independent gene clusters, two atypical groups of genes, whose expression level was highly modulated in committed cells only. Computer based analyses led to the characterization of different sub-types of Pluri and Lifind genes, and revealed their differential modulation by Oct4 or Nanog master genes. Individual knock down of a selection of Pluri and Lifind genes leads to weak changes in the expression of early differentiation markers, in cell growth conditions in which these master genes are still expressed. CONCLUSION: We have identified different sets of LIF-regulated genes depending upon the cell state (reversible or irreversible commitment), which allowed us to present a novel global view of LIF responses. We are also reporting on the identification of genes whose expression is strictly regulated during the commitment step. Furthermore, our studies identify sub-networks of genes with a restricted expression in pluripotent ES cells, whose down regulation occurs while the master knot (composed of OCT4, SOX2 and NANOG) is still expressed and which might be down-regulated together for driving cells towards differentiation.

Marat Gorivodsky, Mahua Mukhopadhyay, Michaela Wilsch-Bräuninger, Matthew Phillips, Andreas Teufel, Changmee Kim, Nasir Malik, Wieland B. Huttner, Heiner Westphal
Intraflagellar transport protein 172 is essential for primary cilia formation and plays a vital role in patterning the mammalian brain.
Dev Biol, 325(1) 24-32 (2009)
PDF DOI

IFT172, also known as Selective Lim-domain Binding protein (SLB), is a component of the intraflagellar transport (IFT) complex. In order to evaluate the biological role of the Ift172 gene, we generated a loss-of-function mutation in the mouse. The resulting Slb mutant embryos die between E12.5 and 13.0, and exhibit severe cranio-facial malformations, failure to close the cranial neural tube, holoprosencephaly, heart edema and extensive hemorrhages. Cilia outgrowth in cells of the neuroepithelium is initiated but the axonemes are severely truncated and do not contain visible microtubules. Morphological and molecular analyses revealed a global brain-patterning defect along the dorsal-ventral (DV) and anterior-posterior (AP) axes. We demonstrate that Ift172 gene function is required for early regulation of Fgf8 at the midbrain-hindbrain boundary and maintenance of the isthmic organizer. In addition, Ift172 is required for proper function of the embryonic node, the early embryonic organizer and for formation of the head organizing center (the anterior mesendoderm, or AME). We propose a model suggesting that forebrain and mid-hindbrain growth and AP patterning depends on the early function of Ift172 at gastrulation. Our data suggest that the formation and function of the node and AME in the mouse embryo relies on an indispensable role of Ift172 in cilia morphogenesis and cilia-mediated signaling.

Kuixing Zhang, Fangwen Rao, Brinda K. Rana, Jiaur R. Gayen, Federico Calegari, Angus King, Patrizia Rosa, Wieland B. Huttner, Mats Stridsberg, Manjula Mahata, Sucheta Vaingankar, Vafa Mahboubi, Rany M Salem, Juan L Rodriguez-Flores, Maple M Fung, Douglas W Smith, Nicholas J Schork, Michael G Ziegler, Laurent Taupenot, Sushil K Mahata, Daniel T O'Connor
Autonomic function in hypertension: Role of genetic variation at the catecholaminestorage vesicle protein chromogranin B (CHGB).
Circ Cardiovasc Genet, 2(1) 46-56 (2009)
PDF DOI

RATIONALE: Hypertension is a complex trait with deranged autonomic control of the circulation. Chromogranin B (CHGB) is the most abundant core protein in human catecholamine secretory vesicles, playing an important role in their biogenesis. Does common inter-individual variation at the CHGB locus contribute to phenotypic variation in CHGB and catecholamine secretion, autonomic stability of the circulation, or blood pressure in the population? METHODS AND RESULTS: To probe inter-individual variability in CHGB, we systematically studied polymorphism across the locus by re-sequencing CHGB (~6 kbp footprint spanning the promoter, 5 exons, exon/intron borders, UTRs) in n=160 subjects (2n=320 chromosomes) of diverse biogeographic ancestries. We identified 53 SNPs, of which 22 were common. We then studied n=1182 subjects drawn from the most extreme BP values in the population (highest and lowest 5(th) %iles), typing 4 common polymorphisms spanning the ~14 kbp locus. Sliding-window haplotype analysis indicated BP associations peaking in the 5'/promoter region, and most prominent in men, and a peak effect in the proximal promoter at variant A-261T (A>T), accounting for ~8/~6 mmHg SBP/DBP in males. The promoter allele (A-261) that was a predictor of higher DBP and SBP was also associated with lower circulating/plasma CHGB concentration (CHGB(439-451) epitope) in twin pairs. In twins, the same CHGB variants that were predictors of lower basal CHGB secretion were also associated with exaggerated catecholamine secretion and BP response to environmental (cold) stress; likewise, women displayed increased plasma CHGB(439-451), but decreased catecholamine secretion as well as BP response to environmental stress. The effect of A-261T on CHGB expression was confirmed in chromaffin cells by site-directed mutagenesis on transfected CHGB promoter/luciferase reporter activity, and the allelic effects of A-261T on gene expression were directionally coordinate in cella and in vivo. To confirm these clinical associations experimentally, we undertook targeted homozygous (-/-) ablation of the mouse Chgb gene; knockout mice displayed substantially increased BP, by ~20/~18 mmHg SBP/DBP, confirming the mechanistic basis of our findings in humans. CONCLUSIONS: We conclude that common genetic variation at the CHGB locus, especially in the proximal promoter, influences CHGB expression, and later catecholamine secretion and the early heritable responses to environmental stress, eventuating in changes in resting/basal BP in the population. Both the early (gene expression) and late (population BP) consequences of CHGB variation are sex-dependent. The results point to new molecular strategies for probing autonomic control of the circulation, and ultimately the susceptibility to and pathogenesis of cardiovascular disease states such as hypertension.

Gaspare Benenati, Sider Penkov, Thomas Müller-Reichert, Eugeni V. Entchev, Teymuras V. Kurzchalia
Two cytochrome P450s in Caenorhabditis elegans are essential for the organization of eggshell, correct execution of meiosis and the polarization of embryo.
Mech Dev, 126(5-6) 382-393 (2009)
PDF DOI

The role of lipids in the process of embryonic development of Caenorhabditis elegans is still poorly understood. Cytochrome P450s, a class of lipid-modifying enzymes, are good candidates to be involved in the production or degradation of lipids essential for development. We investigated two highly similar cytochrome P450s in C. elegans, cyp-31A2 and cyp-31A3, that are homologs of the gene responsible for Bietti crystalline corneoretinal dystrophy in humans. Depletion of both cytochromes either by RNAi or using a double deletion mutant, led to the failure of establishing the correct polarity of the embryo and to complete the extrusion of the polar bodies during meiosis. In addition, the egg became osmotic sensitive and permeable to dyes. The phenotype of cyp-31A2 or cyp-31A3 is very similar to a class of mutants that have polarization and osmotic defects (POD), thus the genes were renamed to pod-7 and pod-8, respectively. Electron microscopic analysis demonstrated that the activity of pod-7/pod-8 is crucial for the proper assembly of the eggshell and, in particular, for the production of its lipid-rich layer. Using a complementation with lipid extracts, we show that POD-7/POD-8 function together with a NADPH cytochrome P450 reductase, coded by emb-8, and are involved in the production of lipid(s) required for eggshell formation.

Elena Bonzón-Kulichenko, Dominik Schwudke, Nilda Gallardo, Eduardo Moltó, Teresa Fernández-Agulló, Andrej Shevchenko, Antonio Andrés
Central leptin regulates total ceramide content and sterol regulatory element binding protein-1C proteolytic maturation in rat white adipose tissue.
Endocrinology, 150(1) 169-178 (2009)
PDF DOI

Obesity and type 2 diabetes are associated with insulin and leptin resistance, and increased ceramide contents in target tissues. Because the adipose tissue has become a central focus in these diseases, and leptin-induced increases in insulin sensitivity may be related to effects of leptin on lipid metabolism, we investigated herein whether central leptin was able to regulate total ceramide levels and the expression of enzymes involved in ceramide metabolism in rat white adipose tissue (WAT). After 7 d central leptin treatment, the total content of ceramides was analyzed by quantitative shotgun lipidomics mass spectrometry. The effects of leptin on the expression of several enzymes of the sphingolipid metabolism, sterol regulatory element binding protein (SREBP)-1c, and insulin-induced gene 1 (INSIG-1) in this tissue were studied. Total ceramide levels were also determined after surgical WAT denervation. Central leptin infusion significantly decreased both total ceramide content and the long-chain fatty acid ceramide species in WAT. Concomitant with these results, leptin decreased the mRNA levels of enzymes involved in de novo ceramide synthesis (SPT-1, LASS2, LASS4) and ceramide production from sphingomyelin (SMPD-1/2). The mRNA levels of enzymes of ceramide degradation (Asah1/2) and utilization (sphingomyelin synthase, ceramide kinase, glycosyl-ceramide synthase, GM3 synthase) were also down-regulated. Ceramide-lowering effects of central leptin were prevented by local autonomic nervous system denervation of WAT. Finally, central leptin treatment markedly increased INSIG-1 mRNA expression and impaired SREBP-1c activation in epididymal WAT. These observations indicate that in vivo central leptin, acting through the autonomic nervous system, regulates total ceramide levels and SREBP-1c proteolytic maturation in WAT, probably contributing to improve the overall insulin sensitivity.

Elizabeth Rach, Hsiang-Yu Yuan, William H Majoros, Pavel Tomancák, Uwe Ohler
Motif composition, conservation and condition-specificity of single and alternative transcription start sites in the Drosophila genome.
Genome Biol, 10(7) Art. No. R73 (2009)
PDF DOI

BACKGROUND: Transcription initiation is a key component in the regulation of gene expression. mRNA 5' full-length sequencing techniques have enhanced our understanding of mammalian transcription start sites (TSSs), revealing different initiation patterns on a genomic scale. RESULTS: To identify TSSs in Drosophila melanogaster, we applied a hierarchical clustering strategy on available 5' expressed sequence tags (ESTs) and identified a high quality set of 5,665 TSSs for approximately 4,000 genes. We distinguished two initiation patterns: 'peaked' TSSs, and 'broad' TSS cluster groups. Peaked promoters were found to contain location-specific sequence elements; conversely, broad promoters were associated with non-location-specific elements. In alignments across other Drosophila genomes, conservation levels of sequence elements exceeded 90% within the melanogaster subgroup, but dropped considerably for distal species. Elements in broad promoters had lower levels of conservation than those in peaked promoters. When characterizing the distributions of ESTs, 64% of TSSs showed distinct associations to one out of eight different spatiotemporal conditions. Available whole-genome tiling array time series data revealed different temporal patterns of embryonic activity across the majority of genes with distinct alternative promoters. Many genes with maternally inherited transcripts were found to have alternative promoters utilized later in development. Core promoters of maternally inherited transcripts showed differences in motif composition compared to zygotically active promoters. CONCLUSIONS: Our study provides a comprehensive map of Drosophila TSSs and the conditions under which they are utilized. Distinct differences in motif associations with initiation pattern and spatiotemporal utilization illustrate the complex regulatory code of transcription initiation.

Eric Lécuyer, Pavel Tomancák
Mapping the gene expression universe.
Curr Opin Genet Dev, 18(6) 506-512 (2008)
PDF DOI

Methods to globally survey gene expression provide valuable insights into gene function during development. In particular, comprehensive in situ hybridization studies have demonstrated that gene expression patterns are extraordinarily diverse and new imaging techniques have been introduced to capture these patterns with higher resolution at the tissue, cellular, and subcellular levels. The analysis of massive image databases can be greatly facilitated by computer vision techniques once annotated image sets reach the crucial mass sufficient to train the computer in pattern recognition. Ultimately, genome-wide atlases of gene expression during development will record gene activity in living animals with at least cellular resolution and in the context of morphogenetic events. These emerging datasets will lead to great advances in the field of comparative genomics and revolutionize our ability to decipher and model developmental processes for a variety of organisms.

Chantilly Munson, Jan Huisken, Nana Bit-Avragim, Taiyi Kuo, P D Dong, Elke A Ober, Heather Verkade, Salim Abdelilah-Seyfried, Didier Y.R. Stainier
Regulation of neurocoel morphogenesis by Pard6 gamma b.
Dev Biol, 324(1) 41-54 (2008)
DOI

The Par3/Par6/aPKC protein complex plays a key role in the establishment and maintenance of apicobasal polarity, a cellular characteristic essential for tissue and organ morphogenesis, differentiation and homeostasis. During a forward genetic screen for liver and pancreas mutants, we identified a pard6gammab mutant, representing the first known pard6 mutant in a vertebrate organism. pard6gammab mutants exhibit defects in epithelial tissue development as well as multiple lumens in the neural tube. Analyses of the cells lining the neural tube cavity, or neurocoel, in wildtype and pard6gammab mutant embryos show that lack of Pard6gammab function leads to defects in mitotic spindle orientation during neurulation. We also found that the PB1 (aPKC-binding) and CRIB (Cdc-42-binding) domains and the KPLG amino acid sequence within the PDZ domain (Pals1-and Crumbs binding) are not required for Pard6gammab localization but are essential for its function in neurocoel morphogenesis. Apical membranes are reduced, but not completely absent, in mutants lacking the zygotic, or both the maternal and zygotic, function of pard6gammab, leading us to examine the localization and function of the three additional zebrafish Pard6 proteins. We found that Pard6alpha, but not Pard6beta or Pard6gammaa, could partially rescue the pard6gammab(s441) mutant phenotypes. Altogether, these data indicate a previously unappreciated functional diversity and complexity within the vertebrate pard6 gene family.

Agnes Tóth-Petróczy, Christopher J Oldfield, István Simon, Yuichiro Takagi, A. Keith Dunker, Vladimir N Uversky, Monika Fuxreiter
Malleable machines in transcription regulation: the mediator complex.
PLoS Comput Biol, 4(12) Art. No. e1000243 (2008)
Open Access DOI

The Mediator complex provides an interface between gene-specific regulatory proteins and the general transcription machinery including RNA polymerase II (RNAP II). The complex has a modular architecture (Head, Middle, and Tail) and cryoelectron microscopy analysis suggested that it undergoes dramatic conformational changes upon interactions with activators and RNAP II. These rearrangements have been proposed to play a role in the assembly of the preinitiation complex and also to contribute to the regulatory mechanism of Mediator. In analogy to many regulatory and transcriptional proteins, we reasoned that Mediator might also utilize intrinsically disordered regions (IDRs) to facilitate structural transitions and transmit transcriptional signals. Indeed, a high prevalence of IDRs was found in various subunits of Mediator from both Saccharomyces cerevisiae and Homo sapiens, especially in the Tail and the Middle modules. The level of disorder increases from yeast to man, although in both organisms it significantly exceeds that of multiprotein complexes of a similar size. IDRs can contribute to Mediator's function in three different ways: they can individually serve as target sites for multiple partners having distinctive structures; they can act as malleable linkers connecting globular domains that impart modular functionality on the complex; and they can also facilitate assembly and disassembly of complexes in response to regulatory signals. Short segments of IDRs, termed molecular recognition features (MoRFs) distinguished by a high protein-protein interaction propensity, were identified in 16 and 19 subunits of the yeast and human Mediator, respectively. In Saccharomyces cerevisiae, the functional roles of 11 MoRFs have been experimentally verified, and those in the Med8/Med18/Med20 and Med7/Med21 complexes were structurally confirmed. Although the Saccharomyces cerevisiae and Homo sapiens Mediator sequences are only weakly conserved, the arrangements of the disordered regions and their embedded interaction sites are quite similar in the two organisms. All of these data suggest an integral role for intrinsic disorder in Mediator's function.

Jeremiah J. Zartman, Nir Yakoby, Christopher A. Bristow, Xiaofeng Zhou, Karin Schlichting, Christian Dahmann, Stanislav Y. Shvartsman
Cad74A is regulated by BR and is required for robust dorsal appendage formation in Drosophila oogenesis.
Dev Biol, 322(2) 289-301 (2008)
PDF DOI

Drosophila egg development is an established model for studying epithelial patterning and morphogenesis, but the connection between signaling pathways and egg morphology is still incompletely understood. We have identified a non-classical cadherin, Cad74A, as a putative adhesion gene that bridges epithelial patterning and morphogenesis in the follicle cells. Starting in mid-oogenesis, Cad74A is expressed in the follicle cells that contact the oocyte, including the border cells and most of the columnar follicle cells. However, Cad74A is repressed in two dorsolateral patches of follicle cells, which participate in the formation of tubular respiratory appendages. We show genetically that Cad74A is downstream of the EGFR and BMP signaling pathways and is repressed by the Zn-finger transcription factor Broad. The correlation of Cad74A repression in the cells that bend out of the plane of the follicular epithelium is preserved across Drosophila species and mutant backgrounds exhibiting a range of eggshell phenotypes. Complete removal of Cad74A from the follicle cells causes defects in dorsal appendage formation. Ectopic expression of Cad74A in the roof cells results in shortened, flattened appendages due to the hindered migration of the roof cells. Based on these results, we propose that Cad74A is part of the adhesive machinery that enables robust dorsal appendage formation, and as such provides a link between the patterning of the follicle cells and eggshell morphogenesis.

Normund Jabs, I Franklin, M B Brenner, J Gromada, Napoleone Ferrara, C B Wollheim, Eckhard Lammert
Reduced insulin secretion and content in VEGF-a deficient mouse pancreatic islets.
Exp Clin Endocrinol Diabetes, 116 Suppl 1 46-49 (2008)
PDF DOI

Mice, deficient for vascular endothelial growth factor VEGF-A in pancreatic islets, have reduced insulin gene expression levels and an impaired glucose tolerance. Here, we investigated whether VEGF-A was required for physiological glucose-stimulated insulin secretion and insulin content. We performed in situ pancreas perfusions and islet perifusions on mice lacking VEGF-A in the pancreatic epithelium in order to study their ability to secrete insulin in response to glucose. We identified insulin secretion defects in the pancreata of VEGF-A deficient mice, including a delayed and blunted response to glucose. Islet perifusion experiments revealed a missing first phase and weaker second phase of insulin secretion, in two of three VEGF-A deficient mice. On average, insulin content in VEGF-A deficient islets was significantly reduced when compared with control islets. We conclude that VEGF-A is required in pancreatic islets for normal glucose-stimulated insulin secretion and physiological insulin content. Thus, VEGF-A is a key factor for pancreatic islet function.

Zhenglin Yang, Yali Chen, Concepcion Lillo, Jeremy Chien, Zhengya Yu, Michel Michaelides, Martin Klein, Kim A Howes, Yang Li, Yuuki Kaminoh, Haoyu Chen, Chao Zhao, Yuhong Chen, Youssef Tawfik Al-Sheikh, Goutam Karan, Denis Corbeil, Pascal Escher, Shin Kamaya, Chunmei Li, Samantha Johnson, Jeanne M Frederick, Yu Zhao, Changguan Wang, D Joshua Cameron, Wieland B. Huttner, Daniel F Schorderet, Frances L Munier, Anthony T Moore, David G Birch, Wolfgang Baehr, David M Hunt, David S Williams, Kang Zhang
Mutant prominin 1 found in patients with macular degeneration disrupts photoreceptor disk morphogenesis in mice.
J Clin Invest, 118(8) 2908-2916 (2008)
PDF DOI

Familial macular degeneration is a clinically and genetically heterogeneous group of disorders characterized by progressive central vision loss. Here we show that an R373C missense mutation in the prominin 1 gene (PROM1) causes 3 forms of autosomal-dominant macular degeneration. In transgenic mice expressing R373C mutant human PROM1, both mutant and endogenous PROM1 were found throughout the layers of the photoreceptors, rather than at the base of the photoreceptor outer segments, where PROM1 is normally localized. Moreover, the outer segment disk membranes were greatly overgrown and misoriented, indicating defective disk morphogenesis. Immunoprecipitation studies showed that PROM1 interacted with protocadherin 21 (PCDH21), a photoreceptor-specific cadherin, and with actin filaments, both of which play critical roles in disk membrane morphogenesis. Collectively, our results identify what we believe to be a novel complex involved in photoreceptor disk morphogenesis and indicate a possible role for PROM1 and PCDH21 in macular degeneration.

Karin Schlichting, Christian Dahmann
Hedgehog and Dpp signaling induce cadherin Cad86C expression in the morphogenetic furrow during Drosophila eye development.
Mech Dev, 125(8) 712-728 (2008)
PDF DOI

During Drosophila eye development, cell differentiation is preceded by the formation of a morphogenetic furrow, which progresses across the epithelium from posterior to anterior. Cells within the morphogenetic furrow are apically constricted and shortened along their apical-basal axis. However, how these cell shape changes and, thus, the progression of the morphogenetic furrow are controlled is not well understood. Here we show that cells simultaneously lacking Hedgehog and Dpp signal transduction fail to shorten and do not enter the morphogenetic furrow. Moreover, we have identified a gene, cadherin Cad86C, which is highly expressed in cells of the leading flank of the morphogenetic furrow. Ectopic activation of either the Hedgehog or Dpp signal transduction pathway results in elevated Cad86C expression. Conversely, simultaneous loss of both Hedgehog and Dpp signal transduction leads to decreased Cad86C expression. Finally, ectopic expression of Cad86C in either eye-antennal imaginal discs or wing imaginal discs results in apical constriction and shortening of cells. We conclude that Hedgehog and Dpp signaling promote the shortening of cells within the morphogenetic furrow. Induction of Cad86C expression might be one mechanism through which Hedgehog and Dpp promote these cell shape changes.

Javier Martinez, Karla M. Neugebauer
El Sur también existe: processing RNA in the Argentine Patagonia. Meetings on 'Cell Biology, Signaling and Alternative Splicing' and 'Gene Expression and RNA Processing'.
EMBO Rep, 9(6) 516-520 (2008)
PDF DOI

Ina Poser, Mihail Sarov, James R A Hutchins, Jean-Karim Hériché, Yusuke Toyoda, Andrei I. Pozniakovsky, Daniela Weigl, Anja Nitzsche, Björn Hegemann, Alexander W. Bird, Laurence Pelletier, Ralf Kittler, Sujun Hua, Ronald Naumann, Martina Augsburg, Martina M Sykora, Helmut Hofemeister, Youming Zhang, Kim Nasmyth, Kevin P White, Steffen Dietzel, Karl Mechtler, Richard Durbin, A. Francis Stewart, Jan-Michael Peters, Frank Buchholz, Anthony A. Hyman
BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.
Nat Methods, 5(5) 409-415 (2008)
PDF DOI

The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.

Lars Demmel^*, Maike Gravert^*, Ebru Ercan, Bianca Habermann, Thomas Müller-Reichert, Viktoria Kukhtina, Volker Haucke, Thorsten Baust, Marc Sohrmann, Yannis Kalaidzidis, Christian Klose, Mike Beck, Matthias Peter, Christiane Walch-Solimena
The clathrin adaptor Gga2p is a phosphatidylinositol 4-phosphate effector at the Golgi exit.
Mol Biol Cell, 19(5) 1991-2002 (2008)
PDF DOI

Phosphatidylinositol 4-phosphate (PI(4)P) is a key regulator of membrane transport required for the formation of transport carriers from the trans-Golgi network (TGN). The molecular mechanisms of PI(4)P signaling in this process are still poorly understood. In a search for PI(4)P effector molecules, we performed a screen for synthetic lethals in a background of reduced PI(4)P and found the gene GGA2. Our analysis uncovered a PI(4)P-dependent recruitment of the clathrin adaptor Gga2p to the TGN during Golgi-to-endosome trafficking. Gga2p recruitment to liposomes is stimulated both by PI(4)P and the small GTPase Arf1p in its active conformation, implicating these two molecules in the recruitment of Gga2p to the TGN, which ultimately controls the formation of clathrin-coated vesicles. PI(4)P binding occurs through a phosphoinositide-binding signature within the N-terminal VHS domain of Gga2p resembling a motif found in other clathrin interacting proteins. These data provide an explanation for the TGN-specific membrane recruitment of Gga2p.

Neil C Chi, Robin M Shaw, Benno Jungblut, Jan Huisken, Tania Ferrer, Rima Arnaout, Ian Scott, Dimitris Beis, Tong Xiao, Herwig Baier, Lily Y Jan, Martin Tristani-Firouzi, Didier Y.R. Stainier
Genetic and physiologic dissection of the vertebrate cardiac conduction system.
PLoS Biol, 6(5) 109-109 (2008)
Open Access DOI

Vertebrate hearts depend on highly specialized cardiomyocytes that form the cardiac conduction system (CCS) to coordinate chamber contraction and drive blood efficiently and unidirectionally throughout the organism. Defects in this specialized wiring system can lead to syncope and sudden cardiac death. Thus, a greater understanding of cardiac conduction development may help to prevent these devastating clinical outcomes. Utilizing a cardiac-specific fluorescent calcium indicator zebrafish transgenic line, Tg(cmlc2:gCaMP)(s878), that allows for in vivo optical mapping analysis in intact animals, we identified and analyzed four distinct stages of cardiac conduction development that correspond to cellular and anatomical changes of the developing heart. Additionally, we observed that epigenetic factors, such as hemodynamic flow and contraction, regulate the fast conduction network of this specialized electrical system. To identify novel regulators of the CCS, we designed and performed a new, physiology-based, forward genetic screen and identified for the first time, to our knowledge, 17 conduction-specific mutations. Positional cloning of hobgoblin(s634) revealed that tcf2, a homeobox transcription factor gene involved in mature onset diabetes of the young and familial glomerulocystic kidney disease, also regulates conduction between the atrium and the ventricle. The combination of the Tg(cmlc2:gCaMP)(s878) line/in vivo optical mapping technique and characterization of cardiac conduction mutants provides a novel multidisciplinary approach to further understand the molecular determinants of the vertebrate CCS.

Chong Hyun Shin, Won-Suk Chung, Sung-Kook Hong, Elke A Ober, Heather Verkade, Holly A Field, Jan Huisken, Didier Y.R. Stainier
Multiple roles for Med12 in vertebrate endoderm development.
Dev Biol, 317(2) 467-479 (2008)
DOI

In zebrafish, the endoderm originates at the blastula stage from the most marginal blastomeres. Through a series of complex morphogenetic movements and differentiation events, the endodermal germ layer gives rise to the epithelial lining of the digestive tract as well as its associated organs such as the liver, pancreas, and swim bladder. How endodermal cells differentiate into distinct cell types such as hepatocytes or endocrine and exocrine pancreatic cells remains a major question. In a forward genetic screen for genes regulating endodermal organ development, we identified mutations at the shiri locus that cause defects in the development of a number of endodermal organs including the liver and pancreas. Detailed phenotypic analyses indicate that these defects are partially due to a reduction in endodermal expression of the hairy/enhancer of split-related gene, her5, at mid to late gastrulation stages. Using the Tg(0.7her5:EGFP)(ne2067) line, we show that her5 is expressed in the endodermal precursors that populate the pharyngeal region as well as the organ-forming region. We also find that knocking down her5 recapitulates some of the endodermal phenotypes of shiri mutants, further revealing the role of her5 in endoderm development. Positional cloning reveals that shiri encodes Med12, a regulatory subunit of the transcriptional Mediator complex recently associated with two human syndromes. Additional studies indicate that Med12 modulates the ability of Casanova/Sox32 to induce sox17 expression. Thus, detailed phenotypic analyses of embryos defective in a component of the Mediator complex have revealed new insights into discrete aspects of vertebrate endoderm development, and provide possible explanations for the craniofacial and digestive system defects observed in humans with mutations in MED12.

Tobias Bollenbach^*, Periklis Pantazis^*, Anna Kicheva^*, Christian Bökel, Marcos González-Gaitán, Frank Jülicher
Precision of the Dpp gradient.
Development, 135(6) 1137-1146 (2008)
PDF DOI

Morphogen concentration gradients provide positional information by activating target genes in a concentration-dependent manner. Recent reports show that the gradient of the syncytial morphogen Bicoid seems to provide precise positional information to determine target gene domains. For secreted morphogenetic ligands, the precision of the gradients, the signal transduction and the reliability of target gene expression domains have not been studied. Here we investigate these issues for the TGF-beta-type morphogen Dpp. We first studied theoretically how cell-to-cell variability in the source, the target tissue, or both, contribute to the variations of the gradient. Fluctuations in the source and target generate a local maximum of precision at a finite distance to the source. We then determined experimentally in the wing epithelium: (1) the precision of the Dpp concentration gradient; (2) the precision of the Dpp signaling activity profile; and (3) the precision of activation of the Dpp target gene spalt. As captured by our theoretical description, the Dpp gradient provides positional information with a maximal precision a few cells away from the source. This maximal precision corresponds to a positional uncertainly of about a single cell diameter. The precision of the Dpp gradient accounts for the precision of the spalt expression range, implying that Dpp can act as a morphogen to coarsely determine the expression pattern of target genes.

Elly M. Tanaka, Gilbert Weidinger
Micromanaging regeneration.
Genes Dev, 22(6) 700-705 (2008)
PDF DOI

Appendage regeneration is defined by rapid changes in gene expression that achieve dramatic developmental effects, suggesting involvement of microRNAs (miRNAs). Here, we find dynamic regulation of many miRNAs during zebrafish fin regeneration. In particular, miR-133 levels are high in uninjured fins but low during regeneration. When regeneration was blocked by Fibroblast growth factor (Fgf) receptor inhibition, high miR-133 levels were quickly restored. Experimentally increasing amounts of miR-133 attenuated fin regeneration. Conversely, miR-133 antagonism during Fgf receptor inhibition accelerated regeneration through increased proliferation within the regeneration blastema. The Mps1 kinase, an established positive regulator of blastemal proliferation, is an in vivo target of miR-133. Our findings identify miRNA depletion as a new regulatory mechanism for complex tissue regeneration.

Dominik Schwudke, Asgar Ergin, Kathrin Michael, Sven Volkmar, Bernd Appel, Dorothea Knabner, Antje Konietzny, Eckhard Strauch
Broad-host-range Yersinia phage PY100: genome sequence, proteome analysis of virions, and DNA packaging strategy.
J Bacteriol, 190(1) 332-342 (2008)
PDF DOI

PY100 is a lytic bacteriophage with a broad host range within the genus Yersinia. The phage forms plaques on strains of the three human pathogenic species Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis at 37 degrees C. PY100 was isolated from farm manure and intended to be used in phage therapy trials. PY100 has an icosahedral capsid containing double-stranded DNA and a contractile tail. The genome consists of 50,291 bp and is predicted to contain 93 open reading frames (ORFs). PY100 gene products were found to be homologous to the capsid proteins and proteins involved in DNA metabolism of the enterobacterial phage T1; PY100 tail proteins possess homologies to putative tail proteins of phage AaPhi23 of Actinobacillus actinomycetemcomitans. In a proteome analysis of virion particles, 15 proteins of the head and tail structures were identified by mass spectrometry. The putative gene product of ORF2 of PY100 shows significant homology to the gene 3 product (small terminase subunit) of Salmonella phage P22 that is involved in packaging of the concatemeric phage DNA. The packaging mechanism of PY100 was analyzed by hybridization and sequence analysis of DNA isolated from virion particles. Newly replicated PY100 DNA is cut initially at a pac recognition site, which is located in the coding region of ORF2.

Carlos Quijano, Pavel Tomancák, Jesus Lopez-Marti, Mikita Suyama, Peer Bork, Marco Milan, David Torrents, Miguel Manzanares
Selective maintenance of Drosophila tandemly arranged duplicated genes during evolution.
Genome Biol, 9(12) 176-176 (2008)
PDF DOI

BACKGROUND: The physical organization and chromosomal localization of genes within genomes is known to play an important role in their function. Most genes arise by duplication and move along the genome by random shuffling of DNA segments. Higher order structuring of the genome occurs in eukaryotes, where groups of physically linked genes are co-expressed. However, the contribution of gene duplication to gene order has not been analyzed in detail, as it is believed that co-expression due to recent duplicates would obscure other domains of co-expression. RESULTS: We have catalogued ordered duplicated genes in Drosophila melanogaster, and found that one in five of all genes is organized as tandem arrays. Furthermore, among arrays that have been spatially conserved over longer periods than would be expected on the basis of random shuffling, a disproportionate number contain genes encoding developmental regulators. Using in situ gene expression data for more than half of the Drosophila genome, we find that genes in these conserved clusters are co-expressed to a much higher extent than other duplicated genes. CONCLUSIONS: These results reveal the existence of functional constraints in insects that retain copies of genes encoding developmental and regulatory proteins as neighbors, allowing their co-expression. This co-expression may be the result of shared cis-regulatory elements or a shared need for a specific chromatin structure. Our results highlight the association between genome architecture and the gene regulatory networks involved in the construction of the body plan.

Klaus Huse, Stefan Taudien, Marco Groth, Philip Rosenstiel, Karol Szafranski, Michael Hiller, Jochen Hampe, Kerstin Junker, Jorg Schubert, Stefan Schreiber, Gerd Birkenmeier, Michael Krawczak, Matthias Platzer
Genetic variants of the copy number polymorphic beta-defensin locus are associated with sporadic prostate cancer.
Tumour Biol, 29(2) 83-92 (2008)
PDF DOI

Prostate cancer represents the cancer with the highest worldwide prevalence in men. Chromosome 8p23 has shown suggestive genetic linkage to early-onset familial prostate cancer and is frequently deleted in cancer cells of the urogenital tract. Within this locus some beta-defensin genes (among them DEFB4, DEFB103, DEFB104) are localized, which are arranged in a gene cluster shown to exhibit an extensive copy number variation in the population. This structural variation considerably hampers genetic studies. In a new approach considering both sequence as well as copy number variations we aimed to compare the defensin locus at 8p23 in prostate cancer patients and controls.

Stefanie Schindler, Karol Szafranski, Michael Hiller, Gul Shad Ali, Saiprasad G Palusa, Rolf Backofen, Matthias Platzer, Anireddy S N Reddy
Alternative splicing at NAGNAG acceptors in Arabidopsis thaliana SR and SR-related protein-coding genes.
BMC Genomics, 9 159-159 (2008)
PDF DOI

Several recent studies indicate that alternative splicing in Arabidopsis and other plants is a common mechanism for post-transcriptional modulation of gene expression. However, few analyses have been done so far to elucidate the functional relevance of alternative splicing in higher plants. Representing a frequent and universal subtle alternative splicing event among eukaryotes, alternative splicing at NAGNAG acceptors contributes to transcriptome diversity and therefore, proteome plasticity. Alternatively spliced NAGNAG acceptors are overrepresented in genes coding for proteins with RNA-recognition motifs (RRMs). As SR proteins, a family of RRM-containing important splicing factors, are known to be extensively alternatively spliced in Arabidopsis, we analyzed alternative splicing at NAGNAG acceptors in SR and SR-related genes.

Anna Shevchenko, Assen Roguev, Daniel Schaft, Luke Buchanan, Bianca Habermann, Cagri Sakalar, Henrik Thomas, Nevan J Krogan, Andrej Shevchenko, A. Francis Stewart
Chromatin Central: towards the comparative proteome by accurate mapping of the yeast proteomic environment.
Genome Biol, 9(11) 167-167 (2008)
PDF DOI

BACKGROUND: Understanding the design logic of living systems requires the understanding and comparison of proteomes. Proteomes define the commonalities between organisms more precisely than genomic sequences. Because uncertainties remain regarding the accuracy of proteomic data, several issues need to be resolved before comparative proteomics can be fruitful. RESULTS: The Saccharomyces cerevisiae proteome presents the highest quality proteomic data available. To evaluate the accuracy of these data, we intensively mapped a proteomic environment, termed 'Chromatin Central', which encompasses eight protein complexes, including the major histone acetyltransferases and deacetylases, interconnected by twelve proteomic hyperlinks. Using sequential tagging and a new method to eliminate background, we confirmed existing data but also uncovered new subunits and three new complexes, including ASTRA, which we suggest is a widely conserved aspect of telomeric maintenance, and two new variations of Rpd3 histone deacetylase complexes. We also examined the same environment in fission yeast and found a very similar architecture based on a scaffold of orthologues comprising about two-thirds of all proteins involved, whereas the remaining one-third is less constrained. Notably, most of the divergent hyperlinks were found to be due to gene duplications, hence providing a mechanism for the fixation of gene duplications in evolution. CONCLUSIONS: We define several prerequisites for comparative proteomics and apply them to examine a proteomic environment in unprecedented detail. We suggest that high resolution mapping of proteomic environments will deliver the highest quality data for comparative proteomics.

Hassan Mziaut^*, Stephan Kersting^*, Klaus-Peter Knoch, Wan-Hung Fan, Mirko Trajkovski, Katja Erdmann, Hendrik Bergert, Florian Ehehalt, Hans-Detlev Saeger, Michele Solimena
ICA512 signaling enhances pancreatic beta-cell proliferation by regulating cyclins D through STATs.
Proc Natl Acad Sci U.S.A., 105(2) 674-679 (2008)
PDF DOI

Changes in metabolic demands dynamically regulate the total mass of adult pancreatic beta-cells to adjust insulin secretion and preserve glucose homeostasis. Glucose itself is a major regulator of beta-cell proliferation by inducing insulin secretion and activating beta-cell insulin receptors. Here, we show that islet cell autoantigen 512 (ICA512)/IA-2, an intrinsic tyrosine phosphatase-like protein of the secretory granules, activates a complementary pathway for beta-cell proliferation. On granule exocytosis, the ICA512 cytoplasmic domain is cleaved and the resulting cytosolic fragment (ICA512-CCF) moves into the nucleus where it enhances the levels of phosphorylated STAT5 and STAT3, thereby inducing insulin gene transcription and granule biogenesis. We now show that knockdown of ICA512 decreases cyclin D1 levels and proliferation of insulinoma INS-1 cells, whereas beta-cell regeneration is reduced in partially pancreatectomized ICA512-/- mice. Conversely, overexpression of ICA512-CCF increases both cyclin D1 and D2 levels and INS-1 cell proliferation. Up-regulation of cyclin D1 and D2 by ICA512-CCF is affected by knockdown of STAT3 and STAT5, respectively, whereas it does not require insulin signaling. These results identify ICA512 as a regulator of cyclins D and beta-cell proliferation through STATs and may have implication for diabetes therapy.

Ralf Kittler, Laurence Pelletier, Anne-Kristin Heninger, Mikolaj Slabicki, Mirko Theis, Lukasz Miroslaw, Ina Poser, Steffen Lawo, Hannes Grabner, Karol Kozak, Jan Wagner, Vineeth Surendranath, Constance Richter, Wayne Bowen, Aimee L Jackson, Bianca Habermann, Anthony A. Hyman, Frank Buchholz
Genome-scale RNAi profiling of cell division in human tissue culture cells.
Nat Cell Biol, 9(12) 1401-1412 (2007)
PDF DOI

Cell division is fundamental for all organisms. Here we report a genome-scale RNA-mediated interference screen in HeLa cells designed to identify human genes that are important for cell division. We have used a library of endoribonuclease-prepared short interfering RNAs for gene silencing and have used DNA content analysis to identify genes that induced cell cycle arrest or altered ploidy on silencing. Validation and secondary assays were performed to generate a nine-parameter loss-of-function phenoprint for each of the genes. These phenotypic signatures allowed the assignment of genes to specific functional classes by combining hierarchical clustering, cross-species analysis and proteomic data mining. We highlight the richness of our dataset by ascribing novel functions to genes in mitosis and cytokinesis. In particular, we identify two evolutionarily conserved transcriptional regulatory networks that govern cytokinesis. Our work provides an experimental framework from which the systematic analysis of novel genes necessary for cell division in human cells can begin.

Imke Listerman, Anita S. Bledau, Inna Grishina, Karla M. Neugebauer
Extragenic accumulation of RNA polymerase II enhances transcription by RNA polymerase III.
PLoS Genet, 3(11) 212-212 (2007)
PDF DOI

Recent genomic data indicate that RNA polymerase II (Pol II) function extends beyond conventional transcription of primarily protein-coding genes. Among the five snRNAs required for pre-mRNA splicing, only the U6 snRNA is synthesized by RNA polymerase III (Pol III). Here we address the question of how Pol II coordinates the expression of spliceosome components, including U6. We used chromatin immunoprecipitation (ChIP) and high-resolution mapping by PCR to localize both Pol II and Pol III to snRNA gene regions. We report the surprising finding that Pol II is highly concentrated approximately 300 bp upstream of all five active human U6 genes in vivo. The U6 snRNA, an essential component of the spliceosome, is synthesized by Pol III, whereas all other spliceosomal snRNAs are Pol II transcripts. Accordingly, U6 transcripts were terminated in a Pol III-specific manner, and Pol III localized to the transcribed gene regions. However, synthesis of both U6 and U2 snRNAs was alpha-amanitin-sensitive, indicating a requirement for Pol II activity in the expression of both snRNAs. Moreover, both Pol II and histone tail acetylation marks were lost from U6 promoters upon alpha-amanitin treatment. The results indicate that Pol II is concentrated at specific genomic regions from which it can regulate Pol III activity by a general mechanism. Consequently, Pol II coordinates expression of all RNA and protein components of the spliceosome.

Hiroshi Qadota, Makiko Inoue, Takao Hikita, Mathias Köppen, Jeffrey D Hardin, Mutsuki Amano, Donald G Moerman, Kozo Kaibuchi
Establishment of a tissue-specific RNAi system in C. elegans.
Gene, 400(1-2) 166-173 (2007)
PDF DOI

In C. elegans, mosaic analysis is a powerful genetic tool for determining in which tissue or specific cells a gene of interest is required. For traditional mosaic analysis, a loss-of-function mutant and a genomic fragment that can rescue the mutant phenotype are required. Here we establish an easy and rapid mosaic system using RNAi (RNA mediated interference), using a rde-1 mutant that is resistant to RNAi. Tissue-specific expression of the wild type rde-1 cDNA in rde-1 mutants limits RNAi sensitivity to a specific tissue. We established hypodermal-and muscle-specific RNAi systems by expressing rde-1 cDNA under the control of the lin-26 and hlh-1 promoters, respectively. We confirmed tissue-specific RNAi using two assays: (1) tissue-specific knockdown of GFP expression, and (2) phenocopy of mutations in essential genes that were previously known to function in a tissue-specific manner. We also applied this system to an essential gene, ajm-1, expressed in hypodermis and gut, and show that lethality in ajm-1 mutants is due to loss of expression in hypodermal cells. Although we demonstrate tissue-specific RNAi in hypodermis and muscle, this method could be easily applied to other tissues.

Eric Lécuyer, Hiroyuki Yoshida, Neela Parthasarathy, Christina Alm, Tomas Babak, Tanja Cerovina, Timothy R Hughes, Pavel Tomancák, Henry M Krause
Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.
Cell, 131(1) 174-187 (2007)
PDF DOI

Although subcellular mRNA trafficking has been demonstrated as a mechanism to control protein distribution, it is generally believed that most protein localization occurs subsequent to translation. To address this point, we developed and employed a high-resolution fluorescent in situ hybridization procedure to comprehensively evaluate mRNA localization dynamics during early Drosophila embryogenesis. Surprisingly, of the 3370 genes analyzed, 71% of those expressed encode subcellularly localized mRNAs. Dozens of new and striking localization patterns were observed, implying an equivalent variety of localization mechanisms. Tight correlations between mRNA distribution and subsequent protein localization and function, indicate major roles for mRNA localization in nucleating localized cellular machineries. A searchable web resource documenting mRNA expression and localization dynamics has been established and will serve as an invaluable tool for dissecting localization mechanisms and for predicting gene functions and interactions.

Ingmar H Riedel-Kruse, Claudia Müller, Andrew C. Oates
Synchrony dynamics during initiation, failure, and rescue of the segmentation clock.
Science, 317(5846) 1911-1915 (2007)
PDF DOI

The "segmentation clock" is thought to coordinate sequential segmentation of the body axis in vertebrate embryos. This clock comprises a multicellular genetic network of synchronized oscillators, coupled by intercellular Delta-Notch signaling. How this synchrony is established and how its loss determines the position of segmentation defects in Delta and Notch mutants are unknown. We analyzed the clock's synchrony dynamics by varying strength and timing of Notch coupling in zebra-fish embryos with techniques for quantitative perturbation of gene function. We developed a physical theory based on coupled phase oscillators explaining the observed onset and rescue of segmentation defects, the clock's robustness against developmental noise, and a critical point beyond which synchrony decays. We conclude that synchrony among these genetic oscillators can be established by simultaneous initiation and self-organization and that the segmentation defect position is determined by the difference between coupling strength and noise.

Thomas Wendl^*, Dejan Adzic^*, Jeffrey J Schoenebeck, Steffen Scholpp, Michael Brand, Deborah Yelon, Klaus B Rohr
Early developmental specification of the thyroid gland depends on han-expressing surrounding tissue and on FGF signals.
Development, 134(15) 2871-2879 (2007)
PDF DOI

The thyroid is an endocrine gland in all vertebrates that develops from the ventral floor of the anterior pharyngeal endoderm. Unravelling the molecular mechanisms of thyroid development helps to understand congenital hypothyroidism caused by the absence or reduction of this gland in newborn humans. Severely reduced or absent thyroid-specific developmental genes concomitant with the complete loss of the functional gland in the zebrafish hands off (han, hand2) mutant reveals the han gene as playing a novel, crucial role in thyroid development. han-expressing tissues surround the thyroid primordium throughout development. Fate mapping reveals that, even before the onset of thyroid-specific developmental gene expression, thyroid precursor cells are in close contact with han-expressing cardiac lateral plate mesoderm. Grafting experiments show that han is required in surrounding tissue, and not in a cell-autonomous manner, for thyroid development. Loss of han expression in the branchial arches and arch-associated cells after morpholino knock-down of upstream regulator genes does not impair thyroid development, indicating that other han-expressing structures, most probably cardiac mesoderm, are responsible for the thyroid defects in han mutants. The zebrafish ace (fgf8) mutant has similar thyroid defects as han mutants, and chemical suppression of fibroblast growth factor (FGF) signalling confirms that this pathway is required for thyroid development. FGF-soaked beads can restore thyroid development in han mutants, showing that FGFs act downstream of or in parallel to han. These data suggest that loss of FGF-expressing tissue in han mutants is responsible for the thyroid defects.

Thomas J. F. Nieland, Jared T Shaw, Firoz A Jaipuri, Zoltan Maliga, Jay L Duffner, Angela N Koehler, Monty Krieger
Influence of HDL-cholesterol-elevating drugs on the in vitro activity of the HDL receptor SR-BI.
J Lipid Res, 48(8) 1832-1845 (2007)
PDF DOI

Treatment of atherosclerotic disease often focuses on reducing plasma LDL-cholesterol or increasing plasma HDL-cholesterol. We examined in vitro the effects on HDL receptor [scavenger receptor class B type I (SR-BI)] activity of three classes of clinical and experimental plasma HDL-cholesterol-elevating compounds: niacin, fibrates, and HDL376. Fenofibrate (FF) and HDL376 were potent (IC(50) approximately 1 microM), direct inhibitors of SR-BI-mediated lipid transport in cells and in liposomes reconstituted with purified SR-BI. FF, a prodrug, was a more potent inhibitor of SR-BI than an activator of peroxisome proliferator-activated receptor alpha, a target of its active fenofibric acid (FFA) derivative. Nevertheless, FFA, four other fibrates (clofibrate, gemfibrozil, ciprofibrate, and bezafibrate), and niacin had little, if any, effect on SR-BI, suggesting that they do not directly target SR-BI in vivo. However, similarities of HDL376 treatment and SR-BI gene knockout on HDL metabolism in vivo (increased HDL-cholesterol and HDL particle sizes) and structure-activity relationship analysis suggest that SR-BI may be a target of HDL376 in vivo. HDL376 and other inhibitors may help elucidate SR-BI function in diverse mammalian models and determine the therapeutic potential of SR-BI-directed pharmaceuticals.

Rima Arnaout, Tania Ferrer, Jan Huisken, Kenneth Spitzer, Didier Y.R. Stainier, Martin Tristani-Firouzi, Neil C Chi
Zebrafish model for human long QT syndrome.
Proc Natl Acad Sci U.S.A., 104(27) 11316-11321 (2007)
DOI

Long QT syndrome (LQTS) is a disorder of ventricular repolarization that predisposes affected individuals to lethal cardiac arrhythmias. To date, an appropriate animal model of inherited LQTS does not exist. The zebrafish is a powerful vertebrate model used to dissect molecular pathways of cardiovascular development and disease. Because fundamental electrical properties of the zebrafish heart are remarkably similar to those of the human heart, the zebrafish may be an appropriate model for studying human inherited arrhythmias. Here we describe the molecular, cellular, and electrophysiological basis of a zebrafish mutant characterized by ventricular asystole. Genetic mapping and direct sequencing identify the affected gene as kcnh2, which encodes the channel responsible for the rapidly activating delayed rectifier K(+) current (I(Kr)). We show that complete loss of functional I(Kr) in embryonic hearts leads to ventricular cell membrane depolarization, inability to generate action potentials (APs), and disrupted calcium release. A small hyperpolarizing current restores spontaneous APs, implying wild-type function of other ionic currents critical for AP generation. Heterozygous fish manifest overt cellular and electrocardiographic evidence for delayed ventricular repolarization. Our findings provide insight into the pathogenesis of homozygous kcnh2 mutations and expand the use of zebrafish mutants as a model system to study human arrhythmias.

Christine A. Fargeas, Wieland B. Huttner, Denis Corbeil
Nomenclature of prominin-1 (CD133) splice variants - an update.
Tissue Antigens, 69(6) 602-606 (2007)
PDF DOI

Prominin-1 (CD133), a pentaspan membrane glycoprotein that constitutes an important cell surface marker of various, either normal or cancerous, stem cell populations is widely used to isolate or characterize such cells in different systems. Occurring throughout the metazoan evolution with a remarkably conserved genomic organization, it may be expressed as different splice variants with distinctive characteristics. A rational nomenclature has been proposed earlier for their consistent designation across species. Although generally accepted, it seems to be misunderstood in view of the recent report of novel prominin-1 complementary DNAs in rhesus monkey and humans with improper naming. As this may lead to confusion, we have reexamined the genomic organization of prominin-1 in various primates to provide an update that should further clarify the rationale of the nomenclature for prominin-1 gene products. This report comprises (i) the determination of the genomic organization of prominin-1 gene in two non-human primates, i.e. Macaca mulatta and Pan troglodytes, commonly used in research, (ii) the mapping of a new exon that creates an alternative cytoplasmic C-terminal end of prominin-1, (iii) the identification of various potential PDZ-binding domains generated by alternative cytoplasmic C-terminal tails, suggesting that different prominin-1 splice variants might interact with distinct protein partners, and (iv) a summing up of the different prominin-1 splice variants.

Jim Swoger, Peter Verveer, Klaus Greger, Jan Huisken, Ernst H K Stelzer
Multi-view image fusion improves resolution in three-dimensional microscopy.
Opt express, 15(13) 8029-8042 (2007)
Open Access

A non-blind, shift-invariant image processing technique that fuses multi-view three-dimensional image data sets into a single, high quality three-dimensional image is presented. It is effective for 1) improving the resolution and isotropy in images of transparent specimens, and 2) improving the uniformity of the image quality of partially opaque samples. This is demonstrated with fluorescent samples such as Drosophila melanogaster and Medaka embryos and pollen grains imaged by Selective Plane Illumination Microscopy (SPIM). The application of the algorithm to SPIM data yields high-resolution images of organ structure and gene expression, in some cases at a sub-cellular level, throughout specimens ranging from several microns up to a millimeter in size.

Hiroshi Kikuta, Mary A Laplante, Pavla Navratilova, Anna Z Komisarczuk, Pär G Engström, David Fredman, Altuna Akalin, Mario Caccamo, Ian Sealy, Kerstin Howe, Julien Ghislain, Guillaume Pezeron, Philippe Mourrain, Staale Ellingsen, Andrew C. Oates, Christine Thisse, Bernard Thisse, Isabelle Foucher, Birgit Adolf, Andrea Geling, Boris Lenhard, Thomas S. Becker
Genomic regulatory blocks encompass multiple neighboring genes and maintain conserved synteny in vertebrates.
Genome Res, 17(5) 545-555 (2007)
PDF DOI

We report evidence for a mechanism for the maintenance of long-range conserved synteny across vertebrate genomes. We found the largest mammal-teleost conserved chromosomal segments to be spanned by highly conserved noncoding elements (HCNEs), their developmental regulatory target genes, and phylogenetically and functionally unrelated "bystander" genes. Bystander genes are not specifically under the control of the regulatory elements that drive the target genes and are expressed in patterns that are different from those of the target genes. Reporter insertions distal to zebrafish developmental regulatory genes pax6.1/2, rx3, id1, and fgf8 and miRNA genes mirn9-1 and mirn9-5 recapitulate the expression patterns of these genes even if located inside or beyond bystander genes, suggesting that the regulatory domain of a developmental regulatory gene can extend into and beyond adjacent transcriptional units. We termed these chromosomal segments genomic regulatory blocks (GRBs). After whole genome duplication in teleosts, GRBs, including HCNEs and target genes, were often maintained in both copies, while bystander genes were typically lost from one GRB, strongly suggesting that evolutionary pressure acts to keep the single-copy GRBs of higher vertebrates intact. We show that loss of bystander genes and other mutational events suffered by duplicated GRBs in teleost genomes permits target gene identification and HCNE/target gene assignment. These findings explain the absence of evolutionary breakpoints from large vertebrate chromosomal segments and will aid in the recognition of position effect mutations within human GRBs.

Eberhard Krausz
High-content siRNA screening.
Mol Biosyst, 3(4) 232-240 (2007)
PDF DOI

Very recent developments in instrumentation and image analysis have made microscopy applicable to high-throughput screening (HTS). For 'High-Content Screening' modern automated microscopy systems provide a throughput of up to 100,000 (confocal) images, with amazingly high resolution, of cells fluorescently stained using multiple colours that are imaged simultaneously during the screen. Image analysis tools provide multi-parametric pattern extraction and quantification on-the-fly. Big pharmaceutical companies have presented image-based screens of more than 100,000 compounds, while academia has published data on large RNA interference screens for functional genomics. Numerous whole-genome sequencing projects have been completed and published. Gene annotation is still in flux. Nevertheless, about 23,000 human genes have been reliably annotated. Additionally, gene expression array technologies and proteomics have added further data on molecules present in cells and tissues. The major challenge of the present and future is to unravel the detailed function of all these gene products and their interaction. One way to gain insight, is to design oligonucleotides that induce lack-of-function phenotypes by specifically inhibiting protein production.

Sunita S Shankaran^*, Dirk Sieger^*, Christian Schröter, Carmen Czepe, Marie-Christin Pauly, Mary A Laplante, Thomas S. Becker, Andrew C. Oates, Martin Gajewski
Completing the set of h/E(spl) cyclic genes in zebrafish: her12 and her15 reveal novel modes of expression and contribute to the segmentation clock.
Dev Biol, 304(2) 615-632 (2007)
PDF DOI

Somitogenesis is the key developmental process that lays down the framework for a metameric body in vertebrates. Somites are generated from the un-segmented presomitic mesoderm (PSM) by a pre-patterning process driven by a molecular oscillator termed the segmentation clock. The Delta-Notch intercellular signaling pathway and genes belonging to the hairy (h) and Enhancer of split (E(spl))-related (h/E(spl)) family of transcriptional repressors are conserved components of this oscillator. A subset of these genes, called cyclic genes, is characterized by oscillating mRNA expression that sweeps anteriorly like a wave through the embryonic PSM. Periodic transcriptional repression by H/E(spl) proteins is thought to provide a critical part of a negative feedback loop in the oscillatory process, but it is an open question how many cyclic h/E(spl) genes are involved in the somitogenesis clock in any species, and what distinct roles they might play. From a genome-wide search for h/E(spl) genes in the zebrafish, we previously estimated a total of five cyclic members. Here we report that one of these, the mHes5 homologue her15 actually exists as a very recently duplicated gene pair. We investigate the expression of this gene pair and analyse its regulation and activity in comparison to the paralogous her12 gene, and the other cyclic h/E(spl) genes in the zebrafish. The her15 gene pair and her12 display novel and distinct expression features, including a caudally restricted oscillatory domain and dynamic stripes of expression in the rostral PSM that occur at the future segmental borders. her15 expression stripes demarcate a unique two-segment interval in the rostral PSM. Mutant, morpholino, and inhibitor studies show that her12 and her15 expression in the PSM is regulated by Delta-Notch signaling in a complex manner, and is dependent on her7, but not her1 function. Morpholino-mediated her12 knockdown disrupts cyclic gene expression, indicating that it is a non-redundant core component of the segmentation clock. Over-expression of her12, her15 or her7 disrupts cyclic gene expression and somite border formation, and structure function analysis of Her7 indicates that DNA binding, but not Groucho-recruitment seems to be important in this process. Thus, the zebrafish has five functional cyclic h/E(spl) genes, which are expressed in a distinct spatial configuration. We propose that this creates a segmentation oscillator that varies in biochemical composition depending on position in the PSM.

Ralf Kittler, Vineeth Surendranath, Anne-Kristin Heninger, Mikolaj Slabicki, Mirko Theis, Gabriele Putz, Kristin Franke, Antonio Caldarelli, Hannes Grabner, Karol Kozak, Jan Wagner, Effi Rees, Bernd Korn, Corina Frenzel, Christoph Sachse, Birte Sönnichsen, Jun Guo, Janell Schelter, Julja Burchard, Peter S Linsley, Aimee L Jackson, Bianca Habermann, Frank Buchholz
Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies.
Nat Methods, 4(4) 337-344 (2007)
PDF DOI

RNA interference (RNAi) has become an important technique for loss-of-gene-function studies in mammalian cells. To achieve reliable results in an RNAi experiment, efficient and specific silencing triggers are required. Here we present genome-wide data sets for the production of endoribonuclease-prepared short interfering RNAs (esiRNAs) for human, mouse and rat. We used an algorithm to predict the optimal region for esiRNA synthesis for every protein-coding gene of these three species. We created a database, RiDDLE, for retrieval of target sequences and primer information. To test this in silico resource experimentally, we generated 16,242 esiRNAs that can be used for RNAi screening in human cells. Comparative analyses with chemically synthesized siRNAs demonstrated a high silencing efficacy of esiRNAs and a 12-fold reduction of downregulated off-target transcripts as detected by microarray analysis. Hence, the presented esiRNA libraries offer an efficient, cost-effective and specific alternative to presently available mammalian RNAi resources.

Christopher D Smith, Robert C Edgar, Mark D Yandell, Douglas R Smith, Susan E Celniker, Eugene W Myers, Gary H Karpen
Improved repeat identification and masking in Dipterans.
Gene, 389(1) 1-9 (2007)
PDF DOI

Repetitive sequences are a major constituent of many eukaryote genomes and play roles in gene regulation, chromosome inheritance, nuclear architecture, and genome stability. The identification of repetitive elements has traditionally relied on in-depth, manual curation and computational determination of close relatives based on DNA identity. However, the rapid divergence of repetitive sequence has made identification of repeats by DNA identity difficult even in closely related species. Hence, the presence of unidentified repeats in genome sequences affects the quality of gene annotations and annotation-dependent analyses (e.g. microarray analyses). We have developed an enhanced repeat identification pipeline using two approaches. First, the de novo repeat finding program PILER-DF was used to identify interspersed repetitive elements in several recently finished Dipteran genomes. Repeats were classified, when possible, according to their similarity to known elements described in Repbase and GenBank, and also screened against annotated genes as one means of eliminating false positives. Second, we used a new program called RepeatRunner, which integrates results from both RepeatMasker nucleotide searches and protein searches using BLASTX. Using RepeatRunner with PILER-DF predictions, we masked repeats in thirteen Dipteran genomes and conclude that combining PILER-DF and RepeatRunner greatly enhances repeat identification in both well-characterized and un-annotated genomes.

Hanchuan Peng, Fuhui Long, Jie Zhou, Garmay Leung, Michael B Eisen, Eugene W Myers
Automatic image analysis for gene expression patterns of fly embryos.
BMC Cell Biol, 8 Suppl 1 7-7 (2007)
Open Access PDF DOI

Staining the mRNA of a gene via in situ hybridization (ISH) during the development of a D. melanogaster embryo delivers the detailed spatio-temporal pattern of expression of the gene. Many biological problems such as the detection of co-expressed genes, co-regulated genes, and transcription factor binding motifs rely heavily on the analyses of these image patterns. The increasing availability of ISH image data motivates the development of automated computational approaches to the analysis of gene expression patterns.

Pavel Tomancák, Benjamin P Berman, Amy Beaton, Richard Weiszmann, Elaine Kwan, Volker Hartenstein, Susan E Celniker, Gerald M Rubin
Global analysis of patterns of gene expression during Drosophila embryogenesis.
Genome Biol, 8(7) 145-145 (2007)
Open Access PDF DOI

BACKGROUND: Cell and tissue specific gene expression is a defining feature of embryonic development in multi-cellular organisms. However, the range of gene expression patterns, the extent of the correlation of expression with function, and the classes of genes whose spatial expression are tightly regulated have been unclear due to the lack of an unbiased, genome-wide survey of gene expression patterns. RESULTS: We determined and documented embryonic expression patterns for 6,003 (44%) of the 13,659 protein-coding genes identified in the Drosophila melanogaster genome with over 70,000 images and controlled vocabulary annotations. Individual expression patterns are extraordinarily diverse, but by supplementing qualitative in situ hybridization data with quantitative microarray time-course data using a hybrid clustering strategy, we identify groups of genes with similar expression. Of 4,496 genes with detectable expression in the embryo, 2,549 (57%) fall into 10 clusters representing broad expression patterns. The remaining 1,947 (43%) genes fall into 29 clusters representing restricted expression, 20% patterned as early as blastoderm, with the majority restricted to differentiated cell types, such as epithelia, nervous system, or muscle. We investigate the relationship between expression clusters and known molecular and cellular-physiological functions. CONCLUSION: Nearly 60% of the genes with detectable expression exhibit broad patterns reflecting quantitative rather than qualitative differences between tissues. The other 40% show tissue-restricted expression; the expression patterns of over 1,500 of these genes are documented here for the first time. Within each of these categories, we identified clusters of genes associated with particular cellular and developmental functions.

Karen Echeverri, Andrew C. Oates
Coordination of symmetric cyclic gene expression during somitogenesis by Suppressor of Hairless involves regulation of retinoic acid catabolism.
Dev Biol, 301(2) 388-403 (2007)
PDF DOI

Vertebrate embryos faithfully produce bilaterally symmetric somites that give rise to repetitive body structures such as vertebrae and skeletal muscle. Body segmentation is regulated by a cyclic gene expression system, containing the Delta-Notch pathway and targets, which generates bilaterally symmetric oscillations across the Pre-Somitic Mesoderm (PSM). The position of the forming somite boundary is controlled by interaction of this oscillator with a determination front comprised of opposing gradients of FGF and retinoic acid (RA) signalling. Disruption of RA production leads to asymmetries in cyclic gene expression, but the link between RA and the oscillator is unknown. In somitogenesis, Notch signalling activates target genes through the transcription factor Suppressor of Hairless (Su(H)). Here, we report that two Su(H) genes coordinate bilaterally symmetric positioning of somite boundaries in the zebrafish embryo. Combined Su(H) gene knockdown caused defects in visceral left/right asymmetry, neurogenic lateral inhibition, and symmetrical failure of the segmentation oscillator. However, by selectively down-regulating Su(H)2 or Su(H)1 function using specific antisense morpholinos, we observed asymmetric defects in anterior or posterior somite boundaries, respectively. These morphological abnormalities were reflected by underlying asymmetric cyclic gene expression waves in the presomitic mesoderm, indicating a key role for Su(H) in coordinating the left-right symmetry of this process. Strikingly, expression of the RA-degrading enzyme cyp26a1 in the tailbud was controlled by Su(H) activity, and morpholino knockdown of cyp26a1 alone caused asymmetric cyclic dlc expression, suggesting that excess RA in the tailbud may contribute to the cyclic asymmetries. Indeed, exogenous RA was sufficient to generate asymmetric expression of all cyclic genes. Our observations indicate that one element of the Notch signalling pathway, Su(H), is required for control of RA metabolism in the tailbud and that this regulation is involved in bilateral symmetry of cyclic gene expression and somitogenesis.

B. Tucker, C Hepperle, D Kortschak, B Rainbird, S Wells, Andrew C. Oates, Michael Lardelli
Zebrafish Angiotensin II Receptor-like 1a (agtrl1a) is expressed in migrating hypoblast, vasculature, and in multiple embryonic epithelia.
Gene Expr Patterns, 7(3) 258-265 (2007)
PDF DOI

The human gene AGTRL1 is an angiotensin II receptor-like gene expressed in vasculature, which acts as the receptor for the small peptide APELIN, and a co-receptor for Human Immunodeficiency Virus. Mammalian AGTRL1 has been shown to modulate cardiac contractility, venous and arterial dilation, and endothelial cell migration in vitro, but no role in the development of the vasculature, or other tissues, has been described. We report the identification and expression of the zebrafish ortholog of the human gene AGTRL1. Zebrafish agtrl1a is first expressed before epiboly in dorsal precursors. During epiboly it is expressed in the enveloping layer, yolk syncytial layer and migrating mesendoderm. During segmentation stages, expression is observed in epithelial structures such as adaxial cells, border cells of the newly formed somites, developing lens, otic vesicles and venous vasculature.

Yan Dong, Aliona Bogdanova, Bianca Habermann, Wolfgang Zachariae, Julie Ahringer
Identification of the C. elegans anaphase promoting complex subunit Cdc26 by phenotypic profiling and functional rescue in yeast.
BMC Dev Biol, 7 Art. No. 19 (2007)
Open Access PDF DOI

BACKGROUND: RNA interference coupled with videorecording of C. elegans embryos is a powerful method for identifying genes involved in cell division processes. Here we present a functional analysis of the gene B0511.9, previously identified as a candidate cell polarity gene in an RNAi videorecording screen of chromosome I embryonic lethal genes. RESULTS: Whereas weak RNAi inhibition of B0511.9 causes embryonic cell polarity defects, strong inhibition causes embryos to arrest in metaphase of meiosis I. The range of defects induced by RNAi of B0511.9 is strikingly similar to those displayed by mutants of anaphase-promoting complex/cyclosome (APC/C) components. Although similarity searches did not reveal any obvious homologue of B0511.9 in the non-redundant protein database, we found that the N-terminus shares a conserved sequence pattern with the N-terminus of the small budding yeast APC/C subunit Cdc26 and its orthologues from a variety of other organisms. Furthermore, we show that B0511.9 robustly complements the temperature-sensitive growth defect of a yeast cdc26Delta mutant. CONCLUSION: These data demonstrate that B0511.9 encodes the C. elegans APC/C subunit CDC-26.

Tiago R Magalhães, Jessica Palmer, Pavel Tomancák, Katherine S Pollard
Transcriptional control in embryonic Drosophila midline guidance assessed through a whole genome approach.
BMC neuroscience, 8 Art. No. 59 (2007)
Open Access PDF DOI

BACKGROUND: During the development of the Drosophila central nervous system the process of midline crossing is orchestrated by a number of guidance receptors and ligands. Many key axon guidance molecules have been identified in both invertebrates and vertebrates, but the transcriptional regulation of growth cone guidance remains largely unknown. It is established that translational regulation plays a role in midline crossing, and there are indications that transcriptional regulation is also involved. To investigate this issue, we conducted a genome-wide study of transcription in Drosophila embryos using wild type and a number of well-characterized Drosophila guidance mutants and transgenics. We also analyzed a previously published microarray time course of Drosophila embryonic development with an axon guidance focus. RESULTS: Using hopach, a novel clustering method which is well suited to microarray data analysis, we identified groups of genes with similar expression patterns across guidance mutants and transgenics. We then systematically characterized the resulting clusters with respect to their relevance to axon guidance using two complementary controlled vocabularies: the Gene Ontology (GO) and anatomical annotations of the Atlas of Pattern of Gene Expression (APoGE) in situ hybridization database. The analysis indicates that regulation of gene expression does play a role in the process of axon guidance in Drosophila. We also find a strong link between axon guidance and hemocyte migration, a result that agrees with mounting evidence that axon guidance molecules are co-opted in vertebrate vascularization. Cell cyclin activity in the context of axon guidance is also suggested from our array data. RNA and protein expression patterns of cell cyclins in axon guidance mutants and transgenics support this possible link. CONCLUSION: This study provides important insights into the regulation of axon guidance in vivo.

Eric Marois, Suzanne Eaton
RNAi in the Hedgehog signaling pathway: pFRiPE, a vector for temporally and spatially controlled RNAi in Drosophila
In: Hedgehog signaling protocols. (Eds.) Jamila I. Horabin Methods in molecular biology ; 397.,Totowa, USA,Humana Press (2007),115-128 Ch. 10
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RNA interference (RNAi) has become an irreplaceable tool for reverse genetics in plants and animals. The universality and specificity of this phenomenon allows silencing of virtually any chosen gene to examine its involvement in biologicalRNA interference (RNAi) has become an irreplaceable tool for reverse genetics in plants and animals. The universality and specificity of this phenomenon allows silencing of virtually any processes. Many strategies exist to reduce the expression of a particular gene using RNAi. Some rely on delivering directly to cells the approximately 21-nucleotide long interfering double-stranded RNA (dsRNA) species that are central mediators of the silencing process. Others rely on the transgenic expression of longer dsRNA molecules, leaving it to the cellular machinery to process these hairpins into short active dsRNA. In this chapter, we describe a transgenic method to deplete a chosen protein from a specific Drosophila tissue following induction of long dsRNA. It was used to uncover the role of lipidic particles in Hedgehog signaling by silencing lipophorin in the fat body (1), and we routinely use it to deplete specific proteins from wing imaginal disc subdomains (2). The method, certainly not restricted to the study of Hedgehog signaling, allows fast and efficient construction of a plasmid incorporating various Drosophila genetic tools to allow heat-shock-induced expression of dsRNA at the desired time and in the desired tissue. For protocols involving injection of in vitro synthesized dsRNA in embryos to study Hedgehog signaling, see for example (3). For genomic screens to identify Hedgehog pathway components in tissue culture cells by transfection of small interfering RNAs, see refs. (4,5).

Jörg Cammenga, Birte Niebuhr, Stefan Horn, Ulla Bergholz, Gabriele Putz, Frank Buchholz, Jürgen Löhler, Carol Stocking
RUNX1 DNA-binding mutants, associated with minimally differentiated acute myelogenous leukemia, disrupt myeloid differentiation.
Cancer Res, 67(2) 537-545 (2007)
PDF DOI

Mutations in the RUNX1 gene are found at high frequencies in minimally differentiated acute myelogenous leukemia. In addition to null mutations, many of the mutations generate Runx1 DNA-binding (RDB) mutants. To determine if these mutants antagonize wild-type protein activity, cDNAs were transduced into murine bone marrow or human cord blood cells using retroviral vectors. Significantly, the RDB mutants did not act in a transdominant fashion in vivo to disrupt Runx1 activity in either T-cell or platelet development, which are highly sensitive to Runx1 dosage. However, RDB mutant expression impaired expansion and differentiation of the erythroid compartment in which Runx1 expression is normally down-regulated, showing that a RDB-independent function is incompatible with erythroid differentiation. Significantly, both bone marrow progenitors expressing RDB mutants or deficient for Runx1 showed increased replating efficiencies in vitro, accompanied by the accumulation of myeloblasts and dysplastic progenitors, but the effect was more pronounced in RDB cultures. Disruption of the interface that binds CBFbeta, an important cofactor of Runx1, did not impair RDB mutant replating activity, arguing against inactivation of Runx1 function by CBFbeta sequestration. We propose that RDB mutants antagonize Runx1 function in early progenitors by disrupting a critical balance between DNA-binding-independent and DNA-binding-dependent signaling.

Cagri Sakalar
Roles of H2A.z in Fission Yeast Chromatin
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2007)
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Covalent histone modifications such as methylation, acetylation as well as differential incorporation of histone variants are shown to coincide with different chromatin compartments and mark active or repressed genes. Msc1 is one of the seven JmjC Domain Proteins (JDPs) in Fission Yeast. JDPs are known to function in chromatin and some act as histone demethylases. We found that Msc1 is a member of Swr1 Complex which is known to exchange histone H2A variant H2A.z in nucleosomes. We purified H2A.z as a member of Swr1 Complex and its interaction with Swr1 Complex depends on Swr1. We’ve shown that histone H4 Lysine 20 trimethylation (H4 K20 Me3) is lost in h2A.z and msc1 deletion strains and these strains are sensitive to UV. Deletion strain of h2A.z is sensitive to Camptothecin. Histones H3 and H4 are obtained in Msc1 and H2A.z purifications and we’ve shown that histone H4 from these purifications has low level of Lysine 16 acetylation (H4 K16 Ac). Deletion strains of h2A.z, swr1 and msc1 are shown to be sensitive to TSA, a histone deacetylase (HDAC) inhibitor suggesting that H2A.z cooperates with HDACs. TSA treatment of wild type cells cause an increase in H4 K16 Ac and a decrease in H4 K20 Me3. Gene expression profiles of h2A.z, swr1 and msc1 are significantly similar and upregulated genes in deletion strains localize at chromosome ends (a region of 160 kb for each end). The number of stress or meiotic inducible genes is increased in deletion strains suggesting that H2A.z has a role in regulation of inducible genes. We suggest that H2A.z, in cooperation with HDACs, functions in regulation of chromatin accessibility of inducible promoters.

Vinciane Grimard
MAPK9: Ein neuer Regulator des Triglyzerid-Stoffwechsels, entdeckt durch siRNA Screening
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2007)

Lipid homeostasis is an essential factor for proper function both at the level of a cell and of an organism. Dysregulation of this process is responsible for some of the major health concerns of our societies such as obesity and atherosclerosis. Considering the wide variety of lipids and the high dynamic of the system, it is clear that regulation is required to keep the appropriate balance between the different lipids. Although a few regulation pathways are already characterized, some others still wait to be discovered. In order to unravel new players involved in lipid regulation, a screening procedure was developed combining RNA interference in Hela cells and thin layer chromatography. Thereby, it is possible to monitor modifications of lipid composition resulting from siRNA knock-down. Furthermore, the potential of mass spectrometry as a lipid analysis tool in large-scale studies was evaluated. This method was then applied to an essential family of regulatory proteins, the kinases. Lipid composition of 600 kinases knock-downs was analyzed. Mostly, variations in triglyceride and cholesterol levels were observed, suggesting that these lipids are more subject to variation in the cells. Unfortunately, it appears that the screen suffered from a high-rate of off-targets effects, implying that most of the phenotypes observed can’t reliably be linked to the corresponding kinase knock-downs. However, several interesting conclusions can still be derived from this screen. First, it was observed that several siRNA induce a decrease in cholesterol, which is coupled to accumulation of a new lipid. Several lines of evidence suggest that this new lipid is in fact a methylated sterol precursor such as lanosterol or demethyllanosterol. Previously, HMG-CoA reductase was considered as the rate-limiting enzyme of cholesterol biosynthesis and the major regulated step of this process. These data show that another major regulation step occurs more downstream in the pathway at the level of methylated cholesterol precursors. Furthermore, MAPK9 was identified as a new regulator of triglyceride homeostasis at the cellular level. Upon MAPK9 knock-down, an increase in triglyceride content was observed both by thin layer chromatography and mass spectrometry. Accordingly, these cells present an increase in lipid droplets, the cellular organelles responsible for triglyceride storage. Sty1 was also identified as the functional homolog of MAPK9 in S. pombe for this process, as similar increase in triglyceride and lipid droplets is observed in a deletion strain for this gene. Although more detailed studies will be necessary to unravel the molecular mechanism of this process, these data suggest the evolutionary conserved implication of the MAP kinase pathway in the regulation of lipid storage both in humans and in yeasts.

Anna Natalia Grzyb
Characterisation of Y-Box protein 3 (MSY3) in the developing murine central nervous system
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2007)
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Neurons, astrocytes and oligodendrocytes of the central nervous system (CNS) arise from a common pool of multipotent neuroepithelial progenitor cells lining the walls of the neural tube. Initially, neuroepithelial cells undergo symmetric proliferative divisions, thereby expanding the progenitor pool and determining the size of brain compartments. At the onset of neurogenesis, a subset of progenitors switch to asymmetric or terminal symmetric neurogenic divisions. Maintenance of progenitor cell population throughout the period of neurogenesis is essential to generate the full diversity of neuronal cell types and proper histological pattern. However, the mechanisms responsible for the maintenance of progenitor cells proliferation are far from being fully understood. The family of Y-box proteins is involved in control of proliferation and transformation in various normal and pathological cellular systems, and therefore was considered as a candidate to exert such a function. Y-box proteins have a capacity to bind DNA and RNA, thereby controlling gene expression from transcription to translation. This study aimed to examine the expression of mouse Y-box protein 3 (MSY3) in the developing nervous system and elucidate its putative role in regulation of proliferation of progenitor cells. As presented in this work, the MSY3 protein in the embryonic CNS is expressed solely in progenitor cells and not neurons. Moreover, as shown by two independent approaches: morphologically, i.e. using immunofluorescence and confocal microscopy, and biochemically, MSY3 expression is downregulated concomitantly with the spatiotemporal progression of neurogenesis. Interestingly, in preliminary results it was shown that MSY3 is expressed in Dcx-positive transient amplifying precursors in germinal zones of the adult brain, and in EGF-dependent neurospheres. To evaluate whether MSY3 could regulate the neurogenesis, the levels of the MSY3 protein in the progenitors were acutely downregulated or elevated by electroporation of RNAi or MSY3 expression plasmids, respectively. Neither premature reduction of MSY3 in the neuroepithelium (E9.5-E11.5) nor prolonged expression at the developmental stage when this protein is endogenously downregulated (E10.5-14.5) did affect proliferation versus the cell cycle exit of progenitors. Moreover, in Notch1-deficient progenitors in the cerebellar anlage, which exhibit precocious differentiation, MSY3 was not prematurely downregulated, suggesting that MSY3 also is not an early marker of differentiation. Differential centrifugation, immunoprecipitation and polysomal analysis performed in this study revealed that the MSY3 protein in the developing embryo, as well as in Neuro-2A cells, is associated with RNA. On a sucrose density gradient MSY3 co-fractionates with ribosomes and actively translating polysomes, suggesting that it might have a role in regulation of translation. However, downregulation or overexpression of MSY3 in the Neuro-2A cell line did not affect global translation rates. Other researchers suggested that the MSY3 protein has the redundant function with Y-box protein 1 (YB-1). Accordingly, in our system the MSY3 protein could be co-immunoprecipitated with YB-1. Importantly, developmentally regulated expression of MSY3 is not a hallmark of general translation apparatus, as several other proteins involved in translation did not show similar downregulation. To summarise, this work showed that the MSY3 protein is a marker of proliferation of progenitor cells in the embryonic and adult brain, being absent from neurons. Discovery of the molecular mechanism by which MSY3 exerts its role in the cell could provide the link between the translational machinery and proliferation.

Yongli Zhang, Corey L Smith, Anjanabha Saha, Stephan W. Grill, Shirley Mihardja, Steven B Smith, Bradley R Cairns, Craig L Peterson, Carlos Bustamante
DNA translocation and loop formation mechanism of chromatin remodeling by SWI/SNF and RSC.
Mol Cell, 24(4) 559-568 (2006)
PDF DOI

ATP-dependent chromatin-remodeling complexes (remodelers) modulate gene transcription by regulating the accessibility of highly packaged genomic DNA. However, the molecular mechanisms involved at the nucleosomal level in this process remain controversial. Here, we monitor the real-time activity of single ySWI/SNF or RSC complexes on single, stretched nucleosomal templates under tensions above 1 pN forces. We find that these remodelers can translocate along DNA at rates of approximately 13 bp/s and generate forces up to approximately 12 pN, producing DNA loops of a broad range of sizes (20-1200 bp, average approximately 100 bp) in a nucleosome-dependent manner. This nucleosome-specific activity differs significantly from that on bare DNA observed under low tensions and suggests a nucleosome-remodeling mechanism through intranucleosomal DNA loop formation. Such loop formation may provide a molecular basis for the biological functions of remodelers.

Eva-Maria Krämer-Albers, Katja Gehrig-Burger, Christoph Thiele, Jacqueline Trotter, Klaus-Armin Nave
Perturbed interactions of mutant proteolipid protein/DM20 with cholesterol and lipid rafts in oligodendroglia: implications for dysmyelination in spastic paraplegia.
J Neurosci, 26(45) 11743-11752 (2006)
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Missense mutations in the human PLP1 gene lead to dysmyelinating diseases with a broad range of clinical severity, ranging from severe Pelizaeus-Merzbacher disease (PMD) to milder spastic paraplegia type 2 (SPG-2). The molecular pathology has been generally attributed to endoplasmic reticulum (ER) retention of misfolded proteolipid protein (PLP) (and its splice isoform DM20) and induction of the unfolded protein response. As opposed to previous studies of heterologous expression systems, we have analyzed PLP/DM20 trafficking in oligodendroglial cells, thereby revealing differences between PMD and SPG-2-associated PLP/DM20 isoforms. PLP(A242V) and DM20(A242V) (jimpy-msd in mice), associated with severe PMD-like phenotype in vivo, were not only retained in the ER but also interfered with oligodendroglial process formation. In contrast, glial cells expressing SPG-2-associated PLP(I186T) or DM20(I186T) (rumpshaker in mice) developed processes, and mutant PLP/DM20 reached a late endosomal/lysosomal compartment. Unexpectedly, PLP/DM20 with either substitution exhibited impaired cholesterol binding, and the association with lipid raft microdomains was strongly reduced. Turnover analysis demonstrated that mutant PLP was rapidly degraded in oligodendroglial cells, with half-lives for PLP > PLP(I186T) > PLP(A242V). Protein degradation was specifically sensitive to proteasome inhibition, although PLP/DM20(I186T) degradation was also affected by inhibition of lysosomal enzymes. We conclude that, in addition to ER retention and unfolded protein response (UPR) induction, impaired cholesterol binding and lipid raft association are characteristic cellular defects of PLP1-missense mutations. Mutant protein is rapidly cleared and does not accumulate in oligodendroglial cells. Whereas UPR-induced cell death governs the PMD phenotype of the msd mutation, we propose that impaired cholesterol and lipid raft interaction of the rsh protein may contribute to the dysmyelination observed in SPG-2.

Agnes Toth-Petroczy, Agnes Szilagyi, Zsolt Ronai^#, Maria Sasvari-Szekely, András Guttman^#
Validation of a tentative microsatellite marker for the dopamine D4 receptor gene by capillary gel electrophoresis.
J Chromatogr A, 1130(2) 201-205 (2006)
DOI

Two to four-basepair-short tandem repeats (i.e. microsatellites) are broadly utilized as genetic markers for mapping disease loci in whole genome search analyses. Based on their close vicinity on chromosome 11, the D11S1984 microsatellite was anticipated as a tentative marker for the dopamine D4 receptor gene. A capillary gel electrophoresis based genotype analysis method and an in-house made computational tool was developed for the analysis of the D11S1984 microsatellite marker to examine a healthy Hungarian population of n=106. The data obtained did not suggest significant linkage between the D11S1984 marker and the DRD4 gene.

Mihail Sarov, Susan Schneider, Andrei I. Pozniakovsky, Assen Roguev, Susanne Ernst, Youming Zhang, Anthony A. Hyman, A. Francis Stewart
A recombineering pipeline for functional genomics applied to Caenorhabditis elegans.
Nat Methods, 3(10) 839-844 (2006)
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We present a new concept in DNA engineering based on a pipeline of serial recombineering steps in liquid culture. This approach is fast, straightforward and facilitates simultaneous processing of multiple samples in parallel. We validated the approach by generating green fluorescent protein (GFP)-tagged transgenes from Caenorhabditis briggsae genomic clones in a multistep pipeline that takes only 4 d. The transgenes were engineered with minimal disturbance to the natural genomic context so that the correct level and pattern of expression will be secured after transgenesis. An example transgene for the C. briggsae ortholog of lin-59 was used for ballistic transformation in Caenorhabditis elegans. We show that the cross-species transgene is correctly expressed and rescues RNA interference (RNAi)-mediated knockdown of the endogenous C. elegans gene. The strategy that we describe adapts the power of recombineering in Escherichia coli for fluent DNA engineering to a format that can be directly scaled up for genomic projects.

David Stanĕk, Karla M. Neugebauer
The Cajal body: a meeting place for spliceosomal snRNPs in the nuclear maze.
Chromosoma, 115(5) 343-354 (2006)
PDF DOI

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) are essential pre-mRNA splicing factors that consist of small nuclear RNAs (snRNAs) complexed with specific sets of proteins. A considerable body of evidence has established that snRNP assembly is accomplished after snRNA synthesis in the nucleus through a series of steps involving cytoplasmic and nuclear phases. Recent work indicates that snRNPs transiently localize to the Cajal body (CB), a nonmembrane-bound inclusion present in the nuclei of most cells, for the final steps in snRNP maturation, including snRNA base modification, U4/U6 snRNA annealing, and snRNA-protein assembly. Here, we review these findings that suggest a crucial role for CBs in the spliceosome cycle in which production of new snRNPs--and perhaps regenerated snRNPs after splicing--is promoted by the concentration of substrates in this previously mysterious subnuclear organelle. These insights allow us to speculate on the role of nuclear bodies in regulating the dynamics of RNP assembly to maintain a functional pool of factors available for key steps in gene expression.

Imke Listerman, Aparna K Sapra, Karla M. Neugebauer
Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells.
Nat Struct Mol Biol, 13(9) 815-822 (2006)
PDF DOI

Coupling between transcription and RNA processing is a key gene regulatory mechanism. Here we use chromatin immunoprecipitation to detect transcription-dependent accumulation of the precursor mRNA (pre-mRNA) splicing factors hnRNP A1, U2AF65 and U1 and U5 snRNPs on the intron-containing human FOS gene. These factors were poorly detected on intronless heat-shock and histone genes, a result that opposes direct recruitment by RNA polymerase II (Pol II) or the cap-binding complex in vivo. However, an observed RNA-dependent interaction between U2AF65 and active forms of Pol II may stabilize U2AF65 binding to intron-containing nascent RNA. We establish chromatin-RNA immunoprecipitation and show that FOS pre-mRNA is cotranscriptionally spliced. Notably, the topoisomerase I inhibitor camptothecin, which stalls elongating Pol II, increased cotranscriptional splicing factor accumulation and splicing in parallel. This provides direct evidence for a kinetic link between transcription, splicing factor recruitment and splicing catalysis.

Manuel A Fernández^*, Cecilia Albor^*, Mercedes Ingelmo-Torres, Susan J. Nixon, Charles Ferguson, Teymuras V. Kurzchalia, Francesc Tebar, Carlos Enrich, Robert G. Parton, Albert Pol
Caveolin-1 is essential for liver regeneration.
Science, 313(5793) 1628-1632 (2006)
PDF DOI

Liver regeneration is an orchestrated cellular response that coordinates cell activation, lipid metabolism, and cell division. We found that caveolin-1 gene-disrupted mice (cav1-/- mice) exhibited impaired liver regeneration and low survival after a partial hepatectomy. Hepatocytes showed dramatically reduced lipid droplet accumulation and did not advance through the cell division cycle. Treatment of cav1-/- mice with glucose (which is a predominant energy substrate when compared to lipids) drastically increased survival and reestablished progression of the cell cycle. Thus, caveolin-1 plays a crucial role in the mechanisms that coordinate lipid metabolism with the proliferative response occurring in the liver after cellular injury.

Nadine Vastenhouw, Karin Brunschwig, Kristy L Okihara, Fritz Müller, Marcel Tijsterman, Ronald H A Plasterk
Gene expression: long-term gene silencing by RNAi.
Nature, 442(7105) 882-882 (2006)
DOI

Small RNA molecules participate in a variety of activities in the cell: in a process known as RNA interference (RNAi), double-stranded RNA triggers the degradation of messenger RNA that has a matching sequence; the small RNA intermediates of this process can also modify gene expression in the nucleus. Here we show that a single episode of RNAi in the nematode Caenorhabditis elegans can induce transcriptional silencing effects that are inherited indefinitely in the absence of the original trigger. Our findings may prove useful in the ongoing development of RNAi to treat disease.

Imke Listerman
On co-transcriptional splicing and U6 snRNA biogenesis
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2006)

Messenger RNA (mRNA) is transcribed by RNA polymerase II (Pol II) and has to undergo multiple processing events before it can be translated into a protein: a cap structure is added to its 5’ end, noncoding, intervening sequences (introns) are removed and coding exons are ligated together and a poly(A) tail is added to its 3’end. Splicing, the process of intron removal, is carried out in the spliceosome, a megacomplex comprehending up to 300 proteins. The core components of the spliceosome that directly interact with the pre-mRNA are the small nuclear ribonucleoprotein particles (snRNPs). They consist of one of the U-rich snRNAs U1, U2, U4, U5 or U6 together with several particle-specific proteins and core proteins. All mRNA processing events can occur co-transcriptionally, i.e. while the RNA is still attached to the gene via Pol II. The in vivo studies of co-transcriptional RNA processing events had been possible only in special biological systems by immunoelectron microscopy and only recently, Chromatin Immunoprecipitation (ChIP) made it possible to investigate cotranscriptional splicing factor assembly on genes. My thesis work is divided into two parts: Part I shows that the core components of the splicing machinery are recruited co-transcriptionally to mammalian genes in vivo by ChIP. The co-transcriptional splicing factor recruitment is dependent on active transcription and the presence of introns in genes. Furthermore, a new assay was developed that allows for the first time the direct monitoring of co-transcriptional splicing in human cells. The topoisomerase I inhibitor camptothecin increases splicing factor accumulation on the c-fos gene as well as co-transcriptional splicing levels, which provides direct evidence that co-transcriptional splicing events depend on the kinetics of RNA synthesis. Part II of the thesis is aimed to investigate whether Pol II has a functional role in the biogenesis of the U6 snRNA, which is the RNA part of the U6 snRNP involved in splicing. Pol III had been shown to transcribe the U6 snRNA gene, but ChIP experiments revealed that Pol II is associated with all the active U6 snRNA gene promoters. Pol II inhibition studies uncovered that U6 snRNA expression and probably 3’end formation is dependent on Pol II.

Gerlinde Reim, Michael Brand
Maternal control of vertebrate dorsoventral axis formation and epiboly by the POU domain protein Spg/Pou2/Oct4.
Development, 133(14) 2757-2770 (2006)
PDF DOI

Dorsoventral (DV) axis formation of the vertebrate embryo is controlled by the maternal genome and is subsequently refined zygotically. In the zygote, repression of ventralizing Bmp activity on the dorsal side through chordin and noggin is crucial for establishment of a dorsally located organizer. This interplay generates a zygotic Bmp activity gradient that defines distinct positional values along the DV axis. The maternal processes that control expression of the zygotic genes implicated in DV patterning are largely unknown. spiel-ohne-grenzen (spg/pou2) is a maternally and zygotically expressed zebrafish gene that encodes the POU domain transcription factor Pou2, an ortholog of mammalian Oct4/Pou5f1. We show that embryos that are genetically depleted of both maternal and zygotic pou2 function (MZspg) exhibit extreme DV patterning defects and, independently, a blastoderm-specific arrest of epiboly. Dorsal tissues expand to the ventral side at the expense of ventrolateral tissue in MZspg embryos. Dorsally expressed Bmp-antagonists, such as Chd and Nog1, and Gsc are ectopically activated at ventral levels in MZspg. Lack of ventral specification is apparent very early, suggesting that maternal processes are affected in MZspg. Indeed, maternal pou2 function is necessary to initiate zygotic expression of ventrally expressed genes such as bmp2b and bmp4, and for proper activation of bmp7, vox, vent and eve1. A constitutively active Alk8-TGFbeta-receptor can ectopically induce bmp2b and bmp4 and rescues the dorsalization of MZspg. This indicates that pou2 acts upstream of Alk8, a maternally provided receptor implicated in the activation of zygotic bmp2b and bmp4 transcription. Consistent with this possibility, Bmp gene misexpression can rescue MZspg embryos, indicating that TGFbeta-mediated signal transduction itself is intact in absence of Pou2. Inhibition of Fgf signaling, another pathway with early dorsalizing activity, can also restore and even ventralize MZspg embryos. The requirement for pou2 to initiate bmp2b expression can therefore be bypassed by releasing the repressive function of Fgf signaling upon bmp2b transcription. In transplantation experiments, we find that dorsalized cells from prospective ventrolateral regions of MZspg embryos are non cell-autonomously respecified to a ventral fate within wild-type host embryos. Analysis of pou2 mRNA injected MZspg embryos shows that pou2 is required on the ventral side of cleavage stage embryos. Based on the maternal requirement for pou2 in ventral specification, we propose that ventral specification employs an active, pou2-dependent maternal induction step, rather than a default ventralizing program.

Mirko Trajkovski, Hassan Mziaut, Peter E Schwarz, Michele Solimena
Genes of type 2 diabetes in beta cells.
Endocrinol Metab Clin North Am, 35(2) 357-369 (2006)
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Type 2 diabetes is a complex polygenic metabolic disorder of epidemic proportions. This review provides a brief overview of the susceptibility genes in type 2 diabetes that primarily affect pancreatic 3 cells, with emphasis on their function and most relevant polymorphisms. We focus on calpain 10, the only susceptibility gene identified thus far through a positional cloning approach in subjects with diabetes.

Karla M. Neugebauer
Please hold--the next available exon will be right with you.
Nat Struct Mol Biol, 13(5) 385-386 (2006)
PDF DOI

A major challenge in gene expression is to understand how precursor messenger RNA (pre-mRNA) processing events are integrated with transcription. A recent study suggests that distant exons in nascent RNA are held together during transcription, promoting accurate splicing independent of intron fate.

Vinzenz Link, Lara Carvalho, Irinka Castanon, Petra Stockinger, Andrej Shevchenko, Carl-Philipp Heisenberg
Identification of regulators of germ layer morphogenesis using proteomics in zebrafish.
J Cell Sci, 119(Pt 10) 2073-2083 (2006)
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During vertebrate gastrulation, a well-orchestrated series of morphogenetic changes leads to the formation of the three germ layers: the ectoderm, mesoderm and endoderm. The analysis of gene expression patterns during gastrulation has been central to the identification of genes involved in germ layer formation. However, many proteins are regulated on a translational or post-translational level and are thus undetectable by gene expression analysis. Therefore, we developed a 2D-gel-based comparative proteomic approach to target proteins involved in germ layer morphogenesis during zebrafish gastrulation. Proteomes of ectodermal and mesendodermal progenitor cells were compared and 35 significantly regulated proteins were identified by mass spectrometry, including several proteins with predicted functions in cytoskeletal organization. A comparison of our proteomic results with data obtained in an accompanying microarray-based gene expression analysis revealed no significant overlap, confirming the complementary nature of proteomics and transcriptomics. The regulation of ezrin2, which was identified based on a reduction in spot intensity in mesendodermal cells, was independently validated. Furthermore, we show that ezrin2 is activated by phosphorylation in mesendodermal cells and is required for proper germ layer morphogenesis. We demonstrate the feasibility of proteomics in zebrafish, concluding that proteomics is a valuable tool for analysis of early development.

Hassan Mziaut, Mirko Trajkovski, Stephan Kersting, Armin Ehninger, Anke Altkrüger, Regis P. Lemaitre, Darja Schmidt, Hans-Detlev Saeger, Myung-Shik Lee, David N. Drechsel, Stefan Müller, Michele Solimena
Synergy of glucose and growth hormone signalling in islet cells through ICA512 and STAT5.
Nat Cell Biol, 8(5) 435-445 (2006)
PDF DOI

Nutrients and growth hormones promote insulin production and the proliferation of pancreatic beta-cells. An imbalance between ever-increasing metabolic demands and insulin output causes diabetes. Recent evidence indicates that beta-cells enhance insulin gene expression depending on their secretory activity. This signalling pathway involves a catalytically inactive receptor tyrosine phosphatase, ICA512, whose cytoplasmic tail is cleaved on glucose-stimulated exocytosis of insulin secretory granules and then moves into the nucleus, where it upregulates insulin transcription. Here, we show that the cleaved cytosolic fragment of ICA512 enhances the transcription of secretory granule genes (including its own gene) by binding to tyrosine phosphorylated signal transducers and activators of transcription (STAT) 5 and preventing its dephosphorylation. Sumoylation of ICA512 by the E3 SUMO ligase PIASy, in turn, may reverse this process by decreasing the binding of ICA512 to STAT5. These findings illustrate how the exocytosis of secretory granules, through a retrograde pathway that sustains STAT activity, converges with growth hormone signalling to induce adaptive changes in beta-cells in response to metabolic demands.

Catherine M Burgess, Dirk Jan Slotboom, Eric R Geertsma, Ria H Duurkens, B Poolman, Douwe van Sinderen
The riboflavin transporter RibU in Lactococcus lactis: molecular characterization of gene expression and the transport mechanism.
J Bacteriol, 188(8) 2752-2760 (2006)
DOI

This study describes the characterization of the riboflavin transport protein RibU in the lactic acid bacterium Lactococcus lactis subsp. cremoris NZ9000. RibU is predicted to contain five membrane-spanning segments and is a member of a novel transport protein family, not described in the Transport Classification Database. Transcriptional analysis revealed that ribU transcription is downregulated in response to riboflavin and flavin mononucleotide (FMN), presumably by means of the structurally conserved RFN (riboflavin) element located between the transcription start site and the start codon. An L. lactis strain carrying a mutated ribU gene exhibits altered transcriptional control of the riboflavin biosynthesis operon ribGBAH in response to riboflavin and FMN and does not consume riboflavin from its growth medium. Furthermore, it was shown that radiolabeled riboflavin is not taken up by the ribU mutant strain, in contrast to the wild-type strain, directly demonstrating the involvement of RibU in riboflavin uptake. FMN and the toxic riboflavin analogue roseoflavin were shown to inhibit riboflavin uptake and are likely to be RibU substrates. FMN transport by RibU is consistent with the observed transcriptional regulation of the ribGBAH operon by external FMN. The presented transport data are consistent with a uniport mechanism for riboflavin translocation and provide the first detailed molecular and functional analysis of a bacterial protein involved in riboflavin transport.

Ganka Nikolova, Normund Jabs, Irena Konstantinova, Anna Domogatskaya, Karl Tryggvason, Lydia Sorokin, Reinhard Fässler, Guoqiang Gu, Hans-Peter Gerber, Napoleone Ferrara, Douglas A Melton, Eckhard Lammert
The vascular basement membrane: a niche for insulin gene expression and Beta cell proliferation.
Dev Cell, 10(3) 397-405 (2006)
PDF DOI

Endocrine pancreatic beta cells require endothelial signals for their differentiation and function. However, the molecular basis for such signals remains unknown. Here, we show that beta cells, in contrast to the exocrine pancreatic cells, do not form a basement membrane. Instead, by using VEGF-A, they attract endothelial cells, which form capillaries with a vascular basement membrane next to the beta cells. We have identified laminins, among other vascular basement membrane proteins, as endothelial signals, which promote insulin gene expression and proliferation in beta cells. We further demonstrate that beta1-integrin is required for the beta cell response to the laminins. The proposed mechanism explains why beta cells must interact with endothelial cells, and it may apply to other cellular processes in which endothelial signals are required.

Angela Huebner, Philipp Mann, Elvira Rohde, Angela M Kaindl, Martin Witt, Paul Verkade, Sibylle Jakubiczka, Mario Menschikowski, Gisela Stoltenburg-Didinger, Katrin Koehler
Mice lacking the nuclear pore complex protein ALADIN show female infertility but fail to develop a phenotype resembling human triple A syndrome.
Mol Cell Biol, 26(5) 1879-1887 (2006)
PDF DOI

Triple A syndrome is a human autosomal recessive disorder characterized by adrenal insufficiency, achalasia, alacrima, and neurological abnormalities affecting the central, peripheral, and autonomic nervous systems. In humans, this disease is caused by mutations in the AAAS gene, which encodes ALADIN, a protein that belongs to the family of WD-repeat proteins and localizes to nuclear pore complexes. To analyze the function of the gene in the context of the whole organism and in an attempt to obtain an animal model for human triple A syndrome, we generated mice lacking a functional Aaas gene. The Aaas-/- animals were found to be externally indistinguishable from their wild-type littermates, although their body weight was on the average lower than that of wild-type mice. Histological analysis of various tissues failed to reveal any differences between Aaas-/- and wild-type mice. Aaas-/- mice exhibit unexpectedly mild abnormal behavior and only minor neurological deficits. Our data show that the lack of ALADIN in mice does not lead to a triple A syndrome-like disease. Thus, in mice either the function of ALADIN differs from that in humans, its loss can be readily compensated for, or additional factors, such as environmental conditions or genetic modifiers, contribute to the disease.

Steffen Scholpp, Olivia Wolf, Michael Brand, Andrew Lumsden
Hedgehog signalling from the zona limitans intrathalamica orchestrates patterning of the zebrafish diencephalon.
Development, 133(5) 855-864 (2006)
PDF DOI

Midway between the anterior neural border and the midbrain-hindbrain boundary, two well-known local signalling centres in the early developing brain, is a further transverse boundary with putative signalling properties -- the zona limitans intrathalamica (ZLI). Here, we describe formation of the ZLI in zebrafish in relation to expression of sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh), and to development of the forebrain regions that flank the ZLI: the prethalamus and thalamus. We find that enhanced Hh signalling increases the size of prethalamic and thalamic gene expression domains, whereas lack of Hh signalling leads to absence of these domains. In addition, we show that shh and twhh display both unique and redundant functions during diencephalic patterning. Genetic ablation of the basal plate shows that Hh expression in the ZLI alone is sufficient for diencephalic differentiation. Furthermore, acquisition of correct prethalamic and thalamic gene expression is dependent on direct Hh signalling. We conclude that proper maturation of the diencephalon requires ZLI-derived Hh signalling.

Karin Schlichting^*, Michaela Wilsch-Bräuninger^*, Fabio Demontis^*, Christian Dahmann
Cadherin Cad99C is required for normal microvilli morphology in Drosophila follicle cells.
J Cell Sci, 119(Pt 6) 1184-1195 (2006)
PDF DOI

Microvilli are actin-filled membranous extensions common to epithelial cells. Several proteins have been identified that localize to microvilli. However, most of these proteins are dispensable for the normal morphogenesis of microvilli. Here, we show by immunoelectron microscopy that the non-classical cadherin Cad99C localizes to microvilli of Drosophila ovarian follicle cells. Loss of Cad99C function leads to disorganized and abnormal follicle cell microvilli. Conversely, overexpression of Cad99C in follicle cells results in large bundles of microvilli. Furthermore, altered microvilli morphology correlates with defects in the assembly of the vitelline membrane, an extracellular layer secreted by follicle cells that is part of the eggshell. Finally, we provide evidence that Cad99C is the homolog of vertebrate protocadherin 15. Mutations in the gene encoding protocadherin 15 lead to the disorganization of stereocilia, which are microvilli-derived extensions of cochlear hair cells, and deafness (Usher syndrome type 1F). Our data suggest an essential role for Cad99C in microvilli morphogenesis that is important for follicle cell function. Furthermore, these results indicate that insects and vertebrates use related cadherins to organize microvilli-like cellular extensions.

Klaus-Peter Knoch, Ronny Meisterfeld, Stephan Kersting, Hendrik Bergert, Anke Altkrüger, Carolin Wegbrod, Melanie Jäger, Hans-Detlev Saeger, Michele Solimena
cAMP-dependent phosphorylation of PTB1 promotes the expression of insulin secretory granule proteins in beta cells.
Cell Metab, 3(2) 123-134 (2006)
PDF DOI

Glucose stimulates the exocytosis of insulin secretory granules of pancreatic beta cells. Granule stores are quickly refilled by activation of posttranscriptional mechanisms that enhance the biosynthesis of granule components. Rapid replacement of granules is important to sustain insulin secretion, since new granules appear to be preferentially released. Posttranscriptional regulation of granule biogenesis includes the glucose-induced nucleocytoplasmic translocation of polypyrimidine tract binding protein 1 (PTB1), which binds mRNAs encoding granule proteins, and thus promotes their stabilization and translation. Glucagon-like peptide 1 (GLP-1) potentiates glucose-stimulated insulin gene expression and secretion by increasing cAMP levels in beta cells. Here, we show that elevation of cAMP levels causes the protein kinase A-dependent phosphorylation and nucleocytoplasmic translocation of PTB1, thereby preventing the rapid degradation of insulin mRNA and enhancing the expression of various granule proteins. Taken together, these findings identify PTB1 as a common downstream target of glucose and GLP-1 for the posttranscriptional upregulation of granule biogenesis.

Gabriele Putz, Andrea Rosner, Ina Nuesslein, Nicole Schmitz, Frank Buchholz
AML1 deletion in adult mice causes splenomegaly and lymphomas.
Oncogene, 25(6) 929-939 (2006)
PDF DOI

AML1 (RUNX1) encodes a DNA-binding subunit of the CBF transcription factor family and is required for the establishment of definitive hematopoiesis. AML1 is one of the most frequently mutated genes associated with human acute leukemia, suggesting that genetic alterations of the gene contribute to leukemogenesis. Here, we report the analysis of mice carrying conditional AML1 knockout alleles that were inactivated using the Cre/loxP system. AML1 was deleted in adult mice by inducing Cre activity to replicate AML1 deletions found in human MDS, familial platelet disorder and rare de novo human AML. At a latency of 2 months after induction, the thymus was reduced in size and frequently populated by immature double negative thymocytes, indicating defective T-lymphocyte maturation, resulting in lymphatic diseases with 50% penetrance, including atypical hyperplasia and thymic lymphoma. Metastatic lymphomas to the liver and the meninges were observed. Mice also developed splenomegaly with an expansion of the myeloid compartment. Increased Howell-Jolly body counts indicated splenic hypofunction. Thrombocytopenia occurred due to immaturity of mini-megakaryocytes in the bone marrow. Together with mild lymphocytopenia in the peripheral blood and increased fractions of immature cells in the bone marrow, AML1 deficient mice display features of a myelodysplastic syndrome, suggesting a preleukemic state.

Michael Hiller, Klaus Huse, Karol Szafranski, Niels Jahn, Jochen Hampe, Stefan Schreiber, Rolf Backofen, Matthias Platzer
Single-nucleotide polymorphisms in NAGNAG acceptors are highly predictive for variations of alternative splicing.
Am J Hum Genet, 78(2) 291-302 (2006)
PDF DOI

Aberrant or modified splicing patterns of genes are causative for many human diseases. Therefore, the identification of genetic variations that cause changes in the splicing pattern of a gene is important. Elsewhere, we described the widespread occurrence of alternative splicing at NAGNAG acceptors. Here, we report a genomewide screen for single-nucleotide polymorphisms (SNPs) that affect such tandem acceptors. From 121 SNPs identified, we extracted 64 SNPs that most likely affect alternative NAGNAG splicing. We demonstrate that the NAGNAG motif is necessary and sufficient for this type of alternative splicing. The evolutionarily young NAGNAG alleles, as determined by the comparison with the chimpanzee genome, exhibit the same biases toward intron phase 1 and single-amino acid insertion/deletions that were already observed for all human NAGNAG acceptors. Since 28% of the NAGNAG SNPs occur in known disease genes, they represent preferable candidates for a more-detailed functional analysis, especially since the splice relevance for some of the coding SNPs is overlooked. Against the background of a general lack of methods for identifying splice-relevant SNPs, the presented approach is highly effective in the prediction of polymorphisms that are causal for variations in alternative splicing.

Lidia Sobkow, Hans-Henning Epperlein, Stephan Herklotz, Werner L. Straube, Elly M. Tanaka
A germline GFP transgenic axolotl and its use to track cell fate: dual origin of the fin mesenchyme during development and the fate of blood cells during regeneration.
Dev Biol, 290(2) 386-397 (2006)
PDF DOI

The development of transgenesis in axolotls is crucial for studying development and regeneration as it would allow for long-term cell fate tracing as well as gene expression analysis. We demonstrate here that plasmid injection into the one-cell stage axolotl embryo generates mosaic transgenic animals that display germline transmission of the transgene. The inclusion of SceI meganuclease in the injections (Thermes, V., Grabher, C., Ristoratore, F., Bourrat, F., Choulika, A., Wittbrodt, J., Joly, J.S., 2002. I-SceI meganuclease mediates highly efficient transgenesis in fish. Mech. Dev. 118, 91-98) resulted in a higher percentage of F0 animals displaying strong expression throughout the body. This represents the first demonstration in the axolotl of germline transmission of a transgene. Using this technique we have generated a germline transgenic animal expressing GFP ubiquitously in all tissues examined. We have used this animal to study cell fate in the dorsal fin during development. We have uncovered a contribution of somite cells to dorsal fin mesenchyme in the axolotl, which was previously assumed to derive solely from neural crest. We have also studied the role of blood during tail regeneration by transplanting the ventral blood-forming region from GFP+ embryos into unlabeled hosts. During tail regeneration, we do not observe GFP+ cells contributing to muscle or nerve, suggesting that during tail regeneration blood stem cells do not undergo significant plasticity.

Hanchuan Peng, Fuhui Long, Michael B Eisen, Eugene W Myers
Clustering gene expression patterns of fly embryos
In: Proceedings in 3rd IEEE International Symposium on Biomedical Imaging: Macro to Nano (2006), Piscataway, N.J., IEEE (2006), 1144-1147
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Ganka Nikolova
The vascular basement membrane : a niche for insuline gene expression and beta cell proliferation
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2006)
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Endocrine pancreatic beta cells depend on endothelial signals for their differentiation and

Eberhard Krausz
Challenges in High-Content siRNA Screening
European Pharmaceutical Review, 6 13-20 (2006)
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Huge progress has been made, both in RNA interference technology applied to mammalian cells and in automated microscopy to analyse gene functions upon silencing in the cellular context. Large-scale siRNA screens have been published recently, mainly applying assays that gain multi-parametric information on biological processes. It is a long way to establish an infrastructure that allows high-content siRNA screening, and in this article the major challenges are summarised.

Karen Oegema^#, Anthony A. Hyman^#
Cell division.
WormBook : the online review of C. elegans biology, Jan 19 1-40 (2006)
PDF DOI

The C. elegans embryo is a powerful model system for studying the mechanics of metazoan cell division. Its primary advantage is that the architecture of the syncytial gonad makes it possible to use RNAi to generate oocytes whose cytoplasm is reproducibly (typically >95%) depleted of targeted essential gene products via a process that does not depend exclusively on intrinsic protein turnover. The depleted oocytes can then be analyzed as they attempt their first mitotic division following fertilization. Here we outline the characteristics that contribute to the usefulness of the C. elegans embryo for cell division studies. We provide a timeline for the first embryonic mitosis and highlight some of its key features. We also summarize some of the recent discoveries made using this system, particularly in the areas of nuclear envelope assembly/dissassembly, centrosome dynamics, formation of the mitotic spindle, kinetochore assembly, chromosome segregation, and cytokinesis.

Jessica Dessimoz, Robert Opoka, Jennifer J Kordich, Anne Grapin-Botton, James M Wells
FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo.
Mech Dev, 123(1) 42-55 (2006)
DOI

At the end of gastrulation in avians and mammals, the endoderm germ layer is an undetermined sheet of cells. Over the next 24-48 h, endoderm forms a primitive tube and becomes regionally specified along the anterior-posterior axis. Fgf4 is expressed in gastrulation and somite stage embryos in the vicinity of posterior endoderm that gives rise to the posterior gut. Moreover, the posterior endoderm adjacent to Fgf4-expressing mesoderm expresses the FGF-target genes Sprouty1 and 2 suggesting that endoderm respond to an FGF signal in vivo. Here, we report the first evidence suggesting that FGF4-mediated signaling is required for establishing gut tube domains along the A-P axis in vivo. At the gastrula stage, exposing endoderm to recombinant FGF4 protein results in an anterior shift in the Pdx1 and CdxB expression domains. These expression domains remain sensitive to FGF4 levels throughout early somite stages. Additionally, FGF4 represses the anterior endoderm markers Hex1 and Nkx2.1 and disrupts foregut morphogenesis. FGF signaling directly patterns endoderm and not via a secondary induction from another germ layer, as shown by expression of dominant-active FGFR1 specifically in endoderm, which results in ectopic anterior expression of Pdx1. Loss-of-function studies using the FGF receptor antagonist SU5402 demonstrate that FGF signaling is necessary for establishing midgut gene expression and for maintaining gene expression boundaries between the midgut and hindgut from gastrulation through somitogenesis. Moreover, FGF signaling in the primitive streak is necessary to restrict Hex1 expression to anterior endoderm. These data show that FGF signaling is critical for patterning the gut tube by promoting posterior and inhibiting anterior endoderm cell fate.

Wieland B. Huttner, Yoichi Kosodo
Symmetric versus asymmetric cell division during neurogenesis in the developing vertebrate central nervous system.
Curr Opin Cell Biol, 17(6) 648-657 (2005)
PDF DOI

The type and number of cell divisions of neuronal progenitors determine the number of neurons generated during the development of the vertebrate central nervous system. Over the past several years, there has been substantial progress in characterizing the various kinds of neuronal progenitors and the types of symmetric and asymmetric divisions they undergo. The understanding of the cell-biological basis of symmetric versus asymmetric progenitor cell division has been consolidated, and the molecular machinery controlling these divisions is beginning to be unravelled. Other recent advances include comparative studies of brain development in rodents and primates, as well as the identification of gene mutations in humans that affect the balance between the various types of cell division of neuronal progenitors.

Ricardo Zayas, Alvaro Hernández, Bianca Habermann, Yuying Wang, Joel M Stary, Phillip A Newmark
The planarian Schmidtea mediterranea as a model for epigenetic germ cell specification: analysis of ESTs from the hermaphroditic strain.
Proc Natl Acad Sci U.S.A., 102(51) 18491-18496 (2005)
PDF DOI

Freshwater planarians have prodigious regenerative abilities that enable them to form complete organisms from tiny body fragments. This plasticity is also exhibited by the planarian germ cell lineage. Unlike many model organisms in which germ cells are specified by localized determinants, planarian germ cells appear to be specified epigenetically, arising postembryonically from stem cells. The planarian Schmidtea mediterranea is well suited for investigating the mechanisms underlying epigenetic germ cell specification. Two strains of S. mediterranea exist: a hermaphroditic strain that reproduces sexually and an asexual strain that reproduces by means of transverse fission. To date, expressed sequence tags (ESTs) have been generated only from the asexual strain. To develop molecular reagents for studying epigenetic germ cell specification, we have sequenced 27,161 ESTs from two developmental stages of the hermaphroditic strain of S. mediterranea; this collection of ESTs represents approximately 10,000 unique transcripts. blast analysis of the assembled ESTs showed that 66% share similarity to sequences in public databases. We annotated the assembled ESTs using Gene Ontology terms as well as conserved protein domains and organized them in a relational database. To validate experimentally the Gene Ontology annotations, we used whole-mount in situ hybridization to examine the expression patterns of transcripts assigned to the biological process "reproduction." Of the 53 genes in this category, 87% were expressed in the reproductive organs. In addition to its utility for studying germ cell development, this EST collection will be an important resource for annotating the planarian genome and studying this animal's amazing regenerative abilities.

William H J Norton, Johan Ledin, Heiner Grandel, Carl J Neumann
HSPG synthesis by zebrafish Ext2 and Extl3 is required for Fgf10 signalling during limb development.
Development, 132(22) 4963-4973 (2005)
PDF DOI

Heparan sulphate proteoglycans (HSPGs) are known to be crucial for signalling by the secreted Wnt, Hedgehog, Bmp and Fgf proteins during invertebrate development. However, relatively little is known about their effect on developmental signalling in vertebrates. Here, we report the analysis of daedalus, a novel zebrafish pectoral fin mutant. Positional cloning identified fgf10 as the gene disrupted in daedalus. We find that fgf10 mutants strongly resemble zebrafish ext2 and extl3 mutants, which encode glycosyltransferases required for heparan sulphate biosynthesis. This suggests that HSPGs are crucial for Fgf10 signalling during limb development. Consistent with this proposal, we observe a strong genetic interaction between fgf10 and extl3 mutants. Furthermore, application of Fgf10 protein can rescue target gene activation in fgf10, but not in ext2 or extl3 mutants. By contrast, application of Fgf4 protein can activate target genes in both ext2 and extl3 mutants, indicating that ext2 and extl3 are differentially required for Fgf10, but not Fgf4, signalling during limb development. This reveals an unexpected specificity of HSPGs in regulating distinct vertebrate Fgfs.

Alexander Picker, Michael Brand
Fgf signals from a novel signaling center determine axial patterning of the prospective neural retina.
Development, 132(22) 4951-4962 (2005)
PDF DOI

Axial eye patterning determines the positional code of retinal ganglion cells (RGCs), which is crucial for their topographic projection to the midbrain. Several asymmetrically expressed determinants of retinal patterning are known, but it is unclear how axial polarity is first established. We find that Fgf signals, including Fgf8, determine retinal patterning along the nasotemporal (NT) axis during early zebrafish embryogenesis: Fgf8 induces nasal and/or suppresses temporal retinal cell fates; and inhibition of all Fgf-receptor signaling leads to complete retinal temporalization and concomitant loss of all nasal fates. Misprojections of RGCs with Fgf-dependent alterations in retinal patterning to the midbrain demonstrate the importance of this early patterning process for late topographic map formation. The crucial period of Fgf-dependent patterning is at the onset of eye morphogenesis. Fgf8 expression, the restricted temporal requirement for Fgf-receptor signaling and target gene expression at this stage suggests that the telencephalic primordium is the source of Fgf8 and acts as novel signaling center for non-autonomous axial patterning of the prospective neural retina.

Ralf Kittler, Anne-Kristin Heninger, Kristin Franke, Bianca Habermann, Frank Buchholz
Production of endoribonuclease-prepared short interfering RNAs for gene silencing in mammalian cells.
Nat Methods, 2(10) 779-784 (2005)
PDF DOI

Jessica Dessimoz, Claude Bonnard, Joerg Huelsken, Anne Grapin-Botton
Pancreas-specific deletion of beta-catenin reveals Wnt-dependent and Wnt-independent functions during development.
Curr Biol, 15(18) 1677-1683 (2005)
DOI

Mutations and deregulation of adenomatous polyposis coli (APC) and beta-catenin are implicated in specific cancers of the pancreas, but the role of Wnt pathway in normal pancreas development and homeostasis is unknown. This article reports a comprehensive investigation of the activity and the role of the Wnt pathway in pancreas organogenesis. We have used two reporter lines to monitor canonical Wnt pathway activity during development and after birth and demonstrate activity in endocrine cells and in the mesenchyme. We have specifically deleted the beta-catenin gene in the epithelium of the pancreas and duodenum by using Pdx1-Cre mice. In agreement with Wnt pathway activity in pancreatic endocrine cells, we find a reduction in endocrine islet numbers. Our study reveals that beta-catenin deletion also affects cells in which Wnt pathway activity is not detected. Indeed, beta-catenin mutant cells have a competitive disadvantage during development that also affects the exocrine compartment. Moreover, the conditional knockout (KO) mice develop acute edematous pancreatitis perinatally due to the disruption of the epithelial structure of acini. These effects are likely to be due to the function of beta-catenin at the membrane. Mice later recover from pancreatitis and regenerate normal pancreas and duodenal villi from the wild-type (wt) cells that escape beta-catenin deletion.

Oliver Bartsch, Stefanie Schmidt, Marion Richter, Susanne Morlot, Eva Seemanová, Glenis J. Wiebe, Sasan Rasi
DNA sequencing of CREBBP demonstrates mutations in 56% of patients with Rubinstein-Taybi syndrome (RSTS) and in another patient with incomplete RSTS.
Hum Genet, 117(5) 485-493 (2005)
PDF DOI

Rubinstein-Taybi syndrome (RSTS) is a distinct dominant disorder characterized by short stature, typical face, broad angulated thumbs and halluces, and mental retardation. The RSTS can be caused by chromosomal microdeletions and molecular mutations in the CREBBP gene; however, relatively few mutations have been reported to date. Here, we aimed to determine the rate of point mutations and other small molecular lesions in true RSTS and possible mild variants, by using genomic DNA sequencing. A consecutive series of patients including 17 patients from our previous study was investigated. We identified 19 causative mutations of CREBBP in a total of 45 patients representing three different diagnostic groups: (a) 17 mutations in 30 patients with unequivocal RSTS (detection rate 56.6%), (b) two mutations in eight patients with features suggestive of RSTS ("moderate or incomplete RSTS", detection rate 25%), and (c) no mutation in seven patients with undiagnosed syndromes and isolated features of RSTS. In general, the mutations were distributed without hot spots and most were unique; however, three recurrent mutations (R370X, R1664H, and N1978S) were identified. Furthermore, we detected 15 different intragenic polymorphisms, including two non-synonymous coding polymorphisms, L551I and Q2208H. We report not only the highest detection rate (56.6%) of CREBBP mutations in patients with RSTS to date, but also the second missense mutation (N1978S) in a patient with moderate or incomplete RSTS. Previous studies have identified cytogenetic deletions in the CREBBP gene in eight to 12% of patients and very recently, Roelfsema et al. reported EP300 gene mutations in three of 92 (3.3%) patients with either true RSTS or different syndromes resembling RSTS. Our 56.6% detection rate of molecular mutations in CREBBP in patients with unequivocal RSTS supports the new concept that RSTS is a genetically heterogeneous disorder and furthermore, indicates that RSTS may be caused by gene/s other than CREBBP in up to 30% of cases.

Pierre-Henri Puech^#, Anna Taubenberger, Florian Ulrich, Michael Krieg, Daniel J. Müller, Carl-Philipp Heisenberg^#
Measuring cell adhesion forces of primary gastrulating cells from zebrafish using atomic force microscopy.
J Cell Sci, 118(18) 4199-4206 (2005)
PDF DOI

During vertebrate gastrulation, progenitor cells of different germ layers acquire specific adhesive properties that contribute to germ layer formation and separation. Wnt signals have been suggested to function in this process by modulating the different levels of adhesion between the germ layers, however, direct evidence for this is still lacking. Here we show that Wnt11, a key signal regulating gastrulation movements, is needed for the adhesion of zebrafish mesendodermal progenitor cells to fibronectin, an abundant extracellular matrix component during gastrulation. To measure this effect, we developed an assay to quantify the adhesion of single zebrafish primary mesendodermal progenitors using atomic-force microscopy (AFM). We observed significant differences in detachment force and work between cultured mesendodermal progenitors from wild-type embryos and from slb/wnt11 mutant embryos, which carry a loss-of-function mutation in the wnt11 gene, when tested on fibronectin-coated substrates. These differences were probably due to reduced adhesion to the fibronectin substrate as neither the overall cell morphology nor the cell elasticity grossly differed between wild-type and mutant cells. Furthermore, in the presence of inhibitors of fibronectin-integrin binding, such as RGD peptides, the adhesion force and work were strongly decreased, indicating that integrins are involved in the binding of mesendodermal progenitors in our assay. These findings demonstrate that AFM can be used to quantitatively determine the substrate-adhesion of cultured primary gastrulating cells and provide insight into the role of Wnt11 signalling in modulating cell adhesion at the single cell scale.

Kristin C. Gunsalus^*, Hui Ge^*, Aaron J Schetter^*, Debra S Goldberg^*, Jing-Dong J Han, Tong Hao, Gabriel F Berriz, Nicolas Bertin, Jerry Huang, Ling-Shiang Chuang, Ning Li, Ramamurthy Mani, Anthony A. Hyman, Birte Sönnichsen, Christophe J. Echeverri, Frederick P. Roth, Marc Vidal, Fabio Piano
Predictive models of molecular machines involved in Caenorhabditis elegans early embryogenesis.
Nature, 436(7052) 861-865 (2005)
PDF DOI

Although numerous fundamental aspects of development have been uncovered through the study of individual genes and proteins, system-level models are still missing for most developmental processes. The first two cell divisions of Caenorhabditis elegans embryogenesis constitute an ideal test bed for a system-level approach. Early embryogenesis, including processes such as cell division and establishment of cellular polarity, is readily amenable to large-scale functional analysis. A first step toward a system-level understanding is to provide 'first-draft' models both of the molecular assemblies involved and of the functional connections between them. Here we show that such models can be derived from an integrated gene/protein network generated from three different types of functional relationship: protein interaction, expression profiling similarity and phenotypic profiling similarity, as estimated from detailed early embryonic RNA interference phenotypes systematically recorded for hundreds of early embryogenesis genes. The topology of the integrated network suggests that C. elegans early embryogenesis is achieved through coordination of a limited set of molecular machines. We assessed the overall predictive value of such molecular machine models by dynamic localization of ten previously uncharacterized proteins within the living embryo.

Tobias Langenberg, Michael Brand
Lineage restriction maintains a stable organizer cell population at the zebrafish midbrain-hindbrain boundary.
Development, 132(14) 3209-3216 (2005)
PDF DOI

The vertebrate hindbrain is subdivided into segments, termed neuromeres, that are units of gene expression, cell differentiation and behavior. A key property of such segments is that cells show a restricted ability to mix across segment borders -- termed lineage restriction. In order to address segmentation in the midbrain-hindbrain boundary (mhb) region, we have analyzed single cell behavior in the living embryo by acquiring time-lapse movies of the developing mhb region in a transgenic zebrafish line. We traced the movement of hundreds of nuclei, and by matching their position with the expression of a midbrain marker, we demonstrate that midbrain and hindbrain cells arise from two distinct cell populations. Single cell labeling and analysis of the distribution of their progeny shows that lineage restriction is probably established during late gastrulation stages. Our findings suggest that segmentation as an organizing principle in early brain development can be extended to the mhb region. We argue that lineage restriction serves to constrain the position of the mhb organizer cell population.

Janina Görnemann, Kimberly M. Kotovic, Katja Hujer, Karla M. Neugebauer
Cotranscriptional spliceosome assembly occurs in a stepwise fashion and requires the cap binding complex.
Mol Cell, 19(1) 53-63 (2005)
PDF DOI

Coupling between transcription and pre-mRNA splicing is a key regulatory mechanism in gene expression. Here, we investigate cotranscriptional spliceosome assembly in yeast, using in vivo crosslinking to determine the distribution of spliceosome components along intron-containing genes. Accumulation of the U1, U2, and U5 small nuclear ribonucleoprotein particles (snRNPs) and the 3' splice site binding factors Mud2p and BBP was detected in patterns indicative of progressive and complete spliceosome assembly; recruitment of the nineteen complex (NTC) component Prp19p suggests that splicing catalysis is also cotranscriptional. The separate dynamics of the U1, U2, and U5 snRNPs are consistent with stepwise recruitment of individual snRNPs rather than a preformed "penta-snRNP", as recently proposed. Finally, we show that the cap binding complex (CBC) is necessary, but not sufficient, for cotranscriptional spliceosome assembly. Thus, the demonstration of an essential link between CBC and spliceosome assembly in vivo indicates that 5' end capping couples pre-mRNA splicing to transcription.

Andrew C. Oates, Laurel A. Rohde, Robert K Ho
Generation of segment polarity in the paraxial mesoderm of the zebrafish through a T-box-dependent inductive event.
Dev Biol, 283(1) 204-214 (2005)
PDF DOI

The first morphological sign of vertebrate postcranial body segmentation is the sequential production from posterior paraxial mesoderm of blocks of cells termed somites. Each of these embryonic structures is polarized along the anterior/posterior axis, a subdivision first distinguished by marker gene expression restricted to rostral or caudal territories of forming somites. To better understand the generation of segment polarity in vertebrates, we have studied the zebrafish mutant fused somites (fss), because its paraxial mesoderm lacks segment polarity. Previously examined markers of caudal half-segment identity are widely expressed, whereas markers of rostral identity are either missing or dramatically down-regulated, suggesting that the paraxial mesoderm of the fss mutant embryo is profoundly caudalized. These findings gave rise to a model for the formation of segment polarity in the zebrafish in which caudal is the default identity for paraxial mesoderm, upon which is patterned rostral identity in an fss-dependent manner. In contrast to this scheme, the caudal marker gene ephrinA1 was recently shown to be down-regulated in fss embryos. We now show that notch5, another caudal identity marker and a component of the Delta/Notch signaling system, is not expressed in the paraxial mesoderm of early segmentation stage fss embryos. We use cell transplantation to create genetic mosaics between fss and wild-type embryos in order to assay the requirement for fss function in notch5 expression. In contrast to the expression of rostral markers, which have a cell-autonomous requirement for fss, expression of notch5 is induced in fss cells at short range by nearby wild-type cells, indicating a cell-non-autonomous requirement for fss function in this process. These new data suggest that segment polarity is created in a three-step process in which cells that have assumed a rostral identity must subsequently communicate with their partially caudalized neighbors in order to induce the fully caudalized state.

Agathi Papanikolaou, Alexandra Papafotika, Carol Murphy, Thomais Papamarcaki, Orestes Tsolas, Marek Drab, Teymuras V. Kurzchalia, Michael Kasper, Savvas Christoforidis
Cholesterol-dependent lipid assemblies regulate the activity of the ecto-nucleotidase CD39.
J Biol Chem, 280(28) 26406-26414 (2005)
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CD39 (ecto-nucleoside triphosphate diphosphohydrolase-1; E-NTPDase1) is a plasma membrane ecto-enzyme that regulates purinergic receptor signaling by controlling the levels of extracellular nucleotides. In blood vessels this enzyme exhibits a thromboregulatory role through the control of platelet aggregation. CD39 is localized in caveolae, which are plasma membrane invaginations with distinct lipid composition, similar to dynamic lipid microdomains, called rafts. Cholesterol is enriched together with sphingolipids in both rafts and caveolae, as well as in other specialized domains of the membrane, and plays a key role in their function. Here, we examine the potential role of cholesterol-enriched domains in CD39 function. Using polarized Madin-Darby canine kidney (MDCK) cells and caveolin-1 gene-disrupted mice, we show that caveolae are not essential either for the enzymatic activity of CD39 or for its targeting to plasma membrane. On the other hand, flotation experiments using detergent-free or detergent-based approaches indicate that CD39 associates, at least in part, with distinct lipid assemblies. In the apical membrane of MDCK cells, which lacks caveolae, CD39 is localized in microvilli, which are also cholesterol and raft-dependent membrane domains. Interfering with cholesterol levels using drugs that either deplete or sequester membrane cholesterol results in a strong inhibition of the enzymatic and anti-platelet activity of CD39. The effects of cholesterol depletion are completely reversed by replenishment of membranes with pure cholesterol, but not by cholestenone. These data suggest a functional link between the localization of CD39 in cholesterol-rich domains of the membrane and its role in thromboregulation.

Dong Ji Zhang, Qi Wang, Jie Wei, Gyulnar Baimukanova, Frank Buchholz, A. Francis Stewart, Xiaohong Mao, Nigel Killeen
Selective expression of the Cre recombinase in late-stage thymocytes using the distal promoter of the Lck gene.
J Immunol, 174(11) 6725-6731 (2005)
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Transgenic mouse lines were generated that express the Cre recombinase under the control of the distal promoter of the mouse Lck gene. Cre recombination in four of these lines of transgenic mice was characterized at the single cell level using ROSA26-regulated loxP-Stop-loxP-betageo and loxP-Stop-loxP-YFP reporter mouse lines. Two of the lines showed T cell-restricted Cre recombination, whereas the other two also expressed Cre in B cells, NK cells, and monocytes. Cre recombination began at a late stage of T cell development (at or after up-regulation of the TCR during positive selection) in the two T cell-restricted lines. Lines of mice that express the Cre recombinase at late stages of thymocyte development are of value for determining the impact of mutations on T cell function in the absence of complicating effects on early thymocyte selection.

Roded Sharan, Eugene W Myers
A motif-based framework for recognizing sequence families.
Bioinformatics, 21 Suppl 1 387-393 (2005)
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Many signals in biological sequences are based on the presence or absence of base signals and their spatial combinations. One of the best known examples of this is the signal identifying a core promoter--the site at which the basal transcription machinery starts the transcription of a gene. Our goal is a fully automatic pattern recognition system for a family of sequences, which simultaneously discovers the base signals, their spatial relationships and a classifier based upon them.

Roman H Szczepanowski^*, Renata Filipek^*, Matthias Bochtler
Crystal structure of a fragment of mouse ubiquitin-activating enzyme.
J Biol Chem, 280(23) 22006-22011 (2005)
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Protein ubiquitination requires the sequential activity of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-ligase (E3). The ubiquitin-transfer machinery is hierarchically organized; for every ubiquitin-activating enzyme, there are several ubiquitin-conjugating enzymes, and most ubiquitin-conjugating enzymes can in turn interact with multiple ubiquitin ligases. Despite the central role of ubiquitin-activating enzyme in this cascade, a crystal structure of a ubiquitin-activating enzyme is not available. The enzyme is thought to consist of an adenylation domain, a catalytic cysteine domain, a four-helix bundle, and possibly, a ubiquitin-like domain. Its adenylation domain can be modeled because it is clearly homologous to the structurally known adenylation domains of the activating enzymes for the small ubiquitin-like modifier (SUMO) and for the protein encoded by the neuronal precursor cell-expressed, developmentally down-regulated gene 8 (NEDD8). Low sequence similarity and vastly different domain lengths make modeling difficult for the catalytic cysteine domain that results from the juxtaposition of two catalytic cysteine half-domains. Here, we present a biochemical and crystallographic characterization of the two half-domains and the crystal structure of the larger, second catalytic cysteine half-domain of mouse ubiquitin-activating enzyme. We show that the domain is organized around a conserved folding motif that is also present in the NEDD8- and SUMO-activating enzymes, and we propose a tentative model for full-length ubiquitin-activating enzyme.

Sylke Winkler, Anja Schwabedissen, Dana Backasch, Christian Bökel, Claudia Seidel, Stefanie Bönisch, Maximilian Fürthauer, Antje Kuhrs, Laura Cobreros, Michael Brand, Marcos González-Gaitán
Target-selected mutant screen by TILLING in Drosophila.
Genome Res, 15(5) 718-723 (2005)
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The availability of the full Drosophila genomic DNA sequence prompts the development of a method to efficiently obtain mutations in genes of interest identified by their sequence homologies or biochemically. To date, molecularly characterized mutations have been generated in around 6000 of the approximately 15,000 annotated fly genes, of which around one-third are essential for viability. To obtain mutations in essential and nonessential genes of interest, we took a reverse genetics approach, based on the large-scale detection of point mutations by Cel-I-mediated heteroduplex cleavage. A library of genomic DNA from 2086 EMS-mutagenized lines was established. The library was screened for mutations in three genes. A total of 6.1 Mb were screened, and 44 hits were found in two different mutagenesis conditions. Optimal conditions yielded an average of one mutation every 156 kb. For an essential gene tested, five of 25 mutations turned out to cause lethality, confirming that EMS mutagenesis leads to high frequency of gene inactivation. We thereby established that Cel-I-mediated TILLING can be used to efficiently obtain mutations in genes of interest in Drosophila.

Gert Dandanell, Roman H Szczepanowski, Borys Kierdaszuk, David Shugar, Matthias Bochtler
Escherichia coli purine nucleoside phosphorylase II, the product of the xapA gene.
J Mol Biol, 348(1) 113-125 (2005)
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Purine nucleoside phosphorylases (PNPs, E. C. 2.4.2.1) use orthophosphate to cleave the N-glycosidic bond of beta-(deoxy)ribonucleosides to yield alpha-(deoxy)ribose 1-phosphate and the free purine base. Escherichia coli PNP-II, the product of the xapA gene, is similar to trimeric PNPs in sequence, but has been reported to migrate as a hexamer and to accept xanthosine with comparable efficiency to guanosine and inosine, the usual physiological substrates for trimeric PNPs. Here, we present a detailed biochemical characterization and the crystal structure of E.coli PNP-II. In three different crystal forms, PNP-II trimers dimerize, leading to a subunit arrangement that is qualitatively different from the "trimer of dimers" arrangement of conventional high molecular mass PNPs. Crystal structures are compatible with similar binding modes for guanine and xanthine, with a preference for the neutral over the monoanionic form of xanthine. A single amino acid exchange, tyrosine 191 to leucine, is sufficient to convert E.coli PNP-II into an enzyme with the specificity of conventional trimeric PNPs, but the reciprocal mutation in human PNP, valine 195 to tyrosine, does not elicit xanthosine phosphorylase activity in the human enzyme.

Kieran Brickley, Miriam J Smith, Mike Beck, F Anne Stephenson
GRIF-1 and OIP106, members of a novel gene family of coiled-coil domain proteins: association in vivo and in vitro with kinesin.
J Biol Chem, 280(15) 14723-14732 (2005)
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Gamma-aminobutyric acid(A) receptor-interacting factor (GRIF-1) is a 913-amino acid protein proposed to function as a GABA(A) receptor beta(2) subunit-interacting, trafficking protein. GRIF-1 shares approximately 44% amino acid sequence identity with O-linked N-acetylglucosamine transferase interacting protein 106, OIP106. Both proteins contain predicted coiled-coil domains and probably constitute a novel gene family. The Drosophila orthologue of this family of proteins may be Milton. Milton shares approximately 44% amino acid homology with GRIF-1. Milton is proposed to function in kinesin-mediated transport of mitochondria to nerve terminals. We report here that GRIF-1 and OIP106 also associate with kinesin and mitochondria. Following expression in human embryonic kidney 293 cells, both GRIF-1 and OIP106 were shown by co-immunoprecipitation to be specifically associated with an endogenous kinesin heavy chain species of 115 kDa and exogenous KIF5C. Association of GRIF-1 with kinesin was also evident in native brain and heart tissue. In the brain, anti-GRIF-1-(8-633) antibodies specifically co-immunoprecipitated two kinesin-immunoreactive species with molecular masses of 118 and 115 kDa, and in the heart, one kinesin-immunoreactive species, 115 kDa, was immunoprecipitated. Further studies revealed that GRIF-1 was predominantly associated with KIF5A in the brain and with KIF5B in both the heart and in HEK 293 cells. Yeast two-hybrid interaction assays and immunoprecipitations showed that GRIF-1 associated directly with KIF5C with the GRIF-1/KIF5C interaction domain localized to GRIF-1-(124-283). These results further support a role for GRIF-1 and OIP106 in protein and/or organelle transport in excitable cells in a manner analogous to glutamate receptor-interacting-protein 1, in the motor-dependent transport of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate glutamate excitatory neurotransmitter receptors to dendrites.

Andrew C. Oates, Claudia Müller, Robert K Ho
Cooperative function of deltaC and her7 in anterior segment formation.
Dev Biol, 280(1) 133-149 (2005)
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Segmentation of paraxial mesoderm in vertebrates is regulated by a genetic oscillator that manifests as a series of wavelike or cyclic gene expression domains in the embryo. In zebrafish, this oscillator involves members of the Delta/Notch intercellular signaling pathway, and its down-stream targets, the Her family of transcriptional repressors. Loss of function of any one of the genes of this system, such as her7, gives rise to segmentation defects in the posterior trunk and tail, concomitant with a disruption of cyclic expression domains, indicating that the oscillator is required for posterior segmentation. Control of segmentation in the anterior trunk, and its relationship to that of the posterior is, however, not yet well understood. A combined loss of the cyclic Her genes her1 and her7 disrupts segmentation of both anterior and posterior paraxial mesoderm, indicating that her genes function redundantly in anterior segmentation. To test whether this anterior redundancy is specific to the her gene family, or alternatively is a more global feature of the segmentation oscillator, we looked at anterior segmentation after morpholino knock down of the cyclic cell-surface Notch ligand deltaC (dlc), either alone or in combination with her7, or other Delta/Notch pathway genes. We find that dlc is required for coherence of wavelike expression domains of cyclic genes her1 and her7 and maintenance of their expression levels, as well as for cyclic transcription of dlc itself, confirming that dlc is a component of the segmentation oscillator. Dose dependent, posteriorly-restricted segmentation defects were seen in the dlc knock down, and in combination with the deltaD or notch1a mutants. However, combined reduction of function of dlc and her7 results in defective segmentation of both anterior and posterior paraxial mesoderm, and a failure of cyclic expression domains to initiate, similar to loss of both her genes. Thus, anterior segmentation requires the functions of both her and delta family members in a parallel manner, suggesting that the segmentation oscillator operates in paraxial mesoderm along the entire vertebrate axis.

Karin Schlichting, Fabio Demontis, Christian Dahmann
Cadherin Cad99C is regulated by Hedgehog signaling in Drosophila.
Dev Biol, 279(1) 142-154 (2005)
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The subdivision of the Drosophila wing imaginal disc into anterior and posterior compartments requires a transcriptional response to Hedgehog signaling. However, the genes regulated by Hedgehog signal transduction that mediate the segregation of anterior and posterior cells have not been identified. Here, we molecularly characterize the previously predicted gene cad99C and show that it is regulated by Hedgehog signaling. Cad99C encodes a transmembrane protein with a molecular weight of approximately 184 kDa that contains 11 cadherin repeats in its extracellular domain and a conserved type I PDZ-binding site at its C-terminus. The levels of cad99C RNA and protein are low throughout the wing imaginal disc. However, in the pouch region, these levels are elevated in a strip of anterior cells along the A/P boundary where the Hedgehog signal is transduced. Ectopic expression of Hedgehog, or the Hedgehog-regulated transcription factor Cubitus interruptus, induces high-level expression of Cad99C. Conversely, blocking Hedgehog signal transduction by either inactivating Smoothened or Cubitus interruptus reduces high-level Cad99C expression. Finally, by analyzing mutant clones of cells, we show that Cad99C is not essential for cell segregation at the A/P boundary. We conclude that cad99C is a novel Hedgehog-regulated gene encoding a member of the cadherin superfamily in Drosophila.

Birte Sönnichsen, L B Koski, A Walsh, P Marschall, B Neumann, Michael Brehm, Anne-Marie Alleaume, J. Artelt, P Bettencourt, E Cassin, M Hewitson, Christian Holz, M Khan, S Lazik, Claudia Martin, Bert Nitzsche, Martine Ruer, Joanne Stamford, Maria Winzi, R Heinkel, M Röder, J Finell, H Häntsch, S J M Jones, M Jones, Fabio Piano, Kristin C. Gunsalus, Karen Oegema, Pierre Gönczy, A Coulson, Anthony A. Hyman, C J Echeverri
Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans.
Nature, 434(7032) 462-469 (2005)
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A key challenge of functional genomics today is to generate well-annotated data sets that can be interpreted across different platforms and technologies. Large-scale functional genomics data often fail to connect to standard experimental approaches of gene characterization in individual laboratories. Furthermore, a lack of universal annotation standards for phenotypic data sets makes it difficult to compare different screening approaches. Here we address this problem in a screen designed to identify all genes required for the first two rounds of cell division in the Caenorhabditis elegans embryo. We used RNA-mediated interference to target 98% of all genes predicted in the C. elegans genome in combination with differential interference contrast time-lapse microscopy. Through systematic annotation of the resulting movies, we developed a phenotypic profiling system, which shows high correlation with cellular processes and biochemical pathways, thus enabling us to predict new functions for previously uncharacterized genes.

Ralf Kittler, Laurence Pelletier, Chunling Ma, Ina Poser, Steffi Fischer, Anthony A. Hyman, Frank Buchholz
RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells.
Proc Natl Acad Sci U.S.A., 102(7) 2396-2401 (2005)
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RNA interference (RNAi) is a widely used method for analysis of gene function in tissue culture cells. However, to date there has been no reliable method for testing the specificity of any particular RNAi experiment. The ideal experiment is to rescue the phenotype by expression of the target gene in a form refractory to RNAi. The transgene should be expressed at physiological levels and with its different splice variants. Here, we demonstrate that expression of murine bacterial artificial chromosomes in human cells provides a reliable method to create RNAi-resistant transgenes. This strategy should be applicable to all eukaryotes and should therefore be a standard technology for confirming the specificity of RNAi. We show that this technique can be extended to allow the creation of tagged transgenes, expressed at physiological levels, for the further study of gene function.

Arndt Friedrich Siekmann, Michael Brand
Distinct tissue-specificity of three zebrafish ext1 genes encoding proteoglycan modifying enzymes and their relationship to somitic Sonic hedgehog signaling.
Dev Dyn, 232(2) 498-505 (2005)
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Proteins of the EXT (Exostosin) 1 family are known for their role in human disease. Mutations in EXT1 cause hereditary multiple exostoses (HME), benign outgrowths of the bones, and therefore were classed as tumor suppressors. More recently, their role during embryonic development of Drosophila and mouse was addressed, revealing important functions of EXT1 genes in major signaling pathways. Here, we report the isolation of three zebrafish members of the EXT1 family, which we named ext1a, ext1b, and ext1c, respectively. They are expressed in restricted temporal and spatial domains during development. Both ext1a and ext1b are provided maternally and expressed during gastrulation: ext1a in the neurectoderm and ext1b in the embryonic midline and in the involuting mesendoderm of the germ ring. During somitogenesis stages, transcripts of all three ext genes can be found in the somitic mesoderm. Furthermore, ext1a is expressed in the dorsal neural tube. These expression domains become more pronounced at 24 hr postfertilization (hpf). At 48 hpf, ext1 genes are present in the brain, while somitic expression ceases. Zebrafish have three members of the EXT1 family, in contrast to only one EXT1 gene in mammals or Xenopus, consistent with the occurrence of partial genome duplications in the teleost lineage. Our expression analysis reveals that the three ext genes have distinct expression patterns, reflecting functional divergence after duplication. In addition, expression of ext1a and ext1c responds to elevated and reduced levels of Sonic hedgehog (shh) signaling in the somites, whereas expression of ext1b does not. This suggests a differential relationship between the shh pathway and individual ext gene function in zebrafish.

Mirko Trajkovski
Regulated secretion and gene expression in ß-cells [beta-cells]: link through : ICA512
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2005)

Eberhard Krausz^#, Anne-Kristin Heninger, Ralf Kittler, Annett Lohmann, Ina Poser, Jan Wagner, Kristin Franke, Karol Kozak, Hannes Grabner, Frank Buchholz^#
Eine Genom-weite RNA-Interferenz-Bibliothek für die funktionelle Charakterisierung aller Gene in kultivierten, menschlichen Zellen
BIOspektrum, 11(4) 436-441 (2005)
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Frank Buchholz, David N. Drechsel, Martine Ruer, Ralf Kittler
Production of siRNA in vitro by enzymatic digestion of double-stranded RNA
In: Gene Silencing by RNA Interference : Technology and Application. (Eds.) Muhammad Sohail,Boca Raton, USA,CRC Press (2005),87-99 Ch. 6
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RNA interference (RNAi) is a powerful tool for gene function studies in a variety of

Frank Buchholz
Functional Genomics in Mammalian Cells by RNAi: Opportunities and Challenges
Laborwelt, 6(2) 18-21 (2005)
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Recent technological advances in biological sciences have extended the repertoire of experimentation towards systematic analysis of gene functions. The sequencing of whole genomes has provided scientists with the blueprint of life, and whole genome gene expression array analyses provide a general view of the expression states of cells or tissues. A relatively recent technological addition for systematic analyses has been the discoveryof RNAi and its scaling to whole genome analyses. Here, I discuss recent advances that have been made in RNAi screening in mammalian cells and present new possibilities and challeges that this technology offers.

Esther Schnapp, Elly M. Tanaka
Quantitative evaluation of morpholino-mediated protein knockdown of GFP, MSX1, and PAX7 during tail regeneration in Ambystoma mexicanum.
Dev Dyn, 232(1) 162-170 (2005)
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Vertebrate regeneration is a fascinating but poorly understood biological phenomena. Urodele amphibians such as Ambystoma mexicanum (the axolotl) can functionally regenerate complex body structures such as the limb and tail, including the spinal cord, throughout life. So far, molecular studies on regeneration have been limited due to the paucity of tools for knocking-down gene and protein function. In this article, we quantitatively assessed the ability of morpholinos to specifically down-regulate protein expression in both cultured urodele cells and in vivo. We focused on the down-regulation of green fluorescent protein and two axolotl proteins, MSX1 and PAX7. Our data show that the expression of these proteins can be efficiently reduced by morpholinos. MSX1 has been hypothesized to be involved in muscle dedifferentiation based on experiments using cultured myotubes. Our studies in in vivo muscle fibers so far have shown no influence of overexpressing or down-regulating MSX1 on the dedifferentiation process.

Christoph Sachse, Eberhard Krausz, Andrea Krönke, Michael Hannus, Andrew Walsh, Anne Grabner, Dmitriy Ovcharenko, David Dorris, Claude Trudel, Birte Sönnichsen, Christophe J. Echeverri
High throughput RNA interference strategies for target discovery and validation using syntheic short interfering RNAs: functional genomics investigations of biological pathways
In: RNA interference. (Eds.) David R. Engelke, John J. Rossi Methods in enzymology ; 392.,Amsterdam, Netherlands,Elsevier (2005),242-277 Ch. 15
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During the past five years, RNA interference (RNAi) has emerged as arguably the best functional genomics tool available to date, providing direct, causal links between individual genes and loss-of-function phenotypes through robust, broadly applicable, and readily upscalable methodologies. Originally applied experimentally in C. elegans and Drosophila, RNAi is now widely used in mammalian cell systems also. The development of commercially available libraries of short interfering RNAs (siRNAs) and other RNAi silencing reagents targeting entire classes of human genes provide the opportunity to carry out genome-scale screens to discover and characterize gene functions directly in human cells. A key challenge of these studies, also faced by earlier genomics or proteomics approaches, resides in reaching an optimal balance between the necessarily high throughput and the desire to achieve the same level of detailed analysis that is routine in conventional small-scale studies. This chapter discusses technical aspects of how to perform such screens, what parameters to monitor, and which readouts to apply. Examples of homogenous assays and multiplexed high-content microscopy-based screens are demonstrated.

Karla M. Neugebauer, Kimberly M. Kotovic, Jennifer A. Geiger, David Stanĕk
Pre-mRNA Processing in the Nuclear Landscape
In: Visions of the Cell Nucleus. (Eds.) Stephan Diekmann, Peter Hemmerich,Stevenson Ranch, USA,American Scientific Publishers (2005),106-119 Ch. 7
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All eukaryotic protein-coding genes are transcribed by RNA polymerase II (pol II), and each mRNA is the product not only of transcription but a variety of pre-mRNA processing events. In humans, every pre-mRNA acquires a methyl-guanosine cap at its 5` end, and nearly every transcript is internally spliced and polyadenylated at its 3` end. The purpose of this chapter is to place these three major pre-mRNA processing steps within the context of the three dimensional space of the cell nucleus. Because capping, splicing and polyadenylation at least begin during RNA synthesis, these reactions occur largely at sites of gene transcription, which are distributed throughout the nucleus and not localized to particular domains or substructures. Splicing and polyadenylation often continue post-transcriptionally, most likely in the interchromatin space. In addition, pre-mRNA processing factors are components of a number of subnuclear structures, such as Cajal Bodies and Cleavage Bodies, suggesting that some functions related to pre-mRNA processing are compartmentalized within the nuclear landscape.

Ralf Kittler, Gabriele Putz, Laurence Pelletier, Ina Poser, Anne-Kristin Heninger, David N. Drechsel, Steffi Fischer, Irena Konstantinova, Bianca Habermann, Hannes Grabner, Marie-Laure Yaspo, Heinz Himmelbauer, Bernd Korn, Karla M. Neugebauer, Maria Teresa Pisabarro, Frank Buchholz
An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division.
Nature, 432(7020) 1036-1040 (2004)
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RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.

Mirko Trajkovski, Hassan Mziaut, Anke Altkrüger, Joke Ouwendijk, Klaus-Peter Knoch, Stefan Müller, Michele Solimena
Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in {beta}-cells.
J Cell Biol, 167(6) 1063-1074 (2004)
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Islet cell autoantigen 512 (ICA512)/IA-2 is a receptor tyrosine phosphatase-like protein associated with the insulin secretory granules (SGs) of pancreatic beta-cells. Here, we show that exocytosis of SGs and insertion of ICA512 in the plasma membrane promotes the Ca(2+)-dependent cleavage of ICA512 cytoplasmic domain by mu-calpain. This cleavage occurs at the plasma membrane and generates an ICA512 cytosolic fragment that is targeted to the nucleus, where it binds the E3-SUMO ligase protein inhibitor of activated signal transducer and activator of transcription-y (PIASy) and up-regulates insulin expression. Accordingly, this novel pathway directly links regulated exocytosis of SGs and control of gene expression in beta-cells, whose impaired insulin production and secretion causes diabetes.

Benjamin M Hogan, Michael P Hunter, Andrew C. Oates, Meredith O Crowhurst, Nathan E Hall, Joan K Heath, Victoria E Prince, Graham J Lieschke
Zebrafish gcm2 is required for gill filament budding from pharyngeal ectoderm.
Dev Biol, 276(2) 508-522 (2004)
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The pharyngeal arches give rise to multiple organs critical for diverse processes, including the thymus, thyroid and parathyroids. Several molecular regulators of thymus and thyroid organogenesis are strikingly conserved between mammals and zebrafish. However, land animals have parathyroids whereas fish have gills. The murine transcription factor Glial cells missing 2 (Gcm2) is expressed specifically in the parathyroid primordium in the endodermal epithelium of the third pharyngeal pouch, and in both mice and humans is required for normal development of parathyroid glands. The molecular regulation of fish gill organogenesis remains to be described. We report the expression of gcm2 in the zebrafish pharyngeal epithelium and a requirement for Hox group 3 paralogs for gcm2 expression. Strikingly, zebrafish gcm2 is expressed in the ectodermal portion of the pharyngeal epithelium and is required for the development of the gill filament buds, precursors of fish-specific gill filaments. This study identifies yet another role for a GCM gene in embryonic development and indicates a role for gcm2 during the evolution of divergent pharyngeal morphologies.

Henry Haeberle^*, Mika Fujiwara^*, Jody Chuang, Michael M Medina, Mayuri V Panditrao, Susanne Bechstedt, Jonathon Howard, Ellen A Lumpkin
Molecular profiling reveals synaptic release machinery in Merkel cells.
Proc Natl Acad Sci U.S.A., 101(40) 14503-14508 (2004)
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Merkel cell-neurite complexes are somatosensory receptors that initiate the perception of gentle touch. The role of epidermal Merkel cells within these complexes is disputed. To ask whether Merkel cells are genetically programmed to be excitable cells that may participate in touch reception, we purified Merkel cells from touch domes and used DNA microarrays to compare gene expression in Merkel cells and other epidermal cells. We identified 362 Merkel-cell-enriched transcripts, including neuronal transcription factors, presynaptic molecules, and ion-channel subunits. Antibody staining of skin sections showed that Merkel cells are immunoreactive for presynaptic proteins, including piccolo, Rab3C, vesicular glutamate transporter 2, and cholecystokinin 26-33. These data indicate that Merkel cells are poised to release glutamate and neuropeptides. Finally, by using Ca(2+) imaging, we discovered that Merkel cells have L- and P/Q-type voltage-gated Ca(2+) channels, which have been shown to trigger vesicle release at synapses. These results demonstrate that Merkel cells are excitable cells and suggest that they release neurotransmitters to shape touch sensitivity.

Steffen Scholpp, Michael Brand
Endocytosis controls spreading and effective signaling range of Fgf8 protein.
Curr Biol, 14(20) 1834-1841 (2004)
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Secreted signaling molecules released from a restricted source are of great importance during embryonic development because they elicit induction, proliferation, differentiation, and patterning events in target cells . Fgf8 is a member of the fibroblast growth factor family with key inductive functions during vertebrate development of, for example, the forebrain , midbrain , cerebellum , heart , inner ear , and mesoderm . However, the mechanism by which the signaling range of Fgf8 is controlled in a field of target cells is unknown. We studied Fgf8 as a potential morphogen in the nascent neuroectoderm of living zebrafish embryos. We find that spreading of epitope-tagged Fgf8 through target tissue is carefully controlled by endocytosis and subsequent degradation in lysosomes, or "restrictive clearance," from extracellular spaces. If internalization is inhibited, Fgf8 protein accumulates extracellularly, spreads further, and activates target gene expression over a greater distance. Conversely, enhanced internalization increases Fgf8 uptake and shortens its effective signaling range. Our results suggest that Fgf8 spreads extracellularly by a diffusion-based mechanism and demonstrate that target cells can actively influence, through endocytosis and subsequent degradation, the availability of Fgf8 ligand to other target cells.

Gijs van Haaften, Nadine Vastenhouw, Ellen A A Nollen, Ronald H A Plasterk, Marcel Tijsterman
Gene interactions in the DNA damage-response pathway identified by genome-wide RNA-interference analysis of synthetic lethality.
Proc Natl Acad Sci U.S.A., 101(35) 12992-12996 (2004)
DOI

Here, we describe a systematic search for synthetic gene interactions in a multicellular organism, the nematode Caenorhabditis elegans. We established a high-throughput method to determine synthetic gene interactions by genome-wide RNA interference and identified genes that are required to protect the germ line against DNA double-strand breaks. Besides known DNA-repair proteins such as the C. elegans orthologs of TopBP1, RPA2, and RAD51, eight genes previously unassociated with a double-strand-break response were identified. Knockdown of these genes increased sensitivity to ionizing radiation and camptothecin and resulted in increased chromosomal nondisjunction. All genes have human orthologs that may play a role in human carcinogenesis.

Laurel A. Rohde, Andrew C. Oates, Robert K Ho
A crucial interaction between embryonic red blood cell progenitors and paraxial mesoderm revealed in spadetail embryos.
Dev Cell, 7(2) 251-262 (2004)
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Zebrafish embryonic red blood cells (RBCs) develop in trunk intermediate mesoderm (IM), and early macrophages develop in the head, suggesting that local microenvironmental cues regulate differentiation of these two blood lineages. spadetail (spt) mutant embryos, which lack trunk paraxial mesoderm (PM) due to a cell-autonomous defect in tbx16, fail to produce embryonic RBCs but retain head macrophage development. In spt mutants, initial hematopoietic gene expression is absent in trunk IM, although endothelial and pronephric expression is retained, suggesting that early blood progenitor development is specifically disrupted. Using cell transplantation, we reveal that spt is required cell autonomously for early hematopoietic gene expression in trunk IM. Further, we uncover an interaction between embryonic trunk PM and blood progenitors that is essential for RBC development. Importantly, our data identify a hematopoietic microenvironment that allows embryonic RBC production in the zebrafish.

Andreas Henschel, Frank Buchholz, Bianca Habermann
DEQOR: a web-based tool for the design and quality control of siRNAs.
Nucleic Acids Res, 32(Web Server issue) 113-120 (2004)
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RNA interference (RNAi) is a powerful tool for inhibiting the expression of a gene by mediating the degradation of the corresponding mRNA. The basis of this gene-specific inhibition is small, double-stranded RNAs (dsRNAs), also referred to as small interfering RNAs (siRNAs), that correspond in sequence to a part of the exon sequence of a silenced gene. The selection of siRNAs for a target gene is a crucial step in siRNA-mediated gene silencing. According to present knowledge, siRNAs must fulfill certain properties including sequence length, GC-content and nucleotide composition. Furthermore, the cross-silencing capability of dsRNAs for other genes must be evaluated. When designing siRNAs for chemical synthesis, most of these criteria are achievable by simple sequence analysis of target mRNAs, and the specificity can be evaluated by a single BLAST search against the transcriptome of the studied organism. A different method for raising siRNAs has, however, emerged which uses enzymatic digestion to hydrolyze long pieces of dsRNA into shorter molecules. These endoribonuclease-prepared siRNAs (esiRNAs or 'diced' RNAs) are less variable in their silencing capabilities and circumvent the laborious process of sequence selection for RNAi due to a broader range of products. Though powerful, this method might be more susceptible to cross-silencing genes other than the target itself. We have developed a web-based tool that facilitates the design and quality control of siRNAs for RNAi. The program, DEQOR, uses a scoring system based on state-of-the-art parameters for siRNA design to evaluate the inhibitory potency of siRNAs. DEQOR, therefore, can help to predict (i) regions in a gene that show high silencing capacity based on the base pair composition and (ii) siRNAs with high silencing potential for chemical synthesis. In addition, each siRNA arising from the input query is evaluated for possible cross-silencing activities by performing BLAST searches against the transcriptome or genome of a selected organism. DEQOR can therefore predict the probability that an mRNA fragment will cross-react with other genes in the cell and helps researchers to design experiments to test the specificity of esiRNAs or chemically designed siRNAs. DEQOR is freely available at http://cluster-1.mpi-cbg.de/Deqor/deqor.html.

Jeanette L Ducut Sigala, Virginie Bottero, David B Young, Andrej Shevchenko, Frank Mercurio, Inder M Verma
Activation of transcription factor NF-kappaB requires ELKS, an IkappaB kinase regulatory subunit.
Science, 304(5679) 1963-1967 (2004)
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The nuclear factor-kappa B (NF-kappaB) family of transcription factors plays a seminal role in inflammation, apoptosis, development, and cancer. Modulation of NF-kappaB-mediated gene expression in response to diverse signals is coordinated by the IkappaB kinase (IKK) complex. We identified ELKS, an essential regulatory subunit of the IKK complex. Silencing ELKS expression by RNA interference blocked induced expression of NF-kappaB target genes, including the NF-kappaB inhibitor IkappaBalpha and proinflammatory genes such as cyclo-oxygenase 2 and interleukin 8. These cells were also not protected from apoptosis in response to cytokines. ELKS likely functions by recruiting IkappaBalpha to the IKK complex and thus serves a regulatory function for IKK activation.

Sebastian Schuck^*, Aki Manninen^*, Masanori Honsho, Joachim Füllekrug, Kai Simons
Generation of single and double knockdowns in polarized epithelial cells by retrovirus-mediated RNA interference.
Proc Natl Acad Sci U.S.A., 101(14) 4912-4917 (2004)
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RNA interference (RNAi) is a ubiquitous mechanism of eukaryotic gene regulation that can be exploited for specific gene silencing. Retroviruses have been successfully used for stable expression of short hairpin RNAs in mammalian cells, leading to persistent inhibition of gene expression by RNAi. Here, we apply retrovirus-mediated RNAi to epithelial Madin-Darby canine kidney cells, whose properties limit the applicability of other RNAi methods. We demonstrate efficient suppression of a set of 13 target genes by retroviral coexpression of short hairpin RNAs and a selectable marker. We characterize the resulting knockdown cell populations with regard to composition and stability, and examine the usefulness of proposed guidelines for choosing RNAi target sequences. Finally, we show that this system can be used to simultaneously target two genes, giving rise to double knockdowns. Thus, retrovirus-mediated RNAi is a convenient method for gene silencing in Madin-Darby canine kidney cells, and is likely to be applicable to virtually any mammalian cell.

Jan Krönke, Ralf Kittler, Frank Buchholz, Marc P Windisch, Thomas Pietschmann, Ralf Bartenschlager, Michael Frese
Alternative approaches for efficient inhibition of hepatitis C virus RNA replication by small interfering RNAs.
J Virol, 78(7) 3436-3446 (2004)
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Persistent infection with hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. It has recently been shown that HCV RNA replication is susceptible to small interfering RNAs (siRNAs), but the antiviral activity of siRNAs depends very much on their complementarity to the target sequence. Thus, the high degree of sequence diversity between different HCV genotypes and the rapid evolution of new quasispecies is a major problem in the development of siRNA-based gene therapies. For this study, we developed two alternative strategies to overcome these obstacles. In one approach, we used endoribonuclease-prepared siRNAs (esiRNAs) to simultaneously target multiple sites of the viral genome. We show that esiRNAs directed against various regions of the HCV coding sequence as well as the 5' nontranslated region (5' NTR) efficiently block the replication of subgenomic and genomic HCV replicons. In an alternative approach, we generated pseudotyped retroviruses encoding short hairpin RNAs (shRNAs). A total of 12 shRNAs, most of them targeting highly conserved sequence motifs within the 5' NTR or the early core coding region, were analyzed for their antiviral activities. After the transduction of Huh-7 cells containing a subgenomic HCV replicon, we found that all shRNAs targeting sequences in domain IV or nearby coding sequences blocked viral replication. In contrast, only one of seven shRNAs targeting sequences in domain II or III had a similar degree of antiviral activity, indicating that large sections of the NTRs are resistant to RNA interference. Moreover, we show that naive Huh-7 cells that stably expressed certain 5' NTR-specific shRNAs were largely resistant to a challenge with HCV replicons. These results demonstrate that the retroviral transduction of HCV-specific shRNAs provides a new possibility for antiviral intervention.

Federico Calegari, Anne-Marie Marzesco, Ralf Kittler, Frank Buchholz, Wieland B. Huttner
Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture.
Differentiation, 72(2-3) 92-102 (2004)
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In mammals, embryonic development is more difficult to analyze than in non-mammalian species because this development occurs in utero. Interestingly, whole embryo culture allows the normal development of mouse post-implantation embryos for up to 2 days in vitro. One limitation of this technology has been the difficulty of performing loss-of-gene function studies in this system. RNA interference (RNAi), whereby double-stranded RNA molecules suppress the expression of complementary genes, has rapidly become a widely used tool for gene function analyses. We have combined the technologies of mouse whole embryo culture and RNAi to allow the molecular dissection of developmental processes. Here, we review the manipulation by topical injection followed by directional electroporation of endoribonuclease-prepared siRNA to demonstrate that this technology may be useful to knock down genes in a tissue- and region-specific manner in several organs of the developing mouse embryo.

Wulf Haubensak, Alessio Attardo, Winfried Denk, Wieland B. Huttner
Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis.
Proc Natl Acad Sci U.S.A., 101(9) 3196-3201 (2004)
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Neurons of the mammalian CNS are thought to originate from progenitors dividing at the apical surface of the neuroepithelium. Here we use mouse embryos expressing GFP from the Tis21 locus, a gene expressed throughout the neural tube in most, if not all, neuron-generating progenitors, to specifically reveal the cell divisions that produce CNS neurons. In addition to the apical, asymmetric divisions of neuroepithelial (NE) cells that generate another NE cell and a neuron, we find, from the onset of neurogenesis, a second population of progenitors that divide in the basal region of the neuroepithelium and generate two neurons. Basal progenitors are most frequent in the telencephalon, where they outnumber the apically dividing neuron-generating NE cells. Our observations reconcile previous data on the origin and lineage of CNS neurons and show that basal, rather than apical, progenitors are the major source of the neurons of the mammalian neocortex.

Muriel Rhinn, Klaus Lun, Michaela Werner, Angela Simeone, Michael Brand
Isolation and expression of the homeobox gene Gbx1 during mouse development.
Dev Dyn, 229(2) 334-339 (2004)
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In zebrafish, gbx1 and otx2 are among the earliest genes expressed in the neuroectoderm, dividing it into an anterior and a posterior domain with a common border that marks the midbrain-hindbrain boundary (MHB) primordium. Here, we describe the sequence and expression pattern of Gbx1 in mouse. The first transcripts are found at embryonic day 7.75 in the hindbrain. Later on, expression of Gbx1 is detectable in the hindbrain (rhombomeres 2 to 7), spinal cord, optic vesicles, and in the ventral telencephalon. In mouse, Gbx1 expression is not observed at the MHB as is the case during early zebrafish development. We suggest that an evolutionary switch occurred: in mouse Gbx2 is involved in the early specification of the MHB primordium, whereas in zebrafish, gbx1 is required instead of gbx2.

Marta Miaczynska, Savvas Christoforidis, Angelika Giner, Anna Shevchenko, Sandrine Uttenweiler-Joseph, Bianca Habermann, Matthias Wilm, Robert G. Parton, Marino Zerial
APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment.
Cell, 116(3) 445-456 (2004)
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Signals generated in response to extracellular stimuli at the plasma membrane are transmitted through cytoplasmic transduction cascades to the nucleus. We report the identification of a pathway directly linking the small GTPase Rab5, a key regulator of endocytosis, to signal transduction and mitogenesis. This pathway operates via APPL1 and APPL2, two Rab5 effectors, which reside on a subpopulation of endosomes. In response to extracellular stimuli such as EGF and oxidative stress, APPL1 translocates from the membranes to the nucleus where it interacts with the nucleosome remodeling and histone deacetylase multiprotein complex NuRD/MeCP1, an established regulator of chromatin structure and gene expression. Both APPL1 and APPL2 are essential for cell proliferation and their function requires Rab5 binding. Our findings identify an endosomal compartment bearing Rab5 and APPL proteins as an intermediate in signaling between the plasma membrane and the nucleus.

Effi Rees, Bernd Korn, Frank Buchholz
Genfunktionsanalyse im Genom-weiten Maßstab mit 'small interfering' RNAs
Laborwelt, 5(1) 21-22 (2004)
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Die RNA-Interferenz (RNAi) ist eine wirkungsvolle Methode zur Untersuchung von Genfunktionen und ermöglicht den „knock-down“ von Genen in einer Vielzahl von Organismen (z.B. Drosophila, Caenorhabditis, Mus musculus) sowie in menschlichen Zellinien. Ein initialer Schritt bei der RNAi ist das Prozessieren der doppelsträngigen RNA (dsRNA) in kurze Stücke, die sogenannten "small interfering" RNAs (siRNA)2. Die siRNA bildet mit einem intrazellulären Proteinkomplex den sogenannten RNA-induced Silencing Complex (RISC); dieser bindet an den antisense-Strang des siRNA 21-23mers und schneidet die mRNA in der Mitte des Hybrides4. Da die gespaltene mRNA sehr instabil ist, wird sie rasch abgebaut und führt zu einem „gene silencing“ auf posttranskriptioneller Ebene.

Bianca Habermann, Anne-Gaelle Bebin, Stephan Herklotz, Michael Volkmer, Kay Eckelt, Kerstin Pehlke, Hans-Henning Epperlein, Hans Konrad Schackert, Glenis J. Wiebe, Elly M. Tanaka
An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries.
Genome Biol, 5(9) 67-67 (2004)
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BACKGROUND: The ambystomatid salamander, Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum. RESULTS: Two cDNA libraries, one made from stage 18-22 embryos and the other from day-6 regenerating tail blastemas, generated 17,352 sequences. From the sequenced ESTs, 6,377 contigs were assembled that probably represent 25% of the expressed genes in this organism. Sequence comparison revealed significant homology to entries in the NCBI non-redundant database. Further examination of this gene set revealed the presence of genes involved in important cell and developmental processes, including cell proliferation, cell differentiation and cell-cell communication. On the basis of these data, we have performed phylogenetic analysis of key cell-cycle regulators. Interestingly, while cell-cycle proteins such as the cyclin B family display expected evolutionary relationships, the cyclin-dependent kinase inhibitor 1 gene family shows an unusual evolutionary behavior among the amphibians. CONCLUSIONS: Our analysis reveals the importance of a comprehensive sequence set from a representative of the Caudata and illustrates that the EST sequence database is a rich source of molecular, developmental and regeneration studies. To aid in data mining, the ESTs have been organized into an easily searchable database that is freely available online.

Michael Hiller, Rolf Backofen, Stephan Heymann, Anke Busch, Timo Mika Glaesser, Johann-Christoph Freytag
Efficient prediction of alternative splice forms using protein domain homology.
In Silico Biol, 4(2) 195-208 (2004)

Alternative splicing can yield manifold different mature mRNAs from one precursor. New findings indicate that alternative splicing occurs much more often than previously assumed. A major goal of functional genomics lies in elucidating and characterizing the entire spectrum of alternative splice forms. Existing approaches such as EST-alignments focus only on the mRNA sequence to detect alternative splice forms. They do not consider function and characteristics of the resulting proteins. One important example of such functional characterization is homology to a known protein domain family. A powerful description of protein domains are profile Hidden Markov models (HMM) as stored in the Pfam database. In this paper we address the problem of identifying the splice form with the highest similarity to a protein domain family. Therefore, we take into consideration all possible splice forms. As demonstrated here for a number of genes, this homology based approach can be used successfully for predicting partial gene structures. Furthermore, we present some novel splice form predictions with high-scoring protein domain homology and point out that the detection of splice form specific protein domains helps to answer questions concerning hereditary diseases. Simple approaches based on a BLASTP search cannot be applied here, since the number of possible splice forms increases exponentially with the number of exons. To this end, we have developed an efficient polynomial-time algorithm, called ASFPred (Alternative Splice Form Prediction). This algorithm needs only a set of exons as input.

József Jászai, Frank Reifers, Alexander Picker, Tobias Langenberg, Michael Brand
Isthmus-to-midbrain transformation in the absence of midbrain-hindbrain organizer activity.
Development, 130(26) 6611-6623 (2003)
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In zebrafish acerebellar (ace) embryos, because of a point mutation in fgf8, the isthmic constriction containing the midbrain-hindbrain boundary (MHB) organizer fails to form. The mutants lack cerebellar development by morphological criteria, and they appear to have an enlarged tectum, showing no obvious reduction in the tissue mass at the dorsal mesencephalic/metencephalic alar plate. To reveal the molecular identity of the tissues located at equivalent rostrocaudal positions along the neuraxis as the isthmic and cerebellar primordia in wild-types, we undertook a detailed analysis of ace embryos. In ace mutants, the appearance of forebrain and midbrain specific marker genes (otx2, dmbx1, wnt4) in the caudal tectal enlargement reveals a marked rostralized gene expression profile during early somitogenesis, followed by the lack of early and late cerebellar-specific gene expression (zath1/atoh1, gap43, tag1/cntn2, neurod, zebrin II). The Locus coeruleus (LC) derived from rostral rhombomere 1 is also absent in the mutants. A new interface between otx2 and epha4a suggests that the rostralization stops at the caudal part of rhombomere 1. The mesencephalic basal plate is also affected in the mutant embryos, as indicated by the caudal expansion of the diencephalic expression domains of epha4a, zash1b/ashb, gap43 and tag1/cntn2, and by the dramatic reduction of twhh expression. No marked differences are seen in cell proliferation and apoptotic patterns around the time the rostralization of gene expression becomes evident in the mutants. Therefore, locally distinct cell proliferation and cell death is unlikely to be the cause of the fate alteration of the isthmic and cerebellar primordia in the mutants. Dil cell-lineage labeling of isthmic primordial cells reveals that cells, at the location equivalent of the wild-type MHB, give rise to caudal tectum in ace embryos. This suggests that a caudalto-rostral transformation leads to the tectal expansion in the mutants. Fgf8-coated beads are able to rescue morphological MHB formation, and elicit the normal molecular identity of the isthmic and cerebellar primordium in ace embryos. Taken together, our analysis reveals that cells of the isthmic and cerebellar primordia acquire a more rostral, tectal identity in the absence of the functional MHB organizer signal Fgf8.

Steffen Scholpp, Michael Brand
Integrity of the midbrain region is required to maintain the diencephalic-mesencephalic boundary in zebrafish no isthmus/pax2.1 mutants.
Dev Dyn, 228(3) 313-322 (2003)
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Initial anterior-posterior patterning of the neural tube into forebrain, midbrain, and hindbrain primordia occurs already during gastrulation, in response to signals patterning the gastrula embryo. After the initial establishment, further development within each brain part is thought to proceed largely independently of the others. However, mechanisms should exist that ensure proper delineation of brain subdivisions also at later stages; such mechanisms are, however, poorly understood. In zebrafish no isthmus mutant embryos, inactivation of the pax2.1 gene leads to a failure of the midbrain and isthmus primordium to develop normally from the gastrula stage onward (Lun and Brand [1998] Development 125:3049-3062). Here, we report that, after the initially correct establishment during gastrulation stages, the neighbouring forebrain primordium and, partially, the hindbrain primordium expand into the misspecified midbrain territory in no isthmus mutant embryos. The expansion is particularly evident for the posterior part of the diencephalon and less so for the first rhombomeric segment, the territories immediately abutting the midbrain/isthmus primordium. The nucleus of the posterior commissure is expanded in size, and marker genes of the forebrain and rhombomere 1 expand progressively into the misspecified midbrain primordium, eventually resulting in respecification of the midbrain primordium. We therefore suggest that the genetic program controlled by Pax2.1 is not only involved in initiating but also in maintaining the identity of midbrain and isthmus cells to prevent them from assuming a forebrain or hindbrain fate.

Tobias Langenberg, Michael Brand, Mark S Cooper
Imaging brain development and organogenesis in zebrafish using immobilized embryonic explants.
Dev Dyn, 228(3) 464-474 (2003)
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Owing to its optical clarity and rapid rate of development, the zebrafish embryo is an ideal model system for studying the cellular mechanics of organogenesis. Unfortunately, extended time-lapse recordings of zebrafish embryos are often disrupted by the extension and straightening of the embryonic axis, as well as movement artifacts associated with developing musculature. In addition, the embryo's massive yolk cell often prevents optical access to tissues of interest. To circumvent these imaging problems, we have developed a procedure to deflate and mechanically remove the yolk cell. A "paralyzing" agent, AMP-PNP (a membrane-impermeant nonhydrolyzable analog of ATP), is first injected into the embryo's contractile yolk cell. The yolk cell is then removed using sharpened tungsten needles. Deyolked embryos, or organ rudiments explanted from them, are then immobilized on a microscope coverslip using a thin plasma clot. This plasma clot immobilization allows novel mountings of the explants so that ventral, lateral, and even cross-sectional fields of views are possible using high numerical aperture objectives. We show that isolated head rudiments undergo normal morphogenesis and gene expression for at least 1 day after being explanted into organotypic culture. These procedures can be used to study the cellular mechanics of organogenesis in "deyolked" embryos, as well as in tissues explanted from green fluorescent protein transgenic animals.

Elly M. Tanaka
Cell differentiation and cell fate during urodele tail and limb regeneration.
Curr Opin Genet Dev, 13(5) 497-501 (2003)
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One of the most striking natural examples of adult tissue plasticity in vertebrates is limb and tail regeneration in urodele amphibians. In this setting, amputation triggers the destabilization of cell differentiation and the production of progenitor cells that extensively proliferate and pattern themselves to recreate a perfect replica of the missing part. A precise understanding of which cells dedifferentiate and how plastic they become has recently begun to emerge. Furthermore, information on which developmental gene programs are activated upon injury is becoming better understood. These studies indicate that, upon injury, an unusual cohort of genes are co-expressed. The future challenge will be to link the systems for studying dedifferentiation with activation of gene expression to understand on a molecular level how cells are 'pushed backward' to regenerate a complex structure such as a limb or tail.

Ralf Kittler, Frank Buchholz
RNA interference: gene silencing in the fast lane.
Semin Cancer Biol, 13(4) 259-265 (2003)
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Sequencing of whole genomes has provided new perspectives into the blueprints of diverse organisms. Knowing the sequences, however, does not always tell us much about the function of the genes that regulate development and homeostasis. RNA interference (RNAi) is becoming the method of choice for gene function analysis in cells and whole organisms. Here we review the approaches available to perform RNAi experiments in mammalian cells and in mice. We discuss usage of RNAi in cancer research and as a possible therapeutic tool for cancer treatment.

Muriel Rhinn^*, Klaus Lun^*, Angel Amores, Yi-Lin Yan, John H Postlethwait, Michael Brand
Cloning, expression and relationship of zebrafish gbx1 and gbx2 genes to Fgf signaling.
Mech Dev, 120(8) 919-936 (2003)
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The organizer at the midbrain-hindbrain boundary (MHB) forms at the interface between Otx2 and Gbx2 expressing cell populations, but how these gene expression domains are set up and integrated with the remaining machinery controlling MHB development is unclear. Here we report the isolation, mapping, chromosomal synteny and spatiotemporal expression of gbx1 and gbx2 in zebrafish. We focus in particular on the expression of these genes during development of the midbrain-hindbrain territory. Our results suggest that these genes function in this area in a complex fashion, as evidenced by their highly dynamic expression patterns and relation to Fgf signaling. Analysis of gbx1 and gbx2 expression during formation of the MHB in mutant embryos for pax2.1, fgf8 and pou2 (noi, ace, spg), as well as Fgf-inhibition experiments, show that gbx1 acts upstream of these genes in MHB development. In contrast, gbx2 activation requires ace (fgf8) function, and in the hindbrain primordium, also spg (pou2). We propose that in zebrafish, gbx genes act repeatedly in MHB development, with gbx1 acting during the positioning period of the MHB at gastrula stages, and gbx2 functioning after initial formation of the MHB, from late gastrulation stages onwards. Transplantation studies furthermore reveal that at the gastrula stage, Fgf8 signals from the hindbrain primordium into the underlying mesendoderm. Apart from the general involvement of gbx genes in MHB development reported also in other vertebrates, these results emphasize that early MHB development can be divided into multiple steps with different genetic requirements with respect to gbx gene function and Fgf signaling. Moreover, our results provide an example for switching of a specific gene function of gbx1 versus gbx2 between orthologous genes in zebrafish and mammals.

Kimberly M. Kotovic, Daniel Lockshon, Lamia Boric, Karla M. Neugebauer
Cotranscriptional recruitment of the U1 snRNP to intron-containing genes in yeast.
Mol Cell Biol, 23(16) 5768-5779 (2003)
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Evidence that pre-mRNA processing events are temporally and, in some cases, mechanistically coupled to transcription has led to the proposal that RNA polymerase II (Pol II) recruits pre-mRNA splicing factors to active genes. Here we address two key questions raised by this proposal: (i) whether the U1 snRNP, which binds to the 5' splice site of each intron, is recruited cotranscriptionally in vivo and, (ii) if so, where along the length of active genes the U1 snRNP is concentrated. Using chromatin immunoprecipitation (ChIP) in yeast, we show that elevated levels of the U1 snRNP were specifically detected in gene regions containing introns and downstream of introns but not along the length of intronless genes. In contrast to capping enzymes, which bind directly to Pol II, the U1 snRNP was poorly detected in promoter regions, except in genes harboring promoter-proximal introns. Detection of the U1 snRNP was dependent on RNA synthesis and was abolished by intron removal. Microarray analysis revealed that intron-containing genes were preferentially selected by ChIP with the U1 snRNP. Thus, U1 snRNP accumulation at genes correlated with the presence and position of introns, indicating that introns are necessary for cotranscriptional U1 snRNP recruitment and/or retention.

Folker Spitzenberger, Massimo Pietropaolo, Paul Verkade, Bianca Habermann, Sandra Lacas-Gervais, Hassan Mziaut, Susan Pietropaolo, Michele Solimena
Islet cell autoantigen of 69 kDa is an arfaptin-related protein associated with the Golgi complex of insulinoma INS-1 cells.
J Biol Chem, 278(28) 26166-26173 (2003)
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Islet cell autoantigen of 69 kDa (ICA69) is a cytosolic protein of still unknown function. Involvement of ICA69 in neurosecretion has been suggested by the impairment of acetylcholine release at neuromuscular junctions upon mutation of its homologue gene ric-19 in C. elegans. In this study, we have further investigated the localization of ICA69 in neurons and insulinoma INS-1 cells. ICA69 was enriched in the perinuclear region, whereas it did not co-localize with markers of synaptic vesicles/synaptic-like microvesicles. Confocal microscopy and subcellular fractionation in INS-1 cells showed co-localization of ICA69 with markers of the Golgi complex and, to a minor extent, with immature insulin-containing secretory granules. The association of ICA69 with these organelles was confirmed by immunoelectron microscopy. Virtually no ICA69 immunogold labeling was observed on secretory granules near the plasma membrane, suggesting that ICA69 dissociates from secretory granule membranes during their maturation. In silico sequence and structural analyses revealed that the N-terminal region of ICA69 is similar to the region of arfaptins that interacts with ARF1, a small GTPase involved in vesicle budding at the Golgi complex and immature secretory granules. ICA69 is therefore a novel arfaptin-related protein that is likely to play a role in membrane trafficking at the Golgi complex and immature secretory granules in neurosecretory cells.

Mike Beck, Volker Bigl, Steffen Rossner
Guinea pigs as a nontransgenic model for APP processing in vitro and in vivo.
Neurochem Res, 28(3-4) 637-644 (2003)
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Alzheimer's disease (AD) is characterized, amongst others, by the appearance of vascular and parenchymal beta-amyloid deposits in brain. Such aggregates are mainly composed of beta-amyloid peptides, which are derived by proteolytic processing of a larger amyloid precursor protein (APP). APP is highly conserved among mammalian species, but experimental studies in rodents are often hampered by the humble APP-processing in the amyloidogenic pathway and by the inability of rodent beta-amyloid peptides to form higher molecular aggregates such as soluble oligomers and insoluble beta-amyloid plaques. Thus, there is need for in vitro and in vivo model systems that allow identification of factors that increase amyloidogenic APP processing and accelerate beta-amyloid plaque formation and testing the potency of pharmacological manipulations to ameliorate beta-amyloid load in brain. Transgenic mice that overexpress human APP containing AD-associated mutations that favor the amyloidogenic pathway of APP processing represent such a model. However, mutations of the APP gene are not frequent in AD and, therefore, the mechanisms of beta-amyloid plaque formation, the composition of beta-amyloid plaques, and the accompanying tissue response in brain of these animals may be different from that in AD. In contrast, guinea pigs express beta-amyloid peptides of the human sequence and appear to represent a more physiological model to examine the long-term effects of experimental manipulations on APP processing and beta-amyloid plaque formation in vivo. Additionally, APP processing in guinea pig primary neuronal cultures has been shown to be similar to cultures of human origin. In this article we highlight the advantages and limitations of using guinea pigs as experimental models to study APP processing.

Christine A. Fargeas, Mareike Florek, Wieland B. Huttner, Denis Corbeil
Characterization of prominin-2, a new member of the prominin family of pentaspan membrane glycoproteins.
J Biol Chem, 278(10) 8586-8596 (2003)
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Prominin/CD133 is a 115/120-kDa integral membrane glycoprotein specifically associated with plasma membrane protrusions in epithelial and non-epithelial cells including neuroepithelial and hematopoietic stem cells. Here we report the identification as well as molecular and cell biological characterization of mouse, rat, and human prominin-2, a 112-kDa glycoprotein structurally related to prominin (referred to as prominin-1). Although the amino acid identity between prominin-2 and prominin-1 is low (<30%), their genomic organization is strikingly similar, suggesting an early gene duplication event. Like prominin-1, prominin-2 exhibits a characteristic membrane topology with five transmembrane segments and two large glycosylated extracellular loops. Upon its ectopic expression in Chinese hamster ovary cells as a green fluorescent protein fusion chimera, prominin-2 was also found to be associated with plasma membrane protrusions, as revealed by its co-localization with prominin-1, suggesting a related role. Consistent with this, prominin-2 shows a similar tissue distribution to prominin-1, being highly expressed in the adult kidney and detected all along the digestive tract as well as in various other epithelial tissues. However, in contrast to prominin-1, prominin-2 was not detected in the eye, which perhaps explains why a loss-of function mutation in the human prominin-1 gene causes retinal degeneration but no other obvious pathological signs. Finally, we present evidence for the existence of a family of pentaspan membrane proteins, the prominins, which are conserved in evolution.

A Bernardo Carvalho, Maria D Vibranovski, Joseph W Carlson, Susan E Celniker, Roger A Hoskins, Gerald M Rubin, Granger G Sutton, Mark D Adams, Eugene W Myers, Andrew Clark
Y chromosome and other heterochromatic sequences of the Drosophila melanogaster genome: how far can we go?
Genetica, 117(2-3) 227-237 (2003)
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Whole genome shotgun assemblies have proven remarkably successful in reconstructing the bulk of euchromatic genes, with the only limit appearing to be determined by the sequencing depth. For genes imbedded in heterochromatin, however, the low cloning efficiency of repetitive sequences, combined with the computational challenges, demand that additional clues be used to annotate the sequences. One approach that has proven very successful in identifying protein coding genes in Y-linked heterochromatin of Drosophila melanogaster has been to make a BLASTable database of the small, unmapped contigs and fragments leftover at the end of a shotgun assembly, and to attempt to capture these by blasting with an appropriate query sequence. This approach often yields a staggered alignment of contigs from the unmapped set to the query sequence, as though the disjoint contigs represent small portions of the gene. Further inspection frequently shows that the contigs are broken by very large, heterochromatic introns. Methods of this sort are being expanded to make best use of all available clues to determine which unmapped contigs are associated with genes. These include use of EST libraries, and, in the case of the Y chromosome, testing of male specific genes and reduced shotgun depth of relevant contigs. It appears much more hopeful than anyone would have imagined that whole genome shotgun assemblies can recover the great bulk of even heterochromatic genes.

Karen Echeverri, Elly M. Tanaka
Electroporation as a tool to study in vivo spinal cord regeneration.
Dev Dyn, 226(2) 418-425 (2003)
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Tailed amphibians such as axolotls and newts have the unique ability to fully regenerate a functional spinal cord throughout life. Where the cells come from and how they form the new structure is still poorly understood. Here, we describe the development of a technique that allows the visualization of cells in the living animal during spinal cord regeneration. A microelectrode needle is inserted into the lumen of the spinal cord and short rapid pulses are applied to transfer the plasmids encoding the green or red fluorescent proteins into ependymal cells close to the plane of amputation. The use of small, transparent axolotls permits imaging with epifluorescence and differential interference contrast microscopy to track the transfected cells as they contribute to the spinal cord. This technique promises to be useful in understanding how neural progenitors are recruited to the regenerating spinal cord and opens up the possibility of testing gene function during this process.

Jan Modregger, Anne A Schmidt, Brigitte Ritter, Wieland B. Huttner, Markus Plomann
Characterization of Endophilin B1b, a brain-specific membrane-associated lysophosphatidic acid acyl transferase with properties distinct from endophilin A1.
J Biol Chem, 278(6) 4160-4167 (2003)
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We have characterized mammalian endophilin B1, a novel member of the endophilins and a representative of their B subgroup. The endophilins B show the same domain organization as the endophilins A, which contain an N-terminal domain responsible for lipid binding and lysophosphatidic acid acyl transferase activity, a central coiled-coil domain for oligomerization, a less conserved linker region, and a C-terminal Src homology 3 (SH3) domain. The endophilin B1 gene gives rise to at least three splice variants, endophilin B1a, which shows a widespread tissue distribution, and endophilins B1b and B1c, which appear to be brain-specific. Endophilin B1, like endophilins A, binds to palmitoyl-CoA, exhibits lysophosphatidic acid acyl transferase activity, and interacts with dynamin, amphiphysins 1 and 2, and huntingtin. However, in contrast to endophilins A, endophilin B1 does not bind to synaptojanin 1 and synapsin 1, and overexpression of its SH3 domain does not inhibit transferrin endocytosis. Consistent with this, immunofluorescence analysis of endophilin B1b transfected into fibroblasts shows an intracellular reticular staining, which in part overlaps with that of endogenous dynamin. Upon subcellular fractionation of brain and transfected fibroblasts, endophilin B1 is largely recovered in association with membranes. Together, our results suggest that the action of the endophilins is not confined to the formation of endocytic vesicles from the plasma membrane, with endophilin B1 being associated with, and presumably exerting a functional role at, intracellular membranes.

Christine A. Fargeas, Denis Corbeil, Wieland B. Huttner
AC133 antigen, CD133, prominin-1, prominin-2, etc.: prominin family gene products in need of a rational nomenclature.
Stem Cells, 21(4) 506-508 (2003)
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Sophie Léger, Michael Brand
Fgf8 and Fgf3 are required for zebrafish ear placode induction, maintenance and inner ear patterning.
Mech Dev, 119(1) 91-108 (2002)
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The vertebrate inner ear develops from initially 'simple' ectodermal placode and vesicle stages into the complex three-dimensional structure which is necessary for the senses of hearing and equilibrium. Although the main morphological events in vertebrate inner ear development are known, the genetic mechanisms controlling them are scarcely understood. Previous studies have suggested that the otic placode is induced by signals from the chordamesoderm and the hindbrain, notably by fibroblast growth factors (Fgfs) and Wnt proteins. Here we study the role of Fgf8 as a bona-fide hindbrain-derived signal that acts in conjunction with Fgf3 during placode induction, maintenance and otic vesicle patterning. Acerebellar (ace) is a mutant in the fgf8 gene that results in a non-functional Fgf8 product. Homozygous mutants for acerebellar (ace) have smaller ears that typically have only one otolith, abnormal semi-circular canals, and behavioral defects. Using gene expression markers for the otic placode, we find that ace/fgf8 and Fgf-signaling are required for normal otic placode formation and maintenance. Conversely, misexpression of fgf8 or Fgf8-coated beads implanted into the vicinity of the otic placode can increase ear size and marker gene expression, although competence to respond to the induction appears restricted. Cell transplantation experiments and expression analysis suggest that Fgf8 is required in the hindbrain in the rhombomere 4-6 area to restore normal placode development in ace mutants, in close neighbourhood to the forming placode, but not in mesodermal tissues. Fgf3 and Fgf8 are expressed in hindbrain rhombomere 4 during the stages that are critical for placode induction. Joint inactivation of Fgf3 and Fgf8 by mutation or antisense-morpholino injection causes failure of placode formation and results in ear-less embryos, mimicking the phenotype we observe after pharmacological inhibition of Fgf-signaling. Fgf8 and Fgf3 together therefore act during induction and differentiation of the ear placode. In addition to the early requirement for Fgf signaling, the abnormal differentiation of inner ear structures and mechanosensory hair cells in ace mutants, pharmacological inhibition of Fgf signaling, and the expression of fgf8 and fgf3 in the otic vesicle demonstrate independent Fgf function(s) during later development of the otic vesicle and lateral line organ. We furthermore addressed a potential role of endomesomerm by studying mzoep mutant embryos that are depleted of head endomesodermal tissue, including chordamesoderm, due to a lack of Nodal-pathway signaling. In these embryos, early placode induction proceeds largely normally, but the ear placode extends abnormally to midline levels at later stages, suggesting a role for the midline in restricting placode development to dorsolateral levels. We suggest a model of zebrafish inner ear development with several discrete steps that utilize sequential Fgf signals during otic placode induction and vesicle patterning.

Konstantinos Anastassiadis, Jinhyun Kim, Nathalie Daigle, R. Sprengel, Hans R Schöler, A F Stewart
A predictable ligand regulated expression strategy for stably integrated transgenes in mammalian cells in culture.
Gene, 298(2) 159-172 (2002)
DOI

Several strategies for regulated stable transgene expression in mammalian cells have been described. These strategies have different strengths and weaknesses, however they all share a common problem, namely predictability in application. Here we address this problem using the leading strategy for ligand inducible transgene expression, the tetracycline repressor system. Initially, we found the best stable clone out of 48 examined showed only 6-fold inducibility. Hence we looked for additions and modifications that improve the chances of a successful outcome. We document three important aspects; first, use of a mammalian codon-optimized tetracycline repressor gene; second, addition of a steroid hormone receptor ligand binding domain to the tetracycline repressor-virion protein 16 fusion protein activator; third, flanking the tet-operator/transgene cassette with insulator elements from the chicken beta-globin locus. By inclusion of these three design features, 18/18 clones showed low basal and highly inducible (>50 x) expression.

Carl-Philipp Heisenberg
Wnt signalling: refocusing on Strabismus.
Curr Biol, 12(19) 657-659 (2002)
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Vertebrate homologues of the Strabismus/van Gogh (stbm/vang) gene have been implicated in patterning and morphogenesis during gastrulation. Recent work shows that stbm/vang is mutated in zebrafish trilobite mutants and that stbm/vang is required for morphogenesis but not patterning during zebrafish gastrulation.

Thomas Wendl, Klaus Lun, Marina Mione, Jack Favor, Michael Brand, Stephen W. Wilson, Klaus B Rohr
pax2.1 is required for the development of thyroid follicles in zebrafish.
Development, 129(15) 3751-3760 (2002)
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The thyroid gland is an organ primarily composed of endoderm-derived follicular cells. Although disturbed embryonic development of the thyroid gland leads to congenital hypothyroidism in humans and mammals, the underlying principles of thyroid organogenesis are largely unknown. In this study, we introduce zebrafish as a model to investigate the molecular and genetic mechanisms that control thyroid development. Marker gene expression suggests that the molecular pathways of early thyroid development are essentially conserved between fish and mammals. However during larval stages, we find both conserved and divergent features of development compared with mammals. A major difference is that in fish, we find evidence for hormone production not only in thyroid follicular cells, but also in an anterior non-follicular group of cells. We show that pax2.1 and pax8, members of the zebrafish pax2/5/8 paralogue group, are expressed in the thyroid primordium. Whereas in mice, only Pax8 has a function during thyroid development, analysis of the zebrafish pax2.1 mutant no isthmus (noi(-/-)) demonstrates that pax2.1 has a role comparable with mouse Pax8 in differentiation of the thyroid follicular cells. Early steps of thyroid development are normal in noi(-/-), but later expression of molecular markers is lost and the formation of follicles fails. Interestingly, the anterior non-follicular site of thyroid hormone production is not affected in noi(-/-). Thus, in zebrafish, some remaining thyroid hormone synthesis takes place independent of the pathway leading to thyroid follicle formation. We suggest that the noi(-/-) mutant serves as a new zebrafish model for hypothyroidism.

Dun Yang, Frank Buchholz, Zhongdong Huang, Andrei Goga, Chih-Ying Chen, Frances M Brodsky, J Michael Bishop
Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells.
Proc Natl Acad Sci U.S.A., 99(15) 9942-9947 (2002)
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Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. Because different siRNAs of the same gene have variable silencing capacities, RNA interference with synthetic siRNA is inefficient and cost intensive, especially for functional genomic studies. Here we report the use of Escherichia coli RNase III to cleave double-stranded RNA (dsRNA) into endoribonuclease-prepared siRNA (esiRNA) that can target multiple sites within an mRNA. esiRNA recapitulates the potent and specific inhibition by long dsRNA in Drosophila S2 cells. In contrast to long dsRNA, esiRNA mediates effective RNA interference without apparent nonspecific effect in cultured mammalian cells. We found that sequence-specific interference by esiRNA and the nonspecific IFN response activated by long dsRNA are independent pathways in mammalian cells. esiRNA works by eliciting the destruction of its cognate mRNA. Because of its simplicity and potency, this approach is useful for analysis of mammalian gene functions.

Patrick Y Muller, Harald Janovjak, André R Miserez, Zuzana Dobbie
Processing of gene expression data generated by quantitative real-time RT-PCR.
BioTechniques, 32(6) 1372-1374 (2002)

Quantitative real-time PCR represents a highly sensitive and powerful technique for the quantitation of nucleic acids. It has a tremendous potential for the high-throughput analysis of gene expression in research and routine diagnostics. However, the major hurdle is not the practical performance of the experiments themselves but rather the efficient evaluation and the mathematical and statistical analysis of the enormous amount of data gained by this technology, as these functions are not included in the software provided by the manufacturers of the detection systems. In this work, we focus on the mathematical evaluation and analysis of the data generated by quantitative real-time PCR, the calculation of the final results, the propagation of experimental variation of the measured values to the final results, and the statistical analysis. We developed a Microsoft Excel-based software application coded in Visual Basic for Applications, called Q-Gene, which addresses these points. Q-Gene manages and expedites the planning, performance, and evaluation of quantitative real-time PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex quantitative real-time PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility.

Susanne Kaitna, Heinke Schnabel, Ralf Schnabel, Anthony A. Hyman, Michael Glotzer
A ubiquitin C-terminal hydrolase is required to maintain osmotic balance and execute actin-dependent processes in the early C. elegans embryo.
J Cell Sci, 115(Pt 11) 2293-2302 (2002)
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In the early Caenorhabditis elegans embryo, establishment of cell polarity and cytokinesis are both dependent upon reorganization of the actin cytoskeleton. Mutations in the cyk-3 gene cause maternal effect embryonic lethality. Embryos produced by homozygous cyk-3 mutant animals become multinucleate. We have further analyzed the cyk-3 mutant phenotype and have found that cyk-3 mutant embryos fail to properly polarize the actin cytoskeleton and fail to segregate germline determinants. In addition, they fail to assemble an intact cleavage furrow. However, we have found that cyk-3 mutant embryos are intrinsically defective in osmotic regulation and that the cytokinesis defects can be partially rescued by providing osmotic support. The cyk-3 gene has been identified and found to encode a ubiquitin C-terminal hydrolase that is active against model substrates. These data indicate that the deubiquitination of certain substrates by CYK-3 is crucial for cellular osmoregulation. Defects in osmoregulation appear to indirectly affect actin-dependent processes.

Heiner Grandel, Klaus Lun, Gerd-Jörg Rauch, Muriel Rhinn, Tatjana Piotrowski, Corinne Houart, Paolo Sordino, Axel M Küchler, Stefan Schulte-Merker, Robert Geisler, Nigel Holder, Stephen W. Wilson, Michael Brand
Retinoic acid signalling in the zebrafish embryo is necessary during pre-segmentation stages to pattern the anterior-posterior axis of the CNS and to induce a pectoral fin bud.
Development, 129(12) 2851-2865 (2002)
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A number of studies have suggested that retinoic acid (RA) is an important signal for patterning the hindbrain, the branchial arches and the limb bud. Retinoic acid is thought to act on the posterior hindbrain and the limb buds at somitogenesis stages in chick and mouse embryos. Here we report a much earlier requirement for RA signalling during pre-segmentation stages for proper development of these structures in zebrafish. We present evidence that a RA signal is necessary during pre-segmentation stages for proper expression of the spinal cord markers hoxb5a and hoxb6b, suggesting an influence of RA on anteroposterior patterning of the neural plate posterior to the hindbrain. We report the identification and expression pattern of the zebrafish retinaldehyde dehydrogenase2 (raldh2/aldh1a2) gene. Raldh2 synthesises retinoic acid (RA) from its immediate precursor retinal. It is expressed in a highly ordered spatial and temporal fashion during gastrulation in the involuting mesoderm and during later embryogenesis in paraxial mesoderm, branchial arches, eyes and fin buds, suggesting the involvement of RA at different times of development in different functional contexts. Mapping of the raldh2 gene reveals close linkage to no-fin (nof), a newly discovered mutant lacking pectoral fins and cartilaginous gill arches. Cloning and functional tests of the wild-type and nof alleles of raldh2 reveal that nof is a raldh2 mutant. By treating nof mutants with RA during different time windows and by making use of a retinoic acid receptor antagonist, we show that RA signalling during pre-segmentation stages is necessary for anteroposterior patterning in the CNS and for fin induction to occur.

Darinel Ortiz^*, Martina Medkova^*, Christiane Walch-Solimena, Peter Novick
Ypt32 recruits the Sec4p guanine nucleotide exchange factor, Sec2p, to secretory vesicles; evidence for a Rab cascade in yeast.
J Cell Biol, 157(6) 1005-1015 (2002)
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SEC2 is an essential gene required for polarized growth of the yeast Saccharomyces cerevisiae. It encodes a protein of 759 amino acids that functions as a guanine nucleotide exchange factor for the small GTPase Sec4p, a regulator of Golgi to plasma membrane transport. Activation of Sec4p by Sec2p is needed for polarized transport of vesicles to exocytic sites. Temperature-sensitive (ts) mutations in sec2 and sec4 result in a tight block in secretion and the accumulation of secretory vesicles randomly distributed in the cell. The proper localization of Sec2p to secretory vesicles is essential for its function and is largely independent of Sec4p. Although the ts mutation sec2-78 does not affect nucleotide exchange activity, the protein is mislocalized. Here we present evidence that Ypt31/32p, members of Rab family of GTPases, regulate Sec2p function. First, YPT31/YPT32 suppress the sec2-78 mutation. Second, overexpression of Ypt31/32p restores localization of Sec2-78p. Third, Ypt32p and Sec2p interact biochemically, but Sec2p has no exchange activity on Ypt32p. We propose that Ypt32p and Sec4p act as part of a signaling cascade in which Ypt32p recruits Sec2p to secretory vesicles; once on the vesicle, Sec2p activates Sec4p, enabling the polarized transport of vesicles to the plasma membrane.

Richard J Mural, Mark D Adams, Eugene W Myers, Hamilton O Smith, George L Gabor Miklos, Ron Wides, Aaron L. Halpern, Peter W Li, Granger G Sutton, Joe Nadeau, Steven L Salzberg, Robert A Holt, Chinnappa Kodira, Fu Lu, Lin Chen, Zuoming Deng, Carlos C Evangelista, Weiniu Gan, Thomas J Heiman, Jiayin Li, Zhenya Li, Gennady V Merkulov, Natalia V Milshina, Ashwinikumar K Naik, Rong Qi, Bixiong Chris Shue, Aihui Wang, Jian Wang, Xin Wang, Xianghe Yan, Jane Ye, Shibu Yooseph, Qi Zhao, Liansheng Zheng, Shiaoping C Zhu, Kendra Biddick, R A Bolanos, A L Delcher, I M Dew, D P Fasulo, M J Flanigan, Daniel H. Huson, S A Kravitz, Jason R Miller, C M Mobarry, Knut Reinert, K A Remington, Qinyu Zhang, Xiangqun H Zheng, D R Nusskern, Zhongwu Lai, Yiding Lei, Wenyan Zhong, Alison Yao, Ping Guan, Rui-Ru Ji, Zhiping Gu, Zhen-Yuan Wang, Fei Zhong, Chunlin Xiao, Chia-Chien Chiang, Mark D Yandell, Jennifer R Wortman, Peter G Amanatides, Susanne L Hladun, Eric C Pratts, Jeffery E Johnson, Kristina L Dodson, Kerry J Woodford, Cheryl A Evans, Barry Gropman, Douglas B Rusch, Eli Venter, Mei Wang, Thomas J Smith, Jarrett T Houck, Donald E Tompkins, Charles Haynes, Debbie Jacob, Soo H Chin, David R Allen, Carl E Dahlke, Robert Sanders, Kelvin Li, Xiangjun Liu, Alex Levitsky, William H Majoros, Quan Chen, Ashley C Xia, John R Lopez, Michael T Donnelly, Matthew H Newman, Anna Glodek, Cheryl L Kraft, Marc Nodell, Feroze Ali, Hui-Jin An, Danita Baldwin-Pitts, Karen Y Beeson, Shuang Cai, Mark Carnes, Amy Carver, Parris M Caulk, Angela Center, Yen-Hui Chen, Ming-Lai Cheng, My D Coyne, Michelle Crowder, Steven Danaher, Lionel B Davenport, Raymond Desilets, Susanne M Dietz, Lisa Doup, Patrick Dullaghan, Steven Ferriera, Carl R Fosler, Harold C Gire, Andres Gluecksmann, Jeannine D Gocayne, Jonathan Gray, Brit Hart, Jason Haynes, Jeffrey Hoover, Tim Howland, Chinyere Ibegwam, Mena Jalali, David Johns, Leslie Kline, Daniel S Ma, Steven MacCawley, Anand Magoon, Felecia Mann, David May, Tina C McIntosh, Somil Mehta, Linda Moy, Mee C Moy, Brian J Murphy, Sean D Murphy, Keith A Nelson, Zubeda Nuri, Kimberly A Parker, Alexandre C Prudhomme, Vinita N Puri, Hina Qureshi, John C Raley, Matthew S Reardon, Megan A Regier, Yu-Hui C Rogers, Deanna L Romblad, Jakob Schutz, John L Scott, Richard Scott, Cynthia D Sitter, Michella Smallwood, Andrew Sprague, Erin Stewart, Renee Strong, Ellen Suh, Karena Sylvester, Reginald Thomas, Ni Ni Tint, Christopher Tsonis, Gary Wang, George Wang, Monica S Williams, Sherita M Williams, Sandra M Windsor, Keriellen Wolfe, Mitchell M Wu, Jayshree Zaveri, Kabir Chaturvedi, Andrei E Gabrielian, Zhaoxi Ke, Jingtao Sun, Gangadharan Subramanian, J Craig Venter, Cynthia Pfannkoch, Mary Barnstead, Lisa D Stephenson
A comparison of whole-genome shotgun-derived mouse chromosome 16 and the human genome.
Science, 296(5573) 1661-1671 (2002)
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The high degree of similarity between the mouse and human genomes is demonstrated through analysis of the sequence of mouse chromosome 16 (Mmu 16), which was obtained as part of a whole-genome shotgun assembly of the mouse genome. The mouse genome is about 10% smaller than the human genome, owing to a lower repetitive DNA content. Comparison of the structure and protein-coding potential of Mmu 16 with that of the homologous segments of the human genome identifies regions of conserved synteny with human chromosomes (Hsa) 3, 8, 12, 16, 21, and 22. Gene content and order are highly conserved between Mmu 16 and the syntenic blocks of the human genome. Of the 731 predicted genes on Mmu 16, 509 align with orthologs on the corresponding portions of the human genome, 44 are likely paralogous to these genes, and 164 genes have homologs elsewhere in the human genome; there are 14 genes for which we could find no human counterpart.

József Jászai, Michael Brand
Cloning and expression of Ventrhoid, a novel vertebrate homologue of the Drosophila EGF pathway gene rhomboid.
Mech Dev, 113(1) 73-77 (2002)
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In Drosophila melanogaster, the seven-pass transmembrane protein Rhomboid (Rho) is a crucial positive modulator of EGF signaling playing a substantial role in patterning of the ventral neuroectoderm and fate specification of neuroblasts. Here, we describe the cloning and expression pattern of Ventrhoid (Vrho), the novel evolutionarily conserved vertebrate cDNA related to fruit fly rho. Most importantly, like rho in Drosophila, Vrho is also expressed in a spatially restricted manner. Vrho expression is most prominent along the developing ventral neural tube, and is also detectable in the ventral forebrain, prospective diencephalon, otic vesicles, mandibular arches, cranial sensory placodes, last formed pair of somites and hindgut in midgestational mouse embryos.

Yuri Voziyanov, A F Stewart, Makkuni Jayaram
A dual reporter screening system identifies the amino acid at position 82 in Flp site-specific recombinase as a determinant for target specificity.
Nucleic Acids Res, 30(7) 1656-1663 (2002)
Open Access DOI

We have developed a dual reporter screen in Escherichia coli for identifying variants of the Flp site-specific recombinase that have acquired reactivity at an altered target site (mFRT). In one reporter, the lacZalpha gene segment is flanked by mFRTs in direct orientation. In the other, the red fluorescence protein (RFP) gene is flanked by the native FRTs. Hence, the color of a colony on an X-gal indicator plate indicates the recombination potential of the variant Flp protein expressed in it: blue if no recombination or only FRT recombination occurs, red if only mFRT recombination occurs and white if both FRT and mFRT recombinations occur. The scheme was validated by identification and in vivo characterization of Flp variants that show either relaxed specificity (active on FRT and mFRT) or moderately shifted specificity toward mFRT. We find that alteration of Lys-82 to Met, Thr, Arg or His enables the corresponding Flp variants to recombine FRT sites as well as altered FRT sites containing a substitution of G-C by C-G at position 1 of the Flp binding element (mFRT11). In contrast, wild-type Flp has no detectable activity on mFRT11. When Lys-82 is replaced by Tyr, the resulting Flp variant shows a small but reproducible preference for mFRT11 over FRT. However, this preference for mFRT11 is nearly lost when Tyr-82 is substituted by Phe.

Shawn Burgess^*, Gerlinde Reim^*, Wenbiao Chen, Nancy Hopkins, Michael Brand
The zebrafish spiel-ohne-grenzen (spg) gene encodes the POU domain protein Pou2 related to mammalian Oct4 and is essential for formation of the midbrain and hindbrain, and for pre-gastrula morphogenesis.
Development, 129(4) 905-916 (2002)
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In early embryonic development, the brain is divided into three main regions along the anteroposterior axis: the forebrain, midbrain and hindbrain. Through retroviral insertional mutagenesis and chemical mutagenesis experiments in zebrafish, we have isolated mutations that cause abnormal hindbrain organization and a failure of the midbrain-hindbrain boundary (MHB) to form, a region that acts as an organizer for the adjacent brain regions. The mutations fail to complement the spiel-ohne-grenzen (spg) mutation, which causes a similar phenotype, but for which the affected gene is unknown. We show through genetic mapping, cloning of the proviral insertion site and allele sequencing that spg mutations disrupt pou2, a gene encoding the Pou2 transcription factor. Based on chromosomal synteny, phylogenetic sequence comparison, and expression and functional data, we suggest that pou2 is the zebrafish ortholog of mouse Oct3/Oct4 and human POU5F1. For the mammalian genes, a function in brain development has so far not been described. In the absence of functional pou2, expression of markers for the midbrain, MHB and the hindbrain primordium (pax2.1, wnt1, krox20) are severely reduced, correlating with the neuroectoderm-specific expression phase of pou2. Injection of pou2 mRNA restores these defects in spg mutant embryos, but does not activate these markers ectopically, demonstrating a permissive role for pou2. Injections of pou2-morpholinos phenocopy the spg phenotype at low concentration, further proving that spg encodes pou2. Two observations suggest that pou2 has an additional earlier function: higher pou2-morpholino concentrations specifically cause a pre-gastrula arrest of cell division and morphogenesis, and expression of pou2 mRNA itself is reduced in spg-homozygous embryos at this stage. These experiments suggest two roles for pou2. Initially, Pou2 functions during early proliferation and morphogenesis of the blastomeres, similar to Oct3/4 in mammals during formation of the inner cell mass. During zebrafish brain formation, Pou2 then functions a second time to activate gene expression in the midbrain and hindbrain primordium, which is reflected at later stages in the specific lack in spg embryos of the MHB and associated defects in the mid- and hindbrain.

Carl-Philipp Heisenberg, Masazumi Tada
Wnt signalling: a moving picture emerges from van gogh.
Curr Biol, 12(4) 126-128 (2002)
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Recent studies on vertebrate homologues of the van gogh/strabismus (vang/stbm) gene, a key player in planar cell polarity signalling in Drosophila, show that vang/stbm is involved in patterning and morphogenesis during vertebrate gastrulation where it modulates two distinct Wnt signals.

Gerlinde Reim, Michael Brand
Spiel-ohne-grenzen/pou2 mediates regional competence to respond to Fgf8 during zebrafish early neural development.
Development, 129(4) 917-933 (2002)
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Neural patterning of the vertebrate brain starts within the ectoderm during gastrulation and requires the activity of organizer cell populations in the neurectoderm. One such organizer is located at the prospective midbrain-hindbrain boundary (MHB) and controls development of the midbrain and the anterior hindbrain via the secreted signaling molecule Fgf8. However, little is known about how the ability of neural precursors to respond to Fgf8 is regulated. We have studied the function of the zebrafish spiel-ohne-grenzen (spg) gene in early neural development. Genetic mapping and molecular characterization presented in the accompanying paper revealed that spg mutations disrupt the pou2 gene, which encodes a POU domain transcription factor that is specifically expressed in the MHB primordium, and is orthologous to mammalian Oct3/Oct4. We show that embryos homozygous for spg/pou2 have severe defects in development of the midbrain and hindbrain primordium. Key molecules that function in the formation of the MHB, such as pax2.1, spry4, wnt1, her5, eng2 and eng3, and in hindbrain development, such as krox20, gbx2, fkd3 and pou2, are all abnormal in spg mutant embryos. By contrast, regional definition of the future MHB in the neuroectoderm by complementary expression of otx2 and gbx1, before the establishment of the complex regulatory cascade at the MHB, is normal in spg embryos. Moreover, the Fgf8 and Wnt1 signaling pathways are activated normally at the MHB but become dependent on spg towards the end of gastrulation. Therefore, spg plays a crucial role both in establishing and in maintaining development of the MHB primordium. Transplantation chimeras show that normal spg function is required within the neuroectoderm but not the endomesoderm. Importantly, gain-of-function experiments by mRNA injection of fgf8 and pou2 or Fgf8 bead implantations, as well as analysis of spg-ace double mutants show that spg embryos are insensitive to Fgf8, although Fgf receptor expression and activity of the downstream MAP kinase signaling pathway appear intact. We suggest that spg/pou2 is a transcription factor that mediates regional competence to respond to Fgf8 signaling.

Pavel Tomancák, Amy Beaton, Richard Weiszmann, Elaine Kwan, ShengQiang Shu, Suzanna E Lewis, Stephen Richards, Michael Ashburner, Volker Hartenstein, Susan E Celniker, Gerald M Rubin
Systematic determination of patterns of gene expression during Drosophila embryogenesis.
Genome Biol, 3(12) 88-88 (2002)
Open Access PDF

BACKGROUND: Cell-fate specification and tissue differentiation during development are largely achieved by the regulation of gene transcription. RESULTS: As a first step to creating a comprehensive atlas of gene-expression patterns during Drosophila embryogenesis, we examined 2,179 genes by in situ hybridization to fixed Drosophila embryos. Of the genes assayed, 63.7% displayed dynamic expression patterns that were documented with 25,690 digital photomicrographs of individual embryos. The photomicrographs were annotated using controlled vocabularies for anatomical structures that are organized into a developmental hierarchy. We also generated a detailed time course of gene expression during embryogenesis using microarrays to provide an independent corroboration of the in situ hybridization results. All image, annotation and microarray data are stored in publicly available database. We found that the RNA transcripts of about 1% of genes show clear subcellular localization. Nearly all the annotated expression patterns are distinct. We present an approach for organizing the data by hierarchical clustering of annotation terms that allows us to group tissues that express similar sets of genes as well as genes displaying similar expression patterns. CONCLUSIONS: Analyzing gene-expression patterns by in situ hybridization to whole-mount embryos provides an extremely rich dataset that can be used to identify genes involved in developmental processes that have been missed by traditional genetic analysis. Systematic analysis of rigorously annotated patterns of gene expression will complement and extend the types of analyses carried out using expression microarrays.

Benjamin P Berman, Pavel Tomancák, Yutaka Nibu, Barret D Pfeiffer, Susan E Celniker, Michael Levine, Gerald M Rubin, Michael B Eisen
Exploiting transcription factor binding site clustering to identify cis-regulatory modules involved in pattern formation in the Drosophila genome.
Proc Natl Acad Sci U.S.A., 99(2) 757-762 (2002)
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A major challenge in interpreting genome sequences is understanding how the genome encodes the information that specifies when and where a gene will be expressed. The first step in this process is the identification of regions of the genome that contain regulatory information. In higher eukaryotes, this cis-regulatory information is organized into modular units [cis-regulatory modules (CRMs)] of a few hundred base pairs. A common feature of these cis-regulatory modules is the presence of multiple binding sites for multiple transcription factors. Here, we evaluate the extent to which the tendency for transcription factor binding sites to be clustered can be used as the basis for the computational identification of cis-regulatory modules. By using published DNA binding specificity data for five transcription factors active in the early Drosophila embryo, we identified genomic regions containing unusually high concentrations of predicted binding sites for these factors. A significant fraction of these binding site clusters overlap known CRMs that are regulated by these factors. In addition, many of the remaining clusters are adjacent to genes expressed in a pattern characteristic of genes regulated by these factors. We tested one of the newly identified clusters, mapping upstream of the gap gene giant (gt), and show that it acts as an enhancer that recapitulates the posterior expression pattern of gt.

D R Shimshek, J Kim, M R Hübner, D J Spergel, Frank Buchholz, E Casanova, A. Francis Stewart, P H Seeburg, R. Sprengel
Codon-improved Cre recombinase (iCre) expression in the mouse.
Genesis, 32(1) 19-26 (2002)
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By applying the mammalian codon usage to Cre recombinase, we improved Cre expression, as determined by immunoblot and functional analysis, in three different mammalian cell lines. The improved Cre (iCre) gene was also designed to reduce the high CpG content of the prokaryotic coding sequence, thereby reducing the chances of epigenetic silencing in mammals. Transgenic iCre expressing mice were obtained with good frequency, and in these mice loxP-mediated DNA recombination was observed in all cells expressing iCre. Moreover, iCre fused to two estrogen receptor hormone binding domains for temporal control of Cre activity could also be expressed in transgenic mice. However, Cre induction after administration of tamoxifen yielded only low Cre activity. Thus, whereas efficient activation of Cre fusion proteins in the brain needs further improvements, our studies indicate that iCre should facilitate genetic experiments in the mouse.

Rüdiger Gerlach, Mike Beck, Ulrike Zeitschel, Volker Seifert
MK 801 attenuates c-Fos and c-Jun expression after in vitro ischemia in rat neuronal cell cultures but not in PC 12 cells
Neuro res, 24(7) 725-729 (2002)
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Cellular homeostatic adaptation to cerebral ischemia is complex and contains changes in receptor mediated gene expression and signaling pathways. The proteins of the immediate early genes c-Fos and c-Jun are thought to be involved in coupling neuronal excitation to target gene expression, due to formation of heterodimers and binding to the AP1 promotor region.We used an in vitro model to compare ischemia induced c-Fos and c-Jun expression in rat neuronal cell cultures and nerve growth factor (NGF) differentiatedPC 12 cells. Since activation of glutamate receptors is known to mediate ischemic injury we determined the effect of the noncompetitive N-methyl-D-aspartate(NMDA) receptor antagonistMK 801 on c-Fos and c-Jun expression in both cell culture systems during ischemia. Neuron rich cultures and NGF differentiated PC 12 cells were exposed to sublethal in vitro ischemia using an hypoxic chamber �ushed with argon/CO2 (95%/5%). C-Fos and c-Jun mRNA expression was analyzed by competitive reverse transcription–polymerase chain reaction using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal standard. One hour of in vitro ischemia signi´cantly increased c-Fos and c-Jun mRNA levels in both cell culture systems. In neuron rich cultures a 10-fold (c-Fos) and 7-fold (c-Jun) mRNA increase was observed. The mRNA rise was less pronounced in PC 12 cells (5.5-fold and 2-fold) for c-Fos and c-Jun, respectively. The addition of MK 801 signi´cantly reduced the expression of c-Fos and c-Jun mRNA in neuronal cultures, whereas no effectwas detectable in PC 12 cells. Since MK 801 failed to reduce the c-Fos and c-Jun expression in NGF differentiatedPC 12 cells different signaling pathways may initiate c-Fos and c-Jun expression in both cell culture systems.

André Bachmann, M Schneider, Eva Theilenberg, Ferdi Grawe, Elisabeth Knust
Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity.
Nature, 414(6864) 638-643 (2001)
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The polarized architecture of epithelial cells depends on the highly stereotypic distribution of cellular junctions and other membrane-associated protein complexes. In epithelial cells of the Drosophila embryo, three distinct domains subdivide the lateral plasma membrane. The most apical one comprises the subapical complex (SAC). It is followed by the zonula adherens (ZA) and, further basally, by the septate junction. A core component of the SAC is the transmembrane protein Crumbs, the cytoplasmic domain of which recruits the PDZ-protein Discs Lost into the complex. Cells lacking crumbs or the functionally related gene stardust fail to organize a continuous ZA and to maintain cell polarity. Here we show that stardust provides an essential component of the SAC. Stardust proteins colocalize with Crumbs and bind to the carboxy-terminal amino acids of its cytoplasmic tail. We introduce two different Stardust proteins here: one MAGUK protein, characterized by a PDZ domain, an SH3 domain and a guanylate kinase domain; and a second isoform comprising only the guanylate kinase domain. The Stardust proteins represent versatile candidates as structural and possibly regulatory constituents of the SAC, a crucial element in the control of epithelial cell polarity.

W W M Pim Pijnappel, Daniel Schaft, Assen Roguev, A Shevchenko, H Tekotte, Matthias Wilm, Guillaume Rigaut, Bertrand Séraphin, Rein Aasland, A F Stewart
The S. cerevisiae SET3 complex includes two histone deacetylases, Hos2 and Hst1, and is a meiotic-specific repressor of the sporulation gene program.
Genes Dev, 15(22) 2991-3004 (2001)
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Set3 is one of two proteins in the yeast Saccharomyces cerevisiae that, like Drosophila Trithorax, contains both SET and PHD domains. We found that Set3 forms a single complex, Set3C, with Snt1, YIL112w, Sif2, Cpr1, and two putative histone deacetylases, Hos2 and NAD-dependent Hst1. Set3C includes NAD-dependent and independent deacetylase activities when assayed in vitro. Homology searches suggest that Set3C is the yeast analog of the mammalian HDAC3/SMRT complex. Set3C represses genes in early/middle of the yeast sporulation program, including the key meiotic regulators ime2 and ndt80. Whereas Hos2 is only found in Set3C, Hst1 is also present in a complex with Sum1, supporting previous characterizations of Hst1 and Sum1 as repressors of middle sporulation genes during vegetative growth. However, Hst1 is not required for meiotic repression by Set3C, thus implying that Set3C (-Hst1) and not Hst1-Sum1, is the meiotic-specific repressor of early/middle sporulation genes.

Marek Drab, Paul Verkade, Marlies Elger, Michael Kasper, Matthias Lohn, Birgit Lauterbach, Jan Menne, Carsten Lindschau, Fanny Mende, Friedrich C. Luft, Aandreas Schedl, Hermann Haller, Teymuras V. Kurzchalia
Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice.
Science, 293(5539) 2449-2452 (2001)
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Caveolae are plasma membrane invaginations that may play an important role in numerous cellular processes including transport, signaling, and tumor suppression. By targeted disruption of caveolin-1, the main protein component of caveolae, we generated mice that lacked caveolae. The absence of this organelle impaired nitric oxide and calcium signaling in the cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic tone. In addition, the lungs of knockout animals displayed thickening of alveolar septa caused by uncontrolled endothelial cell proliferation and fibrosis, resulting in severe physical limitations in caveolin-1-disrupted mice. Thus, caveolin-1 and caveolae play a fundamental role in organizing multiple signaling pathways in the cell.

Pierre Gönczy^*, Jean-Michel Bellanger^*, Matthew Kirkham, Andrei Pozniakovski, Karine Baumer, Jennifer B. Phillips, Anthony A. Hyman
zyg-8, a gene required for spindle positioning in C. elegans, encodes a doublecortin-related kinase that promotes microtubule assembly.
Dev Cell, 1(3) 363-375 (2001)
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Proper spindle positioning is essential for spatial control of cell division. Here, we show that zyg-8 plays a key role in spindle positioning during asymmetric division of one-cell stage C. elegans embryos by promoting microtubule assembly during anaphase. ZYG-8 harbors a kinase domain and a domain related to Doublecortin, a microtubule-associated protein (MAP) affected in patients with neuronal migration disorders. Sequencing of zyg-8 mutant alleles demonstrates that both domains are essential for function. ZYG-8 binds to microtubules in vitro, colocalizes with microtubules in vivo, and promotes stabilization of microtubules to drug or cold depolymerization in COS-7 cells. Our findings demonstrate that ZYG-8 is a MAP crucial for proper spindle positioning in C. elegans, and indicate that the function of the Doublecortin domain in modulating microtubule dynamics is conserved across metazoan evolution.

H Masselink, Nadine Vastenhouw, René Bernards
B-myb rescues ras-induced premature senescence, which requires its transactivation domain.
Cancer Lett, 171(1) 87-101 (2001)

B-myb, a ubiquitously expressed member of the myb gene family, is highly regulated throughout the cell cycle and appears to be required for cell cycle progression. In contrast to its relatives A-myb, c-myb, and v-myb, no transforming activity of B-myb has been reported thus far. We report here that B-myb can rescue senescence induced by an activated ras oncogene in rodent cells in vitro. We show that transformation by B-Myb involves its ability to activate transcription. Similar to other oncogenic transcription factors, such as c-Myc and E2F, we show that B-Myb also has repression activity. We demonstrate that the C-terminus of B-Myb can function as a repressor of transcription, that B-Myb interacts with the repressor molecules BS69 and N-CoR and that the repression function, like the transactivation domain, contributes to B-myb transformation.

Florian Raible, Michael Brand
Tight transcriptional control of the ETS domain factors Erm and Pea3 by Fgf signaling during early zebrafish development.
Mech Dev, 107(1-2) 105-117 (2001)
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Several molecules of the Fibroblast growth factor family have been implicated in the development of the vertebrate brain, but the effectors of these molecules remain largely unknown. Here we study Erm and Pea3, two ETS domain transcription factors, and show that their expression correlates closely with the domains of fgf8 and fgf3 expression. In situ hybridization analysis in wild-type and acerebellar (ace) mutant embryos defective for fgf8 demonstrates a requirement of Fgf8 for normal expression levels of erm and pea3 transcripts in and close to various domains of Fgf8 action, including the prospective midbrain-hindbrain region, the somites, the neural crest, the forebrain, and developing eyes. Morpholino-oligomer-assisted gene knock-down experiments targeted against fgf8 and fgf3 suggest that Fgf3 and Fgf8 are co-regulators of these genes in the early forebrain anlage. Furthermore, inhibition of Fgf signaling by overexpression of sprouty4 or application of the Fgf inhibitor SU5402 leads to a loss of all erm and pea3 expression domains. Conversely, ectopically provided fgf3 mRNA or implanted beads coated with Fgf8 elicit ectopic transcription of erm and pea3. Both activation and loss of transcripts can be observed within short time frames. We conclude that both the transcriptional onset and maintenance of these factors are tightly coupled to Fgf signaling and propose that erm and pea3 transcription is a direct readout of cells to Fgf levels. Given the knowledge that has accumulated on the posttranslational control of ETS domain factors and their combinatorial interactions with other transcription factors, we suggest that the close coupling of erm and pea3 transcription to Fgf signaling might serve to integrate Fgf signaling with other signals to establish refined patterns in embryonic development.

Martin Klemke, Ralph H. Kehlenbach, Wieland B. Huttner
Two overlapping reading frames in a single exon encode interacting proteins--a novel way of gene usage.
EMBO J, 20(14) 3849-3860 (2001)
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The >1 kb XL-exon of the rat XLalphas/Galphas gene encodes the 37 kDa XL-domain, the N-terminal half of the 78 kDa neuroendocrine-specific G-protein alpha-subunit XLalphas. Here, we describe a novel feature of the XL-exon, the presence of an alternative >1 kb open reading frame (ORF) that completely overlaps with the ORF encoding the XL-domain. The alternative ORF starts 32 nucleotides downstream of the start codon for the XL-domain and is terminated by a stop codon exactly at the end of the XL-exon. The alternative ORF encodes ALEX, a very basic (pI 11.8), proline-rich protein of 356 amino acids. Both XLalphas and ALEX are translated from the same mRNA. Like XLalphas, ALEX is expressed in neuroendocrine cells and tightly associated with the cytoplasmic leaflet of the plasma membrane. Remarkably, ALEX binds to the XL-domain of XLalphas. Our results reveal a mechanism of gene usage that is without precedent in mammalian genomes.

Fabian Feiguin, Michael Hannus, Marek Mlodzik, Suzanne Eaton
The ankyrin repeat protein Diego mediates Frizzled-dependent planar polarization.
Dev Cell, 1(1) 93-101 (2001)
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During planar polarization of the Drosophila wing epithelium, the homophilic adhesion molecule Flamingo localizes to proximal/distal cell boundaries in response to Frizzled signaling; perturbing Frizzled signaling alters Flamingo distribution, many cell diameters distant, by a mechanism that is not well understood. This work identifies a tissue polarity gene, diego, that comprises six ankyrin repeats and colocalizes with Flamingo at proximal/distal boundaries. Diego is specifically required for polarized accumulation of Flamingo and drives ectopic clustering of Flamingo when overexpressed. Our data suggest that Frizzled acts through Diego to promote local clustering of Flamingo, and that clustering of Diego and Flamingo in one cell nonautonomously propagates to others.

Isato Araki, Michael Brand
Morpholino-induced knockdown of fgf8 efficiently phenocopies the acerebellar (ace) phenotype.
Genesis, 30(3) 157-159 (2001)
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Morpholino-Induced Knockdown of fgf8 Efficiently Phenocopies the Acerebellar (Ace) Phenotype Isato Araki and Michael Brand* Max-Planck-Institute for Molecular Cell Biology and Genetics, Dresden, Germany Received 1 June 2001; Accepted 18 June 2001 Homozygous acerebellar (ace) embryos lack their cerebellum and the midbrain hindbrain boundary (MHB) organizer, and in addition have defects in forebrain and heart development (Brand et al., 1996; Picker et al., 1999; Reifers et al., 1998, Shanmugalingam et al. (2000); Raible and Brand, 2001; Araki and Brand, unpublished data), suggesting that this may be because of functional redundancy between Fgfs. Because morpholinos might help to resolve such issues, we sought to phenocopy the known defects caused by absence of Ace/Fgf8 through injection of a morpholino against fgf8 (MO-fgf8). In spite of the complexity of the ace phenotype, we find that MO-fgf8 efficiently and uniformly phenocopies the ace mutant in MHB, forebrain, and heart development. We designed an antisense morpholino against fgf8, covering the translational start codon (Fig. 1J). After injections into one- to eight-cell-stage wild-type embryos, we find that MO-fgf8 effectively phenocopies the ace phenotype. We used between 0.5 and 4 g/ l of MOfgf8 for the injection (Table 1), which delivers between 1.6 and 12.6 ng of MO-fgf8 per embryo. Morphologically, embryos at the 24-h stage injected with 1 4 g/ l lacked the cerebellum and the MHB organizer, as do ace mutants at the same stage (Fig. 1A I). A morpholino with four mismatched base pairs against fgf8 (control MO in Fig. 1J) had no effect, nor did it show any nonspecific effects at the same concentration (Table 1). To examine how closely the ace phenotype is mimicked, we stained the injected embryos with a probe for pax2.1 which reveals several of the tissues where Fgf8 functions. pax2.1 expression at the MHB is initially normal in ace mutants, but is not properly maintained (Reifers et al., 1998; Lun and Brand, 1998). At 24 h, MO-fgf8 injected embryos either lacked pax2.1 expression at the MHB completely, or it was reduced to a small dorsal patch, as seen in ace mutants at the same stage (Fig. 1D I and not shown; Reifers et al., 1998). Because ace mutants show defects also in forebrain and heart development, we analyzed with molecular markers whether MO-fgf8 injection can phenocopy the ace mutant also in these tissues. Ten hours after fertilization, injected embryos have reduced and perturbed expression of emx1, an early telencephalic marker, as in ace mutants (Fig. 1K M; Shanmugalingam et al., 2000). Similarly, at 24 h expression of pax2.1 in the optic stalk is reduced, and in the optic chiasm is reduced or missing (not shown), as found previously for ace mutants (Shanmugalingam et al., 2000). Injected embryos stained with nkx2.5, an early marker for heart primordium, showed downregulation of nkx2.5, as is observed in ace mutants (Fig. 1N P; Reifers et al., 2000a). We did not detect any nonspecific effect of MO-fgf8. We conclude that MO-fgf8 injection efficiently phenocopies the known loss-of-function phenotype of acerebellar in MHB, telencephalon, optic stalk, and heart development, thus validating the usefulness of this method. Because morpholino injection is thought to prevent translation (which we assume to be true, but have not tested, for fgf8), these findings also support (Reifers et al., 1998) that ace is a null allele. To further test this notion, we examined whether the phenotype of homozygous acerebellar mutants can be enhanced by Mo-fgf8 injection. Morphologically and after examining pax2.1 expression in injected embryos at the tailbud, two-somite, five-somite, and 24-h stage, we did not observe a difference between ace homozygotes and their wild-type siblings in the same clutch (Table 1 and not shown), showing that the ace phenotype cannot be further enhanced and therefore most likely represents the null phenotype. Morpholinos against other fgf mR-NAs alone or in combination with Mo-fgf8 will help to resolve the problem of redundancy that is often found with signaling by Fgfs. ACKNOWLEDGMENTS We thank Florian Raible and Steffen Scholpp for helpful comments, and Muriel Rhinn for technical advice. I.A. is supported by the Alexander von Humboldt Stiftung. This work was supported by grants from the Deutsche Forschungsgemeinschaft, the EU Biotech Program, and the Max-Planck Society to M.B. LITERATURE CITED Brand M, Heisenberg C-P, Jiang Y-J, Beuchle D, Lun K, van Eeden FJM, Furutani-Seiki M, Granato M, Haffter P, Hammerschmidt M, Kane Contract grant sponsors: Alexander von Humboldt Stiftung, Deutsche Forschungsgemeinschaft, EU Biotech Program, and Max-Planck Society. * Correspondence to: Michael Brand, Max-Planck-Institute for Molecular Cell Biology and Genetics, Pfotenhauerstr 108, D-01307 Dresden, Germany. E-mail: brand@mpi-cbg.de 2001 Wiley-Liss, Inc. genesis 30:157 159 (2001) FIG. 1. Injection of a morpholino against fgf8 phenocopies the ace phenotype. (A K) MO-fgf8 phenocopied ace phenotype in the midbrain hindbrain boundary (MHB). Injected embryos or ace embryos lack the structure of MHB (arrowheads in A), the conspicuous constriction at MHB (compare arrowheads in G and H/I), and pax2.1 expression at MHB. The embryos were injected with MO-fgf8 at 2 g/ l in this figure. The embryos in A, D, and G are wild type; the embryos in B, E, and H are ace mutants. The embryos in D I were stained with probe for pax2.1 mRNA. The pictures in A F are lateral view, whereas the ones in G I are dorsal view. In A I, embryos were fixed at 24 h after fertilization. (J) The nucleotide/amino acid sequence around the initiation codon (underlined) of fgf8 cDNA and the sequences of the morpholino against fgf8 (MO-fgf8) and its negative control (control MO) are shown. The control-MO contains four single nucleotide exchanges (underlined) that are predicted to lead to a strongly reduced target binding (Gene Tools, LLC, Corvallis, OR). (K P) MO-fgf8 phenocopied ace phenotype also in other tissues. The embryos in K and N are wild type; the embryos in L and O are ace mutant; the embryos in M and P are MO-fgf8-injected. The embryos were stained with an emx1 probe and a nkx2.5 probe in K M and N P, respectively. The pictures in K P are dorsal views. Embryos in K M were fixed at 10 h after fertilization; at 12 h in N P. All embryos in this figure are oriented with rostral to the left. OS, optic stalk; OV, otic vesicle; TC, tectum. Methods: Crystallized morpholino oligos (Gene Tools, LLC) were dissolved in water at 40 g/ l, and this stock solution was stored at 20 C. The working solution consisted of 5 mM HEPES (pH 7.5), 0.1% phenol red and the appropriate concentration of morpholinos. Injection was performed between one-cell to eight-cell stage. The injected/control embryos were raised at 28 C. Table 1 Dose Response to a Morpholino Against Fgf8 Morpholino Concentration ( g/ l) Dose (ng) n pax2.1 staining at MHB Absent Reduced Normal MO-Fgf8 0.5 1.6 30 20 (67%) 9 (30%) 1 3.1 103 50 (49%) 53 (51%) 0 2 6.3 101 60 (59%) 41 (41%) 0 4 12.6 24 10 (42%) 14 (58%) 0 4 12.6 7 7 (100%) 0 0 Injection into ace/ace homozygotes 2 6.3 63 58 (93%) 5 (8%) 0 Control MO 2 6.3 22 0 0 22 (100%) 4 12.6 19 0 0 19 (100%) Mock 0 0 23 0 0 23 (100%) The readout was in situ hybridizations with pax2.1 probe at 24 h after fertilization. 158 ARAKI AND BRAND DA, Kelsh RN, Mullins MC, Odenthal J, Nu sslein-Volhard C. 1996. Mutations in zebrafish genes affecting the formation of the boundary between midbrain and hindbrain. Development 123:179 190. Lun K, Brand M. 1998. A series of no isthmus (noi) alleles of the zebrafish pax2.1 gene reveals multiple signaling events in development of the midbrain-hindbrain boundary. Development 125: 3049 3062. Picker A, Brennan C, Reifers F, Clarke JD, Holder N, Brand M. 1999. Requirement for the zebrafish mid-hindbrain boundary in midbrain polarisation, mapping and confinement of the retinotectal projection. Development 126:2967 2978. Raible F, Brand M. 2001. Tight transcriptional control of the ETS domain factors Erm and Pea3 by FGF signaling during early zebrafish nervous system development. Mech Dev (in press). Reifers F, Bo hli H, Walsh EC, Crossley PH, Stainier DYR, Brand M. 1998. Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis. Development 125:2381 2395. Reifers F, Walsh EC, Le ger S, Stainier DYR, Brand M. 2000a. Induction and differentiation of the zebrafish heart requires fibroblast growth factor 8 (fgf8/acerebellar). Development 127:225 235. Reifers F, Adams J, Mason IJ, Schulte-Merker S, Brand M. 2000b. Overlapping and distinct functions provided by Fgf17, a new zebrafish member of the Fgf8/17/18 subgroup of Fgfs. Mech Dev 99:39 49. Shanmugalingam S, Houart C, Picker A, Reifers F, Macdonald R, Barth A, Griffin K, Brand M, Wilson SW. 2000. Ace/Fgf8 is required for forebrain commissure formation and patterning of the telencephalon. Development 127:2549 2561. 159 KNOCKDOWN OF FGF8 PHENOCOPIES ACE PHENOTYPE

Maximilian Fürthauer, Frank Reifers, Michael Brand, Bernard Thisse, Christine Thisse
sprouty4 acts in vivo as a feedback-induced antagonist of FGF signaling in zebrafish.
Development, 128(12) 2175-2186 (2001)
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In looking for novel factors involved in the regulation of the fibroblast growth factor (FGF) signaling pathway, we have isolated a zebrafish sprouty4 gene, based on its extensive similarities with the expression patterns of both fgf8 and fgf3. Through gain- and loss-of-function experiments, we demonstrate that Fgf8 and Fgf3 act in vivo to induce the expression of Spry4, which in turn can inhibit activity of these growth factors. When overexpressed at low doses, Spry4 induces loss of cerebellum and reduction in size of the otic vesicle, thereby mimicking the fgf8/acerebellar mutant phenotype. Injections of high doses of Spry4 cause ventralization of the embryo, an opposite phenotype to the dorsalisation induced by overexpression of Fgf8 or Fgf3. Conversely we have shown that inhibition of Spry4 function through injection of antisense morpholino oligonucleotide leads to a weak dorsalization of the embryo, the phenotype expected for an upregulation of Fgf8 or Fgf3 signaling pathway. Finally, we show that Spry4 interferes with FGF signaling downstream of the FGF receptor 1 (FGFR1). In addition, our analysis reveals that signaling through FGFR1/Ras/mitogen-activated protein kinase pathway is involved, not in mesoderm induction, but in the control of the dorsoventral patterning via the regulation of bone morphogenetic protein (BMP) expression.

Stanny Berghs, Franco Ferracci, Elena Maksimova, Shannon Gleason, Nancy Leszczynski, Margaret H. Butler, Pietro De Camilli, Michele Solimena
Autoimmunity to beta IV spectrin in paraneoplastic lower motor neuron syndrome.
Proc Natl Acad Sci U.S.A., 98(12) 6945-6950 (2001)
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Paraneoplastic neurological disorders may result from autoimmunity directed against antigens shared by the affected neurons and the associated cancer cells. We have recently reported the case of a woman with breast cancer and paraneoplastic lower motor neuron syndrome whose serum contained autoantibodies directed against axon initial segments and nodes of Ranvier of myelinated axons, including the axons of motoneurons. Here, we show that major targets of the autoantibodies of this patient are betaIVSigma1 spectrin and betaIV spectrin 140, two isoforms of the novel betaIV spectrin gene, as well as a neuronal surface epitope yet to be identified. Partial improvement of the neurological symptoms following cancer removal was associated with a drastic reduction in the titer of the autoantibodies against betaIV spectrin and nodal antigens in general, consistent with the autoimmune pathogenesis of the paraneoplastic lower motor neuron syndrome. The identification of betaIV spectrin isoforms and surface nodal antigens as novel autoimmune targets in lower motor neuron syndrome provide new insights into the pathogenesis of this severe neurological disease.

Tom Van de Putte, An Zwijsen, Olivier Lonnoy, Vladimir Rybin, Miranda Cozijnsen, Annick Francis, Veerle Baekelandt, Christine A. Kozak, Marino Zerial, Danny Huylebroeck
Mice with a homozygous gene trap vector insertion in mgcRacGAP die during pre-implantation development.
Mech Dev, 102(1-2) 33-44 (2001)
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In a phenotypic screen in mice using a gene trap approach in embryonic stem cells, we have identified a recessive loss-of-function mutation in the mgcRacGAP gene. Maternal protein is present in the oocyte, and mgcRacGAP gene transcription starts at the four-cell stage and persists throughout mouse pre-implantation development. Total mgcRacGAP deficiency results in pre-implantation lethality. Such E3.5 embryos display a dramatic reduction in cell number, but undergo compaction and form a blastocoel. At E3.0-3.5, binucleated blastomeres in which the nuclei are partially interconnected are frequently observed, suggesting that mgcRacGAP is required for normal mitosis and cytokinesis in the pre-implantation embryo. All homozygous mutant blastocysts fail to grow out on fibronectin-coated substrates, but a fraction of them can still induce decidual swelling in vivo. The mgcRacGAP mRNA expression pattern in post-implantation embryos and adult mouse brain suggests a role in neuronal cells. Our results indicate that mgcRacGAP is essential for the earliest stages of mouse embryogenesis, and add evidence that CYK-4-like proteins also play a role in microtubule-dependent steps in the cytokinesis of vertebrate cells. In addition, the severe phenotype of null embryos indicates that mgcRacGAP is functionally non-redundant and cannot be substituted by other GAPs during early cleavage of the mammalian embryo.

Anne Grapin-Botton, A R Majithia, Douglas A Melton
Key events of pancreas formation are triggered in gut endoderm by ectopic expression of pancreatic regulatory genes.
Genes Dev, 15(4) 444-454 (2001)
DOI

The mechanisms by which the epithelium of the digestive tract and its associated glands are specified are largely unknown. One clue is that several transcription factors are expressed in specific regions of the endoderm prior to and during organogenesis. Pdx-1, for example, is expressed in the duodenum and pancreas and Pdx-1 inactivation results in an arrest of pancreatic development after buds formation. Similarly, ngn3 is transiently expressed in the developing pancreas and a knockout results in the absence of endocrine cells. This paper focuses on the question of whether these and other transcription factors, known to be necessary for pancreatic development, are also sufficient to drive a program of pancreatic organogenesis. Using in ovo electroporation of chick embryos, we show that ectopic expression of Pdx-1 or ngn3 causes cells to bud out of the epithelium like pancreatic progenitors. The Pdx-1-expressing cells extinguish markers for other nonpancreatic regions of the endoderm and initiate, but do not complete, pancreatic cytodifferentiation. Ectopic expression of ngn3 is sufficient to turn endodermal cells of any region into endocrine cells that form islets expressing glucagon and somatostatin in the mesenchyme. The results suggest that simple gene combinations could be used in stem cells to achieve specific endodermal tissue differentiation.

Denis Corbeil, Katja Röper, Christine A. Fargeas, Angret Joester, Wieland B. Huttner
Prominin: a story of cholesterol, plasma membrane protrusions and human pathology.
Traffic, 2(2) 82-91 (2001)
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Prominin is the first identified member of a novel family of polytopic membrane proteins conserved throughout the animal kingdom. It has an unusual membrane topology, containing five transmembrane domains and two large glycosylated extracellular loops. In mammals, prominin is expressed in various embryonic and adult epithelial cells, as well as in nonepithelial cells, such as hematopoietic stem cells. At the subcellular level, prominin is selectively localized in microvilli and other plasma membrane protrusions, irrespective of cell type. At the molecular level, prominin specifically interacts with membrane cholesterol and is a marker of a novel type of cholesterol-based lipid 'raft'. A frameshift mutation in the human prominin gene, which results in a truncated protein that is no longer transported to the cell surface, is associated with retinal degeneration. Given that prominin is concentrated in the plasma membrane evaginations at the base of the outer segment of rod photoreceptor cells, which are essential precursor structures in the biogenesis of photoreceptive disks, it is proposed that prominin has a role in the generation of plasma membrane protrusions, their lipid composition and organization and their membrane-to-membrane interactions.

J C Venter, M D Adams, E W Myers, P W Li, Richard J Mural, Granger G Sutton, H O Smith, Mark D Yandell, Cheryl A Evans, Robert A Holt, Jeannine D Gocayne, Peter G Amanatides, R M Ballew, Daniel H. Huson, Jennifer R Wortman, Qinyu Zhang, Chinnappa Kodira, X H Zheng, L Chen, M Skupski, G Subramanian, P D Thomas, J Zhang, George L Gabor Miklos, C Nelson, Samuel Broder, Andrew Clark, Joe Nadeau, V A McKusick, N Zinder, A J Levine, R J Roberts, M Simon, C Slayman, Michael W Hunkapiller, R A Bolanos, A L Delcher, I M Dew, D P Fasulo, M J Flanigan, Liliana Florea, Aaron L. Halpern, Sridhar Hannenhalli, S A Kravitz, S Levy, C M Mobarry, Knut Reinert, K A Remington, J Abu-Threideh, E M Beasley, Kendra Biddick, V Bonazzi, R C Brandon, M Cargill, I Chandramouliswaran, Rosane Charlab, Kabir Chaturvedi, Zuoming Deng, V Di Francesco, P Dunn, K Eilbeck, Carlos C Evangelista, Andrei E Gabrielian, Weiniu Gan, W Ge, F Gong, Z Gu, Ping Guan, T J Heiman, M E Higgins, Rui-Ru Ji, Z Ke, K A Ketchum, Z Lai, Y Lei, Z Li, J Li, Y Liang, X Lin, F Lu, Gennady V Merkulov, Natalia V Milshina, H M Moore, Ashwinikumar K Naik, V A Narayan, B Neelam, D R Nusskern, Douglas B Rusch, Steven L Salzberg, W Shao, Bixiong Chris Shue, J Sun, Z Wang, A Wang, X Wang, J Wang, M Wei, Ron Wides, C Xiao, C Yan, Alison Yao, Jane Ye, M Zhan, W Zhang, H Zhang, Q Zhao, L Zheng, F Zhong, W Zhong, S Zhu, Senming Zhao, D Gilbert, S Baumhueter, G Spier, Crystal N. Carter, Anibal Cravchik, T Woodage, F Ali, Hui-Jin An, A Awe, Danita Baldwin, Holly Baden, Mary Barnstead, I Barrow, Karen Y Beeson, D Busam, Amy Carver, Angela Center, M L Cheng, L Curry, Steven Danaher, Lionel B Davenport, Raymond Desilets, Susanne M Dietz, Kristina L Dodson, Lisa Doup, Steven Ferriera, N Garg, Andres Gluecksmann, Brit Hart, J Haynes, C Haynes, C Heiner, Susanne L Hladun, D Hostin, Jarrett T Houck, Tim Howland, Chinyere Ibegwam, J Johnson, Francis Kalush, Leslie Kline, S Koduru, A Love, F Mann, David May, S McCawley, T McIntosh, I McMullen, M Moy, L Moy, B Murphy, K Nelson, Cynthia Pfannkoch, Eric C Pratts, V Puri, Hina Qureshi, Matthew S Reardon, R Rodriguez, Y H Rogers, Deanna L Romblad, B Ruhfel, R Scott, Cynthia D Sitter, Michella Smallwood, E Stewart, Renee Strong, E Suh, R Thomas, Ni Ni Tint, S Tse, C Vech, G Wang, J Wetter, S Williams, M Williams, Sandra M Windsor, E Winn-Deen, Keriellen Wolfe, Jayshree Zaveri, K Zaveri, Josep F Abril, R Guigó, M J Campbell, K V Sjolander, B Karlak, A Kejariwal, H Mi, B Lazareva, T Hatton, A Narechania, K Diemer, A Muruganujan, N Guo, S Sato, V Bafna, Sorin Istrail, Ross Lippert, R Schwartz, Brian Walenz, Shibu Yooseph, D Allen, A Basu, J Baxendale, L Blick, M Caminha, J Carnes-Stine, Parris M Caulk, Y H Chiang, My D Coyne, Carl E Dahlke, A Mays, M Dombroski, Michael T Donnelly, D Ely, S Esparham, Carl R Fosler, Harold C Gire, S Glanowski, K Glasser, Anna Glodek, M Gorokhov, K Graham, Barry Gropman, M Harris, J Heil, S Henderson, Jeffrey Hoover, D Jennings, C M Jordan, J Jordan, J Kasha, L Kagan, C Kraft, Alex Levitsky, M Lewis, X Liu, J Lopez, D Ma, William H Majoros, J McDaniel, S Murphy, Matthew H Newman, T Nguyen, N Nguyen, Marc Nodell, S Pan, J Peck, M Peterson, W Rowe, Robert Sanders, J Scott, M Simpson, T Smith, Andrew Sprague, T Stockwell, R Turner, E Venter, M Wang, M Wen, D Wu, M Wu, Ashley C Xia, A Zandieh, X Zhu
The sequence of the human genome.
Science, 291(5507) 1304-1351 (2001)
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A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

K Ladjali-Mohammedi, Anne Grapin-Botton, M A Bonnin, N M Le Douarin
Distribution of HOX genes in the chicken genome reveals a new segment of conservation between human and chicken.
Cytogenet. Cell Genet., 92(1-2) 157-161 (2001)
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Homeobox genes play an important role in the regulation of early embryonic development. They represent a family of evolutionarily highly conserved transcription factors. In this work, several genes that belong to the four HOX gene clusters are assigned by in situ hybridization to four distinct chicken chromosomes. The four gene clusters are mapped to 2p2.1 (HOXA), 3q3.1 (HOXB), 1q3.1 (HOXC) and 7q1.3--> q1.4 (HOXD). We confirm partial homologies already detected by genetic mapping between chicken chromosomes 1, 2 and 7 and human chromosomes 12, 7 and 2 and we describe a new conserved segment between chicken chromosome 3 and human chromosome 17. These results represent the first data that confirm the physical linkage between chicken HOX genes and may improve our understanding of phylogenetic relationships and genome evolution.

Frank Reifers, Jan Adams, Ivor J. Mason, Stefan Schulte-Merker, Michael Brand
Overlapping and distinct functions provided by fgf17, a new zebrafish member of the Fgf8/17/18 subgroup of Fgfs.
Mech Dev, 99(1-2) 39-49 (2000)
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Members of the fibroblast growth factor (Fgf) family are important signaling molecules in several inductive and patterning processes, and act as brain organizer-derived signals during formation of the early vertebrate nervous system. We isolated a new member of the Fgf8/17/18 subgroup of Fgfs from the zebrafish, and studied its expression and function during somitogenesis, optic stalk and midbrain-hindbrain boundary (MHB) development. In spite of a slightly higher aminoacid similarity to Fgf8, expression analysis and mapping to a chromosome stretch that is syntenic with mammalian chromosomes shows that this gene is orthologous to mammalian Fgf17. These data provide a further example of conserved chromosomal organization between zebrafish and mammalian genomes. Using an mRNA injection assay, we show that fgf17 can act similar to fgf8 during gastrulation, when fgf17 is not normally expressed. Direct comparison of the expression patterns of fgf17 and fgf8 suggest however a possible cooperation of these Fgfs at later stages in several tissues requiring Fgf signaling. Analysis of zebrafish MHB mutants demonstrates a gene-dosage dependent requirement of fgf17 expression for the no isthmus// pax2.1 gene, showing that no isthmus/pax2.1 functions upstream of fgf17 at the MHB in a haplo-insufficient manner, similar to what has been reported for mammalian pax2 mutants. In contrast, only maintenance of fgf17 expression is disturbed at the MHB of acerebellar/fgf8 mutants. Consistent with a requirement for fgf8 function, implantation of FGF8-soaked beads induces fgf17 expression, and expression is upregulated in aussicht mutants, which display upregulation of the Fgf8 signaling pathway. Taken together, our results argue that Fgf8 and Fgf17 act as hierarchically organized signaling molecules during development of the MHB organizer and possibly other organizers in the developing nervous system.

Pierre Gönczy, Christophe J. Echeverri, Karen Oegema, A Coulson, Steven J. M. Jones, Richard R. Copley, John Duperon, Jeffrey Oegema, Michael Brehm, E Cassin, Eva Hannak, Matthew Kirkham, Silke Pichler, Kathrin Flohrs, A Goessen, Sebastian Leidel, Anne-Marie Alleaume, Cecilie Martin, Nurhan Ozlü, Peer Bork, Anthony A. Hyman
Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III.
Nature, 408(6810) 331-336 (2000)
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Genome sequencing projects generate a wealth of information; however, the ultimate goal of such projects is to accelerate the identification of the biological function of genes. This creates a need for comprehensive studies to fill the gap between sequence and function. Here we report the results of a functional genomic screen to identify genes required for cell division in Caenorhabditis elegans. We inhibited the expression of approximately 96% of the approximately 2,300 predicted open reading frames on chromosome III using RNA-mediated interference (RNAi). By using an in vivo time-lapse differential interference contrast microscopy assay, we identified 133 genes (approximately 6%) necessary for distinct cellular processes in early embryos. Our results indicate that these genes represent most of the genes on chromosome III that are required for proper cell division in C. elegans embryos. The complete data set, including sample time-lapse recordings, has been deposited in an open access database. We found that approximately 47% of the genes associated with a differential interference contrast phenotype have clear orthologues in other eukaryotes, indicating that this screen provides putative gene functions for other species as well.

Maja Adamska, Sophie Léger, Michael Brand, Thorsten Hadrys, Thomas Braun, Eva Bober
Inner ear and lateral line expression of a zebrafish Nkx5-1 gene and its downregulation in the ears of FGF8 mutant, ace.
Mech Dev, 97(1-2) 161-165 (2000)
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An orthologue of the mouse homeobox gene Nkx5-1 was cloned and characterized in the zebrafish. As in the mouse and chick, the zebrafish Nkx5-1 gene is expressed in the ear placode and vesicle and in cells forming the vestibulo-acoustic ganglion. In addition, a novel expression domain, the lateral line, appears in the zebrafish, supporting a common precursor hypothesis for these two organs. In the FGF8 zebrafish mutant ace, expression of Nkx5-1 in the otic structures is diminished. The most significant reduction of zfNkx5-1 expression was observed in cells of the vestibulo-acoustic ganglion.

Sanna Lehtonen, Ari Ora, Vesa M Olkkonen, Lin Geng, Marino Zerial, Stefan Somlo, Eero Lehtonen
In vivo interaction of the adapter protein CD2-associated protein with the type 2 polycystic kidney disease protein, polycystin-2.
J Biol Chem, 275(42) 32888-32893 (2000)
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We identified a developmentally regulated gene from mouse kidney whose expression is up-regulated in metanephrogenic mesenchyme cells when they are induced to differentiate to epithelial cells during kidney organogenesis. The deduced 70.5-kDa protein, originally named METS-1 (mesenchyme-to-epithelium transition protein with SH3 domains), has since been cloned as a CD2-associated protein (CD2AP). CD2AP is strongly expressed in glomerular podocytes, and the absence of CD2AP in mice results in congenital nephrotic syndrome. We have found that METS-1/CD2AP (hereafter referred to as CD2AP) is expressed at lower levels in renal tubular epithelial cells in the adult kidney, particularly in distal nephron segments. Independent yeast two-hybrid screens using the COOH-terminal region of either CD2AP or polycystin-2 as bait identified the COOH termini of polycystin-2 and CD2AP, respectively, as strong interacting partners. This interaction was confirmed in cultured cells by co-immunoprecipitation of endogenous polycystin-2 with endogenous CD2AP and vice versa. CD2AP shows a diffuse reticular cytoplasmic and perinuclear pattern of distribution, similar to polycystin-2, in cultured cells, and the two proteins co-localize by indirect double immunofluorescence microscopy. CD2AP is an adapter molecule that associates with a variety of membrane proteins to organize the cytoskeleton around a polarized site. Such a function fits well with that hypothesized for the polycystin proteins in renal tubular epithelial cells, and the present findings suggest that CD2AP has a role in polycystin-2 function.

Martin Klemke, H. Amalia Pasolli, Ralph H. Kehlenbach, Stefan Offermanns, Günter Schultz, Wieland B. Huttner
Characterization of the extra-large G protein alpha-subunit XLalphas. II. Signal transduction properties.
J Biol Chem, 275(43) 33633-33640 (2000)
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In the preceding paper (Pasolli, H. A., Klemke, M., Kehlenbach, R. H. , Wang, Y., and Huttner, W. B. (2000) J. Biol. Chem. 275, 33622-33632), we report on the tissue distribution and subcellular localization of XLalphas (extra large alphas), a neuroendocrine-specific, plasma membrane-associated protein consisting of a novel 37-kDa XL domain followed by a 41-kDa alphas domain encoded by exons 2-13 of the Galphas gene. Here, we have studied the signal transduction properties of XLalphas. Like Galphas, XLalphas undergoes a conformational change upon binding of GTPgammaS (guanosine 5'-O-(thio)triphosphate), as revealed by its partial resistance to tryptic digestion, which generated the same fragments as in the case of Galphas. Two approaches were used to analyze XLalphas-betagamma interactions: (i) ADP-ribosylation by cholera toxin to detect even weak or transient XLalphas-betagamma interactions and (ii) sucrose density gradient centrifugation to reveal stable heterotrimer formation. The addition of betagamma subunits resulted in an increased ADP-ribosylation of XLalphas as well as an increased sedimentation rate of XLalphas in sucrose density gradients, indicating that XLalphas interacts with the betagamma dimer. Surprisingly, however, XLalphas, in contrast to Galphas, was not activated by the beta2-adrenergic receptor upon reconstitution of S49cyc(-) membranes. Similarly, using photoaffinity labeling of pituitary membranes with azidoanilide-GTP, XLalphas was not activated upon stimulation of pituitary adenylyl cyclase-activating polypeptide (PACAP) receptors or other Galphas-coupled receptors known to be present in these membranes, whereas Galphas was. Despite the apparent inability of XLalphas to undergo receptor-mediated activation, XLalphas-GTPgammaS markedly stimulated adenylyl cyclase in S49cyc(-) membranes. Moreover, transfection of PC12 cells with a GTPase-deficient mutant of XLalphas, XLalphas-Q548L, resulted in a massive increase in adenylyl cyclase activity. Our results suggest that in neuroendocrine cells, the two related G proteins, Galphas and XLalphas, exhibit distinct properties with regard to receptor-mediated activation but converge onto the same effector system, adenylyl cyclase.

V Jantsch-Plunger, Pierre Gönczy, Alper Romano, Heinke Schnabel, Danielle Hamill, Ralf Schnabel, Anthony A. Hyman, Michael Glotzer
CYK-4: A Rho family gtpase activating protein (GAP) required for central spindle formation and cytokinesis.
J Cell Biol, 149(7) 1391-1404 (2000)
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During cytokinesis of animal cells, the mitotic spindle plays at least two roles. Initially, the spindle positions the contractile ring. Subsequently, the central spindle, which is composed of microtubule bundles that form during anaphase, promotes a late step in cytokinesis. How the central spindle assembles and functions in cytokinesis is poorly understood. The cyk-4 gene has been identified by genetic analysis in Caenorhabditis elegans. Embryos from cyk-4(t1689ts) mutant hermaphrodites initiate, but fail to complete, cytokinesis. These embryos also fail to assemble the central spindle. We show that the cyk-4 gene encodes a GTPase activating protein (GAP) for Rho family GTPases. CYK-4 activates GTP hydrolysis by RhoA, Rac1, and Cdc42 in vitro. RNA-mediated interference of RhoA, Rac1, and Cdc42 indicates that only RhoA is essential for cytokinesis and, thus, RhoA is the likely target of CYK-4 GAP activity for cytokinesis. CYK-4 and a CYK-4:GFP fusion protein localize to the central spindle and persist at cell division remnants. CYK-4 localization is dependent on the kinesin-like protein ZEN-4/CeMKLP1 and vice versa. These data suggest that CYK-4 and ZEN-4/CeMKLP1 cooperate in central spindle assembly. Central spindle localization of CYK-4 could accelerate GTP hydrolysis by RhoA, thereby allowing contractile ring disassembly and completion of cytokinesis.

Shantha Shanmugalingam, Corinne Houart, Alexander Picker, Frank Reifers, Rachel Macdonald, A Barth, Kevin Griffin, Michael Brand, Stephen W. Wilson
Ace/Fgf8 is required for forebrain commissure formation and patterning of the telencephalon.
Development, 127(12) 2549-2561 (2000)
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Fibroblast growth factors (Fgfs) form a large family of secreted signalling proteins that have a wide variety of roles during embryonic development. Within the central nervous system (CNS) Fgf8 is implicated in patterning neural tissue adjacent to the midbrain-hindbrain boundary. However, the roles of Fgfs in CNS tissue rostral to the midbrain are less clear. Here we examine the patterning of the forebrain in zebrafish embryos that lack functional Fgf8/Ace. We find that Ace is required for the development of midline structures in the forebrain. In the absence of Ace activity, midline cells fail to adopt their normal morphology and exhibit altered patterns of gene expression. This disruption to midline tissue leads to severe commissural axon pathway defects, including misprojections from the eye to ectopic ipsilateral and contralateral targets. Ace is also required for the differentiation of the basal telencephalon and several populations of putative telencephalic neurons but not for overall regional patterning of forebrain derivatives. Finally, we show that ace expression co-localises with anterior neural plate cells that have previously been shown to have forebrain patterning activity. Removal of these cells leads to a failure in induction of ace expression indicating that loss of Ace activity may contribute to the phenotypes observed when anterior neural plate cells are ablated. However, as ace mutant neural plate cells still retain at least some inductive activity, then other signals must also be produced by the anterior margin of the neural plate.

Arindam Majumdar, Klaus Lun, Michael Brand, Iain A. Drummond
Zebrafish no isthmus reveals a role for pax2.1 in tubule differentiation and patterning events in the pronephric primordia.
Development, 127(10) 2089-2098 (2000)
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Pax genes are important developmental regulators and function at multiple stages of vertebrate kidney organogenesis. In this report, we have used the zebrafish pax2.1 mutant no isthmus to investigate the role for pax2.1 in development of the pronephros. We demonstrate a requirement for pax2.1 in multiple aspects of pronephric development including tubule and duct epithelial differentiation and cloaca morphogenesis. Morphological analysis demonstrates that noi(- )larvae specifically lack pronephric tubules while glomerular cell differentiation is unaffected. In addition, pax2.1 expression in the lateral cells of the pronephric primordium is required to restrict the domains of Wilms' tumor suppressor (wt1) and vascular endothelial growth factor (VEGF) gene expression to medial podocyte progenitors. Ectopic podocyte-specific marker expression in pronephric duct cells correlates with loss of expression of the pronephric tubule and duct-specific markers mAb 3G8 and a Na(+)/K(+) ATPase (&agr;)1 subunit. The results suggest that the failure in pronephric tubule differentiation in noi arises from a patterning defect during differentiation of the pronephric primordium and that mutually inhibitory regulatory interactions play an important role in defining the boundary between glomerular and tubule progenitors in the forming nephron.

Silke Pichler, Pierre Gönczy, Heinke Schnabel, Andrei I. Pozniakovsky, Anthony J. Ashford, Ralf Schnabel, Anthony A. Hyman
OOC-3, a novel putative transmembrane protein required for establishment of cortical domains and spindle orientation in the P(1) blastomere of C. elegans embryos.
Development, 127(10) 2063-2073 (2000)
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Asymmetric cell divisions require the establishment of an axis of polarity, which is subsequently communicated to downstream events. During the asymmetric cell division of the P(1) blastomere in C. elegans, establishment of polarity depends on the establishment of anterior and posterior cortical domains, defined by the localization of the PAR proteins, followed by the orientation of the mitotic spindle along the previously established axis of polarity. To identify genes required for these events, we have screened a collection of maternal-effect lethal mutations on chromosome II of C. elegans. We have identified a mutation in one gene, ooc-3, with mis-oriented division axes at the two-cell stage. Here we describe the phenotypic and molecular characterization of ooc-3. ooc-3 is required for the correct localization of PAR-2 and PAR-3 cortical domains after the first cell division. OOC-3 is a novel putative transmembrane protein, which localizes to a reticular membrane compartment, probably the endoplasmic reticulum, that spans the whole cytoplasm and is enriched on the nuclear envelope and cell-cell boundaries. Our results show that ooc-3 is required to form the cortical domains essential for polarity after cell division.

M D Adams, Susan E Celniker, Robert A Holt, Cheryl A Evans, Jeannine D Gocayne, Peter G Amanatides, Steven E Scherer, P W Li, Roger A Hoskins, R F Galle, Reed A George, S E Lewis, Stephen Richards, Michael Ashburner, S Henderson, Granger G Sutton, Jennifer R Wortman, Mark D Yandell, Qinyu Zhang, L X Chen, R C Brandon, Y H Rogers, R G Blazej, Mark Champe, B D Pfeiffer, Kenneth Wan, C Doyle, E G Baxter, G Helt, C R Nelson, G L Gabor, Josep F Abril, A Agbayani, Hui-Jin An, C Andrews-Pfannkoch, Danita Baldwin, R M Ballew, A Basu, J Baxendale, L Bayraktaroglu, E M Beasley, Karen Y Beeson, P V Benos, Benjamin P Berman, D Bhandari, S Bolshakov, D Borkova, M R Botchan, J Bouck, P Brokstein, P Brottier, K C Burtis, D Busam, H Butler, E Cadieu, Angela Center, I Chandra, J M Cherry, S Cawley, Carl E Dahlke, Lionel B Davenport, P Davies, B de Pablos, A L Delcher, Zuoming Deng, A Mays, I M Dew, Susanne M Dietz, Kristina L Dodson, Lisa Doup, M Downes, S Dugan-Rocha, B C Dunkov, P Dunn, K J Durbin, Carlos C Evangelista, C Ferraz, Steven Ferriera, W Fleischmann, Carl R Fosler, Andrei E Gabrielian, N Garg, W M Gelbart, K Glasser, Anna Glodek, F Gong, J H Gorrell, Z Gu, Ping Guan, M Harris, N L Harris, D Harvey, T J Heiman, J R Hernandez, Jarrett T Houck, D Hostin, K A Houston, Tim Howland, M H Wei, Chinyere Ibegwam, Mena Jalali, Francis Kalush, Gary H Karpen, Z Ke, J A Kennison, K A Ketchum, B E Kimmel, Chinnappa Kodira, C Kraft, S A Kravitz, D Kulp, Z Lai, P Lasko, Y Lei, Alex Levitsky, J Li, Z Li, Y Liang, X Lin, X Liu, B Mattei, T C McIntosh, M P McLeod, D McPherson, Gennady V Merkulov, Natalia V Milshina, C M Mobarry, J Morris, A Moshrefi, S M Mount, M Moy, B Murphy, L Murphy, Donna M Muzny, D L Nelson, D R Nelson, K A Nelson, K Nixon, D R Nusskern, Joanne M Pacleb, M Palazzolo, G S Pittman, S Pan, J Pollard, V Puri, M G Reese, Knut Reinert, K A Remington, R D Saunders, F Scheeler, H Shen, Bixiong Chris Shue, I Sidén-Kiamos, M Simpson, M Skupski, T Smith, E Spier, A C Spradling, Mark Stapleton, Renee Strong, E Sun, Robert R Svirskas, C Tector, R Turner, E Venter, A H Wang, X Wang, Z Y Wang, D A Wassarman, George Weinstock, Jean Weissenbach, S M Williams, S M WoodageT, K C Worley, D Wu, S Yang, Q A Yao, Jane Ye, R F Yeh, J S Zaveri, M Zhan, G Zhang, Q Zhao, L Zheng, X H Zheng, F N Zhong, W Zhong, X Zhou, S Zhu, X Zhu, H O Smith, Richard A Gibbs, E W Myers, G M Rubin, J C Venter
The genome sequence of Drosophila melanogaster.
Science, 287(5461) 2185-2195 (2000)
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The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.

Frank Reifers, Emily C. Walsh, Sophie Léger, Didier Y.R. Stainier, Michael Brand
Induction and differentiation of the zebrafish heart requires fibroblast growth factor 8 (fgf8/acerebellar).
Development, 127(2) 225-235 (2000)
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Vertebrate heart development is initiated from bilateral lateral plate mesoderm that expresses the Nkx2.5 and GATA4 transcription factors, but the extracellular signals specifying heart precursor gene expression are not known. We describe here that the secreted signaling factor Fgf8 is expressed in and required for development of the zebrafish heart precursors, particularly during initiation of cardiac gene expression. fgf8 is mutated in acerebellar (ace) mutants, and homozygous mutant embryos do not establish normal circulation, although vessel formation is only mildly affected. In contrast, heart development, in particular of the ventricle, is severely abnormal in acerebellar mutants. Several findings argue that Fgf8 has a direct function in development of cardiac precursor cells: fgf8 is expressed in cardiac precursors and later in the heart ventricle. Fgf8 is required for the earliest stages of nkx2.5 and gata4, but not gata6, expression in cardiac precursors. Cardiac gene expression is restored in acerebellar mutant embryos by injecting fgf8 RNA, or by implanting a Fgf8-coated bead into the heart primordium. Pharmacological inhibition of Fgf signalling during formation of the heart primordium phenocopies the acerebellar heart phenotype, confirming that Fgf signaling is required independently of earlier functions during gastrulation. These findings show that fgf8/acerebellar is required for induction and patterning of myocardial precursors.

Marion A. Maw, Denis Corbeil, Julia Koch, Andrea Hellwig, Jane C. Wilson-Wheeler, Robyn J. Bridges, Govindasamy Kumaramanickavel, Sheila John, Derek Nancarrow, Katja Röper, Anja Weigmann, Wieland B. Huttner, Michael J. Denton
A frameshift mutation in prominin (mouse)-like 1 causes human retinal degeneration.
Hum Mol Genet, 9(1) 27-34 (2000)
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The disks of vertebrate photoreceptors are produced by outgrowths of the plasma membrane. Hence genes that encode retinal proteins targeted to plasma membrane protrusions represent candidates for inherited retinal degenerations. One such candidate is the gene encoding human prominin (mouse)-like 1 (PROML1, previously known as AC133 antigen) which belongs to the prominin family of 5-transmembrane domain proteins. Murine prominin (prom) shows a strong preference for plasma membrane protrusions in a variety of epithelial cells whereas PROML1 is expressed in retinoblastoma cell lines and adult retina. In the present study, molecular genetic analyses of a pedigree segregating for autosomal recessive retinal degeneration indicated that the affected individuals were homozygous for a nucleotide 1878 deletion in PROML1. This alteration is predicted to result in a frameshift at codon 614 with premature termination of translation. Expression of a similar prom deletion mutant in CHO cells indicated that the truncated protein does not reach the cell surface. Immunocytochemistry revealed that prom is concentrated in the plasma membrane evaginations at the base of the outer segments of rod photoreceptors. These findings suggest that loss of prominin causes retinal degeneration, possibly because of impaired generation of the evaginations and/or impaired conversion of the evaginations to disks.

Pierre Gönczy, Silke Pichler, Matthew Kirkham, Anthony A. Hyman
Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage Caenorhabditis elegans embryo.
J Cell Biol, 147(1) 135-150 (1999)
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We have investigated the role of cytoplasmic dynein in microtubule organizing center (MTOC) positioning using RNA-mediated interference (RNAi) in Caenorhabditis elegans to deplete the product of the dynein heavy chain gene dhc-1. Analysis with time-lapse differential interference contrast microscopy and indirect immunofluorescence revealed that pronuclear migration and centrosome separation failed in one cell stage dhc-1 (RNAi) embryos. These phenotypes were also observed when the dynactin components p50/dynamitin or p150(Glued) were depleted with RNAi. Moreover, in 15% of dhc-1 (RNAi) embryos, centrosomes failed to remain in proximity of the male pronucleus. When dynein heavy chain function was diminished only partially with RNAi, centrosome separation took place, but orientation of the mitotic spindle was defective. Therefore, cytoplasmic dynein is required for multiple aspects of MTOC positioning in the one cell stage C. elegans embryo. In conjunction with our observation of cytoplasmic dynein distribution at the periphery of nuclei, these results lead us to propose a mechanism in which cytoplasmic dynein anchored on the nucleus drives centrosome separation.

Marcos González-Gaitán, Herbert Jäckle
The range of spalt-activating Dpp signalling is reduced in endocytosis-defective Drosophila wing discs.
Mech Dev, 87(1-2) 143-151 (1999)
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Pattern formation along the anterior-posterior (A/P) axis of the developing Drosophila wing depends on Decapentaplegic (Dpp), a member of the conserved transforming growth factor beta (TGFbeta) family of secreted proteins. Dpp is expressed in a stripe along the A/P compartment boundary of the wing imaginal disc and forms a long-range concentration gradient with morphogen-like properties which generates distinct cell fates along the A/P axis. We have monitored Dpp expression and Dpp signalling in endocytosis-mutant wing imaginal discs which develop severe pattern defects specifically along the A/P wing axis. The results show that the size of the Dpp expression domain is expanded in endocytosis-mutant wing discs. However, this expansion did not result in a concomitant expansion of the functional range of Dpp activity but rather its reduction as indicated by the reduced expression domain of the Dpp target gene spalt. The data suggest that clathrin-mediated endocytosis, a cellular process necessary for membrane recycling and vesicular trafficking, participates in Dpp action during wing development. Genetic interaction studies suggest a link between the Dpp receptors and clathrin. Impaired endocytosis does not interfere with the reception of the Dpp signal or the intracellular processing of the mediation of the signal in the responder cells, but rather affects the secretion and/or the distribution of Dpp in the developing wing cells.

N Paricio, Fabian Feiguin, Michael Boutros, Suzanne Eaton, Marek Mlodzik
The Drosophila STE20-like kinase misshapen is required downstream of the Frizzled receptor in planar polarity signaling.
EMBO J, 18(17) 4669-4678 (1999)
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The Drosophila misshapen (msn) gene is a member of the STE20 kinase family. We show that msn acts in the Frizzled (Fz) mediated epithelial planar polarity (EPP) signaling pathway in eyes and wings. Both msn loss- and gain-of-function result in defective ommatidial polarity and wing hair formation. Genetic and biochemical analyses indicate that msn acts downstream of fz and dishevelled (dsh) in the planar polarity pathway, and thus implicates an STE20-like kinase in Fz/Dsh-mediated signaling. This demonstrates that seven-pass transmembrane receptors can signal via members of the STE20 kinase family in higher eukaryotes. We also show that Msn acts in EPP signaling through the JNK (Jun-N-terminal kinase) module as it does in dorsal closure. Although at the level of Fz/Dsh there is no apparent redundancy in this pathway, the downstream effector JNK/MAPK (mitogen-activated protein kinase) module is redundant in planar polarity generation. To address the nature of this redundancy, we provide evidence for an involvement of the related MAP kinases of the p38 subfamily in planar polarity signaling downstream of Msn.

Anne Grapin-Botton, F Cambronero, H L Weiner, M A Bonnin, L Puelles, N M Le Douarin
Patterning signals acting in the spinal cord override the organizing activity of the isthmus.
Mech Dev, 84(1-2) 41-53 (1999)
DOI

The regionalization of the neural tube along the anteroposterior axis is established through the action of patterning signals from the endomesoderm including the organizer. These signals set up a pre-pattern which is subsequently refined through local patterning events. The midbrain-hindbrain junction, or isthmus, is endowed with such an organizing activity. It is able to induce graded expression of the Engrailed protein in the adjacent mesencephalon and rhombencephalon, and subsequently elicits the development of tectal and cerebellar structures. Ectopically grafted isthmus was also shown to induce Engrailed expression in diencephalon and otic and pre-otic rhombencephalon. Fgf8 is a signalling protein which is produced by the isthmus and which is able to mimic most isthmic properties. We show here that the isthmus, when transposed to the level of either rhombomere 8 or the spinal cord, loses its ability to induce Engrailed and cerebellar development in adjacent tissues. This is accompanied by the down-regulation of fgf8 expression in the grafted isthmus and by the up-regulation of a marker of the recipient site, Hoxb-4. Moreover, these changes in gene activity in the transplant are followed by a transformation of the fate of the grafted cells which adjust to their novel environment. These results show that the fate of the isthmus is not determined at 10-somite stage and that the molecular loop of isthmic maintenance can be disrupted by exogenous signals.

Jochen Wittbrodt, Michael Brand
Zebrafisch und Medaka als Modellorganismen der Entwicklungsbiologie
BIOspektrum, 6 437-442 (1999)
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Die Genomsequenzierungen erzeugen eine Fï¿½lle neuer Sequenzdaten aller in den Genomen der verschiedenen Modellorganismen enthaltenen Gene. Wie aber kann die Funktion dieser Gene ermittelt werden, und wie kann die Information zwischen den Organismen verglichen werden, um Entwicklung und Evolution zu verstehen? In den genetisch gut zugï¿½nglichen Invertebraten-Modellorganismen mit ihren etwa 19000 (C. elegans) beziehungsweise rund 12000 Genen (Drosophila) ist das Verstï¿½ndnis von Entwicklungsprozessen bereits weit gediehen. Bei Vertrebraten mit ihren ca. 70000 bis 100000 Genen ist dies aufgrund der grï¿½ï¿½eren Komplexitï¿½t der Entwicklungsprozesse und der grï¿½ï¿½eren Zahl der Gene ungleich schwieriger - die Antworten sind jedoch fï¿½r Grundlagenforschung und Medizin gleichermaï¿½en von Bedeutung. Zwei Modellorganismen der Entwicklunsbiologie, Zebrafisch und Medaka, bieten die Mï¿½glichkeit zur integrierten molekularen, klassich-genetischen und experimentellen Analyse und eignen sich daher hervorragend fï¿½r eine detaillierte mechanistische und schlieï¿½lich vergeichende Analyse des Vertrebraten-Genoms. Als Beispiel diskutieren wir unsere Untersuchungen zur Entwicklung des Auges beziehungsweise der zentralnervï¿½sen Schaltstellen visueller Information im Vorder- und Mittelhirn.

G Couly, Anne Grapin-Botton, P Coltey, B Ruhin, N M Le Douarin
Determination of the identity of the derivatives of the cephalic neural crest: incompatibility between Hox gene expression and lower jaw development.
Development, 125(17) 3445-3459 (1998)

In addition to pigment cells, and neural and endocrine derivatives, the neural crest is characterized by its ability to yield mesenchymal cells. In amniotes, this property is restricted to the cephalic region from the mid-diencephalon to the end of rhombomere 8 (level of somites 4/5). The cephalic neural crest is divided into two domains: an anterior region corresponding to the diencephalon, mesencephalon and metencephalon (r1, r2) in which expression of Hox genes is never observed, and a posterior domain in which neural crest cells exhibit (with a few exceptions) the same Hox code as the rhombomeres from which they originate. By altering the normal distribution of neural crest cells in the branchial arches through appropriate embryonic manipulations, we have investigated the relationships between Hox gene expression and the level of plasticity that neural crest cells display when they are led to migrate to an ectopic environment. We made the following observations. (i) Hox gene expression is not altered in neural crest cells by their transposition to ectopic sites. (ii) Expression of Hox genes by the BA ectoderm does not depend upon an induction by the neural crest. This second finding further supports the concept of segmentation of the cephalic ectoderm into ectomeres (Couly and Le Douarin, 1990). According to this concept, metameres can be defined in large bands of ectoderm including not only the CNS and the neural crest but also the corresponding superficial ectoderm fated to cover craniofacial primordia. (iii) The construction of a lower jaw requires the environment provided by the ectomesodermal components of BA1 or BA2 associated with the Hox gene non-expressing neural crest cells. Hox gene-expressing neural crest cells are unable to yield the lower jaw apparatus including the entoglossum and basihyal even in the BA1 environment. In contrast, the posterior part of the hyoid bone can be constructed by any region of the neural crest cells whether or not they are under the regulatory control of Hox genes. Such is also the case for the neural and connective tissues (including those comprising the cardiovascular system) of neural crest origin, upon which no segmental restriction is imposed. The latter finding confirms the plasticity observed 24 years ago (Le Douarin and Teillet, 1974) for the precursors of the PNS.

Roland Beisswanger, Denis Corbeil, Christian Vannier, Christoph Thiele, Ulrike Dohrmann, Roland Kellner, Keith Ashman, Christof Niehrs, Wieland B. Huttner
Existence of distinct tyrosylprotein sulfotransferase genes: molecular characterization of tyrosylprotein sulfotransferase-2.
Proc Natl Acad Sci U.S.A., 95(19) 11134-11139 (1998)
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Tyrosylprotein sulfotransferase (TPST) is a 54- to 50-kDa integral membrane glycoprotein of the trans-Golgi network found in essentially all tissues investigated, catalyzing the tyrosine O-sulfation of soluble and membrane proteins passing through this compartment. Here we describe (i) an approach to identify the TPST protein, referred to as MSC (modification after substrate crosslinking) labeling, which is based on the crosslinking of a substrate peptide to TPST followed by intramolecular [35S]sulfate transfer from the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (PAPS); and (ii) the molecular characterization of a human TPST, referred to as TPST-2, whose sequence is distinct from that reported [TPST-1; Ouyang, Y.-B., Lane, W. S. & Moore, K. L. (1998) Proc. Natl. Acad. Sci. USA 95, 2896-2901] while this study was in progress. Human TPST-2 is a type II transmembrane protein of 377 aa residues that is encoded by a ubiquitously expressed 1.9-kb mRNA originating from seven exons of a gene located on chromosome 22 (22q12.1). A 304-residue segment in the luminal domain of TPST-2 shows 75% amino acid identity to the corresponding segment of TPST-1, including conservation of the residues implicated in the binding of PAPS. Expression of the TPST-2 cDNA in CHO cells resulted in an approximately 13-fold increase in both TPST protein, as determined by MSC labeling, and TPST activity. A predicted 359-residue type II transmembrane protein in Caenorhabditis elegans with 45% amino acid identity to TPST-2 in a 257-residue segment of the luminal domain points to the evolutionary conservation of the TPST protein family.

Klaus Lun, Michael Brand
A series of no isthmus (noi) alleles of the zebrafish pax2.1 gene reveals multiple signaling events in development of the midbrain-hindbrain boundary.
Development, 125(16) 3049-3062 (1998)
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Generation of cell diversity in the vertebrate central nervous system starts during gastrulation stages in the ectodermal germ layer and involves specialized cell groups, such as the organizer located at the midbrain-hindbrain boundary (MHB). Mutations in the zebrafish no isthmus (noi) gene alter development of the MHB, and affect the pax2.1 gene (formerly pax(zf-b)). Analysis of the structure of pax2.1 reveals at least 12 normal splice variants. The noi alleles can be arranged, by molecular and phenotypic criteria, into a series of five alleles of differing strength, ranging from a null allele to weak alleles. In keeping with a role in development of the MHB organizer, gene expression is already affected in the MHB primordium of the gastrula neural ectoderm in noi mutants. eng3 activation is completely and eng2 activation is strongly dependent on noi function. In contrast, onset of wnt1, fgf8 and her5 expression occurs normally in the null mutants, but is eliminated later on. Our observations suggest that three signaling pathways, involving pax2.1, wnt1 and fgf8, are activated independently in early anterior-posterior patterning of this area. In addition, analysis of the allelic series unexpectedly suggests that noi activity is also required during dorsal-ventral patterning of the MHB in somitogenesis stages, and possibly in a later eng expression phase. We propose that noi/pax2.1 participates in sequential signaling processes as a key integrator of midbrain-hindbrain boundary development.

Peter L. Pfeffer, Thomas Gerster, Klaus Lun, Michael Brand, Meinrad Busslinger
Characterization of three novel members of the zebrafish Pax2/5/8 family: dependency of Pax5 and Pax8 expression on the Pax2.1 (noi) function.
Development, 125(16) 3063-3074 (1998)
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The mammalian Pax2, Pax5 and Pax8 genes code for highly related transcription factors, which play important roles in embryonic development and organogenesis. Here we report the characterization of all members of the zebrafish Pax2/5/8 family. These genes have arisen by duplications before or at the onset of vertebrate evolution. Due to an additional genome amplification in the fish lineage, the zebrafish contains two Pax2 genes, the previously known Pax[b] gene (here renamed as Pax2.1) and a novel Pax2.2 gene. The zebrafish Pax2.1 gene most closely resembles the mammalian Pax2 gene in its expression pattern, as it is transcribed first in the midbrain-hindbrain boundary region, then in the optic stalk, otic system, pronephros and nephric ducts, and lastly in specific interneurons of the hindbrain and spinal cord. Pax2.2 differs from Pax2.1 by the absence of expression in the nephric system and by a delayed onset of transcription in other Pax2.1 expession domains. Pax8 is also expressed in the same domains as Pax2.1, but its transcription is already initiated during gastrulation in the primordia of the otic placode and pronephric anlage, thus identifying Pax8 as the earliest developmental marker of these structures. The zebrafish Pax5 gene, in contrast to its mouse orthologue, is transcribed in the otic system in addition to its prominent expression at the midbrain-hindbrain boundary. The no isthmus (noi) mutation is known to inactivate the Pax2.1 gene, thereby affecting the development of the midbrain-hindbrain boundary region, pronephric system, optic stalk and otic region. Although the different members of the Pax2/5/8 family may potentially compensate for the loss of Pax2.1 function, we demonstrate here that only the expression of the Pax2.2 gene remains unaffected in noi mutant embryos. The expression of Pax5 and Pax8 is either not initiated at the midbrain-hindbrain boundary or is later not maintained in other expression domains. Consequently, the noi mutation of zebrafish is equivalent to combined inactivation of the mouse Pax2 and Pax5 genes with regard to the loss of midbrain-hindbrain boundary development.

Frank Reifers, Heike Böhli, Emily C. Walsh, Phillip H. Crossley, Didier Y.R. Stainier, Michael Brand
Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis.
Development, 125(13) 2381-2395 (1998)
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We describe the isolation of zebrafish Fgf8 and its expression during gastrulation, somitogenesis, fin bud and early brain development. By demonstrating genetic linkage and by analysing the structure of the Fgf8 gene, we show that acerebellar is a zebrafish Fgf8 mutation that may inactivate Fgf8 function. Homozygous acerebellar embryos lack a cerebellum and the midbrain-hindbrain boundary organizer. Fgf8 function is required to maintain, but not initiate, expression of Pax2.1 and other marker genes in this area. We show that Fgf8 and Pax2.1 are activated in adjacent domains that only later become overlapping, and activation of Fgf8 occurs normally in no isthmus embryos that are mutant for Pax2.1. These findings suggest that multiple signaling pathways are independently activated in the midbrain-hindbrain boundary primordium during gastrulation, and that Fgf8 functions later during somitogenesis to polarize the midbrain. Fgf8 is also expressed in a dorsoventral gradient during gastrulation and ectopically expressed Fgf8 can dorsalize embryos. Nevertheless, acerebellar mutants show only mild dorsoventral patterning defects. Also, in spite of the prominent role suggested for Fgf8 in limb development, the pectoral fins are largely unaffected in the mutants. Fgf8 is therefore required in development of several important signaling centers in the zebrafish embryo, but may be redundant or dispensable for others.

A C Sharman, Michael Brand
Evolution and homology of the nervous system: cross-phylum rescues of otd/Otx genes.
Trends Genet, 14(6) 211-214 (1998)
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C O M M E N T Evolution and homology of the nervous system: cross-phylum rescues of otd/Otx genes ANNA C . SHARMAN AND MICHAEL BRAND anna.sharman@urz.uni-heidelberg.de brand@sun0.urz.uni-heidelberg.de I NSTITUTE OF N EUROBIOLOGY , U NIVERSITY OF H EIDELBERG , I N N EUENHEIMER F ELD 345, 69120 H EIDELBERG , G ERMANY . The homeotic or Hox genes were the first gene family to be shown to act in similar, probably homologous, ways in insect and mammalian development. They are thought to form a combinatorial code specifying the identity of different segments. They are clustered in the genome, and expressed in a nested pattern along the anterior posterior axis in an order that is colinear with their chromosomal order 1 . Since then, perhaps a surprisingly high number of developmental gene families have also been shown to have conserved expression patterns over several phyla. One such family is the Otx genes, which like the Hox genes encode homeodomain-containing DNA-binding proteins 2,3 . Flies and amphioxus have a single gene in the family, called orthodenticle (otd ) in Drosophila, while vertebrates typically have two Otx genes. Mouse Otx1 and Otx2, together with another family of homeobox genes, Emx1 and Emx2, are expressed in nested patterns in the fore- and mid-brain 3 , while their fly homologues otd and ems are expressed in the most anterior segments 4 6 . It has been suggested 3,7 that the Otx and Emx genes specify segmental identity in insects and vertebrates, fulfilling a role for the anterior brain similar to that of the Hox genes in the hindbrain and spinal cord. This hypothesis is supported by knockout phenotypes and mutations 8 12 : in Otx2 +/ Otx1 / mice the midbrain and posterior diencephalon are completely missing (interpretation of single Otx mutant phenotypes is complicated in mice by variability, an early role for Otx2 in gastrulation and by redundancy between Otx1 and Otx2). It also appears that in mammals, Otx function influences development of the midbrain hindbrain boundary, known to be an organizer of cell fate in the midbrain and anterior hindbrain 8 13 . In Emx knockouts discrete parts of the forebrain are missing 14,15 . So expression and mutation of Otx/otd and Hox genes show that the anterior posterior (AP) patterning mechanisms in insects and vertebrates share many features. Does this mean that these mechanisms are homologous, that is, that the common ancestor had these mechanisms too? The fact that amphioxus, a primitive chordate, also has anterior Otx expression 2 indicates that the mechanism is quite widespread, although otd expression in echinoderms 16 is quite different and variable. It is first of all necessary to test whether the genes are acting in the same way in both groups of animals. This can be done experimentally by expressing vertebrate genes in flies mutant for their homologues, or vice versa, and asking whether the foreign gene can rescue the mutant phenotype. This has already been done for some Hox genes 17 19 , showing that they can function in an equivalent manner, but it has not until now been tried for the Otx/otd family. A second factor also needs to be considered. The nervous system of vertebrates has been compared to the early ectoderm of insects, at a stage long before the central nervous system (CNS) is formed. A question mark has hung over interpretations of AP patterning similarities: could these two systems really be homologous, when they referred to such different stages of development? Two responses have been given towards the latter question. One (called the auricularia hypothesis) was to propose that the nervous system of chordates is homologous to the entire outer ectoderm of insects while the insect nervous system and the chordate outer ectoderm have arisen independently 20 . This was supported by studies of the anatomy of larval echinoderms (particularly auricularia larvae) and urochordates, which are proposed to be intermediates in the transition between the arthropodand chordate-type body plans, and by the fact that insect Hox genes are expressed in the surface ectoderm, whereas they are not in vertebrates. The nervous systems of insects and vertebrates have long been assumed to have evolved independently, because they are organized differently and are on opposite sides of the embryo 21 . The other response grew out of recent work showing that the dorsoventral axis is in fact patterned by the same set of genes in similar relationships in insects and in vertebrates, but with the dorsoventral axis inverted 21,22 . Many developmental genes, such as the achaete/scute homologues, Nkx2, Msx and Hox homeobox genes and netrins are expressed in similar patterns in insect and vertebrate nervous systems 21,23 . It is a reasonable hypothesis that the dorsoventral axis inverted at some point in evolution, and thus that the nervous systems of insects and vertebrates are homologous (Fig. 1). Which of these two theories is correct? Four recent papers 24 27 address the question of Otx functional equivalence, and supply further evidence for homology of the insect and vertebrate nervous systems. Three of them 24 26 show that the Otx and otd genes have a conserved function, as well as conserved expression patterns. Previously, the expression of otd and ems in the fly nervous system was determined (Ref. 6 and Fig. 2), and the fourth paper 27 finds that Hox gene expression there is more similar to expression of vertebrate Hox genes than was previously suspected. Functional equivalence of Otx and otd genes Two papers describe the overexpression of human OTX1 and OTX2 genes in flies defective in otd function. Nagao et al. 26 found that the human OTX genes could rescue the ocelliless phenotype (oc is a regulatory mutation causing defective otd expression in the fly pupa) just as well as otd could rescue. Not only the final phenotype, but also gene expression TIG J UNE 1998 V OL . 14 N O . 6 211 Copyright 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00 PII: S0168-9525(98)01488-7 C O M M E N T in the eye-antennal imaginal discs, is rescued. Leuzinger et al. 25 used a similar approach to drive transient ubiquitous expression of otd, OTX1 or OTX2, to look at the better-studied embryonic function of otd. According to several criteria, otd expression can rescue the otd mutant phenotype, though in a variable proportion of embryos. OTX2 also rescues, though at a lower frequency than otd, and OTX1 rescues less efficiently still. The third paper 24 goes in the other direction: the mouse Otx1 coding region was replaced by that of Drosophila otd. Many of the defects of Otx1 / mice were rescued by otd, for example several brain regions previously missing are restored. Interestingly, the rescue was less efficient nearer to the MHB, for example the mesencephalon was never completely normal, and cerebellar foliation remained abnormal. In the sense organs, most defects were rescued, but the lateral semicircular canal in the inner ear was not. Hox genes in the fly nervous system Hirth et al. 27 examine the expression and function of the Drosophila homeotic (Hox) genes in the CNS. In the mouse, the group 2 Hox genes have the most anterior expression boundaries, while group 1 Hox genes are expressed in restricted domains, one rhombomere posterior to the group 2 anterior boundary (Ref. 1 and Fig. 2). This lapse in the rule of colinearity has so far only been noted in vertebrates 28 ; in the Drosophila ectoderm, strict colinearity is adhered to. Previous descriptions of homeotic gene expression in the fly nervous system put the anterior boundary of labial (lab ; a group 1 Hox gene) and proboscipedia (pb ; group 2) at the same level, within the deutocerebrum 29 . However, Hirth et al. have re-examined this expression using neural segmental markers, and now show that lab is actually expressed only in the posterior of brain segment b3 (the tritocerebrum), posterior to the anterior boundary of pb expression in segment b2 (the deutocerebrum) (Ref. 27; Fig. 2). The same exception to colinearity therefore occurs in the fly CNS as well as in vertebrates. Homology of nervous systems It is important to realize that the ability of homologous genes to replace each other functionally does not necessarily show that the regions in which they are expressed are homologous. The Otx and otd gene products, for example, are transcription factors. It is quite possible that the ability of OTX proteins to bind a particular DNA sequence has been conserved, while the downstream genes regulated by this binding have altered. This explanation would still allow the proteins to replace each other, but they would be regulating different downstream genes in each animal. The formal possibility has not been ruled out that expression of otd and Otx in the anterior brain could have arisen independently in insects and chordates. However, it is now clear that the similar expression of Otx genes in the two nervous systems has functional relevance; the similarity cannot be explained by the gain of a few enhancer elements, and thus is less likely to have arisen by convergent evolution. These papers also show that, even though Hox and Otx genes are expressed in the early blastoderm in flies, this does not (as the auricularia hypothesis 20 suggests) mean that the ectoderm wall is homologous to the vertebrate neural tube, because the same genes are also expressed in the fly CNS, and in a very similar manner to vertebrates. Together with other evidence 17,22,30,31 , these results might be the final nail in the coffin for theories like the auricularia hypothesis that support an independent origin for the insect and vertebrate nervous systems. Significance of otd/Otx functional equivalence The fly otd gene can replace mouse Otx1 astoundingly well. We would have perhaps expected that Otx1 would have evolved new functions to do with vertebrate-specific developmental programs, that otd could not perform. It appears that only the homeobox and the regulatory elements, plus perhaps some less well conserved regions like the acidic activation domains, are sufficient for most of the functions of Otx1. New expression domains acquired since the arthropod/vertebrate split might account for much of the differences between otd and Otx1; these differences can only be detected experimentally by examining and possibly replacing regulatory elements rather than just coding regions. Replacement of regions of the gene outside the homeobox would test whether these regions, or only the homeobox, is important. Although otd can replace most functions of Otx1, some cannot be replaced. This could simply be a quantitative effect: Acampora et al. 9 have shown that in brain development Otx `dosage' is more important than which individual Otx gene is present, although Otx2 does appear to be more `potent' than Otx1, since the Otx2 mutant has a stronger phenotype. In the rescue experiments of Leuzinger et al. 25 , Otx1 is less effective at rescuing the otd phenotype than Otx2. otd could simply be less TIG J UNE 1998 V OL . 14 N O . 6 212 dpp dpp sog Ventral Dorsal Drosophila Ventral Dorsal Mouse msh vnd vnd ms h ms x Nkx2 Nkx2 msx chordin Bmp4 Bmp4 Midline, netrin + AS-C/ash F IGURE 1. Transverse sections through the fly and vertebrate central nervous system primordia, showing similar dorsoventral regulation of pattern by the sog (short gastrulation)/chordin, dpp (decapentaplegic)/BMP4, Msx/msh, Nkx2/vnd, AS-C (achaete-scute complex)/ash (AS-C homologues) and netrin gene families. (Redrawn from Ref. 23, with extra information from Ref. 22.) C O M M E N T `potent' in the mouse than either Otx1 or Otx2, as is supported by the fact that mice with two copies of Otx2 and no Otx1 show a weaker phenotype than mice 24 with one copy of each gene (Ref. 32). Some of the lack of rescue cannot be explained in this way, however, and could reflect truly novel functions of Otx1 that depend on its coding region. One example is the lateral semicircular canal phenotype of Otx1, which cannot be rescued by otd, suggesting that Otx1 has taken on a role in development of this canal in the inner ear after duplication. Significantly, this canal evolved around the time when the Otx genes are thought to have duplicated, at the origin of jawed vertebrates 33 (agnathan fish have only two semicircular canals, and lack the lateral one 24 ). The evolution of the midbrain hindbrain boundary A second way in which otd cannot replace Otx1 is shown in the graded level of rescue in the brain: the midbrain hindbrain boundary (MHB), midbrain and cerebellum, at the posterior of the Otx domain, are most sensitive to a low level of otd/Otx function and thus can be less easily rescued, while the telencephalon (at the anterior of the Otx domain) is least sensitive 24 . The vertebrate MHB is known to act as an organizer, producing signals that pattern the midbrain and anterior hindbrain 35,36 , but no such organizer has been described in the brain of Drosophila. Like the lateral semicircular canal, the organizer may have arisen early in vertebrate evolution, since lampreys and all jawed vertebrates, but not hagfish or cephalochordates, have an undeniable midbrain and cerebellum 34 . Thus the inability of Drosophila otd to rescue the midbrain hindbrain boundary phenotype of mouse Otx1 may be because the MHB is a new structure that evolved, or was at least greatly elaborated, early in the vertebrate lineage. The Otx genes themselves may have helped in the evolution of the MHB organizer; changing Otx expression patterns could have produced a gap between Otx and Hox expression domains (otd and Hox are adjacent in the fly; Fig. 2), which was then filled by MHB-specific transcription factors and signalling molecules such as EN, PAX2, PAX5, PAX8, WNT1 and FGF8 (Ref. 36). A thorough analysis of the development of this region in a range of vertebrates is clearly needed. Perspectives The papers reviewed here answer some questions while raising others. The list of known similarities between the vertebrate and arthropod nervous systems has been expanded significantly, suggesting that they are probably homologous structures. If this is so, has the auricularia hypothesis been disproved? Enteropneusts, a group of invertebrate chordates, have both a dorsal neural tube and a ventral nerve plexus, and it has been argued 37 that this shows that the vertebrate dorsal neural tube cannot be homologous to the invertebrate ventral nervous system. However, it is possible to argue that they are, if one of the enteropneust nervous systems is an independently-derived novelty, or if the two enteropneust nervous systems arose from a duplication of the single ancestral one. Investigation of expression patterns of developmental genes in enteropneusts may resolve the discrepancy. Additionally, it is still not resolved whether the functional equivalence of genes, as shown by their ability to replace each other, really demonstrates that the genes are doing the same thing in the two animals concerned. It would be interesting, for example, to test whether mammalian Hoxb2 can rescue the phenotype of Drosophila lab mutants (lab being a group 1 Hox gene, Hoxb2 a group 2). So far, genes in the same subfamily have mostly been tested for rescuing ability [otd and Otx1 (Refs 24 26), hh and shh (Ref. 30) or Hoxb1 and labial (Ref. 17)], but this ability may simply show that the important coding-region functions of a whole gene family have remained constant, rather than that two genes are the closest homologues within the family. The ability to rescue should be tested for a range of related genes, not just the ones suspected to be most closely related to each other. Certainly, these studies emphasize the need to compare regulatory elements as much as coding regions between species, since it appears that evolution has relied predominantly on regulatory changes. References 1 McGinnis, W. and Krumlauf, R. (1992) Cell 68, 283 302 2 Williams, N.A. and Holland, P.W.H. (1996) Nature 383, 490 3 Simeone, A. et al. (1992) Nature 358, 687 690 4 Younossi-Hartenstein, A. et al. (1997) Dev. Biol. 182, 270 283 TIG J UNE 1998 V OL . 14 N O . 6 213 1 2 3 4 5 7 6 M P2 P3 T Rhombomeres Mouse 8 P1 B1 B2 B3 S1 S2 S3 ems lab (Hox1) pb (Hox2 ) otd Drosophila Dfd (Hox4 ) Scr (Hox5 ) Antp (Hox6 ) Ubx (Hox7 ) Hoxb1 Hoxb2 Otx2 Otx1 Emx2 Emx1 Hoxb3 Hoxb4 Hoxb5 Hoxb6 Hoxb7 F IGURE 2. Anteroposterior gene expression in the fly and mouse central nervous systems showing Hox, Otx/otd and Emx/ems expression patterns. (Redrawn from Ref. 27, with extra information from Refs 6, 29, 38 and H. Reichert, pers. commun.) Arrow denotes the midbrain hindbrain boundary; B1 B3, brain segments (proto-, deuto- and trito-cerebrum, respectively); S1 S3, mandibular, maxillary and labial segments, respectively; P1 P3, prosomeres in the diencephalon; T, telencephalon; M, mesencephalon. C O M M E N T TIG J UNE 1998 V OL . 14 N O . 6 214 Copyright 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00 PII: S0168-9525(98)01500-5 5 Finkelstein, R. and Boncinelli, E. (1994) Trends Genet. 10, 310 315 6 Hirth, F. et al. (1995) Neuron 15, 769 778 7 Holland, P., Ingham, P. and Krauss, S. (1992) Nature 358, 627 628 8 Matsuo, I. et al. (1995) Genes Dev. 9, 2646 2658 9 Acampora, D. et al. (1997) Development 124, 3639 3650 10 Ang, S.L. et al. (1996) Development 122, 243 252 11 Acampora, D. et al. (1995) Development 121, 3279 3290 12 Acampora, D. et al. (1996) Nat. Genet. 14, 218 222 13 Marin, F. and Puelles, L. (1994) Dev. Biol. 163, 19 37 14 Pellegrini, M. et al. (1996) Development 122, 3893 3898 15 Qiu, M. et al. (1996) Dev. Biol. 178, 174 178 16 Lowe, C.J. and Wray, G.A. (1997) Nature 389, 718 721 17 Lutz, B. et al. (1996) Genes Dev. 10, 176 184 18 Malicki, J., Schughart, K. and McGinnis, W. (1990) Cell 63, 961 967 19 Zhao, J.J., Lazzarini, R.A. and Pick, L. (1993) Genes Dev. 7, 343 354 20 Lacalli, T.C. (1994) Am. Zool. 34, 533 541 21 Arendt, D. and N bler-Jung, K. (1996) BioEssays 18, 255 259 22 DeRobertis, E.M. and Sasai, Y. (1996) Nature 380, 37 40 23 D'Alessio, M. and Frasch, M. (1996) Mech. Dev. 58, 217 231 24 Acampora, D. et al. Development (in press) 25 Leuzinger, S. et al. Development (in press) 26 Nagao, T. et al. Proc. Natl. Acad. Sci. U. S. A. (in press) 27 Hirth, F., Hartmann, B. and Reichert, H. Development (in press) 28 Prince, V.E. et al. (1998) Development 125, 393 406 29 Diederich, R.J. et al. (1989) Genes Dev. 3, 399 414 30 Krauss, S., Concordet, J.P. and Ingham, P.W. (1993) Cell 75, 1431 1444 31 Halder, G., Callaerts, P. and Gehring, W.J. (1995) Science 267, 1788 1792 32 Suda, Y., Matsuo, I., Kuratani, S. and Aizawa, S. (1996) Genes Cells 1, 1031 1044 33 Williams, N.A. and Holland, P.W.H. Mol. Biol. Evol. (in press) 34 Butler, A.B. and Hodos, W. (1996) Comparative Vertebrate Neuroanatomy, Wiley-Liss 35 Marin, F. and Puelles, L. (1995) Eur. J. Neurosci. 7, 1714 1738 36 Joyner, A.L. (1996) Trends Genet. 12, 15 20 37 Peterson, K.J. (1995) Nature 373, 111 112 38 Puelles, L. and Rubenstein, J.L. (1993) Trends Neurosci. 16, 472 479 Last month's issue of Trends in Genetics featured a review of the disease-related potential of the transcriptional cofactors CREB-binding protein (CBP) and the adenovirus E1A-associated protein, p300 (Ref. 1). Shortly after the publication of this review, the combined efforts of David Livingston's laboratory (Harvard Medical School, Boston, MA, USA) and Richard Eckner's laboratory (Univ. of Zu rich, Switzerland) appeared in Cell, describing the deleterious effects of inactivating one or both murine CBP and/or p300 alleles 2 . The results-at-a-glance are presented in Table 1. When a single p300 allele is inactivated, the resultant embryos suffer a significantly reduced viability (up to 55% died in utero, depending on genetic background), although heterozygotes that do survive do not suffer from further p300-insufficiency after birth. Mice homozygous for p300 mutations always die in utero, between days 9 and 11.5 of gestation. These nullizygous embryos are much smaller than their littermates and exhibit severe open neural tube and heart defects. Interestingly, cells removed from the p300 homozygous mutants displayed poor proliferation properties, implying that p300 is required for growth stimulation, an idea that is contrary to the general opinion that CBP and p300 are tumor suppressor proteins. Unlike p300, CBP heterozygous mutant mice, described earlier 3 , manifest skeletal abnormalities consistent with the human congenital Rubinstein Taybi syndrome, in which one CBP allele is inactivated 4 . CBP homozygous mutant mice, however, strongly resemble the p300 mutants, and also die in utero, between days 9 and 11.5 of gestation. Crossing the p300 and CBP heterozygous mutants produced double heterozygous CBP/p300 mutant embryos, which died in utero but otherwise shared phenotypic similarities to both CBP and p300 homozygous mutants. This remarkable result suggests that the two proteins exert certain common embryonic survival functions and that the combined dose of CBP and p300 is critical for mouse embryonic development. Although CBP and p300 are not completely redundant physiologically, these results suggest that a 25% drop in combined CBP/p300 levels (through the loss of one CBP or p300 allele) is enough to interfere seriously with embryonic development, while a 50% drop results invariably in embryonic death. References 1 Giles, R.H., Peters, D.J.M. and Breuning, M.H. (1998) Trends Genet. 14, 178 183 2 Yao, T.P. et al. (1998) Cell 93, 361 372 3 Tanaka, Y. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10215 10220 4 Petrij, F. et al. (1995) Nature 376, 348 351 Rachel H. Giles rachel@ruly46.medfac.leidenuniv.nl Department of Human Genetics, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. Update CBP/p300 transgenic mice T ABLE 1. Mouse models for CBP and p300 mutations CBP p300 Phenotype Refs ++ ++ Normal + ++ Skeletal abnormalities 3 ++ Embryonic lethal 2, 3 ++ + Reduced viability 2 ++ Embryonic lethal 2 + + Embryonic lethal 2 Abbreviations: +, normal allele; , inactive allele.

A M Mager, Anne Grapin-Botton, K Ladjali, D Meyer, C M Wolff, P Stiegler, M A Bonnin, P Remy
The avian fli gene is specifically expressed during embryogenesis in a subset of neural crest cells giving rise to mesenchyme.
Int. J. Dev. Biol., 42(4) 561-572 (1998)

The ets-family of transcription factors is involved in the development of endothelial and hematopoietic cells. Among these genes, fliwas shown to be responsible for erythroblastomas and Ewing's sarcomas. Its involvement in Ewing's sarcoma, a putative neurectodermal tumor, as well as the in situ hybridization studies performed in mice and Xenopus suggested a role in neural crest development. We cloned quail fli cDNA in order to analyze in more detail its expression in neural crest cells, which have been extensively studied in avian species. Fli gene maps on chicken chromosome 1 to band q31->q33. Two RNAs are transcribed, most likely arising from two different promoters. The analysis of its expression in neural crest cells reveals that it is expressed rather late, when the neural crest cells reach their target. Among the various lineages derived from the crest, it is restricted to the mesenchymal one. It is maintained at later stages in the cartilage of neural crest but also of mesodermal origin. In addition, fli is expressed in several mesoderm-derived cells: endothelial cells as well as intermediate and splanchnopleural mesoderm.

Anne Grapin-Botton, M A Bonnin, M Sieweke, N M Le Douarin
Defined concentrations of a posteriorizing signal are critical for MafB/Kreisler segmental expression in the hindbrain.
Development, 125(7) 1173-1181 (1998)

It has been shown by using the quail/chick chimera system that Hox gene expression in the hindbrain is influenced by positional signals arising from the environment. In order to decipher the pathway that leads to Hox gene induction, we have investigated whether a Hox gene regulator, the leucine zipper transcription factor MafB/Kr, is itself transcriptionally regulated by the environmental signals. This gene is normally expressed in rhombomeres (r) 5 and 6 and their associated neural crest. MafB/Kr expression is maintained in r5/6 when grafted into the environment of r3/4. On the contrary, the environment of rhombomeres 7/8 represses MafB/Kr expression. Thus, as previously shown for the expression of Hox genes, MafB/Kr expression is regulated by a posterior-dominant signal, which in this case induces the loss of expression of this gene. We also show that the posterior signal can be transferred to the r5/6 neuroepithelium by posterior somites (somites 7 to 10) grafted laterally to r5/6. At the r4 level, the same somites induce MafB/Kr in r4, leading it to behave like r5/6. The posterior environment regulates MafB/Kr expression in the neural crest as it does in the corresponding hindbrain level, showing that some positional regulatory mechanisms are shared by neural tube and neural crest cells. Retinoic acid beads mimic the effect produced by the somites in repressing MafB/Kr in r5/6 and progressively inducing it more rostrally as its concentration increases. We therefore propose that the MafB/Kr expression domain is defined by a molecule unevenly distributed in the paraxial mesoderm. This molecule would allow the expression of the MafB/Kr gene in a narrow window of concentration by activating its expression at a definite threshold and repressing it at higher levels, accounting for its limited domain of expression in only two rhombomeres. It thus appears that the regulation of MafB/Kr expression in the rhombomeres could be controlled by the same posteriorizing factor(s) as Hox genes.

A Eichmann, Anne Grapin-Botton, L Kelly, T Graf, N M Le Douarin, M Sieweke
The expression pattern of the mafB/kr gene in birds and mice reveals that the kreisler phenotype does not represent a null mutant.
Mech Dev, 65(1-2) 111-122 (1997)
DOI

The recessive mouse mutation kreisler affects hindbrain segmentation and inner ear development in homozygous mice. The mouse gene affected by the mutation was found to encode a basic domain leucine-zipper (bZIP)-type transcription factor of the Maf-family named kr (Cordes, S.P. and Barsh, G.S. (1994) Cell 79, 1025-1034). The avian bZIP transcription factor mafB, which shows high homology to kr, has been identified as an interaction partner of c-Ets 1 (Sieweke, M.H., Tekotte, M.H., Frampton, J. and Graf, T. (1996) Cell 85, 49-60). Here we demonstrate by Southern blot analysis that mafB is the avian homologue of kr, and present a detailed pattern of its expression during avian and murine embryonic development. Consistent with the kreisler phenotype, mafB is expressed in avians in the tissues which are affected by the mouse mutation: rhombomeres 5 and 6 (r5 and r6) and the neural crest derived from these rhombomeres. However, our analysis reveals a variety of additional expression sites: mafB/kr expression persists in vestibular and acoustic nuclei and is also observed in differentiating neurons of the spinal cord and brain stem. Restricted expression sites are found in the mesonephros, the perichondrium, and in the hemopoietic system. Since these expression sites are conserved between mouse and chicken we reexamined homozygous kreisler mice for unrevealed phenotypes in the hemopoietic system. However, peritoneal macrophages from homozygous kreisler mice were found to be functionally normal and still expressed mafB/kr. Other adult tissues examined from homozygous kreisler mice had also not lost mafB/kr expression. Our results thus indicate that the kreisler mutation involves a tissue specific gene inactivation and suggest additional roles for mafB/kr in later developmental and differentiation processes that are not revealed by the mutation.

Julia Pearl Forjanic, Chao-Kung Chen, Herbert Jäckle, Marcos González-Gaitán
Genetic analysis of stomatogastric nervous system development in Drosophila using enhancer trap lines.
Dev Biol, 186(2) 139-154 (1997)
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The stomatogastric nervous system (SNS) of Drosophila melanogaster is a small, simply organized neural circuitry which innervates the anterior enteric system. It is responsible for regulating the passage of food through the pharynx and esophagus and into the midgut. Here we show that the development of the SNS is amenable to genetic dissection. We screened lines from a P-element mutagenesis, selecting those with lacZ reporter gene expression and/or a phenotype in the SNS, associated glia, and garland cells. We report a collection of expression patterns and mutant phenotypes among lines found to have a mutation in genes required for the establishment of the larval SNS. Our results indicate that SNS development depends on pattern organizer genes including components of the Ras/Raf pathway.

Rachel Macdonald, John Scholes, Uwe Strähle, Caroline Brennan, Nigel Holder, Michael Brand, Stephen W. Wilson
The Pax protein Noi is required for commissural axon pathway formation in the rostral forebrain.
Development, 124(12) 2397-2408 (1997)
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No-isthmus (Noi) is a member of the zebrafish Pax family of transcriptional regulators that is expressed in restricted domains of the developing CNS. In the developing eye and optic nerve, the Noi+ cells are primitive glial cells that line the choroid fissure and optic stalk/nerve to its junction with the optic tract. This pattern of Noi expression is retained in the adult, defining the optic nerve astroglia, which wrap the left and right nerves separately at the midline, thus forming the bodily crossed optic chiasm found in fish. In embryos carrying mutations in the noi gene, the choroid fissure fails to close, glial cells of the optic nerve fail to differentiate and optic axons exhibit abnormal trajectories exiting the eye and at the midline of the diencephalon. Optic axons select inappropriate pathways into the contralateral optic nerve, rostrally towards the anterior commissure and along the ipsilateral optic tract. Noi+ cells also border the pathway of axons in the postoptic commissure, which is located adjacent to the optic chiasm. These postoptic commissural axons are defasciculated and also exhibit pathfinding defects in noi- embryos. These results indicate that Noi is required in cells that line the pathways taken by optic and non-optic commissural axons for guidance across the midline of the diencephalon. We find that expression of two members of the Netrin family of axon guidance molecules and the signalling protein Sonic hedgehog is disturbed in noi- embryos, whereas several members of the Eph family of receptors and ligands show no obvious alterations in expression at the diencephalic midline.

Anne Grapin-Botton, M A Bonnin, N M Le Douarin
Hox gene induction in the neural tube depends on three parameters: competence, signal supply and paralogue group.
Development, 124(4) 849-859 (1997)

It has been previously shown that Hox gene expression in the rhombencephalon is controlled by environmental cues. Thus posterior transposition of anterior rhombomeres to the r7/8 level results in the activation of Hox genes of the four first paralog groups and in homeotic transformations of the neuroepithelial fate according to its position along the anteroposterior axis. We demonstrate here that although the anteroposterior levels of r2 to r6 express Hox genes they do not have inducing activity on more anterior territories. If transposed at the posterior rhombencephalon and trunk level, however, the same anterior regions are able to express Hox gene such as Hoxa-2, a-3 or b-4. We also provide evidence that these signals are transferred by two paths: one vertical, arising from the paraxial mesoderm, and one planar, travelling in the neural epithelium. The competence to express Hox genes extends up to the forebrain and midbrain but expression of Hox genes does not preclude Otx2 expression in these territories and results only in slight changes in their phenotypes. Similarly, rhombomeres transplanted to posterior truncal levels turned out to be able to express posterior genes of the first eight paralog groups to the exclusion of others located downstream in the Hox genes genomic clusters. This suggests that the neural tube is divided into large territories characterized by different Hox gene regulatory features.

N M Le Douarin, A Grapin-Botton
Contrôle génétique du développement du rhombencéphale par les gènes Hox étudié chez l'embryon d'oiseau par la méthode des chimères caille-poulet [Genetic control of rhombencephalon development by Hox genes studied in bird embryo by the quail-chick chimera method].
C R Seances Soc Biol Fil, 191(1) 29-42 (1997)

The rhombencephalic neural tube is transiently segmented along the anteroposterior axis into 8 rhombomeres. Each rhombomere, as well as its derived neural crest cells, is characterized by the expression of a specific set of Hox genes which constitute its Hox code. This code is supposed to define the morphogenetic program of these cells according to their position. We took advantage of the quail/chick chimera system to study the regulation of Hox gene expression in neural tube and neural crest cells. We have therefore ectopically transplanted the presumptive territories of the future rhombomeres and studied the evolution of their Hox code. We evidence in the posterior rhombencephalon and the spinal cord a posteriorising signal able to induce Hox gene expression, to repress anterior molecular markers and to control the subsequent development of the neural tube. This signal is conveyed horizontally in the plane of the neuroepithelium and vertically from the mesoderm to the ectoderm. The anteroposterior identity of the neural crest cells seem independent from this inducer after formation of the neural fold.

Eugene W Myers, Sanford Selznick^#, Zheng Zhang^#, Webb Miller^#
Progressive Multiple Alignment with Constraints
J Comput Biol, 3(4) 563-572 (1996)
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A progressive alignment algorithm produces a multi-alignment of a set of sequences by repeatedly aligning pairs of sequences and/or previously gen- erated alignments. We describe a method for guaranteeing that the alignment generated by a progressive alignment strategy satisfies a user-specified collec- tion of constraints about where certain sequence positions should appear rel- ative to others. Given a collection of constraints over sequences whose total length is , our algorithm takes time. An alignment of the -like globin gene clusters of several mammals illustrates the practi- cality of the method.

Anne Grapin-Botton, M A Bonnin, L A McNaughton, Robb Krumlauf, N M Le Douarin
Plasticity of transposed rhombomeres: Hox gene induction is correlated with phenotypic modifications.
Development, 121(9) 2707-2721 (1995)

In this study we have analysed the expression of Hoxb-4, Hoxb-1, Hoxa-3, Hoxb-3, Hoxa-4 and Hoxd-4 in the neural tube of chick and quail embryos after rhombomere (r) heterotopic transplantations within the rhombencephalic area. Grafting experiments were carried out at the 5-somite stage, i.e. before rhombomere boundaries are visible. They were preceeded by the establishment of the precise fate map of the rhombencephalon in order to determine the presumptive territory corresponding to each rhombomere. When a rhombomere is transplanted from a caudal to a more rostral position it expresses the same set of Hox genes as in situ. By contrast in many cases, if rhombomeres are transplanted from rostral to caudal their Hox gene expression pattern is modified. They express genes normally activated at the new location of the explant, as evidenced by unilateral grafting. This induction occurs whether transplantation is carried out before or after rhombomere boundary formation. Moreover, the fate of the cells of caudally transplanted rhombomeres is modified: the rhombencephalic nuclei in the graft develop according to the new location as shown for an r5/6 to r8 transplantation. Transplantation of 5 consecutive rhombomeres (i.e. r2 to r6), to the r8 level leads to the induction of Hoxb-4 in the two posteriormost rhombomeres but not in r2,3,4. Transplantations to more caudal regions (posterior to somite 3) result in some cases in the induction of Hoxb-4 in the whole transplant. Neither the mesoderm lateral to the graft nor the notochord is responsible for the induction. Thus, the inductive signal emanates from the neural tube itself, suggesting that planar signalling and predominance of posterior properties are involved in the patterning of the neural primordium.

T Tabata, Suzanne Eaton, T B Kornberg
The Drosophila hedgehog gene is expressed specifically in posterior compartment cells and is a target of engrailed regulation.
Genes Dev, 6(12B) 2635-2645 (1992)
DOI

cDNAs were isolated that represent transcripts of the Drosophila segment polarity gene, hedgehog (hh). Sequence analysis reveals a motif characteristic of a transmembrane domain, suggesting that the hh protein is membrane-associated. hh expression in epidermal cells is confined to the posterior compartments and coincides precisely with that of engrailed (en). Despite the similar patterns of expression in the cellular blastoderm, hh expression is independent of en, but hh expression becomes sensitive to and dependent on en during the extended germ band stage. The ectopic expression of hh that is normally induced in patched (ptc) mutant embryos does not appear in ptc en double mutants. We discuss these findings in terms of the relationship between en and hh, and the role of the hh function.

S F Altschul, W Gish, W Miller, E W Myers, D J Lipman
Basic local alignment search tool.
J Mol Biol, 215(3) 403-410 (1990)
PDF DOI

A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.

Philippe Chavrier, Robert G. Parton, Hans-Peter Hauri, K Simons, Marino Zerial
Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments.
Cell, 62(2) 317-329 (1990)
DOI

A set of 11 clones encoding putative GTP binding proteins highly homologous to the yeast YPT1/SEC4 gene products have been isolated from an MDCK cell cDNA library. We localized three of the corresponding proteins in mammalian cells by using affinity-purified antibodies in immunofluorescence and immunoelectron microscopy studies. One, the MDCK homolog of rab2, is associated with a structure having the characteristics of an intermediate compartment between the endoplasmic reticulum and the Golgi apparatus. The second, rab5, is located at the cytoplasmic surface of the plasma membrane and on early endosomes, while the third, rab7, is found on late endosomes. These findings provide evidence that members of the YPT1/SEC4 subfamily of GTP binding proteins are localized to specific exocytic and endocytic subcompartments in mammalian cells.

Kontakte

Cell Technology

Jarrells, Julia

Penkova, Vili

Myers

Myers, Gene

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