* joint first author # joint corresponding author

David J Barry, Claudia Gerri, Donald M Bell, Rocco D'Antuono, Kathy K Niakan
GIANI - open-source software for automated analysis of 3D microscopy images.
J Cell Sci, 135(10) Art. No. jcs259511 (2022)
Open Access DOI
The study of cellular and developmental processes in physiologically relevant three-dimensional (3D) systems facilitates an understanding of mechanisms underlying cell fate, disease and injury. While cutting-edge microscopy technologies permit the routine acquisition of 3D datasets, there is currently a limited number of open-source software packages to analyse such images. Here, we describe General Image Analysis of Nuclei-based Images (GIANI; https://djpbarry.github.io/Giani), new software for the analysis of 3D images. The design primarily facilitates segmentation of nuclei and cells, followed by quantification of morphology and protein expression. GIANI enables routine and reproducible batch-processing of large numbers of images, and comes with scripting and command line tools. We demonstrate the utility of GIANI by quantifying cell morphology and protein expression in confocal images of mouse early embryos and by segmenting nuclei from light-sheet microscopy images of the flour beetle embryo. We also validate the performance of the software using simulated data. More generally, we anticipate that GIANI will be a useful tool for researchers in a variety of biomedical fields.

Koji Ando✳︎#, Yu-Huan Shih✳︎, Lwaki Ebarasi, Ann Grosse, Daneal Portman, Ayano Chiba, Kenny Mattonet, Claudia Gerri, Didier Y.R. Stainier, Naoki Mochizuki, Shigetomo Fukuhara, Christer Betsholtz, Nathan D Lawson#
Conserved and context-dependent roles for pdgfrb signaling during zebrafish vascular mural cell development.
Dev Biol, 479 11-22 (2021)
Platelet derived growth factor beta and its receptor, Pdgfrb, play essential roles in the development of vascular mural cells, including pericytes and vascular smooth muscle cells. To determine if this role was conserved in zebrafish, we analyzed pdgfb and pdgfrb mutant lines. Similar to mouse, pdgfb and pdgfrb mutant zebrafish lack brain pericytes and exhibit anatomically selective loss of vascular smooth muscle coverage. Despite these defects, pdgfrb mutant zebrafish did not otherwise exhibit circulatory defects at larval stages. However, beginning at juvenile stages, we observed severe cranial hemorrhage and vessel dilation associated with loss of pericytes and vascular smooth muscle cells in pdgfrb mutants. Similar to mouse, pdgfrb mutant zebrafish also displayed structural defects in the glomerulus, but normal development of hepatic stellate cells. We also noted defective mural cell investment on coronary vessels with concomitant defects in their development. Together, our studies support a conserved requirement for Pdgfrb signaling in mural cells. In addition, these zebrafish mutants provide an important model for definitive investigation of mural cells during early embryonic stages without confounding secondary effects from circulatory defects.

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.

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)
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.

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)
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.

Jessica Guerra✳︎, Paola Chiodelli✳︎, Chiara Tobia, Claudia Gerri, Marco Presta
Long-Pentraxin 3 Affects Primary Cilium in Zebrafish Embryo and Cancer Cells via the FGF System.
Cancers (Basel), 12(7) Art. No. 1756 (2020)
Open Access DOI
Primary cilium drives the left-right asymmetry process during embryonic development. Moreover, its dysregulation contributes to cancer progression by affecting various signaling pathways. The fibroblast growth factor (FGF)/FGF receptor (FGFR) system modulates primary cilium length and plays a pivotal role in embryogenesis and tumor growth. Here, we investigated the impact of the natural FGF trap long-pentraxin 3 (PTX3) on the determination of primary cilium extension in zebrafish embryo and cancer cells. The results demonstrate that down modulation of the PTX3 orthologue ptx3b causes the shortening of primary cilium in zebrafish embryo in a FGF-dependent manner, leading to defects in the left-right asymmetry determination. Conversely, PTX3 upregulation causes the elongation of primary cilium in FGF-dependent cancer cells. Previous observations have identified the PTX3-derived small molecule NSC12 as an orally available FGF trap with anticancer effects on FGF-dependent tumors. In keeping with the non-redundant role of the FGF/FGR system in primary cilium length determination, NSC12 induces the elongation of primary cilium in FGF-dependent tumor cells, thus acting as a ciliogenic anticancer molecule in vitro and in vivo. Together, these findings demonstrate the ability of the natural FGF trap PTX3 to exert a modulatory effect on primary cilium in embryonic development and cancer. Moreover, they set the basis for the design of novel ciliogenic drugs with potential implications for the therapy of FGF-dependent tumors.

Sissy E Wamaitha, Katarzyna J Grybel, Gregorio Alanis-Lobato, Claudia Gerri, Sugako Ogushi, Afshan McCarthy, Shantha K Mahadevaiah, Lyn Healy, Rebecca A Lea, Miriam Molina-Arcas, Liani G Devito, Kay Elder, Phil Snell, Leila Christie, Julian Downward, James M A Turner, Kathy K Niakan
IGF1-mediated human embryonic stem cell self-renewal recapitulates the embryonic niche.
Nat Commun, 11(1) Art. No. 764 (2020)
Open Access DOI
Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche.

Zaheer Ali, Anthony Mukwaya, Antje Biesemeier, Maria Ntzouni, Daniel Ramsköld, Sarantis Giatrellis, Parviz Mammadzada, Renhai Cao, Anton Lennikov, Michele Marass, Claudia Gerri, Camilla Hildesjö, Michael Taylor, Qiaolin Deng, Beatrice Peebo, Luis Del Peso, Anders Kvanta, Rickard Sandberg, Ulrich Schraermeyer, Helder Andre, John F Steffensen, Neil Lagali, Yihai Cao, Julianna Kele, Lasse Dahl Jensen
Intussusceptive Vascular Remodeling Precedes Pathological Neovascularization.
Arterioscler Thromb Vasc Biol, 39(7) 1402-1418 (2019)
Open Access DOI
Objective- Pathological neovascularization is crucial for progression and morbidity of serious diseases such as cancer, diabetic retinopathy, and age-related macular degeneration. While mechanisms of ongoing pathological neovascularization have been extensively studied, the initiating pathological vascular remodeling (PVR) events, which precede neovascularization remains poorly understood. Here, we identify novel molecular and cellular mechanisms of preneovascular PVR, by using the adult choriocapillaris as a model. Approach and Results- Using hypoxia or forced overexpression of VEGF (vascular endothelial growth factor) in the subretinal space to induce PVR in zebrafish and rats respectively, and by analyzing choriocapillaris membranes adjacent to choroidal neovascular lesions from age-related macular degeneration patients, we show that the choriocapillaris undergo robust induction of vascular intussusception and permeability at preneovascular stages of PVR. This PVR response included endothelial cell proliferation, formation of endothelial luminal processes, extensive vesiculation and thickening of the endothelium, degradation of collagen fibers, and splitting of existing extravascular columns. RNA-sequencing established a role for endothelial tight junction disruption, cytoskeletal remodeling, vesicle- and cilium biogenesis in this process. Mechanistically, using genetic gain- and loss-of-function zebrafish models and analysis of primary human choriocapillaris endothelial cells, we determined that HIF (hypoxia-induced factor)-1α-VEGF-A-VEGFR2 signaling was important for hypoxia-induced PVR. Conclusions- Our findings reveal that PVR involving intussusception and splitting of extravascular columns, endothelial proliferation, vesiculation, fenestration, and thickening is induced before neovascularization, suggesting that identifying and targeting these processes may prevent development of advanced neovascular disease in the future. Visual Overview- An online visual overview is available for this article.

Michele Marass✳︎, Arica Beisaw✳︎, Claudia Gerri, Francesca Luzzani, Nana Fukuda, Stefan Günther, Carsten Kuenne, Sven Reischauer, Didier Y.R. Stainier
Genome-wide strategies reveal target genes of Npas4l associated with vascular development in zebrafish.
Development, 146(11) Art. No. dev173427 (2019)
The development of a vascular network is essential to nourish tissues and sustain organ function throughout life. Endothelial cells (ECs) are the building blocks of blood vessels, yet our understanding of EC specification remains incomplete. Zebrafish cloche/npas4l mutants have been used broadly as an avascular model, but little is known about the molecular mechanisms of action of the Npas4l transcription factor. Here, to identify its direct and indirect target genes, we have combined complementary genome-wide approaches, including transcriptome analyses and chromatin immunoprecipitation. The cross-analysis of these datasets indicates that Npas4l functions as a master regulator by directly inducing a group of transcription factor genes that are crucial for hematoendothelial specification, such as etv2, tal1 and lmo2 We also identified new targets of Npas4l and investigated the function of a subset of them using the CRISPR/Cas9 technology. Phenotypic characterization of tspan18b mutants reveals a novel player in developmental angiogenesis, confirming the reliability of the datasets generated. Collectively, these data represent a useful resource for future studies aimed to better understand EC fate determination and vascular development.

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)
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.

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)
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.

Claudia Gerri, Rubén Marín-Juez, Michele Marass, Alora Marks, Hans-Martin Maischein, Didier Y.R. Stainier
Hif-1α regulates macrophage-endothelial interactions during blood vessel development in zebrafish.
Nat Commun, 8 Art. No. 15492 (2017)
Open Access DOI
Macrophages are known to interact with endothelial cells during developmental and pathological angiogenesis but the molecular mechanisms modulating these interactions remain unclear. Here, we show a role for the Hif-1α transcription factor in this cellular communication. We generated hif-1aa;hif-1ab double mutants in zebrafish, hereafter referred to as hif-1α mutants, and find that they exhibit impaired macrophage mobilization from the aorta-gonad-mesonephros (AGM) region as well as angiogenic defects and defective vascular repair. Importantly, macrophage ablation is sufficient to recapitulate the vascular phenotypes observed in hif-1α mutants, revealing for the first time a macrophage-dependent angiogenic process during development. Further substantiating our observations of vascular repair, we find that most macrophages closely associated with ruptured blood vessels are Tnfα-positive, a key feature of classically activated macrophages. Altogether, our data provide genetic evidence that Hif-1α regulates interactions between macrophages and endothelial cells starting with the mobilization of macrophages from the AGM.

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)
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.