* joint first author # joint corresponding author

Adriano Bolondi✳︎, Benjamin K Law✳︎, Helene Kretzmer, Seher Ipek Gassaloglu, René Buschow, Christina Riemenschneider, Dian Yang, Maria Walther, Jesse V Veenvliet#, Alexander Meissner#, Zachary D Smith#, Michelle M Chan
Reconstructing axial progenitor field dynamics in mouse stem cell-derived embryoids.
Dev Cell, 59(12) 1489-1505 (2024)
Open Access DOI
Embryogenesis requires substantial coordination to translate genetic programs to the collective behavior of differentiating cells, but understanding how cellular decisions control tissue morphology remains conceptually and technically challenging. Here, we combine continuous Cas9-based molecular recording with a mouse embryonic stem cell-based model of the embryonic trunk to build single-cell phylogenies that describe the behavior of transient, multipotent neuro-mesodermal progenitors (NMPs) as they commit into neural and somitic cell types. We find that NMPs show subtle transcriptional signatures related to their recent differentiation and contribute to downstream lineages through a surprisingly broad distribution of individual fate outcomes. Although decision-making can be heavily influenced by environmental cues to induce morphological phenotypes, axial progenitors intrinsically mature over developmental time to favor the neural lineage. Using these data, we present an experimental and analytical framework for exploring the non-homeostatic dynamics of transient progenitor populations as they shape complex tissues during critical developmental windows.

Alba Villaronga Luque✳︎, Ryan Savill✳︎, Natalia Lopez-Anguita, Adriano Bolondi, Sumit Garai, Seher Ipek Gassaloglu, Aayush Poddar, Aydan Bulut-Karslioglu, Jesse V Veenvliet
Integrated Molecular-Phenotypic Profiling Reveals Metabolic Control of Morphological Variation in Stembryos.
bioRxiv, Art. No. doi: https://doi.org/10.1101/2023.12.04.569921 (2023)
Open Access DOI
Mammalian stem-cell-based models of embryo development (stembryos) hold great promise in basic and applied research. However, considerable phenotypic variation despite identical culture conditions limits their potential. The biological processes underlying this seemingly stochastic variation are poorly understood. Here, we investigate the roots of this phenotypic variation by intersecting transcriptomic states and morphological history of individual stembryos across stages modeling post-implantation and early organogenesis. Through machine learning and integration of time-resolved single-cell RNA-sequencing with imaging-based quantitative phenotypic profiling, we identify early features predictive of the phenotypic end-state. Leveraging this predictive power revealed that early imbalance of oxidative phosphorylation and glycolysis results in aberrant morphology and a neural lineage bias that can be corrected by metabolic interventions. Collectively, our work establishes divergent metabolic states as drivers of phenotypic variation, and offers a broadly applicable framework to chart and predict phenotypic variation in organoid systems. The strategy can be leveraged to identify and control underlying biological processes, ultimately increasing the reproducibility of in vitro systems.

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.

Berna Sozen#, Deniz Conkar, Jesse V Veenvliet#
Carnegie in 4D? Stem-cell-based models of human embryo development.
Semin Cell Dev Biol, 131 44-57 (2022)
How cells build embryos is still a major mystery. Many unresolved questions require the study of the processes that pattern and shape the embryo in live specimens, in toto, across spatial and temporal scales. In mammalian embryogenesis, this remains a major challenge as the embryo develops in utero, precluding easy accessibility. For human embryos, technical, ethical and legal limitations further hamper the in-depth investigation of embryogenesis, especially beyond gastrulation stages. This has resulted in an over-reliance on model organisms, particularly mice, to understand mammalian development. However, recent efforts show critical differences between rodent and primate embryos, including timing, architecture and transcriptional regulation. Thus, a human-centric understanding of embryogenesis is much needed. To empower this, novel in vitro approaches, which coax human pluripotent stem cells to form embryonic organoids that model embryo development, are pivotal. Here, we summarize these emergent technologies that recapitulate aspects of human development "in a dish". We show how these technologies can provide insights into the molecular, cellular and morphogenetic processes that fuel the formation of a fully formed fetus, and discuss the potential of these platforms to revolutionize our understanding of human development in health and disease. Despite their clear promise, we caution against over-interpreting the extent to which these in vitro platforms model the natural embryo. In particular, we discuss how fate, form and function - a tightly coupled trinity in vivo, can be disconnected in vitro. Finally, we propose how careful benchmarking of existing models, in combination with rational protocol design based on an increased understanding of in vivo developmental dynamics and insights from mouse in vitro models of embryo development, will help guide the establishment of better models of human embryo development.

Natalia López-Anguita, Seher Ipek Gassaloglu, Maximilian Stötzel, Adriano Bolondi, Deniz Conkar, Marina Typou, René Buschow, Jesse V Veenvliet#, Aydan Bulut-Karslioglu#
Hypoxia induces an early primitive streak signature, enhancing spontaneous elongation and lineage representation in gastruloids.
Development, 149(20) Art. No. dev200679 (2022)
Open Access DOI
The cellular microenvironment, together with intrinsic regulators, shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia in vivo and appear to benefit from hypoxic culture in vitro. Yet, how hypoxia influences stem cell transcriptional networks and lineage choices remain poorly understood. Here, we investigated the molecular effects of acute and prolonged hypoxia on embryonic and extra-embryonic stem cells as well as the functional impact on differentiation potential. We find a temporal and cell type-specific transcriptional response including an early primitive streak signature in hypoxic embryonic stem cells mediated by HIF1α. Using a 3D gastruloid differentiation model, we show that hypoxia-induced T expression enables symmetry breaking and axial elongation in the absence of exogenous WNT activation. When combined with exogenous WNT activation, hypoxia enhances lineage representation in gastruloids, as demonstrated by highly enriched signatures of gut endoderm, notochord, neuromesodermal progenitors and somites. Our findings directly link the microenvironment to stem cell function and provide a rationale supportive of applying physiological conditions in models of embryo development.

Jesse V Veenvliet, Pierre-François Lenne, David A Turner, Iftach Nachman, Vikas Trivedi
Sculpting with stem cells: how models of embryo development take shape.
Development, 148(24) Art. No. dev192914 (2021)
Open Access DOI
During embryogenesis, organisms acquire their shape given boundary conditions that impose geometrical, mechanical and biochemical constraints. A detailed integrative understanding how these morphogenetic information modules pattern and shape the mammalian embryo is still lacking, mostly owing to the inaccessibility of the embryo in vivo for direct observation and manipulation. These impediments are circumvented by the developmental engineering of embryo-like structures (stembryos) from pluripotent stem cells that are easy to access, track, manipulate and scale. Here, we explain how unlocking distinct levels of embryo-like architecture through controlled modulations of the cellular environment enables the identification of minimal sets of mechanical and biochemical inputs necessary to pattern and shape the mammalian embryo. We detail how this can be complemented with precise measurements and manipulations of tissue biochemistry, mechanics and geometry across spatial and temporal scales to provide insights into the mechanochemical feedback loops governing embryo morphogenesis. Finally, we discuss how, even in the absence of active manipulations, stembryos display intrinsic phenotypic variability that can be leveraged to define the constraints that ensure reproducible morphogenesis in vivo.

Dennis Schifferl, Manuela Scholze-Wittler, Lars Wittler, Jesse V Veenvliet, Frederic Koch#, Bernhard G Herrmann#
A 37 kb region upstream of brachyury comprising a notochord enhancer is essential for notochord and tail development.
Development, 148(23) Art. No. dev200059 (2021)
Open Access DOI
The node-streak border region comprising notochord progenitor cells (NPCs) at the posterior node and neuro-mesodermal progenitor cells (NMPs) in the adjacent epiblast is the prime organizing center for axial elongation in mouse embryos. The T-box transcription factor brachyury (T) is essential for both formation of the notochord and maintenance of NMPs, and thus is a key regulator of trunk and tail development. The T promoter controlling T expression in NMPs and nascent mesoderm has been characterized in detail; however, control elements for T expression in the notochord have not been identified yet. We have generated a series of deletion alleles by CRISPR/Cas9 genome editing in mESCs, and analyzed their effects in mutant mouse embryos. We identified a 37 kb region upstream of T that is essential for notochord function and tailbud outgrowth. Within that region, we discovered a T-binding enhancer required for notochord cell specification and differentiation. Our data reveal a complex regulatory landscape controlling cell type-specific expression and function of T in NMP/nascent mesoderm and node/notochord, allowing proper trunk and tail development.

Adriano Bolondi, Leah Haut, Seher Ipek Gassaloglu, Polly Burton, Helene Kretzmer, René Buschow, Alexander Meissner#, Bernhard G Herrmann#, Jesse V Veenvliet#
Generation of Mouse Pluripotent Stem Cell-derived Trunk-like Structures: An in vitro Model of Post-implantation Embryogenesis.
Bio Protoc, 11(11) Art. No. e4042 (2021)
Open Access DOI
Post-implantation mammalian embryogenesis involves profound molecular, cellular, and morphogenetic changes. The study of these highly dynamic processes is complicated by the limited accessibility of in utero development. In recent years, several complementary in vitro systems comprising self-organized assemblies of mouse embryonic stem cells, such as gastruloids, have been reported. We recently demonstrated that the morphogenetic potential of gastruloids can be further unlocked by the addition of a low percentage of Matrigel as an extracellular matrix surrogate. This resulted in the formation of highly organized trunk-like structures (TLSs) with a neural tube that is frequently flanked by bilateral somites. Notably, development at the molecular and morphogenetic levels is highly reminiscent of the natural embryo. To facilitate access to this powerful model, here we provide a detailed step-by-step protocol that should allow any lab with access to standard cell culture techniques to implement the culture system. This will provide the user with a means to investigate early mid-gestational mouse embryogenesis at an unprecedented spatiotemporal resolution.

Jesse V Veenvliet, Bernhard G Herrmann
Modeling mammalian trunk development in a dish.
Dev Biol, 474 5-15 (2021)
Mammalian post-implantation development comprises the coordination of complex lineage decisions and morphogenetic processes shaping the embryo. Despite technological advances, a comprehensive understanding of the dynamics of these processes and of the self-organization capabilities of stem cells and their descendants remains elusive. Building synthetic embryo-like structures from pluripotent embryonic stem cells in vitro promises to fill these knowledge gaps and thereby may prove transformative for developmental biology. Initial efforts to model the post-implantation embryo resulted in structures with compromised morphology (gastruloids). Recent approaches employing modified culture media, an extracellular matrix surrogate or extra-embryonic stem cells, however, succeeded in establishing embryo-like architecture. For example, embedding of gastruloids in Matrigel unlocked self-organization into trunk-like structures with bilateral somites and a neural tube-like structure, together with gut tissue and primordial germ cell-like cells. In this review, we describe the currently available models, discuss how these can be employed to acquire novel biological insights, and detail the imminent challenges for improving current models by in vitro engineering.

Natalia Lopez-Anguita, Seher Ipek Gassaloglu, Maximilian Stötzel, Marina Typou, Iiris Virta, Sara Hetzel, René Buschow, Burak Koksal, Derya Atilla, Ronald Maitschke-Rajasekharan, Rui Chen, Alexandra L. Mattei, Ivan Bedzhov, David Meierhofer, Alexander Meissner, Jesse V Veenvliet, Aydan Bulut-Karslioglu
Hypoxia induces a transcriptional early primitive streak signature in pluripotent cells enhancing spontaneous elongation and lineage representation in gastruloids.
bioRxiv, Art. No. https://doi.org/10.1101/2021.07.21.452906 (2021)
Open Access DOI
The cellular microenvironment together with intrinsic regulators shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia in vivo and appear to benefit from hypoxic culture in vitro. Yet, components of the hypoxia response and h o w t h e i r i n t e r p l a y i m p a c t s s t e m c e l l transcriptional networks and lineage choices r e m a i n p o o r l y u n d e r s t o o d . H e r e w e investigated the molecular effects of acute and prolonged hypoxia on distinct embryonic and extraembryonic stem cell types as well as the functional impact on differentiation potential. We find a temporal and cell type-specific transcriptional response including an early primitive streak signature in hypoxic embryonic stem (ES) cells. Using a 3D gastruloid differentiation model, we show that hypoxia-induced T expression enables symmetry breaking and axial elongation in the absence of exogenous WNT activation. Importantly, h y p o x i a a l s o m o d u l a t e s T l e v e l s i n conventional gastruloids and enhances representation of endodermal and neural markers. Mechanistically, we identify Hif1α as a c e n t r a l f a c t o r t h a t m e d i a t e s t h e transcriptional response to hypoxia in balance with epigenetic and metabolic rewiring. Our findings directly link the microenvironment to stem cell function and provide a rationale supportive of applying physiological conditions in models of embryo development.

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)
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.

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.

Emmeke Aarts, Matthijs Verhage, Jesse V Veenvliet, Conor V Dolan, Sophie van der Sluis
A solution to dependency: using multilevel analysis to accommodate nested data.
Nat Neurosci, 17(4) 491-496 (2014)
In neuroscience, experimental designs in which multiple observations are collected from a single research object (for example, multiple neurons from one animal) are common: 53% of 314 reviewed papers from five renowned journals included this type of data. These so-called 'nested designs' yield data that cannot be considered to be independent, and so violate the independency assumption of conventional statistical methods such as the t test. Ignoring this dependency results in a probability of incorrectly concluding that an effect is statistically significant that is far higher (up to 80%) than the nominal α level (usually set at 5%). We discuss the factors affecting the type I error rate and the statistical power in nested data, methods that accommodate dependency between observations and ways to determine the optimal study design when data are nested. Notably, optimization of experimental designs nearly always concerns collection of more truly independent observations, rather than more observations from one research object.

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)
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.

Alexi Nott, Justyna Nitarska, Jesse V Veenvliet, Stephan Schacke, Alwin A H A Derijck, Piotr Sirko, Christian Muchardt, R Jeroen Pasterkamp, Marten P Smidt, Antonella Riccio
S-nitrosylation of HDAC2 regulates the expression of the chromatin-remodeling factor Brm during radial neuron migration.
Proc Natl Acad Sci U.S.A., 110(8) 3113-3118 (2013)
Dynamic epigenetic modifications play a key role in mediating the expression of genes required for neuronal development. We previously identified nitric oxide (NO) as a signaling molecule that mediates S-nitrosylation of histone deacetylase 2 (HDAC2) and epigenetic changes in neurons. Here, we show that HDAC2 nitrosylation regulates neuronal radial migration during cortical development. Bead-array analysis performed in the developing cortex revealed that brahma (Brm), a subunit of the ATP-dependent chromatin-remodeling complex BRG/brahma-associated factor, is one of the genes regulated by S-nitrosylation of HDAC2. In the cortex, expression of a mutant form of HDAC2 that cannot be nitrosylated dramatically inhibits Brm expression. Our study identifies NO and HDAC2 nitrosylation as part of a signaling pathway that regulates cortical development and the expression of Brm in neurons.

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)
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.