Single-cell technologies: a new lens into epigenetic regulation in development

The totipotent zygote gives rise to diverse cell types through a series of well-orchestrated regulatory mechanisms. Epigenetic modifiers play an essential, though still poorly understood, role in the transition from pluripotency towards organogenesis. However, recent advances in single-cell technologies have enabled an unprecedented, high-resolution dissection of this crucial developmental window, highlighting more cell-type-specific functions of these ubiquitous regulators. In this review, we discuss and contextualize several recent studies that explore epigenetic regulation during mouse embryogenesis, emphasizing the opportunities presented by single-cell technologies, in vivo perturbation approaches as well as advanced in vitro models to characterize dynamic developmental transitions

Single-cell technologies: a new lens into epigenetic regulation in development

The totipotent zygote gives rise to diverse cell types through a series of well-orchestrated regulatory mechanisms. Epigenetic modifiers play an essential, though still poorly understood, role in the transition from pluripotency towards organogenesis. However, recent advances in single-cell technologies have enabled an unprecedented, high-resolution dissection of this crucial developmental window, highlighting more cell-type-specific functions of these ubiquitous regulators. In this review, we discuss and contextualize several recent studies that explore epigenetic regulation during mouse embryogenesis, emphasizing the opportunities presented by single-cell technologies, in vivo perturbation approaches as well as advanced in vitro models to characterize dynamic developmental transitions

Lineage tracing of early organogenesis and liver mesenchymal cells with ultrasound-guided in utero nano-injection

Current methods used to investigate embryo development and alter gene expression in mouse embryos are often time consuming and require large numbers of mice. To circumvent these limitations, we were aiming to develop a flexible and efficient method to investigate embryo development and manipulate gene expression in utero. Two tissues of interest to target in vivo are the ectodermal/ neural compartment, and mesoderm. Neurectoderm gives rise to the brain, spinal cord and peripheral nervous system, among others, while mesoderm gives rise to blood, muscle, and mesenchymal cells, among other cell types. Mesenchymal cells in liver, including peri-portal fibroblasts, mesothelial cells and hepatic stellate cells (HSCs), play multiple crucial roles in normal liver development, liver regeneration or liver fibrosis when liver is injured. Mesenchymal cells express both neural and mesenchymal markers, therefore both a neural crest (NC) and mesoderm origin have been proposed. The embryonic origin of HSCs is a long-debated topic in this field due to the lack of specific marker genes and potential convergent differentiation from different origins. To investigate nervous system development, we first hypothesized that by injecting lentivirus into the amniotic cavity (AC) prior to the neural plate closure, the open neural plate should be labeled and therefore label the future brain and spinal cord. In Paper I and II, we developed NEPTUNE (NEural Plate Targeting by in Utero NanoinjEction) to transduce either nervous system with up to 99% efficacy or selectively achieve the expression in specific cell types by using cell-specific MiniPromoters. In Paper III, the first aim was to develop a method to target mesoderm, and then to apply this technology to investigate liver mesenchymal cells. We hypothesized that exclusive labeling of the mesoderm and its progeny could be achieved by injecting into the exocoelomic cavity (ExC) during gastrulation after the segregation of three germ layers and full establishment of two cavities (AC and ExC), since mesoderm is in contact with the ExC. Therefore, we further adapted ultrasound-guided in utero nano-injection to lineage trace mesoderm descendants by injecting a diverse lentivirus barcode library into the ExC at embryonic day (E)7.5. In parallel, we used NEPTUNE, to target ectoderm/neural crest as well as primitive streak (PS) by injecting into AC at E7.5. Embryos were collected at E9.5 and E10.5 to address early organogenesis and the origin of septum transversum mesenchyme (STM), a transit tissue believed to contribute to the mesenchymal compartments of diverse internal organs. E16.5 livers were collected to resolve the clonal relations between different mesenchymal cells in the liver. In summary, during my doctoral studies, we developed two new approaches to target embryonic tissues during development. Ultrasound-guided in utero nano-injection is a flexible and efficient tool to elucidate clonal relations among tissues in early mouse embryo development, as well as for gene manipulation. It significantly minimizes both the financial cost and ethical burdens associated with animal research, in the meantime accelerating the progression from hypothesis to in vivo results

The transcription factor Rreb1 regulates epithelial architecture, invasiveness, and vasculogenesis in early mouse embryos.

Ras-responsive element-binding protein 1 (Rreb1) is a zinc-finger transcription factor acting downstream of RAS signaling. Rreb1 has been implicated in cancer and Noonan-like RASopathies. However, little is known about its role in mammalian non-disease states. Here, we show that Rreb1 is essential for mouse embryonic development. Loss of Rreb1 led to a reduction in the expression of vasculogenic factors, cardiovascular defects, and embryonic lethality. During gastrulation, the absence of Rreb1 also resulted in the upregulation of cytoskeleton-associated genes, a change in the organization of F-ACTIN and adherens junctions within the pluripotent epiblast, and perturbed epithelial architecture. Moreover, Rreb1 mutant cells ectopically exited the epiblast epithelium through the underlying basement membrane, paralleling cell behaviors observed during metastasis. Thus, disentangling the function of Rreb1 in development should shed light on its role in cancer and other diseases involving loss of epithelial integrity

Discovery of directional and nondirectional pioneer transcription factors by modeling DNase profile magnitude and shape

We describe protein interaction quantitation (PIQ), a computational method for modeling the magnitude and shape of genome-wide DNase I hypersensitivity profiles to identify transcription factor (TF) binding sites. Through the use of machine-learning techniques, PIQ identified binding sites for >700 TFs from one DNase I hypersensitivity analysis followed by sequencing (DNase-seq) experiment with accuracy comparable to that of chromatin immunoprecipitation followed by sequencing (ChIP-seq). We applied PIQ to analyze DNase-seq data from mouse embryonic stem cells differentiating into prepancreatic and intestinal endoderm. We identified 120 and experimentally validated eight 'pioneer' TF families that dynamically open chromatin. Four pioneer TF families only opened chromatin in one direction from their motifs. Furthermore, we identified 'settler' TFs whose genomic binding is principally governed by proximity to open chromatin. Our results support a model of hierarchical TF binding in which directional and nondirectional pioneer activity shapes the chromatin landscape for population by settler TFs.National Institutes of Health (U.S.) (Common Fund 5UL1DE019581)National Institutes of Health (U.S.) (Common Fund RL1DE019021)National Institutes of Health (U.S.) (Common Fund 5TL1EB008540)National Institutes of Health (U.S.) (Grant 1U01HG007037)National Institutes of Health (U.S.) (Grant 5P01NS055923

Discovery of non-directional and directional pioneer transcription factors by modeling DNase profile magnitude and shape

Here we describe Protein Interaction Quantitation (PIQ), a computational method that models the magnitude and shape of genome-wide DNase profiles to facilitate the identification of transcription factor (TF) binding sites. Through the use of machine learning techniques, PIQ identified binding sites for >700 TFs from one DNase-seq experiment with accuracy comparable to ChIP-seq for motif-associated TFs (median AUC=0.93 across 303 TFs). We applied PIQ to analyze DNase-seq data from mouse embryonic stem cells differentiating into pre-pancreatic and intestinal endoderm. We identified (n=120) and experimentally validated eight ‘pioneer’ TF families that dynamically open chromatin, enabling other TFs to bind to adjacent DNA. Four pioneer TF families only open chromatin in one direction from their motifs. Furthermore, we identified a class of ‘settler’ TFs whose genomic binding is principally governed by proximity to open chromatin. Our results support a model of hierarchical TF binding in which directional and non-directional pioneer activity shapes the chromatin landscape for population by settler TFs

Impact of hypoxia on embryonic and extraembryonic stem cells and during differentiation via gastruloid formation

Environmental oxygen is crucial for mammalian life. Low oxygen levels –namely hypoxia— occur naturally in the developing embryo and cells adapt to it. Nonetheless, the role of oxygen as a factor influencing stem cell behavior and developmental trajectories is not well understood. In this study, I dissected the effects of acute and prolonged hypoxia in embryonic and extraembryonic stem cells as well as the functional impact on lineage choices and differentiation potential. I show that, while stem cells maintain their cellular identity in hypoxia, low oxygen levels promote a cell type-specific and temporal transcriptional response. Specifically, in embryonic stem (ES) cells, hypoxia selectively induces a transcriptional early primitive streak signature with induction of mesendoderm marker genes, such as Wnt3, T and Eomes, without inducing spontaneous differentiation. Mechanistically, I show that HIF1a activation in normoxia recapitulates the induction of developmental genes as those observed in hypoxia. Additionally, low oxygen levels also alter the epigenetic landscape of ES cells, leading to global DNA demethylation and bivalent chromatin modification rewiring. Last, using a 3D gastruloid differentiation model and in combination with scRNA-seq, I show that hypoxia-induced WNT pathway enables symmetry breaking, polarization and axial elongation in the absence of exogenous WNT activation. When combined with exogenous WNT activation, hypoxia enhances lineage representation by enriching the cell types that are otherwise absent or underrepresented in normoxic gastruloids, such as notochord and gut endoderm, respectively. Moreover, hypoxia provides morphological cues to gut endodermal cells which self-organize in tubular structures reminiscent of the embryonic gut tube. Taken together, these findings reveal the impact of hypoxia on stem cell behavior and during gastrulation where it modulates morphogenesis and cellular composition in 3D gastrulation models. Hence, my investigation provides a direct link between microenvironmental factors and stem cell functions and strongly supports the use of physiologically relevant oxygen levels in models of embryo development

Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids.

Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization1-3. To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral-caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner.This work was supported by an European Research Council Advanced grant (ERC-AdG 742225-IntScOmics), a Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) TOP award (NWOCW 714.016.001), and the Foundation for Fundamental Research on Matter, financially supported by NWO (FOM-14NOISE01) to S.C.v.d.B., A.A., V.v.B., M.B., J.V. and A.v.O., a BBSRC (No. BB/M023370/1 and BB/P003184/1), Newton Trust (INT16.24b) and MRC (MR/R017190/1) grant to A.M.A., a Newnham College Cambridge Junior Research Fellowship to N.M., and a studentship from the Engineering and Physical Sciences Research Council (EPSRC) to P.B.J.. The Cambridge Stem Cell Institute is supported by core funding from the Wellcome Trust and Medical Research Council; J. N. was funded by the University of Cambridge, and K.F.S. by core funding from the Hubrecht Institute. This work is part of the Oncode Institute which is partly financed by the Dutch Cancer Society

Study of the human SOX17 locus and its genetic determinants in definitive endoderm

Embryonic development and organogenesis depend on the precise spatiotemporal expression of specific sets of genes. Precisely controlled gene expression ensures cell state transitions, especially in the early stages of development, as gastrulation. These complex multi-layered cellular processes are orchestrated by the interfacing of the epigenome, 3-dimensional (3D) nuclear organization, cis-regulatory elements (CREs) with transcription factors (TF), and long non-coding RNAs (lncRNAs). In the gastrulating embryo, definitive endoderm is specified from the pluripotent epiblast following a series of regulatory events, including the activation of SOX17, a key TF of that particular germ layer. Although SOX17 has been extensively studied in early embryonic development, the precise control of its activation, the locus, and the epigenetic rules governing its genetic regulatory network (GRN) remains poorly investigated. In my thesis, I in-depth characterized the human SOX17 locus, exploring the relevance and regulatory impact of 3D nuclear organization, its distal CREs, and their activity. I applied a series of loss of function (LOF) and transgenic experiments to dissect the locus at a satisfactory resolution. In particular, I showed SOX17 among a subset of developmental regulators topologically isolated within CTCF-CTCF loop domains and highlighted the importance of gene control in 3D within this type of domain. I pinpointed the relevance of SOX17’s distal CREs and their definitive endoderm-specific interaction and showed this interaction to be highly dependent on CTCF-CTCF loop-formation to guarantee proper gene control. I found CRE-dependent SOX17 gene deregulation associated with poor definitive endoderm differentiation outcome and a stalled “mesendodermal-like” phenotype. Assessing the genetic identity of different CREs, I divulged the presence of a novel lncRNA within the locus, namely LNCSOX17. I fully characterized LNCSOX17 and established its identity as a bona fide lncRNA through a series of genetic perturbations. I demonstrated the importance of LNCSOX17 for forming definitive endoderm and the lack of participation in SOX17 cis-acting gene control. I associated the loss of LNCSOX17 RNA but not its active transcription at the locus with an aberrant endodermal transcriptome, a lack of epithelial-to-mesenchymal transition (EMT), and the hyperactivity of the detrimental definitive endoderm JNK/JUN/AP1 signaling pathway. I found definitive endoderm lacking LNCSOX17 to be functionally impeded in the generation of pancreatic progenitor populations. The studies within this thesis serve as valuable examples to support the functional relevance of 3D nuclear organization and its importance for developmental gene control in cis via CTCF-CTCF loop domain-mediated CRE-promoter contact facilitation. They associate developmental gene expression levels with various phenotypes, identify a so far unknown developmental lncRNA molecule, and imply its relevance for the formation of definitive endoderm. The outlined results advance our knowledge of developmental TF gene-control and its importance for the development of human definitive endoderm

On the origin of vertebrate body plan: Insights from the endoderm using the hourglass model

The vertebrate body plan is thought to be derived during the early Cambrian from a worm-like chordate ancestor. While all three germ layers were clearly involved in this innovation, the role of the endoderm remains elusive. According to the hourglass model, the optimal window for investigating the evolution of vertebrate endoderm-derived structures during cephalochordate development is from the Spemann\u27s organizer stage to the opening of the mouth (Stages 1–7, described herein). Regulatory gene expression, examined during these stages, illustrate that the cephalochordate endoderm is patterned into 12 organ primordia. Early vertebrates inherited at least a portion of 6 of these primordia, while the remainder were lost. Of those that were preserved, we demonstrate that the vertebrate symmetric mouth was built on a vestige of the anterior pre-oral pit, that the pre-existing pharyngeal pouch in this chordate ancestor laid the foundation for the new neural crest cell (NCC)-derived vertebrate-type pharyngeal arches, that the thyroid evolved from the posterior endostyle primordim, that the pancreas was derived from the Pdx1-expressing diverticulum primordium, and the small and large intestines originated with the Cdx1-expressing hindgut rudiments. This investigation uncovers the evolutionary foundations of vertebrate endoderm-derived structures, and demonstrates that the number of organ primordia were reduced during evolution