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

T-REX17 is a transiently expressed non-coding RNA essential for human endoderm formation

Long non-coding RNAs (lncRNAs) have emerged as fundamental regulators in various biological processes, including embryonic development and cellular differentiation. Despite much progress over the past decade, the genome-wide annotation of lncRNAs remains incomplete and many known non-coding loci are still poorly characterized. Here, we report the discovery of a previously unannotated lncRNA that is transcribed 230 kb upstream of the SOX17 gene and located within the same topologically associating domain. We termed it T-REX17 (Transcript Regulating Endoderm and activated by soX17) and show that it is induced following SOX17 activation but its expression is more tightly restricted to early definitive endoderm. Loss of T-REX17 affects crucial functions independent of SOX17 and leads to an aberrant endodermal transcriptome, signaling pathway deregulation and epithelial to mesenchymal transition defects. Consequently, cells lacking the lncRNA cannot further differentiate into more mature endodermal cell types. Taken together, our study identified and characterized T-REX17 as a transiently expressed and essential non-coding regulator in early human endoderm differentiation

Hypoxia induces an early primitive streak signature, enhancing spontaneous elongation and lineage representation in gastruloids

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

Spatiotemporal transcriptomic maps of whole mouse embryos at the onset of organogenesis

Spatiotemporal orchestration of gene expression is required for proper embryonic development. The use of single-cell technologies has begun to provide improved resolution of early regulatory dynamics, including detailed molecular definitions of most cell states during mouse embryogenesis. Here we used Slide-seq to build spatial transcriptomic maps of complete embryonic day (E) 8.5 and E9.0, and partial E9.5 embryos. To support their utility, we developed sc3D, a tool for reconstructing and exploring three-dimensional ‘virtual embryos’, which enables the quantitative investigation of regionalized gene expression patterns. Our measurements along the main embryonic axes of the developing neural tube revealed several previously unannotated genes with distinct spatial patterns. We also characterized the conflicting transcriptional identity of ‘ectopic’ neural tubes that emerge in Tbx6 mutant embryos. Taken together, we present an experimental and computational framework for the spatiotemporal investigation of whole embryonic structures and mutant phenotypes

Dynamic antagonism between key repressive pathways maintains the placental epigenome

DNA and Histone 3 Lysine 27 methylation typically function as repressive modifications and operate within distinct genomic compartments. In mammals, the majority of the genome is kept in a DNA methylated state, whereas the Polycomb repressive complexes regulate the unmethylated CpG-rich promoters of developmental genes. In contrast to this general framework, the extra-embryonic lineages display non-canonical, globally intermediate DNA methylation levels, including disruption of local Polycomb domains. Here, to better understand this unusual landscape’s molecular properties, we genetically and chemically perturbed major epigenetic pathways in mouse trophoblast stem cells. We find that the extra-embryonic epigenome reflects ongoing and dynamic de novo methyltransferase recruitment, which is continuously antagonized by Polycomb to maintain intermediate, locally disordered methylation. Despite its disorganized molecular appearance, our data point to a highly controlled equilibrium between counteracting repressors within extra-embryonic cells, one that can seemingly persist indefinitely without bistable features typically seen for embryonic forms of epigenetic regulation

A CLK3-HMGA2 Alternative Splicing Axis Impacts Human Hematopoietic Stem Cell Molecular Identity throughout Development

While gene expression dynamics have been extensively cataloged during hematopoietic differentiation in the adult, less is known about transcriptome diversity of human hematopoietic stem cells (HSCs) during development. To characterize transcriptional and post-transcriptional changes in HSCs during development, we leveraged high-throughput genomic approaches to profile miRNAs, lincRNAs, and mRNAs. Our findings indicate that HSCs manifest distinct alternative splicing patterns in key hematopoietic regulators. Detailed analysis of the splicing dynamics and function of one such regulator, HMGA2, identified an alternative isoform that escapes miRNA-mediated targeting. We further identified the splicing kinase CLK3 that, by regulating HMGA2 splicing, preserves HMGA2 function in the setting of an increase in let-7 miRNA levels, delineating how CLK3 and HMGA2 form a functional axis that influences HSC properties during development. Collectively, our study highlights molecular mechanisms by which alternative splicing and miRNA-mediated post-transcriptional regulation impact the molecular identity and stage-specific developmental features of human HSCs. Human hematopoietic stem cells (HSCs) display substantial transcriptional diversity during development. Here, we investigated the contribution of alternative splicing to such diversity by analyzing the dynamics of a key hematopoietic regulator, HMGA2. Next, we showed that CLK3, by regulating the splicing pattern of HMGA2, reinforces an HSC-specific program

Generation and molecular characterization of mouse embryonic stem cells derived Trunk-Like Structures

One of the greatest mysteries in developmental biology is how the orchestrated process of cellular differentiation guides a single cell to form a functional organism comprised of complex tissues and organs. A detailed understanding of these events in mammals is obscured by the inaccessibility of embryonic development in utero. In recent years, community efforts have led to the development of stem cell-based models of embryogenesis, able to mimic some cellular and molecular aspects of the early stages of development in vitro. Despite these advances, such models lack proper embryo-like morphogenesis necessary for tissue formation and organ development. To overcome this limitation, I optimized and characterized a protocol to precisely model the morphogenetic changes that occur during gastrulation and early organogenesis. Within only five days, the protocol produces “Trunk-Like Structures (TLS),” self-organized synthetic embryos originating from mouse pluripotent stem cell aggregates treated with chemical signaling molecules (gastruloids) and embedded in an extra-cellular-matrix (ECM) to mirror the in utero environment. TLS morphologically and molecularly resemble in vivo development, displaying a high level of trunk-tissue organization that includes the presence of somites (building blocks of future bones, muscles, and cartilage), a neural tube (future spinal cord), and a gut tube (future gastrointestinal tract). To test TLS similarities to the in vivo embryo, I integrated different single-cell technologies and confirmed the high complexity of these synthetic embryos. Specifically, I observed that TLS progress from their stem cell origin through cell type maturation, producing relevant subtypes of the developing trunk. Unlike in vivo development, the TLS platform allows rapid and tunable genetic and chemical perturbations with the benefits of uninterrupted and continuous observation. As a proof of principle, I utilized CRISPR/Cas9 based genetic ablation to recapitulate a well-studied mutant phenotype. Next, I influenced TLS developmental trajectories by targeted chemical modulation, resulting in the overproduction of somites, a phenotype never observed before in vivo. Finally, I investigated the molecular mechanism responsible for the enhanced tissue morphogenesis observed in TLS. I found that cells’ ability to organize into complex tissues requires the presence of the extra-cellular-matrix, a specific feature we implemented in our protocol. Trunk-Like Structures provide a scalable, tractable, and reproducible platform to study normal and aberrant embryonic development in vitro at an unprecedented spatiotemporal resolution

Topological isolation of developmental regulators in mammalian genomes

Precise control of mammalian gene expression is facilitated through epigenetic mechanisms and nuclear organization. In particular, insulated chromosome structures are important for regulatory control, but the phenotypic consequences of their boundary disruption on developmental processes are complex and remain insufficiently understood. Here, we generated deeply sequenced Hi-C data for human pluripotent stem cells (hPSCs) that allowed us to identify CTCF loop domains that have highly conserved boundary CTCF sites and show a notable enrichment of individual developmental regulators. Importantly, perturbation of such a boundary in hPSCs interfered with proper differentiation through deregulated distal enhancer-promoter activity. Finally, we found that germline variations affecting such boundaries are subject to purifying selection and are underrepresented in the human population. Taken together, our findings highlight the importance of developmental gene isolation through chromosomal folding structures as a mechanism to ensure their proper expression

T-REX17 is a transiently expressed non-coding RNA essential for human endoderm formation

Long non-coding RNAs (lncRNAs) have emerged as fundamental regulators in various biological processes, including embryonic development and cellular differentiation. Despite much progress over the past decade, the genome-wide annotation of lncRNAs remains incomplete and many known non-coding loci are still poorly characterized. Here, we report the discovery of a previously unannotated lncRNA that is transcribed 230 kb upstream of the SOX17 gene and located within the same topologically associating domain. We termed it T-REX17 (Transcript Regulating Endoderm and activated by soX17) and show that it is induced following SOX17 activation but its expression is more tightly restricted to early definitive endoderm. Loss of T-REX17 affects crucial functions independent of SOX17 and leads to an aberrant endodermal transcriptome, signaling pathway deregulation and epithelial to mesenchymal transition defects. Consequently, cells lacking the lncRNA cannot further differentiate into more mature endodermal cell types. Taken together, our study identified and characterized T-REX17 as a transiently expressed and essential non-coding regulator in early human endoderm differentiation