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Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1 −/− chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine. Single-cell profiling is used to create a molecular-level atlas of cell differentiation trajectories during gastrulation and early organogenesis in the mouse.

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes 1 – 5 . Global epigenetic reprogramming accompanies these changes 6 – 8 , but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers. Single-cell mapping of chromatin accessibility, DNA methylation and RNA expression during gastrulation in mouse embryos shows characteristic epigenetic changes that accompany formation of the primary germ layers.

Background Human pluripotent stem cells offer the best available model to study the underlying cellular and molecular mechanisms of human embryonic lineage specification. However, it is not fully understood how individual stem cells exit the pluripotent state and transition towards their respective progenitor states. Results Here, we analyze the transcriptomes of human embryonic stem cell-derived lineage-specific progenitors by single-cell RNA-sequencing (scRNA-seq). We identify a definitive endoderm (DE) transcriptomic signature that leads us to pinpoint a critical time window when DE differentiation is enhanced by hypoxia. The molecular mechanisms governing the emergence of DE are further examined by time course scRNA-seq experiments, employing two new statistical tools to identify stage-specific genes over time (SCPattern) and to reconstruct the differentiation trajectory from the pluripotent state through mesendoderm to DE (Wave-Crest). Importantly, presumptive DE cells can be detected during the transitory phase from Brachyury (T) + mesendoderm toward a CXCR4 + DE state. Novel regulators are identified within this time window and are functionally validated on a screening platform with a T-2A-EGFP knock-in reporter engineered by CRISPR/Cas9. Through loss-of-function and gain-of-function experiments, we demonstrate that KLF8 plays a pivotal role modulating mesendoderm to DE differentiation. Conclusions We report the analysis of 1776 cells by scRNA-seq covering distinct human embryonic stem cell-derived progenitor states. By reconstructing a differentiation trajectory at single-cell resolution, novel regulators of the mesendoderm transition to DE are elucidated and validated. Our strategy of combining single-cell analysis and genetic approaches can be applied to uncover novel regulators governing cell fate decisions in a variety of systems.

Tissue morphogenesis arises from coordinated changes in cell shape driven by actomyosin contractions. Patterns of gene expression regionalize cell behaviours by controlling actomyosin contractility. Here we report two modes of control over Rho1 and myosin II (MyoII) activation in the Drosophila endoderm. First, Rho1–MyoII are induced in a spatially restricted primordium via localized transcription of the G-protein-coupled receptor ligand Fog. Second, a tissue-scale wave of Rho1–MyoII activation and cell invagination progresses anteriorly away from the primordium. The wave does not require sustained gene transcription, and is not governed by regulated Fog delivery. Instead, MyoII inhibition blocks Rho1 activation and propagation, revealing a mechanical feedback driven by MyoII. We find that MyoII activation and invagination in each row of cells drives adhesion to the vitelline membrane mediated by integrins, apical spreading, MyoII activation and invagination in the next row. Endoderm morphogenesis thus emerges from local transcriptional initiation and a mechanically driven cycle of cell deformation. Tissue shape changes in the posterior endoderm of the early Drosophila embryo are driven by actomyosin contractions emerging from a transcriptional induction followed by a mechanically-driven propagation of RhoI–myosin II activation.

Organoids have extensive therapeutic potential and are increasingly opening up new avenues within regenerative medicine. However, their clinical application is greatly limited by the lack of effective GMP-compliant systems for organoid expansion in culture. Here, we envisage that the use of extracellular matrix (ECM) hydrogels derived from decellularized tissues (DT) can provide an environment capable of directing cell growth. These gels possess the biochemical signature of tissue-specific ECM and have the potential for clinical translation. Gels from decellularized porcine small intestine (SI) mucosa/submucosa enable formation and growth of endoderm-derived human organoids, such as gastric, hepatic, pancreatic, and SI. ECM gels can be used as a tool for direct human organoid derivation, for cell growth with a stable transcriptomic signature, and for in vivo organoid delivery. The development of these ECM-derived hydrogels opens up the potential for human organoids to be used clinically. Organoid cultures have been developed from multiple tissues, opening new possibilities for regenerative medicine. Here the authors demonstrate the derivation of GMP-compliant hydrogels from decellularized porcine small intestine which support formation and growth of human gastric, liver, pancreatic and small intestinal organoids.

Here we delineate the ontogeny of the mammalian endoderm by generating 112,217 single-cell transcriptomes, which represent all endoderm populations within the mouse embryo until midgestation. We use graph-based approaches to model differentiating cells, which provides a spatio-temporal characterization of developmental trajectories and defines the transcriptional architecture that accompanies the emergence of the first (primitive or extra-embryonic) endodermal population and its sister pluripotent (embryonic) epiblast lineage. We uncover a relationship between descendants of these two lineages, in which epiblast cells differentiate into endoderm at two distinct time points—before and during gastrulation. Trajectories of endoderm cells were mapped as they acquired embryonic versus extra-embryonic fates and as they spatially converged within the nascent gut endoderm, which revealed these cells to be globally similar but retain aspects of their lineage history. We observed the regionalized identity of cells along the anterior–posterior axis of the emergent gut tube, which reflects their embryonic or extra-embryonic origin, and the coordinated patterning of these cells into organ-specific territories. The developing mouse gut endoderm, mapped at single-cell resolution, reveals trajectories of cell differentiation before and during gastrulation and the emergence of regionalized cell identities along the anterior–posterior axis of the gut tube.

Single-cell RNA sequencing allows us to model cellular state dynamics and fate decisions using expression similarity or RNA velocity to reconstruct state-change trajectories; however, trajectory inference does not incorporate valuable time point information or utilize additional modalities, whereas methods that address these different data views cannot be combined or do not scale. Here we present CellRank 2, a versatile and scalable framework to study cellular fate using multiview single-cell data of up to millions of cells in a unified fashion. CellRank 2 consistently recovers terminal states and fate probabilities across data modalities in human hematopoiesis and endodermal development. Our framework also allows combining transitions within and across experimental time points, a feature we use to recover genes promoting medullary thymic epithelial cell formation during pharyngeal endoderm development. Moreover, we enable estimating cell-specific transcription and degradation rates from metabolic-labeling data, which we apply to an intestinal organoid system to delineate differentiation trajectories and pinpoint regulatory strategies. CellRank 2 is a comprehensive framework of cell fate analysis using large-scale multiview single-cell data.

It is generally accepted that epiblast cells ingress into the primitive streak by epithelial-to-mesenchymal transition (EMT) to give rise to the mesoderm; however, it is less clear how the endoderm acquires an epithelial fate. Here, we used embryonic stem cell and mouse embryo knock‐in reporter systems to combine time-resolved lineage labelling with high-resolution single-cell transcriptomics. This allowed us to resolve the morphogenetic programs that segregate the mesoderm from the endoderm germ layer. Strikingly, while the mesoderm is formed by classical EMT, the endoderm is formed independent of the key EMT transcription factor Snail1 by mechanisms of epithelial cell plasticity. Importantly, forkhead box transcription factor A2 (Foxa2) acts as an epithelial gatekeeper and EMT suppressor to shield the endoderm from undergoing a mesenchymal transition. Altogether, these results not only establish the morphogenetic details of germ layer formation, but also have broader implications for stem cell differentiation and cancer metastasis. Scheibner et al. demonstrate that, during gastrulation in the mouse, epithelial epiblast progenitors upregulate Foxa2 and form the definitive endoderm independently of a full EMT–MET cycle.

Intestinal tissue made in vitro Using a sequence of growth-factor manipulations to mimic embryonic intestinal development in culture, a new study has successfully generated human intestinal tissue in vitro from embryonic and induced pluripotent stem cells. The resulting epithelium was uniformly intestinal, with villus-like structures and progenitor domains, and contained all of the functional cell types of the gut, including brush borders that were indistinguishable from adult intestine. This approach may provide therapeutic benefit for disease studies. Using a temporal series of growth factor manipulations to mimic embryonic intestinal development in culture, this study has successfully directed the differentiation of human pluripotent stem cells (both embryonic stem cells and induced pluripotent stem cells) into intestinal tissue. This approach may provide therapeutic benefit for disease studies. Studies in embryonic development have guided successful efforts to direct the differentiation of human embryonic and induced pluripotent stem cells (PSCs) into specific organ cell types in vitro 1 , 2 . For example, human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells 3 , 4 , 5 , 6 that have therapeutic efficacy in animal models of liver disease 7 , 8 and diabetes 9 , respectively. However, the generation of complex three-dimensional organ tissues in vitro remains a major challenge for translational studies. Here we establish a robust and efficient process to direct the differentiation of human PSCs into intestinal tissue in vitro using a temporal series of growth factor manipulations to mimic embryonic intestinal development 10 . This involved activin-induced definitive endoderm formation 11 , FGF/Wnt-induced posterior endoderm pattering, hindgut specification and morphogenesis 12 , 13 , 14 , and a pro-intestinal culture system 15 , 16 to promote intestinal growth, morphogenesis and cytodifferentiation. The resulting three-dimensional intestinal ‘organoids’ consisted of a polarized, columnar epithelium that was patterned into villus-like structures and crypt-like proliferative zones that expressed intestinal stem cell markers 17 . The epithelium contained functional enterocytes, as well as goblet, Paneth and enteroendocrine cells. Using this culture system as a model to study human intestinal development, we identified that the combined activity of WNT3A and FGF4 is required for hindgut specification whereas FGF4 alone is sufficient to promote hindgut morphogenesis. Our data indicate that human intestinal stem cells form de novo during development. We also determined that NEUROG3, a pro-endocrine transcription factor that is mutated in enteric anendocrinosis 18 , is both necessary and sufficient for human enteroendocrine cell development in vitro . PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development and disease.

Direct lineage reprogramming involves the conversion of cellular identity. Single-cell technologies are useful for deconstructing the considerable heterogeneity that emerges during lineage conversion. However, lineage relationships are typically lost during cell processing, complicating trajectory reconstruction. Here we present ‘CellTagging’, a combinatorial cell-indexing methodology that enables parallel capture of clonal history and cell identity, in which sequential rounds of cell labelling enable the construction of multi-level lineage trees. CellTagging and longitudinal tracking of fibroblast to induced endoderm progenitor reprogramming reveals two distinct trajectories: one leading to successfully reprogrammed cells, and one leading to a ‘dead-end’ state, paths determined in the earliest stages of lineage conversion. We find that expression of a putative methyltransferase, Mettl7a1 , is associated with the successful reprogramming trajectory; adding Mettl7a1 to the reprogramming cocktail increases the yield of induced endoderm progenitors. Together, these results demonstrate the utility of our lineage-tracing method for revealing the dynamics of direct reprogramming. Combinatorial tagging of single cells using expressed DNA barcodes, delivered by a lentiviral vector, is used to track individual cells and reconstruct their lineages and trajectories during cell fate reprogramming.