Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types.

Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence, comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end, here, we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages, including bone, muscle, and heart. We defined the extrinsic signals controlling each binary lineage decision, enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%-99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation, a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively, this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. VIDEO ABSTRACT.

Self-patterning of human stem cells into post-implantation lineages.

Investigating human development is a substantial scientific challenge due to the technical and ethical limitations of working with embryonic samples. In the face of these difficulties, stem cells have provided an alternative to experimentally model inaccessible stages of human development in vitro1-13. Here we show that human pluripotent stem cells can be triggered to self-organize into three-dimensional structures that recapitulate some key spatiotemporal events of early human post-implantation embryonic development. Our system reproducibly captures spontaneous differentiation and co-development of embryonic epiblast-like and extra-embryonic hypoblast-like lineages, establishes key signalling hubs with secreted modulators and undergoes symmetry breaking-like events. Single-cell transcriptomics confirms differentiation into diverse cell states of the perigastrulating human embryo14,15 without establishing placental cell types, including signatures of post-implantation epiblast, amniotic ectoderm, primitive streak, mesoderm, early extra-embryonic endoderm, as well as initial yolk sac induction. Collectively, our system captures key features of human embryonic development spanning from Carnegie stage16 4-7, offering a reproducible, tractable and scalable experimental platform to understand the basic cellular and molecular mechanisms that underlie human development, including new opportunities to dissect congenital pathologies with high throughput.

Primate gastrulation and early organogenesis at single-cell resolution.

Our understanding of human early development is severely hampered by limited access to embryonic tissues. Due to their close evolutionary relationship with humans, nonhuman primates are often used as surrogates to understand human development but currently suffer from a lack of in vivo datasets, especially from gastrulation to early organogenesis during which the major embryonic cell types are dynamically specified. To fill this gap, we collected six Carnegie stage 8-11 cynomolgus monkey (Macaca fascicularis) embryos and performed in-depth transcriptomic analyses of 56,636 single cells. Our analyses show transcriptomic features of major perigastrulation cell types, which help shed light on morphogenetic events including primitive streak development, somitogenesis, gut tube formation, neural tube patterning and neural crest differentiation in primates. In addition, comparative analyses with mouse embryos and human embryoids uncovered conserved and divergent features of perigastrulation development across species-for example, species-specific dependency on Hippo signalling during presomitic mesoderm differentiation-and provide an initial assessment of relevant stem cell models of human early organogenesis. This comprehensive single-cell transcriptome atlas not only fills the knowledge gap in the nonhuman primate research field but also serves as an invaluable resource for understanding human embryogenesis and developmental disorders.

Tracking early mammalian organogenesis - prediction and validation of differentiation trajectories at whole organism scale.

Early organogenesis represents a key step in animal development, during which pluripotent cells diversify to initiate organ formation. Here, we sampled 300,000 single-cell transcriptomes from mouse embryos between E8.5 and E9.5 in 6-h intervals and combined this new dataset with our previous atlas (E6.5-E8.5) to produce a densely sampled timecourse of >400,000 cells from early gastrulation to organogenesis. Computational lineage reconstruction identified complex waves of blood and endothelial development, including a new programme for somite-derived endothelium. We also dissected the E7.5 primitive streak into four adjacent regions, performed scRNA-seq and predicted cell fates computationally. Finally, we defined developmental state/fate relationships by combining orthotopic grafting, microscopic analysis and scRNA-seq to transcriptionally determine cell fates of grafted primitive streak regions after 24 h of in vitro embryo culture. Experimentally determined fate outcomes were in good agreement with computationally predicted fates, demonstrating how classical grafting experiments can be revisited to establish high-resolution cell state/fate relationships. Such interdisciplinary approaches will benefit future studies in developmental biology and guide the in vitro production of cells for organ regeneration and repair.

Formation and segmentation of the vertebrate body axis.

Body axis elongation and segmentation are major morphogenetic events that take place concomitantly during vertebrate embryonic development. Establishment of the final body plan requires tight coordination between these two key processes. In this review, we detail the cellular and molecular as well as the physical processes underlying body axis formation and patterning. We discuss how formation of the anterior region of the body axis differs from that of the posterior region. We describe the developmental mechanism of segmentation and the regulation of body length and segment numbers. We focus mainly on the chicken embryo as a model system. Its accessibility and relatively flat structure allow high-quality time-lapse imaging experiments, which makes it one of the reference models used to study morphogenesis. Additionally, we illustrate conservation and divergence of specific developmental mechanisms by discussing findings in other major embryonic model systems, such as mice, frogs, and zebrafish.

The evolution of gastrulation morphologies.

During gastrulation, early embryos specify and reorganise the topology of their germ layers. Surprisingly, this fundamental and early process does not appear to be rigidly constrained by evolutionary pressures; instead, the morphology of gastrulation is highly variable throughout the animal kingdom. Recent experimental results demonstrate that it is possible to generate different alternative gastrulation modes in single organisms, such as in early cnidarian, arthropod and vertebrate embryos. Here, we review the mechanisms that underlie the plasticity of vertebrate gastrulation both when experimentally manipulated and during evolution. Using the insights obtained from these experiments we discuss the effects of the increase in yolk volume on the morphology of gastrulation and provide new insights into two crucial innovations during amniote gastrulation: the transition from a ring-shaped mesoderm domain in anamniotes to a crescent-shaped domain in amniotes, and the evolution of the reptilian blastoporal plate/canal into the avian primitive streak.

Basement membrane remodelling regulates mouse embryogenesis.

Tissue sculpting during development has been attributed mainly to cellular events through processes such as convergent extension or apical constriction1,2. However, recent work has revealed roles for basement membrane remodelling in global tissue morphogenesis3-5. Upon implantation, the epiblast and extraembryonic ectoderm of the mouse embryo become enveloped by a basement membrane. Signalling between the basement membrane and these tissues is critical for cell polarization and the ensuing morphogenesis6,7. However, the mechanical role of the basement membrane in post-implantation embryogenesis remains unknown. Here we demonstrate the importance of spatiotemporally regulated basement membrane remodelling during early embryonic development. Specifically, we show that Nodal signalling directs the generation and dynamic distribution of perforations in the basement membrane by regulating the expression of matrix metalloproteinases. This basement membrane remodelling facilitates embryo growth before gastrulation. The establishment of the anterior-posterior axis8,9 further regulates basement membrane remodelling by localizing Nodal signalling-and therefore the activity of matrix metalloproteinases and basement membrane perforations-to the posterior side of the embryo. Perforations on the posterior side are essential for primitive-streak extension during gastrulation by rendering the basement membrane of the prospective primitive streak more prone to breaching. Thus spatiotemporally regulated basement membrane remodelling contributes to the coordination of embryo growth, morphogenesis and gastrulation.

A developmental landscape of 3D-cultured human pre-gastrulation embryos.

Our understanding of how human embryos develop before gastrulation, including spatial self-organization and cell type ontogeny, remains limited by available two-dimensional technological platforms1,2 that do not recapitulate the in vivo conditions3-5. Here we report a three-dimensional (3D) blastocyst-culture system that enables human blastocyst development up to the primitive streak anlage stage. These 3D embryos mimic developmental landmarks and 3D architectures in vivo, including the embryonic disc, amnion, basement membrane, primary and primate unique secondary yolk sac, formation of anterior-posterior polarity and primitive streak anlage. Using single-cell transcriptome profiling, we delineate ontology and regulatory networks that underlie the segregation of epiblast, primitive endoderm and trophoblast. Compared with epiblasts, the amniotic epithelium shows unique and characteristic phenotypes. After implantation, specific pathways and transcription factors trigger the differentiation of cytotrophoblasts, extravillous cytotrophoblasts and syncytiotrophoblasts. Epiblasts undergo a transition to pluripotency upon implantation, and the transcriptome of these cells is maintained until the generation of the primitive streak anlage. These developmental processes are driven by different pluripotency factors. Together, findings from our 3D-culture approach help to determine the molecular and morphogenetic developmental landscape that occurs during human embryogenesis.

The extra-embryonic area opaca plays a role in positioning the primitive streak of the early chick embryo.

Classical studies have established that the marginal zone, a ring of extra-embryonic epiblast immediately surrounding the embryonic epiblast (area pellucida) of the chick embryo, is important in setting embryonic polarity by positioning the primitive streak, the site of gastrulation. The more external extra-embryonic region (area opaca) was thought to have only nutritive and support functions. Using experimental embryology approaches, this study reveals three separable functions for this outer region. First, juxtaposition of the area opaca directly onto the area pellucida induces a new marginal zone from the latter; this induced domain is entirely posterior in character. Second, ablation and grafting experiments using an isolated anterior half of the blastoderm and pieces of area opaca suggest that the area opaca can influence the polarity of the adjacent marginal zone. Finally, we show that the loss of the ability of such isolated anterior half-embryos to regulate (re-establish polarity spontaneously) at the early primitive streak stage can be rescued by replacing the area opaca by one from a younger stage. These results uncover new roles of chick extra-embryonic tissues in early development.

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.

Controlled modelling of human epiblast and amnion development using stem cells.

Early human embryonic development involves extensive lineage diversification, cell-fate specification and tissue patterning1. Despite its basic and clinical importance, early human embryonic development remains relatively unexplained owing to interspecies divergence2,3 and limited accessibility to human embryo samples. Here we report that human pluripotent stem cells (hPSCs) in a microfluidic device recapitulate, in a highly controllable and scalable fashion, landmarks of the development of the epiblast and amniotic ectoderm parts of the conceptus, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and primitive streak cells. We further show that amniotic ectoderm-like cells function as a signalling centre to trigger the onset of gastrulation-like events in hPSCs. Given its controllability and scalability, the microfluidic model provides a powerful experimental system to advance knowledge of human embryology and reproduction. This model could assist in the rational design of differentiation protocols of hPSCs for disease modelling and cell therapy, and in high-throughput drug and toxicity screens to prevent pregnancy failure and birth defects.

A single-cell molecular map of mouse gastrulation and early organogenesis.

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.

Spatiotemporal sequence of mesoderm and endoderm lineage segregation during mouse gastrulation.

Anterior mesoderm (AM) and definitive endoderm (DE) progenitors represent the earliest embryonic cell types that are specified during germ layer formation at the primitive streak (PS) of the mouse embryo. Genetic experiments indicate that both lineages segregate from Eomes-expressing progenitors in response to different Nodal signaling levels. However, the precise spatiotemporal pattern of the emergence of these cell types and molecular details of lineage segregation remain unexplored. We combined genetic fate labeling and imaging approaches with single-cell RNA sequencing (scRNA-seq) to follow the transcriptional identities and define lineage trajectories of Eomes-dependent cell types. Accordingly, all cells moving through the PS during the first day of gastrulation express Eomes AM and DE specification occurs before cells leave the PS from Eomes-positive progenitors in a distinct spatiotemporal pattern. ScRNA-seq analysis further suggested the immediate and complete separation of AM and DE lineages from Eomes-expressing cells as last common bipotential progenitor.

Lethal Phenotype-Based Database Screening Identifies Ceramide as a Negative Regulator of Primitive Streak Formation.

In early embryogenesis, the primitive streak (PrS) generates the mesendoderm and is essential for organogenesis. However, because the PrS is a minute and transient tissue, elucidating the mechanism of its formation has been challenging. We performed comprehensive screening of 2 knockout mouse databases based on the fact that failure of PrS formation is lethal. We identified 812 genes involved in various cellular functions and responses that might be linked to PrS formation, with the category of greatest abundance being "Metabolism." In this study, we focused on genes of sphingolipid metabolism and investigated their roles in PrS formation using an in vitro mouse ES cell differentiation system. We show here that elevated intracellular ceramide negatively regulates gene expression essential for PrS formation and instead induces neurogenesis. In addition, sphingosine-1-phosphate (a ceramide derivative) positively regulates neural maturation. Our results indicate that ceramide regulates both PrS formation and the induction of neural differentiation.

Ventricular, atrial, and outflow tract heart progenitors arise from spatially and molecularly distinct regions of the primitive streak.

The heart develops from 2 sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single-cell transcriptomic assay combined with genetic lineage tracing and live imaging, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Moreover, a subset of atrial progenitors are gradually incorporated in posterior locations of the FHF. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract cells originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single-cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are prepatterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function, and disease.

Self-organization of a human organizer by combined Wnt and Nodal signalling.

In amniotes, the development of the primitive streak and its accompanying 'organizer' define the first stages of gastrulation. Although these structures have been characterized in detail in model organisms, the human primitive streak and organizer remain a mystery. When stimulated with BMP4, micropatterned colonies of human embryonic stem cells self-organize to generate early embryonic germ layers 1 . Here we show that, in the same type of colonies, Wnt signalling is sufficient to induce a primitive streak, and stimulation with Wnt and Activin is sufficient to induce an organizer, as characterized by embryo-like sharp boundary formation, markers of epithelial-to-mesenchymal transition and expression of the organizer-specific transcription factor GSC. Moreover, when grafted into chick embryos, human stem cell colonies treated with Wnt and Activin induce and contribute autonomously to a secondary axis while inducing a neural fate in the host. This fulfils the most stringent functional criteria for an organizer, and its discovery represents a milestone in human embryology.

Measurement of junctional tension in epithelial cells at the onset of primitive streak formation in the chick embryo via non-destructive optical manipulation.

Directional cell intercalations of epithelial cells during gastrulation has, in several organisms, been shown to be associated with a planar cell polarity in the organisation of the actin-myosin cytoskeleton and is postulated to reflect directional tension that drives oriented cell intercalations. We have characterised and applied a recently introduced non-destructive optical manipulation technique to measure the tension in individual epithelial cell junctions of cells in various locations and orientations in the epiblast of chick embryos in the early stages of primitive streak formation. Junctional tension of mesendoderm precursors in the epiblast is higher in junctions oriented in the direction of intercalation than in junctions oriented perpendicular to the direction of intercalation and higher than in junctions of other cells in the epiblast. The kinetic data fit best with a simple viscoelastic Maxwell model, and we find that junctional tension, and to a lesser extent viscoelastic relaxation time, are dependent on myosin activity.

Principles of early human development and germ cell program from conserved model systems.

Human primordial germ cells (hPGCs), the precursors of sperm and eggs, originate during weeks 2-3 of early post-implantation development. Using in vitro models of hPGC induction, recent studies have suggested that there are marked mechanistic differences in the specification of human and mouse PGCs. This may be due in part to the divergence in their pluripotency networks and early post-implantation development. As early human embryos are not accessible for direct study, we considered alternatives including porcine embryos that, as in humans, develop as bilaminar embryonic discs. Here we show that porcine PGCs originate from the posterior pre-primitive-streak competent epiblast by sequential upregulation of SOX17 and BLIMP1 in response to WNT and BMP signalling. We use this model together with human and monkey in vitro models simulating peri-gastrulation development to show the conserved principles of epiblast development for competency for primordial germ cell fate. This process is followed by initiation of the epigenetic program and regulated by a balanced SOX17-BLIMP1 gene dosage. Our combinatorial approach using human, porcine and monkey in vivo and in vitro models provides synthetic insights into early human development.

Exploring the roles of FGF/MAPK and cVG1/GDF signalling on mesendoderm induction and convergent extension during chick primitive streak formation.

During primitive streak formation in the chick embryo, cells undergo mesendoderm specification and convergent extension at the same time and in the same cells. Previous work has implicated cVG1 (GDF3) as a key factor for induction of primitive streak identity and positioning the primitive streak, whereas FGF signalling was implicated in regulating cell intercalation via regulation of components of the WNT-planar cell polarity (PCP) pathway. FGF has also been reported to be able to induce a primitive streak (but lacking the most axial derivatives such as notochord/prechordal mesendoderm). These signals emanate from different cell populations in the embryo, so how do they interact to ensure that the same cells undergo both cell intercalation and acquire primitive streak identity? Here we begin to address this question by examining in more detail the ability of the two classes of signals in regulating the two developmental events. Using misexpression of inducers and/or exposure to inhibitors and in situ hybridisation, we study how these two signals regulate expression of Brachyury (TBXT) and PRICKLE1 as markers for the primitive streak and the PCP, respectively. We find that both signals can induce both properties, but while FGF seems to be required for induction of the streak by cVG1, it is not necessary for induction of PRICKLE1. The results are consistent with cVG1 being a common regulator for both primitive streak identity and the initiation of convergent extension that leads to streak elongation.

Prediction and control of symmetry breaking in embryoid bodies by environment and signal integration.

During early embryogenesis, mechanical constraints and localized biochemical signals co-occur around anteroposterior axis determination and symmetry breaking. Their relative roles, however, are hard to tease apart in vivo Using brachyury (Bra), a primitive streak and mesendoderm marker in mouse embryoid bodies (EBs), we studied how contact, biochemical cues and neighboring cell cues affect the positioning of a primitive streak-like locus and thus determine the anteroposterior axis. We show that a Bra-competent layer must be formed in the EB before Bra expression initiates, and that Bra onset locus position is biased by contact points of the EB with its surrounding, probably through modulation of chemical cues rather than by mechanical signaling. We can push or pull Bra onset away from contact points by introducing a separate localized Wnt signal source, or maneuver Bra onset to a few loci or to an isotropic peripheral pattern. Furthermore, we show that Foxa2-positive cells are predictive of the future location of Bra onset, demonstrating an earlier symmetry-breaking event. Our analysis of factors affecting symmetry breaking and spatial fate choice during this developmental process could prove valuable for in vitro differentiation and organoid formation.