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Large-scale cell flow characterizes gastrulation in animal development. In amniote gastrulation, particularly in avian gastrula, a bilateral vortex-like counter-rotating cell flow, called 'polonaise movements', appears along the midline. Here, through experimental manipulations, we addressed relationships between the polonaise movements and morphogenesis of the primitive streak, the earliest midline structure in amniotes. Suppression of the Wnt/planar cell polarity (PCP) signaling pathway maintains the polonaise movements along a deformed primitive streak. Mitotic arrest leads to diminished extension and development of the primitive streak and maintains the early phase of the polonaise movements. Ectopically induced Vg1, an axis-inducing morphogen, generates the polonaise movements, aligned to the induced midline, but disturbs the stereotypical cell flow pattern at the authentic midline. Despite the altered cell flow, induction and extension of the primitive streak are preserved along both authentic and induced midlines. Finally, we show that ectopic axis-inducing morphogen, Vg1, is capable of initiating the polonaise movements without concomitant PS extension under mitotic arrest conditions. These results are consistent with a model wherein primitive streak morphogenesis is required for the maintenance of the polonaise movements, but the polonaise movements are not necessarily responsible for primitive streak morphogenesis. Our data describe a previously undefined relationship between the large-scale cell flow and midline morphogenesis in gastrulation.

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BACKGROUND: The avian node is the equivalent of the amphibian Spemann's organizer, as indicated by its ability to induce a secondary axis, cellular contribution, and gene expression, whereas the node of the mouse, which displays limited inductive capacities, was suggested to be a part of spatially distributed signaling. Furthermore, the structural identity of the mouse node is subject of controversy, while little is known about equivalent structures in other mammals. RESULTS: We analyzed the node and emerging organizer in the pig using morphology and the expression of selected organizer genes prior to and during gastrulation. The node was defined according to the "four-quarter model" based on comparative consideration. The node of the pig displays a multilayered, dense structure that includes columnar epithelium, bottle-like cells in the dorsal part, and mesenchymal cells ventrally. Expression of goosecoid (gsc), chordin, and brachyury, together with morphology, reveal the consecutive emergence of three distinct domains: the gastrulation precursor domain, the presumptive node, and the mature node. Additionally, gsc displays a ventral expression domain prior to epiblast epithelialization. CONCLUSION: Our study defines the morphological and molecular context of the emerging organizer equivalent in the pig and suggests a sequential development of its function.

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During embryonic development, the timing of events at the cellular level must be coordinated across multiple length scales to ensure the formation of a well-proportioned body plan. This is clear during somitogenesis, where progenitors must be allocated to the axis over time whilst maintaining a progenitor population for continued elaboration of the body plan. However, the relative importance of intrinsic and extrinsic signals in timing progenitor addition at the single-cell level is not yet understood. Heterochronic grafts from older to younger embryos have suggested a level of intrinsic timing whereby later staged cells contribute to more posterior portions of the axis. To determine the precise step at which cells are delayed, we performed single-cell transcriptomic analysis on heterochronic grafts of somite progenitors in the chicken embryo. This revealed a previously undescribed cell state within which heterochronic grafted cells are stalled. The delayed exit of older cells from this state correlates with expression of posterior Hox genes. Using grafting and explant culture, we find that both Hox gene expression and the migratory capabilities of progenitor populations are intrinsically regulated at the population level. However, by grafting varied sizes of tissue, we find that small heterochronic grafts disperse more readily and contribute to more anterior portions of the body axis while still maintaining Hox gene expression. This enhanced dispersion is not replicated in explant culture, suggesting that it is a consequence of interaction between host and donor tissue and thus extrinsic to the donor tissue. Therefore, we demonstrate that the timing of cell dispersion and resulting axis contribution is impacted by a combination of both intrinsic and extrinsic cues.

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The microfluidic amniotic sac embryoid (µPASE) is a human pluripotent stem cell (hPSC)-derived multicellular human embryo-like structure with molecular and morphological features resembling the progressive development of the early post-implantation human embryonic sac. The microfluidic device is specifically designed to control the formation of hPSC clusters and expose the clusters to different morphogen environments, allowing the development of µPASEs in a highly controllable, reproducible, and scalable fashion. The µPASE model displays human embryonic developmental landmarks such as lumenogenesis of the epiblast, amniotic cavity formation, and the specification of primordial germ cells and gastrulating cells (or mesendoderm cells). Here, we provide detailed instructions needed to reproduce µPASEs, including the immunofluorescence staining and cell retrieval protocols for characterizing µPASEs obtained under different experimental conditions.

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During mammalian gastrulation, a mass of pluripotent cells surrounded by extraembryonic tissues differentiates into germ layers, mesoderm, endoderm, and ectoderm. The three germ layers are then organized into a body plan with organ rudiments via morphogenetic gastrulation movements of emboly, epiboly, convergence, and extension. Emboly is the most conserved gastrulation movement, whereby mesodermal and endodermal progenitors undergo epithelial-to-mesenchymal transition (EMT) and move via a blastopore/primitive streak beneath the ectoderm. Decades of embryologic, genetic, and molecular studies in invertebrates and vertebrates, delineated a BMP > WNT > NODAL signaling cascade underlying mesoderm and endoderm specification. Advances have been made in the research animals in understanding the cellular and molecular mechanisms underlying gastrulation morphogenesis. In contrast, little is known about human gastrulation, which occurs in utero during the third week of gestation and its investigations face ethical and methodological limitations. This is changing with the unprecedented progress in modeling aspects of human development, using human pluripotent stem cells (hPSCs), including embryonic stem cells (hESC)-based embryo-like models (SCEMs). In one approach, hESCs of various pluripotency are aggregated to self-assemble into structures that resemble pre-implantation or post-implantation embryo-like structures that progress to early gastrulation, and some even reach segmentation and neurulation stages. Another approach entails coaxing hESCs with biochemical signals to generate germ layers and model aspects of gastrulation morphogenesis, such as EMT. Here, we review the recent advances in understanding signaling cascades that direct germ layers specification and the early stages of gastrulation morphogenesis in these models. We discuss outstanding questions, challenges, and opportunities for this promising area of developmental biology.

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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.

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Naive human pluripotent stem cells have the remarkable ability to self-organize into blastocyst-like structures ("blastoids") that model lineage segregation in the pre-implantation embryo. However, the extent to which blastoids can recapitulate the defining features of human post-implantation development remains unexplored. Here, we report that blastoids cultured on thick three-dimensional (3D) extracellular matrices capture hallmarks of early post-implantation development, including epiblast lumenogenesis, rapid expansion and diversification of trophoblast lineages, and robust invasion of extravillous trophoblast cells by day 14. Extended blastoid culture results in the localized activation of primitive streak marker TBXT and the emergence of embryonic germ layers by day 21. We also show that the modulation of WNT signaling alters the balance between epiblast and trophoblast fates in post-implantation blastoids. This work demonstrates that 3D-cultured blastoids offer a continuous and integrated in vitro model system of human embryonic and extraembryonic development from pre-implantation to early gastrulation stages.

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The development process of human embryo until blastocyst is well understood during the past 30 years, however, embryogenesis from blastocyst to pre-gastrulation was still remained a "black box". Limited by research materials and culture technologies, the "black box" is still unopened. We recently established an extended three-dimensional (3D) culture system of human blastocysts (Xiang et al., Nature 577(7791):537-542, 2020). The 3D embryo culture system could enable human blastocyst growing up to early primitive streak anlage stage in vitro. Here, we introduce the detail protocol and notes of culturing human 3D embryos.

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During the early development of Placentalia, a distinctive projection emerges at the posterior embryonic-extraembryonic interface of the conceptus; its fingerlike shape presages maturation into the placental umbilical cord, whose major role is to shuttle fetal blood to and from the chorion for exchange with the mother during pregnancy. Until recently, the biology of the cord's vital vascular anlage, called the body stalk/allantois in humans and simply the allantois in rodents, has been largely unknown. Here, new insights into the development of the mouse allantois are featured, from its origin and mechanism of arterial patterning through its union with the chorion. Key to generating the allantois and its critical functions are the primitive streak and visceral endoderm, which together are sufficient to create the entire fetal-placental connection. Their newly discovered roles at the embryonic-extraembryonic interface challenge conventional wisdom, including the physical limits of the primitive streak, its function as sole purveyor of mesoderm in the mouse, potency of visceral endoderm, and the putative role of the allantois in the germ line. With this working model of allantois development, understanding a plethora of hitherto poorly understood orphan diseases in humans is now within reach. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

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Despite its clinical and fundamental importance, our understanding of early human development remains limited. Stem cell-derived, embryo-like structures (or embryoids) allowing studies of early development without using natural embryos can potentially help fill the knowledge gap of human development. Herein, transcriptome at the single-cell level of a human embryoid model was profiled at different time points. Molecular maps of lineage diversifications from the pluripotent human epiblast toward the amniotic ectoderm, primitive streak/mesoderm, and primordial germ cells were constructed and compared with in vivo primate data. The comparative transcriptome analyses reveal a critical role of NODAL signaling in human mesoderm and primordial germ cell specification, which is further functionally validated. Through comparative transcriptome analyses and validations with human blastocysts and in vitro cultured cynomolgus embryos, we further proposed stringent criteria for distinguishing between human blastocyst trophectoderm and early amniotic ectoderm cells.

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The mechanism governing the transition of human embryonic stem cells (hESCs) toward differentiated cells is only partially understood. To explore this transition, the activity and expression of the ubiquitous phosphatidylinositol 3-kinase (PI3Kα and PI3Kß) were modulated in primed hESCs. The study reports a pathway that dismantles the restraint imposed by the EZH2 polycomb repressor on an essential stemness gene, NODAL, and on transcription factors required to trigger primitive streak formation. The primitive streak is the site where gastrulation begins to give rise to the three embryonic cell layers from which all human tissues derive. The pathway involves a PI3Kß non-catalytic action that controls nuclear/active RAC1 levels, activation of JNK (Jun N-terminal kinase) and nuclear ß-catenin accumulation. ß-Catenin deposition at promoters triggers release of the EZH2 repressor, permitting stemness maintenance (through control of NODAL) and correct differentiation by allowing primitive streak master gene expression. PI3Kß epigenetic control of EZH2/ß-catenin might be modulated to direct stem cell differentiation.

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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.

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Embryo studies have established that the patterning of the mouse gastrula depends on a regulatory network in which the WNT, BMP, and NODAL signaling pathways cooperate, but aspects of their respective contributions remain unclear. Studying their impact on the spatial organization and developmental trajectories of micropatterned epiblast-like cell (EpiLC) colonies, we show that NODAL is required prior to BMP action to establish the mesoderm and endoderm lineages. The presence of BMP then forces NODAL and WNT to support the formation of posterior primitive streak (PS) derivatives, while its absence allows them to promote that of anterior PS derivatives. Also, a Nodal mutation elicits more severe patterning defects in vitro than in the embryo, suggesting that ligands of extra-embryonic origin can rescue them. These results support the implication of a combinatorial process in PS patterning and illustrate how the study of micropatterned EpiLC colonies can complement that of embryos.

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During the last decades, signaling pathways responsible for the initiation of gastrulation in mammalian embryos have been identified. However, the physical rules governing the tissue spatial patterning and the extensive morphogenetic movements occurring during that process are still elusive. Progress on these issues is slowed by the difficulty to record or perturb the patterning events in real time, especially in mammalian embryos that develop in utero. Because they permit easy observation and manipulation, in vitro model systems offer an exciting opportunity to dissect the rules governing the organization of the mammalian gastrula. For instance, it is sufficient to cultivate human embryonic stem cells on micropatterned substrates to reveal their self-organization potential. We present here a method to obtain micropatterned mouse Epiblast Like Cells colonies, providing a convenient way to compare spatial organization of mouse and human pluripotent stem cells and to complement the characterization of mutant embryos in a controlled environment.

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In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos, including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name the 'neighbourhood watch' model) illuminates the classic 'French Flag Problem' and how positional information is interpreted by a sheet of cells in a large developing system.

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In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen's node, which generates neural, mesodermal, and endodermal structures along the midline. While most cells only pass through this region, the node has been suggested to contain a small population of resident stem cells. However, it is unknown whether the rest of the node constitutes an instructive niche that specifies this self-renewal behavior. Here, we use heterotopic transplantation of groups and single cells and show that cells not destined to enter the node can become resident and self-renew. Long-term resident cells are restricted to the posterior part of the node and single-cell RNA-sequencing reveals that the majority of these resident cells preferentially express G2/M phase cell-cycle-related genes. These results provide strong evidence that the node functions as a niche to maintain self-renewal of axial progenitors.

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The formation of the primitive streak (PS) and the subsequent induction of neuroectoderm are hallmarks of gastrulation. Combining an in vitro reconstitution of this process based on mouse embryonic stem cells (mESCs) with a collection of knockouts in reporter mESC lines, we identified retinoic acid (RA) as a critical mediator of early neural induction triggered by TGFß or Wnt signaling inhibition. Single-cell RNA sequencing analysis captured the temporal unfolding of cell type diversification, up to the emergence of somite and neural fates. In the absence of the RA-synthesizing enzyme Aldh1a2, a sensitive RA reporter revealed a hitherto unidentified residual RA signaling that specified neural fate. Genetic evidence showed that the RA-degrading enzyme Cyp26a1 protected PS-like cells from neural induction, even in the absence of TGFß and Wnt antagonists. Overall, we characterized a multi-layered control of RA levels that regulates early neural differentiation in an in vitro PS-like system.

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The anterior-posterior axis is a central element of the body plan and, during amniote gastrulation, forms through several transient domains with specific morphogenetic activities. In the chick, experimentally proven activity of signalling molecules and transcription factors lead to the concept of a 'global positioning system' for initial axis formation whereas in the (mammotypical) rabbit embryo, a series of morphological or molecular domains are part of a putative 'three-anchor-point model'. Because circular expression patterns of genes involved in axis formation exist in both amniote groups prior to, and during, gastrulation and may thus be suited to reconcile these models, the expression patterns of selected genes known in the chick, namely the ones coding for the transcription factors eomes and tbx6, the signalling molecule wnt3 and the wnt inhibitor pkdcc, were analysed in the rabbit embryonic disc using in situ hybridisation and placing emphasis on their germ layer location. Peripheral wnt3 and eomes expression in all layers is found initially to be complementary to central pkdcc expression in the hypoblast during early axis formation. Pkdcc then appears - together with a posterior-anterior gradient in wnt3 and eomes domains - in the epiblast posteriorly before the emerging primitive streak is marked by pkdcc and tbx6 at its anterior and posterior extremities, respectively. Conserved circular expression patterns deduced from some of this data may point to shared mechanisms in amniote axis formation while the reshaping of localised gene expression patterns is discussed as part of the 'three-anchor-point model' for establishing the mammalian body plan.

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INTRODUCTION: The term caudal duplication syndrome (CDS) was first introduced for complex anomalies of the distal caudal end of the trunk. The pathoembryogenesis of CDS is yet unknown, although a few theories have been proposed. We reviewed the previously proposed pathoembryogenetic theories and suggested a new perspective through the common clinical characteristics shown in CDS cases reported in the literature. METHODS: We conducted a systematic literature search of the online database PubMed from October 1993 to October 2020, using the search term "caudal duplication syndrome", according to the first mention of this entity. A total of 17 articles with 23 patients were reviewed. RESULTS: The most common manifestations were the duplication of the distal colon, genitourinary organs, and lower spine. Specifically, the duplicated bladders or uteri contacted their counterpart through a septum, and the duplicated bowels ran parallel. More caudal structures, such as the urethra or anus, were formed separately. The duplication seems to be a result of division by an intervening septum or structure in each part. In addition, duplication was not limited to the structures formed from the caudal cell mass (CCM), such as the distal spine and spinal cord, but also included hindgut structures. Moreover, anomalies involving caudal mesenchymal defects were also present. Considering clinical manifestations that are related to all three germ layers and seemingly the overseptation of these germ layers in CDS patients, with supporting data from animal experiments, events such as late-stage errors involving Hensen's node/the primitive streak and the duplication of the CCM with the hyperplasia of the abnormally located central caudal mesenchyme are probable pathoembryogenetic mechanisms for CDS. The "leakage" of the normal growth power of the caudal mesenchyme into the intervening midline space between the two CCMs and consequent weak lateral and caudal pushes of the caudal mesenchyme may explain the association of caudal agenesis or its related anomalies with CDS. CONCLUSION: We propose a theory that by a molecular interaction, an insult causes late gastrulation phase problems, resulting in ectopic primitive streak formation, and therefore, a duplication of the CCM is induced. Subsequently, the overactivity of abnormally positioned midline mesenchyme between the two CCMs may divide the hindgut derivatives by a central septum. Underactive lateral and caudal pushes of the caudal mesenchyme may lead to an association of features shown in caudal agenesis.

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Stem cell-based embryo models open an unprecedented avenue for modeling embryogenesis, cell lineage differentiation, tissue morphogenesis, and organogenesis in mammalian development. Experimentation on these embryo models can lead to a better understanding of the mechanisms of development and offers opportunities for functional genomic studies of disease-causing mechanisms, identification of therapeutic targets, and preclinical modeling of advanced therapeutics for precision medicine. An immediate challenge is to create embryo models of high fidelity to embryogenesis and organogenesis in vivo, to ensure that the knowledge gleaned is biologically meaningful and clinically relevant.

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