During the epithelial-mesenchymal transition driving mouse embryo gastrulation, cells divide more frequently at the primitive streak, and half of those divisions happen away from the apical pole. These observations suggest that non-apical mitoses might play a role in cell delamination. We aim to uncover and challenge the molecular determinants of mitosis position in different regions of the epiblast through computational modeling and pharmacological treatments of embryos and stem cell-based epiblast spheroids. Blocking basement membrane degradation at the streak has no impact on the asymmetry in mitosis frequency and position. By contrast, disturbance of the actomyosin cytoskeleton or cell cycle dynamics elicits ectopic non-apical mitosis and shows that the streak region is characterized by local relaxation of the actomyosin cytoskeleton and less stringent regulation of cell division. These factors are essential for normal dynamics at the streak and favor cell delamination from the epiblast.

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.
CONCLUSIONS: Our study defines the morphological and molecular context of the emerging organizer equivalent in the pig and suggests a sequential development of its function.

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.

A bilateral body plan is predominant throughout the animal kingdom. Bilaterality of amniote embryos becomes recognizable as midline morphogenesis begins at gastrulation, bisecting an embryonic field into the left and right sides, and left-right asymmetry patterning follows. While a series of laterality genes expressed after the left-right compartmentalization has been extensively studied, the laterality patterning prior to and at the initiation of midline morphogenesis has remained unclear. Here, through a biophysical quantification in a high spatial and temporal resolution, applied to a chick model system, we show that a large-scale bilateral counter-rotating cellular flow, termed as \'polonaise movements\', display left-right asymmetries in early gastrulation. This cell movement starts prior to the formation of the primitive streak, which is the earliest midline structure, and earlier than expression of laterality genes. The cellular flow speed and vorticity unravel the location and timing of the left-right asymmetries. The bilateral flows displayed a Right dominance after six hours since the start of cell movements. Mitotic arrest that diminishes primitive streak formation resulted in changes in the bilateral flow pattern, but the Right dominance persisted. Our data indicate that the left-right asymmetry in amniote gastrula becomes detectable prior to the point when the asymmetric regulation of the laterality signals at the node leads to the left-right patterning. More broadly, our results suggest that physical processes can play an unexpected but significant role in influencing left-right laterality during embryonic development.
UNASSIGNED: Bilaterians are defined by a bilaterally symmetrical body plan. Vertebrates exhibit external bilateral symmetry but display left-right (LR) asymmetry in their internal organs. In amniote embryos, the initiation of LR symmetry breaking is not well understood. Here, we study LR symmetry breaking in the chick embryo due to its easy accessibility and similarity to human development. Our biophysical approaches to quantify cellular flows inferred that LR symmetry breaking occurs prior to the formation of Hensen\'s node, a LR organizer, which serves as a signaling center for LR patterning programs. Our work demonstrates that quantitative biophysical parameters can help unravel the initiation of LR symmetry breaking, suggesting involvement of physical mechanisms in this critical biological patterning process.

BMP signaling is essential for mammalian gastrulation, as it initiates a cascade of signals that control self-organized patterning. As development is highly dynamic, it is crucial to understand how time-dependent combinatorial signaling affects cellular differentiation. Here, we show that BMP signaling duration is a crucial control parameter that determines cell fates upon the exit from pluripotency through its interplay with the induced secondary signal WNT. BMP signaling directly converts cells from pluripotent to extraembryonic fates while simultaneously upregulating Wnt signaling, which promotes primitive streak and mesodermal specification. Using live-cell imaging of signaling and cell fate reporters together with a simple mathematical model, we show that this circuit produces a temporal morphogen effect where, once BMP signal duration is above a threshold for differentiation, intermediate and long pulses of BMP signaling produce specification of mesoderm and extraembryonic fates, respectively. Our results provide a systems-level picture of how these signaling pathways control the landscape of early human development.

The blueprint of the mammalian body plan is laid out during gastrulation, when a trilaminar embryo is formed. This process entails a burst of proliferation, the ingression of embryonic epiblast cells at the primitive streak, and their priming toward primitive streak fates. How these different events are coordinated remains unknown. Here, we developed and characterized a 3D culture of self-renewing mouse embryonic cells that captures the main transcriptional and architectural features of the early gastrulating mouse epiblast. Using this system in combination with microfabrication and in vivo experiments, we found that proliferation-induced crowding triggers delamination of cells that express high levels of the apical polarity protein aPKC. Upon delamination, cells become more sensitive to Wnt signaling and upregulate the expression of primitive streak markers such as Brachyury. This mechanistic coupling between ingression and differentiation ensures that the right cell types become specified at the right place during embryonic development.

Basement membranes (BMs) are sheet-like structures of extracellular matrix (ECM) that provide structural support for many tissues and play a central role in signaling. They are key regulators of cell behavior and tissue functions, and defects in their assembly or composition are involved in numerous human diseases. Due to the differences between human and animal embryogenesis, ethical concerns, legal constraints, the scarcity of human tissue material, and the inaccessibility of the in vivo condition, BM regulation during human embryo development has remained elusive. Using the post-implantation amniotic sac embryoid (PASE), we delineate BM assembly upon post-implantation development and BM disassembly during primitive streak (PS) cell dissemination. Further, we show that the transcription factor Oct4 regulates the expression of BM structural components and receptors and controls BM development by regulating Akt signaling and the small GTPase Rac1. These results represent a relevant step toward a more comprehensive understanding of early human development.

Many amniote vertebrate species including humans can form identical twins from a single embryo, but this only occurs rarely. It has been suggested that the primitive-streak-forming embryonic region emits signals that inhibit streak formation elsewhere but the signals involved, how they are transmitted and how they act has not been elucidated. Here we show that short tracks of calcium firing activity propagate through extraembryonic tissue via gap junctions and prevent ectopic primitive streak formation in chick embryos. Cross-regulation of calcium activity and an inhibitor of primitive streak formation (Bone Morphogenetic Protein, BMP) via NF-κB and NFAT establishes a long-range BMP gradient spanning the embryo. This mechanism explains how embryos of widely different sizes can maintain positional information that determines embryo polarity. We provide evidence for similar mechanisms in two different human embryo models and in Drosophila, suggesting an ancient evolutionary origin.

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.

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.