The vertebrate organizer plays a crucial role in building the main (antero-posterior) axis of the embryo: it neuralizes the surrounding ectoderm, and is the site of emigration for cells making axial and paraxial mesendoderm during elongation. The chick organizer becomes a stem zone at the onset of elongation; it stops recruiting cells from the neighbouring ectoderm and generates all its derivatives from the small number of resident cells it contains at the end of gastrulation stages. Nothing is known about the molecular identity of this stem zone. Here, we specifically labelled long-term resident cells of the organizer and compared their RNA-seq profile to that of the neighbouring cell populations. Screening by reverse transcription-polymerase chain reaction and in situ hybridization identified four genes (WIF1, PTGDS, ThPO and UCKL1) that are upregulated only in the organizer region when it becomes a stem zone and remain expressed there during axial elongation. In experiments specifically labelling the resident cells of the mature organizer, we show that only these cells express these genes. These findings molecularly define the organizer as a stem zone and offer a key to understanding how this zone is set up, the molecular control of its cells\' behaviour and the evolution of axial growth zones.

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

A simple machine is a basic of device that takes mechanical advantage to apply force. Animals and plants self-assemble through the operation of a wide variety of simple machines. Embryos of different species actuate these simple machines to drive the geometric transformations that convert a disordered mass of cells into organized structures with discrete identities and function. These transformations are intrinsically coupled to sequential and overlapping steps of self-organization and self-assembly. The processes of self-organization have been explored through the molecular composition of cells and tissues and their information networks. By contrast, efforts to understand the simple machines underlying self-assembly must integrate molecular composition with the physical principles of mechanics. This primer is concerned with effort to elucidate the operation of these machines, focusing on the \"problem\" of morphogenesis. Advances in understanding self-assembly will ultimately connect molecular-, subcellular-, cellular- and meso-scale functions of plants and animals and their ability to interact with larger ecologies and environmental influences.

Biomechanics in embryogenesis is a dynamic field intertwining the physical forces and biological processes that shape the first days of a mammalian embryo. From the first cell fate bifurcation during blastulation to the complex symmetry breaking and tissue remodeling in gastrulation, mechanical cues appear critical in cell fate decisions and tissue patterning. Recent strides in mouse and human embryo culture, stem cell modeling of mammalian embryos, and biomaterial design have shed light on the role of cellular forces, cell polarization, and the extracellular matrix in influencing cell differentiation and morphogenesis. This chapter highlights the essential functions of biophysical mechanisms in blastocyst formation, embryo implantation, and early gastrulation where the interplay between the cytoskeleton and extracellular matrix stiffness orchestrates the intricacies of embryogenesis and placenta specification. The advancement of in vitro models like blastoids, gastruloids, and other types of embryoids, has begun to faithfully recapitulate human development stages, offering new avenues for exploring the biophysical underpinnings of early development. The integration of synthetic biology and advanced biomaterials is enhancing the precision with which we can mimic and study these processes. Looking ahead, we emphasize the potential of CRISPR-mediated genomic perturbations coupled with live imaging to uncover new mechanosensitive pathways and the application of engineered biomaterials to fine-tune the mechanical conditions conducive to embryonic development. This synthesis not only bridges the gap between experimental models and in vivo conditions to advancing fundamental developmental biology of mammalian embryogenesis, but also sets the stage for leveraging biomechanical insights to inform regenerative medicine.

Cell-fate decisions during mammalian gastrulation are poorly understood outside of rodent embryos. The embryonic disc of pig embryos mirrors humans, making them a useful proxy for studying gastrulation. Here we present a single-cell transcriptomic atlas of pig gastrulation, revealing cell-fate emergence dynamics, as well as conserved and divergent gene programs governing early porcine, primate, and murine development. We highlight heterochronicity in extraembryonic cell-types, despite the broad conservation of cell-type-specific transcriptional programs. We apply these findings in combination with functional investigations, to outline conserved spatial, molecular, and temporal events during definitive endoderm specification. We find early FOXA2 + /TBXT- embryonic disc cells directly form definitive endoderm, contrasting later-emerging FOXA2/TBXT+ node/notochord progenitors. Unlike mesoderm, none of these progenitors undergo epithelial-to-mesenchymal transition. Endoderm/Node fate hinges on balanced WNT and hypoblast-derived NODAL, which is extinguished upon endodermal differentiation. These findings emphasise the interplay between temporal and topological signalling in fate determination during gastrulation.

BACKGROUND: The Wnt signaling pathway is highly conserved in metazoans and regulates a large array of cellular processes including motility, polarity and fate determination, and stem cell homeostasis. Modulation of the actin cytoskeleton via the non-canonical Wnt pathway regulate cell polarity and cell migration that are required for proper vertebrate gastrulation and subsequent neurulation. However, the mechanism(s) of how the non-canonical pathway mediates actin cytoskeleton modulation is not fully understood.
RESULTS: Herein, we characterize the role of the Formin-homology protein; dishevelled associated activator of morphogenesis 2 (Daam2) protein in the Wnt signaling pathway. Co-immunoprecipitation assays confirm the binding of Daam2 to dishevelled2 (Dvl2) as well as the domains within these proteins required for interaction; additionally, the interaction between Daam2 and Dvl2 was Wnt-regulated. Sub-cellular localization studies reveal Daam2 is cytoplasmic and regulates the cellular actin cytoskeleton by modulating actin filament formation. During Xenopus development, a knockdown or loss of Daam2 specifically produces neural tube closure defects indicative of a role in non-canonical signaling. Additionally, our studies did not identify any role for Daam2 in canonical Wnt signaling in mammalian culture cells or the Xenopus embryo.
CONCLUSIONS: Our studies together identify Daam2 as a component of the non-canonical Wnt pathway and Daam2 is a regulator of neural tube morphogenesis during vertebrate development.

Twisted gastrulation (TWSG1) is an evolutionarily conserved secreted glycoprotein which controls signaling by Bone Morphogenetic Proteins (BMPs). TWSG1 binds BMPs and their antagonist Chordin to control BMP signaling during embryonic development, kidney regeneration and cancer. We report crystal structures of TWSG1 alone and in complex with a BMP ligand, Growth Differentiation Factor 5. TWSG1 is composed of two distinct, disulfide-rich domains. The TWSG1 N-terminal domain occupies the BMP type 1 receptor binding site on BMPs, whereas the C-terminal domain binds to a Chordin family member. We show that TWSG1 inhibits BMP function in cellular signaling assays and mouse colon organoids. This inhibitory function is abolished in a TWSG1 mutant that cannot bind BMPs. The same mutation in the Drosophila TWSG1 ortholog Tsg fails to mediate BMP gradient formation required for dorsal-ventral axis patterning of the early embryo. Our studies reveal the evolutionarily conserved mechanism of BMP signaling inhibition by TWSG1.

The coordinated movement of germ layer progenitor cells reaches its peak at the dorsal side, where the Bmp signaling gradient is low, and minimum at the ventral side, where the Bmp gradient is high. This dynamic cell movement is regulated by the interplay of various signaling pathways. The noncanonical Wnt signaling cascade serves as a pivotal regulator of convergence and extension cell movement, facilitated by the activation of small GTPases such as Rho, Rab, and Rac. However, the underlying cause of limited cell movement at the ventral side remains elusive. To explore the functional role of a key regulator in constraining gastrulation cell movement at the ventral side, we investigated the Bmp4-direct target gene, sizzled (szl), to assess its potential role in inhibiting noncanonical Wnt signaling. In our current study, we demonstrated that ectopic expression of szl led to gastrulation defects in a dose-dependent manner without altering cell fate specification. Overexpression of szl resulted in decreased elongation of Activin-treated animal cap and Keller explants. Furthermore, our immunoprecipitation assay unveiled the physical interaction of Szl with noncanonical Wnt ligand proteins (Wnt5 and Wnt11). Additionally, the activation of small GTPases involved in Wnt signaling mediation (RhoA and Rac1) was diminished upon szl overexpression. In summary, our findings suggest that Bmp4 signaling negatively modulates cell movement from the ventral side of the embryo by inducing szl expression during early Xenopus gastrulation.

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.

External bilateral symmetry is a prevalent feature in vertebrates, which emerges during early embryonic development. To begin with, vertebrate embryos are largely radially symmetric before transitioning to bilaterally symmetry, after which, morphogenesis of various bilateral tissues (e.g somites, otic vesicle, limb bud), and structures (e.g palate, jaw) ensue. While a significant amount of work has probed the mechanisms behind symmetry breaking in the left-right axis leading to asymmetric positioning of internal organs, little is known about how bilateral tissues emerge at the same time with the same shape and size and at the same position on the two sides of the embryo. By discussing emergence of symmetry in many bilateral tissues and structures across vertebrate model systems, we highlight that understanding symmetry establishment is largely an open field, which will provide deep insights into fundamental problems in developmental biology for decades to come.