Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometry-based proteomics approach to investigate the global dynamics of (phospho) protein expression during gastruloid differentiation. Our findings revealed many proteins with temporal expression and unique expression profiles for each germ layer, which we also validated using single-cell proteomics technology. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with single-cell RNA sequencing (scRNA-seq) identified a critical role for ZEB2 in mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.

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.

Progress in understanding early human development has been impeded by the scarcity of reference datasets from natural embryos, particularly those with spatial information during crucial stages like gastrulation. We conducted high-resolution spatial transcriptomics profiling on 38,562 spots from 62 transverse sections of an intact Carnegie stage (CS) 8 human embryo. From this spatial transcriptomic dataset, we constructed a 3D model of the CS8 embryo, in which a range of cell subtypes are identified, based on gene expression patterns and positional register, along the anterior-posterior, medial-lateral, and dorsal-ventral axis in the embryo. We further characterized the lineage trajectories of embryonic and extra-embryonic tissues and associated regulons and the regionalization of signaling centers and signaling activities that underpin lineage progression and tissue patterning during gastrulation. Collectively, the findings of this study provide insights into gastrulation and post-gastrulation development of the human embryo.

Patterning and growth are fundamental features of embryonic development that must be tightly coordinated. To understand how metabolism impacts early mesoderm development, we used mouse embryonic stem-cell-derived gastruloids, that co-expressed glucose transporters with the mesodermal marker T/Bra. We found that the glucose mimic, 2-deoxy-D-glucose (2-DG), blocked T/Bra expression and abolished axial elongation in gastruloids. However, glucose removal did not phenocopy 2-DG treatment despite a decline in glycolytic intermediates. As 2-DG can also act as a competitive inhibitor of mannose in protein glycosylation, we added mannose together with 2-DG and found that it could rescue the mesoderm specification both in vivo and in vitro. We further showed that blocking production and intracellular recycling of mannose abrogated mesoderm specification. Proteomics analysis demonstrated that mannose reversed glycosylation of the Wnt pathway regulator, secreted frizzled receptor Frzb. Our study showed how mannose controls mesoderm specification in mouse gastruloids.

Within embryonic development, the occurrence of gastrulation is critical in the formation of multiple germ layers with many differentiative abilities. These cells are instructed through exposure to signalling molecules called morphogens. The secretion of morphogens from a source tissue creates a concentration gradient that allows distinct pattern formation in the receiving tissue. This review focuses on the morphogens Wnt and Fgf in zebrafish development. Wnt has been shown to have critical roles throughout gastrulation, including in anteroposterior patterning and neural posterisation. Fgf is also a vital signal, contributing to involution and mesodermal specification. Both morphogens have also been found to work in finely balanced synergy for processes such as neural induction. Thus, the signalling range of Wnts and Fgfs must be strictly controlled to target the correct target cells. Fgf and Wnts signal to local cells as well as to cells in the distance in a highly regulated way, requiring specific dissemination mechanisms that allow efficient and precise signalling over short and long distances. Multiple transportation mechanisms have been discovered to aid in producing a stable morphogen gradient, including short-range diffusion, filopodia-like extensions called cytonemes and extracellular vesicles, mainly exosomes. These mechanisms are specific to the morphogen that they transport and the intended signalling range. This review article discusses how spreading mechanisms in these two morphogenetic systems differ and the consequences on paracrine signalling, hence tissue patterning.

A comprehensive regulatory network of transcription factors controls the dorsoventral patterning of the body axis in developing vertebrate embryos. Bone morphogenetic protein signaling is essential for activating the Ventx family of homeodomain transcription factors, which regulates embryonic patterning and germ layer identity during Xenopus gastrulation. Although Ventx1.1 and Ventx2.1 of the Xenopus Ventx family have been extensively investigated, Ventx3.2 remains largely understudied. Therefore, this study aimed to investigate the transcriptional regulation of ventx3.2 during the embryonic development of Xenopus. We used goosecoid (Gsc) genome-wide chromatin immunoprecipitation-sequencing data to isolate and replicate the promoter region of ventx3.2. Serial deletion and site-directed mutagenesis were used to identify the cis-acting elements for Gsc and caudal type homeobox 1 (Cdx1) within the ventx3.2 promoter. Cdx1 and Gsc differentially regulated ventx3.2 transcription in this study. Additionally, positive cis-acting and negative response elements were observed for Cdx1 and Gsc, respectively, within the 5\' flanking region of the ventx3.2 promoter. This result was corroborated by mapping the active Cdx1 response element (CRE) and Gsc response element (GRE). Moreover, a point mutation within the CRE and GRE completely abolished the activator and repressive activities of Cdx1 and Gsc, respectively. Furthermore, the chromatin immunoprecipitation-polymerase chain reaction confirmed the direct binding of Cdx1 and Gsc to the CRE and GRE, respectively. Inhibition of Cdx1 and Gsc activities at their respective functional regions, namely, the ventral marginal zone and dorsal marginal zone, reversed their effects on ventx3.2 transcription. These results indicate that Cdx1 and Gsc modulate ventx3.2 transcription in the ventral marginal zone and dorsal marginal zone by directly binding to the promoter region during Xenopus gastrulation.

Studies using early-stage avian embryos have substantially impacted developmental biology, through the availability of simple culture methods and easiness in tissue manipulation. However, the regulations underlying brain and head development, a central issue of developmental biology, have not been investigated systematically. Yoshihi et al. (2022a) devised a technique to randomly label the epiblast cells with a green fluorescent protein before their development into the brain tissue. This technique was combined with grafting a node or node-derived anterior mesendoderm labeled with a cherry-colored fluorescent protein. Then cellular events were live-recorded over 18 hours during the brain and head development. The live imaging-based analyses identified previously undescribed mechanisms central to brain development: all anterior epiblast cells have a potential to develop into the brain tissues and their gathering onto a proximal anterior mesendoderm forms a brain primordium whereas the remaining cells develop into the covering head ectoderm. The analyses also ruled out the direct participation of the node\'s activity in the brain development. Yoshihi et al. (2022a) also demonstrate how the enigmatic data from classical models can be reinterpreted in the new model.This chapter was adapted from Yoshihi K, Iida H, Teramoto M, Ishii Y, Kato K, Kondoh H. (2022b). Epiblast cells gather onto the anterior mesendoderm and initiate brain development without the direct involvement of the node in avian embryos: Insights from broad-field live imaging. Front Cell Dev Biol. 10:1019845. doi: 10.3389/fcell.2022.1019845.

As epiblast cells initiate development into various somatic cells, they undergo a large-scale reorganization, called gastrulation. The gastrulation of the epiblast cells produces three groups of cells: the endoderm layer, the collection of miscellaneous mesodermal tissues, and the ectodermal layer, which includes the neural, epidermal, and associated tissues. Most studies of gastrulation have focused on the formation of the tissues that provide the primary route for cell reorganization, that is, the primitive streak, in the chicken and mouse. In contrast, how gastrulation alters epiblast-derived cells has remained underinvestigated. This chapter highlights the regulation of cell and tissue fate via the gastrulation process. The roles and regulatory functions of neuromesodermal progenitors (NMPs) in the gastrulation process, elucidated in the last decade, are discussed in depth to resolve points of confusion. Chicken and mouse embryos, which form a primitive streak as the site of mesoderm precursor ingression, have been investigated extensively. However, primitive streak formation is an exception, even among amniotes. The roles of gastrulation processes in generating various somatic tissues will be discussed broadly.

The discovery of the amphibian gastrula organizer and its publication by Hans Spemann and Hilde Mangold in 1924 is a foundation of experimental embryology, and has shaped our understanding of embryonic induction and pattern formation in vertebrates until today. The original publication is a piece of scientific art, characterized by the meticulous hand drawings by Hilde Mangold, as well as the text that develops mechanistic concepts of modern embryology. While historic microphotographs of specimens got lost, the original microscope slides and Hilde Mangold\'s laboratory notebook have been secured in embryological collections until today. Here, we make the original data of the six embryonic specimens reported in 1924, as well as the laboratory notebook, available in an accessible digital format. Together, these data shed light on the scientific process that led to the discovery, and should help to make the experiments on the most important signalling center in early vertebrate development transparent for generations of embryologists to come.

In the nascent mesoderm, TBXT expression must be precisely regulated to ensure that cells exit the primitive streak and pattern the anterior-posterior axis, but how varying dosage informs morphogenesis is not well understood. In this study, we define the transcriptional consequences of TBXT dosage reduction during early human gastrulation using human induced pluripotent stem cell models of gastrulation and mesoderm differentiation. Multi-omic single-nucleus RNA and single-nucleus ATAC sequencing of 2D gastruloids comprising wild-type, TBXT heterozygous or TBXT null human induced pluripotent stem cells reveal that varying TBXT dosage does not compromise the ability of a cell to differentiate into nascent mesoderm, but instead directly influences the temporal progression of the epithelial-to-mesenchymal transition with wild type transitioning first, followed by TBXT heterozygous and then TBXT null. By differentiating cells into nascent mesoderm in a monolayer format, we further illustrate that TBXT dosage directly impacts the persistence of junctional proteins and cell-cell adhesions. These results demonstrate that epithelial-to-mesenchymal transition progression can be decoupled from the acquisition of mesodermal identity in the early gastrula and shed light on the mechanisms underlying human embryogenesis.