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Minimal in vitro systems composed of embryonic stem cells (ESCs) have been shown to recapitulate the establishment of the anteroposterior (AP) axis. In contrast to the native embryo, ESC aggregates - such as gastruloids - can break symmetry, which is demarcated by polarization of the mesodermal marker T, autonomously without any localized external cues. However, associated earliest patterning events, such as the spatial restriction of cell fates and concomitant transcriptional changes, remain poorly understood. Here, we dissect the dynamics of AP axis establishment in mouse gastruloids, particularly before external Wnt stimulation. Through single-cell RNA sequencing, we identify key cell state transitions and the molecular signatures of T+ and T- populations underpinning AP polarization. We also show that this process is robust to modifications of aggregate size. Finally, transcriptomic comparison with the mouse embryo indicates that gastruloids develop similar mesendodermal cell types, despite initial differences in their primed pluripotent populations, which adopt a more mesenchymal state in lieu of an epiblast-like transcriptome. Hence, our findings suggest the possibility of alternate ESC states in vivo and in vitro that can converge onto similar cell fates., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Aggregates of stem cells can break symmetry and self-organize into embryo-like structures with complex morphologies and gene expression patterns. Mechanisms including reaction-diffusion Turing patterns and cell sorting have been proposed to explain symmetry breaking but distinguishing between these candidate mechanisms of self-organization requires identifying which early asymmetries evolve into subsequent tissue patterns and cell fates. Here we use synthetic 'signal-recording' gene circuits to trace the evolution of signalling patterns in gastruloids, three-dimensional stem cell aggregates that form an anterior-posterior axis and structures resembling the mammalian primitive streak and tailbud. We find that cell sorting rearranges patchy domains of Wnt activity into a single pole that defines the gastruloid anterior-posterior axis. We also trace the emergence of Wnt domains to earlier heterogeneity in Nodal activity even before Wnt activity is detectable. Our study defines a mechanism through which aggregates of stem cells can form a patterning axis even in the absence of external spatial cues., Competing Interests: Competing interests J.E.T. is a scientific advisor for Prolific Machines and Nereid Therapeutics. B.A. is an advisory board member with options for Arbor Biotechnologies and Tessera Therapeutics, and holds equity in Celsius Therapeutics. H.M.M. is a cofounder and scientific advisor for C16 Biosciences. The remaining authors declare no conflicts of interest., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Gastruloids are a powerful in vitro model of early human development. However, although elongated and composed of all three germ layers, human gastruloids do not morphologically resemble post-implantation human embryos. Here we show that an early pulse of retinoic acid (RA), together with later Matrigel, robustly induces human gastruloids with posterior embryo-like morphological structures, including a neural tube flanked by segmented somites and diverse cell types, including neural crest, neural progenitors, renal progenitors and myocytes. Through in silico staging based on single-cell RNA sequencing, we find that human RA-gastruloids progress further than other human or mouse embryo models, aligning to E9.5 mouse and CS11 cynomolgus monkey embryos. We leverage chemical and genetic perturbations of RA-gastruloids to confirm that WNT and BMP signalling regulate somite formation and neural tube length in the human context, while transcription factors TBX6 and PAX3 underpin presomitic mesoderm and neural crest, respectively. Looking forward, RA-gastruloids are a robust, scalable model for decoding early human embryogenesis., (© 2024. The Author(s).)

Drafting gene regulatory networks (GRNs) requires embryological knowledge pertaining to the cell type families, information on the regulatory genes, causal data from gene knockdown experiments and validations of the identified interactions by cis-regulatory analysis. We use multi-omics involving next-generation sequencing to obtain the necessary information for drafting the Strongylocentrotus purpuratus (Sp) posterior gut GRN. Here, we present an update to the GRN using: (1) a single-cell RNA-sequencing-derived cell atlas highlighting the 2 day-post-fertilization (dpf) sea urchin gastrula cell type families, as well as the genes expressed at the single-cell level; (2) a set of putative cis-regulatory modules and transcription factor-binding sites obtained from chromatin accessibility ATAC-seq data; and (3) interactions directionality obtained from differential bulk RNA sequencing following knockdown of the transcription factor Sp-Pdx1, a key regulator of gut patterning in sea urchins. Combining these datasets, we draft the GRN for the hindgut Sp-Pdx1-positive cells in the 2 dpf gastrula embryo. Overall, our data suggest the complex connectivity of the posterior gut GRN and increase the resolution of gene regulatory cascades operating within it., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Competing interests B.G.B. is a founder, shareholder and advisor of Tenaya Therapeutics and is an advisor for Silver Creek Pharmaceuticals. I.M.-V. is the founder and Chief Executive Officer of Vitra Labs, Inc. T.C.M. is an employee at Genentech and is an advisor for Vitra Labs. The work presented here is not related to the interests of these commercial entities., (© 2024. Published by The Company of Biologists Ltd.)

Convergent extension of the chordamesoderm is the best-examined gastrulation movement in Xenopus. Here we study general features of cell-cell contacts in this tissue by combining depletion of adhesion factors C-cadherin, Syndecan-4, fibronectin, and hyaluronic acid, the analysis of respective contact width spectra and contact angles, and La3+ staining of the pericellular matrix. We provide evidence that like in other gastrula tissues, cell-cell adhesion in the chordamesoderm is largely mediated by different types of pericellular matrix. Specific glycocalyx structures previously identified in Xenopus gastrula tissues are absent in chordamesoderm but other contact types like 10-20 nm wide La3+ stained structures are present instead. Knockdown of any of the adhesion factors reduces the abundance of cell contacts but not the average relative adhesiveness of the remaining ones: a decrease of adhesiveness at low contact widths is compensated by an increase of contact widths and an increase of adhesiveness proportional to width. From the adhesiveness-width relationship, we derive a model of chordamesoderm cell adhesion that involves the interdigitation of distinct pericellular matrix units. Quantitative description of pericellular matrix deployment suggests that reduced contact abundance upon adhesion factor depletion is correlated with excessive accumulation of matrix material in non-adhesive gaps and the loss of some contact types., Competing Interests: The authors declare that they have no conflict of interest., (Copyright: © 2024 Luu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Gastruloids have emerged as highly useful in vitro models of mammalian gastrulation. One of the most striking features of 3D gastruloids is their elongation, which mimics the extension of the embryonic anterior-posterior axis. Although axis extension is crucial for development, the underlying mechanism has not been fully elucidated in mammalian species. Gastruloids provide an opportunity to study this morphogenic process in vitro. Here, we measure and quantify the shapes of elongating gastruloids and show, by Cellular Potts model simulations based on a novel, optimized algorithm, that convergent extension, driven by a combination of active cell crawling and differential adhesion can explain the observed shapes. We reveal that differential adhesion alone is insufficient and also directly observe hallmarks of convergent extension by time-lapse imaging of gastruloids. Finally, we show that gastruloid elongation can be abrogated by inhibition of the Rho kinase pathway, which is involved in convergent extension in vivo. All in all, our study demonstrates, how gastruloids can be used to elucidate morphogenic processes in embryonic development., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 de Jong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Morphogenetic movements during animal development involve repeated making and breaking of cell-cell contacts. Recent biophysical models of cell-cell adhesion integrate adhesion molecule interactions and cortical cytoskeletal tension modulation, describing equilibrium states for established contacts. We extend this emerging unified concept of adhesion to contact formation kinetics, showing that aggregating Xenopus embryonic cells rapidly achieve Ca 2+ -independent low-contact states. Subsequent transitions to cadherin-dependent high-contact states show rapid decreases in contact cortical F-actin levels but slow contact area growth. We developed a biophysical model that predicted contact growth quantitatively from known cellular and cytoskeletal parameters, revealing that elastic resistance to deformation and cytoskeletal network turnover are essential determinants of adhesion kinetics. Characteristic time scales of contact growth to low and high states differ by an order of magnitude, being at a few minutes and tens of minutes, respectively, thus providing insight into the timescales of cell-rearrangement-dependent tissue movements., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Chemical signalling is the primary means by which cells communicate in the embryo. The underlying principle refers to a group of ligand-producing cells and a group of cells that respond to this signal because they express the appropriate receptors 1,2 . In the zebrafish embryo, Wnt5b binds to the receptor Ror2 to trigger the Wnt-planar cell polarity (PCP) signalling pathway to regulate tissue polarity and cell migration 3,4 . However, it remains unclear how this lipophilic ligand is transported from the source cells through the aqueous extracellular space to the target tissue. In this study, we provide evidence that Wnt5b, together with Ror2, is loaded on long protrusions called cytonemes. Our data further suggest that the active Wnt5b-Ror2 complexes form in the producing cell and are handed over from these cytonemes to the receiving cell. Then, the receiving cell has the capacity to initiate Wnt-PCP signalling, irrespective of its functional Ror2 receptor status. On the tissue level, we further show that cytoneme-dependent spreading of active Wnt5b-Ror2 affects convergence and extension in the zebrafish gastrula. We suggest that cytoneme-mediated transfer of ligand-receptor complexes is a vital mechanism for paracrine signalling. This may prompt a reevaluation of the conventional concept of characterizing responsive and non-responsive tissues solely on the basis of the expression of receptors., (© 2023. The Author(s).)

Gastrulation represents a pivotal phase of development and aberrations during this period can have major consequences, from minor anatomical deviations to severe congenital defects. Animal models are used to study gastrulation, however, there is considerable morphological and molecular diversity of gastrula across mammalian species. Here, we provide an overview of the latest research on interspecies developmental control across mammals. This includes single-cell atlases of several mammalian gastrula which have enabled comparisons of the temporal and molecular dynamics of differentiation. These studies highlight conserved cell differentiation regulators and both absolute and relative differences in differentiation dynamics between species. Recent advances in in vitro culture techniques have facilitated the derivation, maintenance and differentiation of cell lines from a range of species and the creation of multi-species models of gastrulation. Gastruloids are three-dimensional aggregates capable of self-organising and recapitulating aspects of gastrulation. Such models enable species comparisons outside the confines of the embryo. We highlight recent in vitro evidence that differentiation processes such as somitogenesis and neuronal maturation scale with known in vivo differences in developmental tempo across species. This scaling is likely due to intrinsic differences in cell biochemistry. We also highlight several studies which provide examples of cell differentiation dynamics being influenced by extrinsic factors, including culture conditions, chimeric co-culture, and xenotransplantation. These collective studies underscore the complexity of gastrulation across species, highlighting the necessity of additional datasets and studies to decipher the intricate balance between intrinsic cellular programs and extrinsic signals in shaping embryogenesis., (© 2023 The Author(s).)

The reciprocal inhibition between two signaling centers, the Spemann organizer (dorsal mesoderm) and ventral region (mesoderm and ectoderm), collectively regulate the overall development of vertebrate embryos. Each center expresses key homeobox transcription factors (TFs) that directly control target gene transcription. Goosecoid (Gsc) is an organizer (dorsal mesoderm)-specific TF known to induce dorsal fate and inhibit ventral/ectodermal specification. Ventx1.1 (downstream of Bmp signaling) induces the epidermal lineage and inhibits dorsal organizer-specific genes from the ventral region. Chordin (Chrd) is an organizer-specific secreted Bmp antagonist whose expression is primarily activated by Gsc. Alternatively, chrd expression is repressed by Bmp/Ventx1.1 in the ventral/epidermal region. However, the regulatory mechanisms underlying the transcription mediated by Gsc and Ventx1.1 remain elusive. Here, we found that the chrd promoter contained two cis-acting response elements that responded negatively to Ventx1.1 and positively to Gsc. In the ventral/ectodermal region, Ventx1.1 was directly bound to the Ventx1.1 response element (VRE) and inhibited chrd transcription. In the organizer region, Gsc was bound to the Gsc response elements (GRE) to activate chrd transcription. The Gsc-mediated positive response on the chrd promoter completely depended on another adjacent Wnt response cis-acting element (WRE), which was the TCF7 (also known as Tcf1) binding element. Site-directed mutagenesis of VRE, GRE, or WRE completely abolished the repressive or activator activity of Ventx1.1 and Gsc, respectively. The ChIP-PCR results confirmed the direct binding of Ventx1.1 and Gsc/Tcf7 to VRE and GRE/WRE, respectively. These results demonstrated that chrd expression is oppositely modulated by homeobox TFs, Ventx1.1, and Gsc/Tcf7 during the embryonic patterning of Xenopus gastrula.

Congenital Heart Disease (CHD) is the most common birth defect and leading cause of infant mortality, yet molecular mechanisms explaining CHD remain mostly unknown. Sequencing studies are identifying CHD candidate genes at a brisk rate including MINK1, a serine/threonine kinase. However, a plausible molecular mechanism connecting CHD and MINK1 is unknown. Here, we reveal that mink1 is required for proper heart development due to its role in left-right patterning. Mink1 regulates canonical Wnt signaling to define the cell fates of the Spemann Organizer and the Left-Right Organizer, a ciliated structure that breaks bilateral symmetry in the vertebrate embryo. To identify Mink1 targets, we applied an unbiased proteomics approach and identified the high mobility group architectural transcription factor, Hmga2. We report that Hmga2 is necessary and sufficient for regulating Spemann's Organizer. Indeed, we demonstrate that Hmga2 can induce Spemann Organizer cell fates even when β-catenin, a critical effector of the Wnt signaling pathway, is depleted. In summary, we discover a transcription factor, Hmga2, downstream of Mink1 that is critical for the regulation of Spemann's Organizer, as well as the LRO, defining a plausible mechanism for CHD., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Wnt11 family proteins are ligands that activate a type of Dishevelled-mediated, non-canonical Wnt signaling pathway. Loss of function causes defects in gastrulation and/or anterior-posterior axis extension in all vertebrates. Non-mammalian vertebrate genomes encode two Wnt11 family proteins whose distinct functions have been unclear. We knocked down Wnt11b and Wnt11, separately and together, in Xenopus laevis. Single morphants exhibited very similar phenotypes of delayed blastopore closure, but they had different phenotypes during the tailbud period. In response to their very similar gastrulation phenotypes, we chose to characterize dual morphants. Using dark field illuminated time-lapse imaging and kymograph analysis, we identified a failure of dorsal blastopore lip maturation that correlated with slower blastopore closure and failure to internalize the endoderm at the dorsal blastopore lip. We connected these externally visible phenotypes to cellular events in the internal tissues by imaging intact fixed embryos stained for anillin and microtubules. We found that the initial extension of the archenteron is correlated with blastopore lip maturation, and archenteron extension is dramatically disrupted by decreased Wnt11 family signaling. We were aided in our interpretation of the immunofluorescence by the novel, membrane proximal location of the cleavage furrow protein anillin in the epithelium of the blastopore lip and early archenteron., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The control of cell-cell adhesion and detachment is essential for collective migration and cell rearrangement. Here, we have used the contact behavior of Xenopus gastrula mesoderm explants migrating directionally on ectoderm conditioned substratum to study the regulation of active cell-cell detachment. When colliding laterally, explants repelled each other, whereas they fused front-to-back when aligned in the direction of migration. For this mesoderm polarization by the substratum, we identified three control modules. First, PDGF-A signaling normally suppresses contact-induced collapse of lamellipodia in a polarized manner. Disruption of PDGF-A function, or of Xwnt6, decreased the polarization of explant contact behavior. Second, the Wnt receptor Xfz7 acted upstream of the kinase Pak1 to control explant fusion independently of PDGF-A-promoted lamellipodia stability. Third, ephrinB1 acted with Dishevelled (Dvl) in front-to-back explant fusion. The second and third modules have been identified previously as regulators of tissue separation at the ectoderm-mesoderm boundary. On non-polarizing, fibronectin-coated substratum, they controlled repulsion between explants in the same way as between tissues during boundary formation. However, explant repulsion/fusion responses were reversed on conditioned substratum by the endogenous guidance cues that also control oriented contact inhibition of lamellipodia. Together, control modules and substratum-bound guidance cues combine preferential front-back adhesion and diminished lateral adhesion of cells to promote collective directional mesoderm migration.

Bioelectricity is defined as endogenous electrical signaling mediated by the dynamic distribution of charged molecules. Recently, increasing evidence has revealed that cellular bioelectric signaling is critical for regulating embryonic development, regeneration, and congenital diseases. However, systematic real-time in vivo dynamic electrical activity monitoring of whole organisms has been limited, mainly due to the lack of a suitable model system and voltage measurement tools for in vivo biology. Here, we addressed this gap by utilizing a genetically stable zebrafish line, Tg ( ubiquitin : ASAP1), and ASAP1 (Accelerated sensor of action potentials 1), a genetically encoded voltage indicator (GEVI). With light-sheet microscopy, we systematically investigated cell membrane potential (Vm) signals during different embryonic stages. We found cells of zebrafish embryos showed local membrane hyperpolarization at the cleavage furrows during the cleavage period of embryogenesis. This signal appeared before cytokinesis and fluctuated as it progressed. In contrast, whole-cell transient hyperpolarization was observed during the blastula and gastrula stages. These signals were generally limited to the superficial blastomere, but they could be detected within the deeper cells during the gastrulation period. Moreover, the zebrafish embryos exhibit tissue-level cell Vm signals during the segmentation period. Middle-aged somites had strong and dynamic Vm fluctuations starting at about the 12-somite stage. These embryonic stage-specific characteristic cellular bioelectric signals suggest that they might play a diverse role in zebrafish embryogenesis that could underlie human congenital diseases.

Left-right symmetry breaking in most studied vertebrates makes use of so-called leftward flow, a mechanism which was studied in detail especially in mouse and Xenopus laevis embryos and is based on rotation of monocilia on specialized epithelial surface designated as left-right organizer or laterality coordinator. However, it has been argued that prior to emergence of leftward flow an additional mechanism operates during early cleavage stages in Xenopus embryo which is based on cytoskeletal processes. Evidence in favour of this early mechanism was supported by left-right abnormalities after chemical inhibition of cytoskeletal protein formin. Here we analyzed temporal dimension of this effect in detail and found that reported abnormalities arise only after treatment at gastrula-neurula stages, i.e. just prior to and during the operation of left-right organizer. Moreover, molecular and morphological analysis of the left-right organizer reveals its abnormal development. Our results strongly indicate that left-right abnormalities reported after formin inhibition cannot serve as support of models based on early symmetry breaking event in Xenopus embryo., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Petri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Gastrulation is a fundamental process during embryonic development, conserved across all multicellular animals [1]. In the majority of metazoans, gastrulation is characterised by large scale morphogenetic remodeling, leading to the conversion of an early pluripotent embryonic cell layer into the three primary 'germ layers': an outer ectoderm, inner endoderm and intervening mesoderm layer. The morphogenesis of these three layers of cells is closely coordinated with cellular diversification, laying the foundation for the generation of the hundreds of distinct specialized cell types in the animal body. The process of gastrulation has for a long time attracted tremendous attention in a broad range of experimental systems ranging from sponges to mice. In humans the process of gastrulation starts approximately 14 days after fertilization and continues for slightly over a week. However our understanding of this important process, as it pertains to human, is limited. Donations of human fetal material at these early stages are exceptionally rare, making it nearly impossible to study human gastrulation directly. Therefore, our understanding of human gastrulation is predominantly derived from animal models such as the mouse [2,3] and from studies of limited collections of fixed whole samples and histological sections of human gastrulae [4-7], some of which date back to over a century ago. More recently we have been gaining valuable molecular insights into human gastrulation using in vitro models of hESCs [8-12] and increasingly, in vitro cultured human and non-human primate embryos [13-16]. However, while methods have been developed to culture human embryos into this stage (and probably beyond), current ethical standards prohibit the culture of human embryos past 14 days again limiting our ability to experimentally probe human gastrulation. This review discusses recent molecular insights from the study of a rare CS 7 human gastrula obtained as a live sample and raises several questions arising from this recent study that it will be interesting to address in the future using emerging models of human gastrulation., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In the primitive vertebrate gastrula, the boundary between ectoderm and mesoderm is formed by Brachet's cleft. Here we examine Brachet's cleft and its control by Eph/ephrin signaling in Xenopus at the ultrastructural level and by visualizing cortical F-actin. We infer cortical tension ratios at tissue surfaces and their interface in normal gastrulae and after depletion of receptors EphB4 and EphA4 and ligands ephrinB2 and ephrinB3. We find that cortical tension downregulation at cell contacts, a normal process in adhesion, is asymmetrically blocked in the ectoderm by Eph/ephrin signals from the mesoderm. This generates high interfacial tension that can prevent cell mixing across the boundary. Moreover, it determines an asymmetric boundary structure that is suited for the respective roles of ectoderm and mesoderm, as substratum and as migratory layers. The Eph and ephrin isoforms also control different cell-cell contact types in ectoderm and mesoderm. Respective changes of adhesion upon isoform depletion affect adhesion at the boundary to different degrees but usually do not prohibit cleft formation. In an extreme case, a new type of cleft-like boundary is even generated where cortical tension is symmetrically increased on both sides of the boundary., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

FGF and anti-BMP signals from the Spemann organizer of mesodermal origin are essential for Xenopus neural development from gastrula ectoderm. However, the detailed cellular and molecular mechanisms of signaling, especially those underlying the neural induction process, are still controversial. We show here that the expression of early neural marker genes such as sox2 and otx2 is suppressed both in vivo and in vitro, when ectoderm cells are loaded with a dominant-negative construct of Ets transcription factors or a translation-blocking antisense FGF2 MO or FGF8 MO, respectively. This indicates that the expression of these FGF signaling molecules in ectoderm cells contributes significantly to neural induction, in contrast to the "neural default model" that has been emphasized, in which anti-BMP signaling from the organizer plays a major role in the neural induction in Xenopus. Our results indicate that cell-autonomous FGF signaling between ectoderm cells, rather than paracrine signaling from organizer cells, directly induces the expression of sox2 and otx2 via the FGF2, FGF8/MAPK/Ets pathway at very low signaling levels. This is independent of inhibiting BMP signaling via the FGF/MAPK/Smad pathway, which has been proposed as the primary pathway for FGF signaling in Xenopus neural induction. Using cultured ectoderm cells, we also obtained results suggesting that the mode of contribution of FGF signaling to neural development is altered during the subsequent neural patterning stage. As we increase the amounts of FGF in the culture medium, anterior neural genes activated at low FGF signaling levels are suppressed, and instead position-specific, more posterior neural genes are activated in a dose-dependent manner via the FGF/MAPK/Ets pathway. These results support the claim that morphogenic FGFs from the organizer diffuse in a paracrine manner through the plane of the induced neuroectoderm, causing anteroposterior patterning of neural tissue., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Chemokines play important roles in early embryogenesis, including morphogenesis and cell differentiation, before the immune system is established. We characterized Xenopus laevis CC-type chemokine receptor 7 S (ccr7.S) to clarify its role during early development. ccr7 transcripts were detected ubiquitously in early embryos. Dorsal overexpression of ccr7.S inhibited gastrulation, and ccr7.S mRNA-injected embryos had short axes and widely opened neural folds. Because the Keller sandwich explants of the injected embryos elongated well, ccr7.S might affect cell migration, but not convergent extension movements. Ventral ccr7.S overexpression induced secondary axes and chrd.1 upregulation in gastrula-stage embryos. Animal cap assays showed increased expression of neural and cement gland marker genes at later stages. Ccr7.S knockdown reduced chrd.1 expression and inhibited gastrulation at the dorsal side. Our findings suggest that ccr7.S plays important roles in morphogenetic movement and cell differentiation., (© 2022 Japanese Society of Developmental Biologists.)

During the gastrula stage of Xenopus laevis, mesodermal cells migrate on the blastocoel roof (BCR) toward the animal pole. In this process, mesodermal cells directly adhere to the BCR via adhesion molecules, such as cadherins, which in turn trigger a repulsive reaction through factors such as Eph/ephrin. Therefore, the mesoderm and BCR repeatedly adhere to and detach from each other, and the frequency of this adhesion is thought to control mesoderm migration. Although knockdown of cadherin or Eph/ephrin causes severe gastrulation defects, these molecules have been reported to contribute not only to boundary formation but also to the internal function of each tissue. Therefore, it is possible that the defect caused by knockdown occurs due to tissue function abnormalities. To address this problem, we developed a method to specifically induce adhesion between different tissues using rapalog (an analog of rapamycin). When adhesion between the BCR and mesoderm was specifically enhanced by rapalog, mesoderm migration was strongly suppressed. Furthermore, we confirmed that rapalog significantly increased the frequency of adhesion between the two tissues. These results support the idea that the adhesion frequency controls mesoderm migration, and demonstrate that our method effectively enhances adhesion between specific tissues in vivo., (© 2022 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Gastrulation is a universal process in the morphogenesis of many animal embryos. Although morphological and molecular events in gastrulation have been well studied, the mechanical driving forces and underlying regulatory mechanisms are not fully understood. Here, we investigated the gastrulation of embryos of a sea urchin, Hemicentrotus pulcherrimus, which involves the invagination of a single-layered vegetal plate into the blastocoel. We observed that omeprazole, a proton pump inhibitor capable of perturbing the left-right asymmetry of sea urchin embryo, induced "partial exogastrulation" where the secondary invagination proceeds outward. During early gastrulation, intracellular apical-basal polarity of F-actin distribution in vegetal half was higher than those in animal half, while omeprazole treatment disturbed the apical-basal polarity of F-actin distribution in vegetal half. Furthermore, gastrulation stopped and even partial exogastrulation did not occur when F-actin polymerization or degradation in whole embryo was partially inhibited via RhoA or YAP1 knockout. A mathematical model of the early gastrulation reproduced the shapes of both normal and exogastrulating embryos using cell-dependent cytoskeletal features based on F-actin. Additionally, such cell position-dependent intracellular F-actin distributions might be regulated by intracellular pH distributions. Therefore, apical-basal polarity of F-actin distribution disrupted by omeprazole may induce the partial exogastrulation via anomalous secondary invagination., (© 2022 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

The planar polarization of cells and subcellular structures is critical for embryonic development. Coordination of this polarity can provide cells a sense of direction in relation to the anterior-posterior and dorsal-ventral body axes. Fly epithelia use a core pathway comprised of transmembrane (Van Gogh/Strabismus, Frizzled, and Flamingo/Starry night) and cytoplasmic (Prickle or Spiny-legs, Dishevelled, and Diego) proteins to communicate directional information between cells and thereby promote the uniform orientation of structures such as hairs. In the zebrafish gastrula, planar polarity underlies complex cellular processes, including directed migration and intercalation, that are required to shape the embryo body. Like other vertebrates, the zebrafish genome encodes homologs of each core protein, and it is well-established that polarized gastrula cell behaviors are regulated by some of them. However, it is unknown whether a conserved six-member core protein pathway regulates planar polarity during zebrafish gastrulation. Here, we review our current understanding of core protein function as it relates to two specific examples of planar polarity, the dorsal convergence of lateral gastrula cells and the mediolateral intercalation of midline cells. We consider the hallmarks of fly planar polarity and discuss data regarding asymmetric protein localization and function, and the intercellular communication of polarity information., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The ascidian larval tail contains muscle cells for swimming. Most of these muscle cells differentiate autonomously. The genetic program behind this autonomy has been studied extensively and the genetic cascade from maternal factors to initiation of expression of a muscle structural gene, Myl.c, has been uncovered; Myl.c expression is directed initially by transcription factor Tbx6-r.b at the 64-cell stage and then by the combined actions of Tbx6-r.b and Mrf from the gastrula to early tailbud stages. In the present study, we showed that transcription of Myl.c continued in late tailbud embryos and larvae, although a fusion protein of Tbx6-r.b and GFP was hardly detectable in late tailbud embryos. A knockdown experiment, reporter assay, and in vitro binding assay indicated that an essential cis-regulatory element of Myl.c that bound Tbx6-r.b in early embryos bound Tbx15/18/22 in late embryos to maintain expression of Myl.c. We also found that Tbx15/18/22 was controlled by Mrf, which constitutes a regulatory loop with Tbx6-r.b. Therefore, our data indicated that Tbx15/18/22 was activated initially under control of this regulatory loop as in the case of Myl.c, and then Tbx15/18/22 maintained the expression of Myl.c after Tbx6-r.b had disappeared. RNA-sequencing of Tbx15/18/22 morphant embryos revealed that many muscle structural genes were regulated similarly by Tbx15/18/22. Thus, the present study revealed the mechanisms of maintenance of transcription of muscle structural genes in late embryos in which Tbx15/18/22 takes the place of Tbx6-r.b., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Brachyury is a T-box family transcription factor and plays pivotal roles in morphogenesis. In sea urchin embryos, Brachyury is expressed in the invaginating endoderm, and in the oral ectoderm of the invaginating mouth opening. The oral ectoderm is hypothesized to serve as a signaling center for oral (ventral)-aboral (dorsal) axis formation and to function as a ventral organizer. Our previous results of a single-cell RNA-seq (scRNA-seq) atlas of early Strongylocentrotus purpuratus embryos categorized the constituent cells into 22 clusters, in which the endoderm consists of three clusters and the oral ectoderm four clusters (Foster et al., 2020). Here we examined which clusters of cells expressed Brachyury in relation to the morphogenesis and the identity of the ventral organizer. Our results showed that cells of all three endoderm clusters expressed Brachyury in blastulae. Based on expression profiles of genes involved in the gene regulatory networks (GRNs) of sea urchin embryos, the three clusters are distinguishable, two likely derived from the Veg2 tier and one from the Veg1 tier. On the other hand, of the four oral-ectoderm clusters, cells of two clusters expressed Brachyury at the gastrula stage and genes that are responsible for the ventral organizer at the late blastula stage, but the other two clusters did not. At a single-cell level, most cells of the two oral-ectoderm clusters expressed organizer-related genes, nearly a half of which coincidently expressed Brachyury. This suggests that the ventral organizer contains Brachyury-positive cells which invaginate to form the stomodeum. This scRNA-seq study therefore highlights significant roles of Brachyury-expressing cells in body-plan formation of early sea urchin embryos, though cellular and molecular mechanisms for how Brachyury functions in these processes remain to be elucidated in future studies., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Morphogens are signaling molecules that convey positional information and dictate cell fates during development. Although ectopic expression in model organisms suggests that morphogen gradients form through diffusion, little is known about how morphogen gradients are created and interpreted during mammalian embryogenesis due to the combined difficulties of measuring endogenous morphogen levels and observing development in utero. Here we take advantage of a human gastruloid model to visualize endogenous Nodal protein in living cells, during specification of germ layers. We show that Nodal is extremely short range so that Nodal protein is limited to the immediate neighborhood of source cells. Nodal activity spreads through a relay mechanism in which Nodal production induces neighboring cells to transcribe Nodal. We further show that the Nodal inhibitor Lefty, while biochemically capable of long-range diffusion, also acts locally to control the timing of Nodal spread and therefore of mesoderm differentiation during patterning. Our study establishes a paradigm for tissue patterning by an activator-inhibitor pair., (© 2022. The Author(s).)

How embryos scale patterning according to size is still not fully understood. Through in silico screening and analysis of reaction-diffusion systems that could be responsible for scaling, we predicted the existence of genes whose expression is sensitive to embryo size and which regulate the scaling of embryonic patterning. To find these scalers, we identified genes with strongly altered expression in half-size Xenopus laevis embryos compared with full-size siblings at the gastrula stage. Among found genes, we investigated the role of matrix metalloproteinase-3 (mmp3), which was most strongly downregulated in half-size embryos. We show that Mmp3 scales dorsal-ventral patterning by degrading the slowly diffusing embryonic inducers Noggin1 and Noggin2 but preventing cleavage of the more rapidly diffusing inducer Chordin via degradation of a Tolloid-type proteinase. In addition to unraveling the mechanism underlying the scaling of dorsal-ventral patterning, this work provides proof of principal for scalers identification in embryos of other species., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Gastrulation is the fundamental process in all multicellular animals through which the basic body plan is first laid down 1-4 . It is pivotal in generating cellular diversity coordinated with spatial patterning. In humans, gastrulation occurs in the third week after fertilization. Our understanding of this process in humans is relatively limited and based primarily on historical specimens 5-8 , experimental models 9-12 or, more recently, in vitro cultured samples 13-16 . Here we characterize in a spatially resolved manner the single-cell transcriptional profile of an entire gastrulating human embryo, staged to be between 16 and 19 days after fertilization. We use these data to analyse the cell types present and to make comparisons with other model systems. In addition to pluripotent epiblast, we identified primordial germ cells, red blood cells and various mesodermal and endodermal cell types. This dataset offers a unique glimpse into a central but inaccessible stage of our development. This characterization provides new context for interpreting experiments in other model systems and represents a valuable resource for guiding directed differentiation of human cells in vitro., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Inhibition of the bone morphogenetic proteins (BMPs) is the primary step toward neuroectoderm formation in vertebrates. In this process, the Spemann organizer of the dorsal mesoderm plays a decisive role by secreting several extracellular BMP inhibitors such as Chordin (Chrd). Chrd physically interacts with BMP proteins and inhibits BMP signaling, which triggers the expression of neural-specific transcription factors (TFs), including Foxd4l1.1. Thus, Chrd induces in a BMP-inhibited manner and promotes neuroectoderm formation. However, the regulatory feedback mechanism of Foxd4l1.1 on mesodermal genes expression during germ-layer specification has not been fully elucidated. In this study, we investigated the regulatory mechanism of Foxd4l1.1 on chrd (a mesodermal gene). We demonstrate that Foxd4l1.1 inhibits chrd expression during neuroectoderm formation in two ways: First, Foxd4l1.1 directly binds to FRE (Foxd4l1.1 response elements) within the chrd promoter region to inhibit transcription. Second, Foxd4l1.1 physically interacts with Smad2 and Smad3, and this interaction blocks Smad2 and Smad3 binding to activin response elements (AREs) within the chrd promoter. Site-directed mutagenesis of FRE within the chrd(-2250) promoter completely abolished repressor activity of the Foxd4l1.1. RT-PCR and reporter gene assay results indicate that Foxd4l1.1 strongly inhibits mesoderm- and ectoderm-specific marker genes to maintain neural fate. Altogether, these results suggest that Foxd4l1.1 negatively regulates chrd transcription by dual mechanism. Thus, our study demonstrates the existence of precise reciprocal regulation of chrd transcription during neuroectoderm and mesoderm germ-layer specification in Xenopus embryos.

The neural crest is a dynamic embryonic structure that plays a major role in the formation of the vertebrate craniofacial skeleton. Neural crest formation is regulated by a complex sequence of events directed by a network of transcription factors working in concert with chromatin modifiers. The high mobility group nucleosome binding protein 1 (Hmgn1) is a nonhistone chromatin architectural protein, associated with transcriptionally active chromatin. Here we report the expression and function of Hmgn1 during Xenopus neural crest and craniofacial development. Hmgn1 is broadly expressed at the gastrula and neurula stages, and is enriched in the head region at the tailbud stage, especially in the eyes and the pharyngeal arches. Hmgn1 knockdown affected the expression of several neural crest specifiers, including sox8, sox10, foxd3, and twist1, while other genes (sox9 and snai2) were only marginally affected. The specificity of this phenotype was confirmed by rescue, where injection of Hmgn1 mRNA was able to restore sox10 expression in morphant embryos. The reduction in neural crest gene expression at the neurula stage in Hmgn1 morphant embryos correlated with a decreased number of sox10- and twist1-positive cells in the pharyngeal arches at the tailbud stage, and hypoplastic craniofacial cartilages at the tadpole stage. These results point to a novel role for Hmgn1 in the control of gene expression essential for neural crest and craniofacial development. Future work will investigate the precise mode of action of Hmgn1 in this context., (© 2021 Wiley Periodicals LLC.)

Background: Single-cell technologies are transforming biomedical research, including the recent demonstration that unspliced pre-mRNA present in single-cell RNA-Seq permits prediction of future expression states. Here we apply this RNA velocity concept to an extended timecourse dataset covering mouse gastrulation and early organogenesis., Results: Intriguingly, RNA velocity correctly identifies epiblast cells as the starting point, but several trajectory predictions at later stages are inconsistent with both real-time ordering and existing knowledge. The most striking discrepancy concerns red blood cell maturation, with velocity-inferred trajectories opposing the true differentiation path. Investigating the underlying causes reveals a group of genes with a coordinated step-change in transcription, thus violating the assumptions behind current velocity analysis suites, which do not accommodate time-dependent changes in expression dynamics. Using scRNA-Seq analysis of chimeric mouse embryos lacking the major erythroid regulator Gata1, we show that genes with the step-changes in expression dynamics during erythroid differentiation fail to be upregulated in the mutant cells, thus underscoring the coordination of modulating transcription rate along a differentiation trajectory. In addition to the expected block in erythroid maturation, the Gata1-chimera dataset reveals induction of PU.1 and expansion of megakaryocyte progenitors. Finally, we show that erythropoiesis in human fetal liver is similarly characterized by a coordinated step-change in gene expression., Conclusions: By identifying a limitation of the current velocity framework coupled with in vivo analysis of mutant cells, we reveal a coordinated step-change in gene expression kinetics during erythropoiesis, with likely implications for many other differentiation processes.

Transforming growth factor (TGF)β/activin superfamily regulates diverse biological processes including germ layer specification and axis patterning in vertebrates. TGFβ/activin leads to phosphorylation of Smad2 and Smad3, followed by regulation of their target genes. Activin treatment also induces the essential organizer gene chordin (chrd). The involvement of Smad2/3 in chrd expression has been unclear as to whether Smad2/3 involvement is direct or indirect and whether any cis-acting response elements for Smad2/3 are present in the proximal or distal regions of its promoter. In the present study, we isolated the -2250 bps portion of the chrd promoter, showing that it contained Smad2/3 direct binding sites at its distal portion, separate from the proximal locations of other organizer genes, goosecoid and cerberus. The pattern of transcription activation for the promoter (-2250 bps) was indistinguishable from that of the endogenous chrd in gastrula Xenopus embryos. Reporter gene assays and site-directed mutagenesis analysis of the chrd promoter mapped two active activin/Smad response elements (ARE1 and ARE2) for Smad2 and Smad3. For a differential chrd induction, Smad2 acted on both ARE1 and ARE2, but Smad3 was only active for ARE2. Collectively, the results demonstrate that the distal region of chrd promoter contains the direct binding cis-acting elements for Smad2 and Smad3, which differentially modulate chrd transcription in gastrula Xenopus embryos., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Markers for the endoderm and mesoderm germ layers are commonly expressed together in the early embryo, potentially reflecting cells' ability to explore potential fates before fully committing. It remains unclear when commitment to a single-germ layer is reached and how it is impacted by external signals. Here, we address this important question in Drosophila , a convenient model system in which mesodermal and endodermal fates are associated with distinct cellular movements during gastrulation. Systematically applying endoderm-inducing extracellular signal-regulated kinase (ERK) signals to the ventral medial embryo-which normally only receives a mesoderm-inducing cue-reveals a critical time window during which mesodermal cell movements and gene expression are suppressed by proendoderm signaling. We identify the ERK target gene huckebein ( hkb ) as the main cause of the ventral furrow suppression and use computational modeling to show that Hkb repression of the mesoderm-associated gene snail is sufficient to account for a broad range of transcriptional and morphogenetic effects. Our approach, pairing precise signaling perturbations with observation of transcriptional dynamics and cell movements, provides a general framework for dissecting the complexities of combinatorial tissue patterning., Competing Interests: The authors declare no competing interest.

Generating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.

Background: Explanted tissues from vertebrate embryos reliably develop in culture and have provided essential paradigms for understanding embryogenesis, from early embryological investigations of induction, to the extensive study of Xenopus animal caps, to the current studies of mammalian gastruloids. Cultured explants of the Xenopus dorsal marginal zone ("Keller" explants) serve as a central paradigm for studies of convergent extension cell movements, yet we know little about the global patterns of gene expression in these explants., Results: In an effort to more thoroughly develop this important model system, we provide here a time-resolved bulk transcriptome for developing Keller explants., Conclusions: The dataset reported here provides a useful resource for those using Keller explants for studies of morphogenesis and provide genome-scale insights into the temporal patterns of gene expression in an important tissue when explanted and grown in culture., (© 2020 American Association of Anatomists.)

Heterodimeric TGF-β ligands outperform homodimers in a variety of developmental, cell culture, and therapeutic contexts; however, the mechanisms underlying this increased potency remain uncharacterized. Here, we use dorsal-ventral axial patterning of the zebrafish embryo to interrogate the BMP2/7 heterodimer signaling mechanism. We demonstrate that differential interactions with BMP antagonists do not account for the reduced signaling ability of homodimers. Instead, we find that while overexpressed BMP2 homodimers can signal, they require two nonredundant type I receptors, one from the Acvr1 subfamily and one from the Bmpr1 subfamily. This implies that all BMP signaling within the zebrafish gastrula, even BMP2 homodimer signaling, requires Acvr1. This is particularly surprising as BMP2 homodimers do not bind Acvr1 in vitro. Furthermore, we find that the roles of the two type I receptors are subfunctionalized within the heterodimer signaling complex, with the kinase activity of Acvr1 being essential, while that of Bmpr1 is not. These results suggest that the potency of the Bmp2/7 heterodimer arises from the ability to recruit both Acvr1 and Bmpr1 into the same signaling complex., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

During Xenopus gastrulation, leading edge mesendoderm (LEM) advances animally as a wedge-shaped cell mass over the vegetally moving blastocoel roof (BCR). We show that close contact across the BCR-LEM interface correlates with attenuated net advance of the LEM, which is pulled forward by tip cells while the remaining LEM frequently separates from the BCR. Nevertheless, lamellipodia persist on the detached LEM surface. They attach to adjacent LEM cells and depend on PDGF-A, cell-surface fibronectin and cadherin. We argue that active cell motility on the LEM surface prevents adverse capillary effects in the liquid LEM tissue as it moves by being pulled. It counters tissue surface-tension effects with oriented cell movement and bulges the LEM surface out to keep it close to the curved BCR without attaching to it. Proximity to the BCR is necessary, in turn, for the maintenance and orientation of lamellipodia that permit mass cell movement with minimal substratum contact. Together with a similar process in epithelial invagination, vertical telescoping, the cell movement at the LEM surface defines a novel type of cell rearrangement: vertical shearing., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

The blastula Chordin- and Noggin-expressing (BCNE) center comprises animal-dorsal and marginal-dorsal cells of the amphibian blastula and contains the precursors of the brain and the gastrula organizer. Previous findings suggested that the BCNE behaves as a homogeneous cell population that only depends on nuclear β-catenin activity but does not require Nodal and later segregates into its descendants during gastrulation. In contrast to previous findings, in this work, we show that the BCNE does not behave as a homogeneous cell population in response to Nodal antagonists. In fact, we found that chordin.1 expression in a marginal subpopulation of notochordal precursors indeed requires Nodal input. We also establish that an animal BCNE subpopulation of cells that express both, chordin.1 and sox2 (a marker of pluripotent neuroectodermal cells), and gives rise to most of the brain, persisted at blastula stage after blocking Nodal. Therefore, Nodal signaling is required to define a population of chordin.1+ cells and to restrict the recruitment of brain precursors within the BCNE as early as at blastula stage. We discuss our findings in Xenopus in comparison to other vertebrate models, uncovering similitudes in early brain induction and delimitation through Nodal signaling., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Bone Morphogenetic Protein (BMP) patterns the dorsal-ventral (DV) embryonic axis in all vertebrates, but it is unknown how cells along the DV axis interpret and translate the gradient of BMP signaling into differential gene activation that will give rise to distinct cell fates. To determine the mechanism of BMP morphogen interpretation in the zebrafish gastrula, we identified 57 genes that are directly activated by BMP signaling. By using Seurat analysis of single-cell RNA sequencing (scRNA-seq) data, we found that these genes are expressed in at least 3 distinct DV domains of the embryo. We distinguished between 3 models of BMP signal interpretation in which cells activate distinct gene expression through interpretation of thresholds of (1) the BMP signaling gradient slope; (2) the BMP signal duration; or (3) the level of BMP signal activation. We tested these 3 models using quantitative measurements of phosphorylated Smad5 (pSmad5) and by examining the spatial relationship between BMP signaling and activation of different target genes at single-cell resolution across the embryo. We found that BMP signaling gradient slope or BMP exposure duration did not account for the differential target gene expression domains. Instead, we show that cells respond to 3 distinct levels of BMP signaling activity to activate and position target gene expression. Together, we demonstrate that distinct pSmad5 threshold levels activate spatially distinct target genes to pattern the DV axis., Competing Interests: The authors have declared that no competing interests exist.

This review highlights the work that my research group has been developing, together with international collaborators, during the last decade. Since we were able to establish the Xenopus laevis experimental model in Brazil, we have been focused on understanding early embryonic patterns regarding neural induction and axes establishment. In this context, the Wnt pathway appears as a major player and has been much explored by us and other research groups. Here, we chose to review three published works which we consider to be landmarks within the course of our research and also within the history of modern findings regarding neural induction and patterning. We intend to show how our series of discoveries, when painted together, tells a story that covers crucial developmental windows of early differentiation paths of anterior neural tissue: 1. establishing the head organizer in contrast to the trunk organizer in the early gastrula; 2. deciding between neural ectoderm and epidermis ectoderm at the blastula/gastrula stages, and 3. the gathering of prechordal unique properties in the late gastrula/early neurula.

Lineage specification is governed by gene regulatory networks (GRNs) that integrate the activity of signaling effectors and transcription factors (TFs) on enhancers. Sox17 is a key transcriptional regulator of definitive endoderm development, and yet, its genomic targets remain largely uncharacterized. Here, using genomic approaches and epistasis experiments, we define the Sox17-governed endoderm GRN in Xenopus gastrulae. We show that Sox17 functionally interacts with the canonical Wnt pathway to specify and pattern the endoderm while repressing alternative mesectoderm fates. Sox17 and β-catenin co-occupy hundreds of key enhancers. In some cases, Sox17 and β-catenin synergistically activate transcription apparently independent of Tcfs, whereas on other enhancers, Sox17 represses β-catenin/Tcf-mediated transcription to spatially restrict gene expression domains. Our findings establish Sox17 as a tissue-specific modifier of Wnt responses and point to a novel paradigm where genomic specificity of Wnt/β-catenin transcription is determined through functional interactions between lineage-specific Sox TFs and β-catenin/Tcf transcriptional complexes. Given the ubiquitous nature of Sox TFs and Wnt signaling, this mechanism has important implications across a diverse range of developmental and disease contexts., Competing Interests: SM, PC, SR, MF, MW, KP, MM, XC, MW, IB, KC, AZ No competing interests declared, (© 2020, Mukherjee et al.)

Gastrulation is generally understood as the morphogenetic processes that result in the spatial organization of the blastomere into the three germ layers, ectoderm, mesoderm and endoderm. This review summarizes our current knowledge of the morphogenetic mechanisms in Drosophila gastrulation. In addition to the events that drive mesoderm invagination and germband elongation, we pay particular attention to other, less well-known mechanisms including midgut invagination, cephalic furrow formation, dorsal fold formation, and mesoderm layer formation. This review covers topics ranging from the identification and functional characterization of developmental and morphogenetic control genes to the analysis of the physical properties of cells and tissues and the control of cell and tissue mechanics of the morphogenetic movements in the gastrula., (Copyright © 2020 Elsevier B.V. All rights reserved.)

During ontogeny, proliferating cells become restricted in their fate through the combined action of cell-type-specific transcription factors and ubiquitous epigenetic machinery, which recognizes universally available histone residues or nucleotides in a context-dependent manner 1,2 . The molecular functions of these regulators are generally well understood, but assigning direct developmental roles to them is hampered by complex mutant phenotypes that often emerge after gastrulation 3,4 . Single-cell RNA sequencing and analytical approaches have explored this highly conserved, dynamic period across numerous model organisms 5-8 , including mouse 9-18 . Here we advance these strategies using a combined zygotic perturbation and single-cell RNA-sequencing platform in which many mutant mouse embryos can be assayed simultaneously, recovering robust  morphological and transcriptional information across a panel of ten essential regulators. Deeper analysis of central Polycomb repressive complex (PRC) 1 and 2 components indicates substantial cooperativity, but distinguishes a dominant role for PRC2 in restricting the germline. Moreover, PRC mutant phenotypes emerge after gross epigenetic and transcriptional changes within the initial conceptus prior to gastrulation. Our experimental framework may eventually lead to a fully quantitative view of how cellular diversity emerges using an identical genetic template and from a single totipotent cell.

The mechanisms used by embryos to pattern tissues across their axes has fascinated developmental biologists since the founding of embryology. Here, using single-cell technology, we interrogate complex patterning defects and define a Hedgehog (Hh)-fibroblast growth factor (FGF) signaling axis required for anterior mesoderm lineage development during gastrulation. Single-cell transcriptome analysis of Hh-deficient mesoderm revealed selective deficits in anterior mesoderm populations, culminating in defects to anterior embryonic structures, including the pharyngeal arches, heart, and anterior somites. Transcriptional profiling of Hh-deficient mesoderm during gastrulation revealed disruptions to both transcriptional patterning of the mesoderm and FGF signaling for mesoderm migration. Mesoderm-specific Fgf4 / Fgf8 double-mutants recapitulated anterior mesoderm defects and Hh-dependent GLI transcription factors modulated enhancers at FGF gene loci. Cellular migration defects during gastrulation induced by Hh pathway antagonism were mitigated by the addition of FGF4 protein. These findings implicate a multicomponent signaling hierarchy activated by Hh ligands from the embryonic node and executed by FGF signals in nascent mesoderm to control anterior mesoderm patterning., Competing Interests: The authors declare no competing interest.

The process that partitions the nascent vertebrate central nervous system into forebrain, midbrain, hindbrain, and spinal cord after neural induction is of fundamental interest in developmental biology, and is known to be dependent on Wnt/β-catenin signaling at multiple steps. Neural induction specifies neural ectoderm with forebrain character that is subsequently posteriorized by graded Wnt signaling: embryological and mutant analyses have shown that progressively higher levels of Wnt signaling induce progressively more posterior fates. However, the mechanistic link between Wnt signaling and the molecular subdivision of the neural ectoderm into distinct domains in the anteroposterior (AP) axis is still not clear. To better understand how Wnt mediates neural AP patterning, we performed a temporal dissection of neural patterning in response to manipulations of Wnt signaling in zebrafish. We show that Wnt-mediated neural patterning in zebrafish can be divided into three phases: (I) a primary AP patterning phase, which occurs during gastrulation, (II) a mes/r1 (mesencephalon-rhombomere 1) specification and refinement phase, which occurs immediately after gastrulation, and (III) a midbrain-hindbrain boundary (MHB) morphogenesis phase, which occurs during segmentation stages. A major outcome of these Wnt signaling phases is the specification of the major compartment divisions of the developing brain: first the MHB, then the diencephalic-mesencephalic boundary (DMB). The specification of these lineage divisions depends upon the dynamic changes of gene transcription in response to Wnt signaling, which we show primarily involves transcriptional repression or indirect activation. We show that otx2b is directly repressed by Wnt signaling during primary AP patterning, but becomes resistant to Wnt-mediated repression during late gastrulation. Also during late gastrulation, Wnt signaling becomes both necessary and sufficient for expression of wnt8b, en2a, and her5 in mes/r1. We suggest that the change in otx2b response to Wnt regulation enables a transition to the mes/r1 phase of Wnt-mediated patterning, as it ensures that Wnts expressed in the midbrain and MHB do not suppress midbrain identity, and consequently reinforce formation of the DMB. These findings integrate important temporal elements into our spatial understanding of Wnt-mediated neural patterning and may serve as an important basis for a better understanding of neural patterning defects that have implications in human health., (Published by Elsevier Inc.)