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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.

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.

When stimulated with a pulse from an exogenous WNT pathway activator, small aggregates of mouse embryonic stem cells (ESCs) can undergo embryo-like axial morphogenesis and patterning along the three major body axes. However, these structures, called gastruloids, currently lack the anterior embryonic regions, such as those belonging to the brain. Here, we describe an approach to generate gastruloids that have a more complete antero-posterior development. We used hydrogel microwell arrays to promote the robust derivation of mouse ESCs into post-implantation epiblast-like (EPI) aggregates in a reproducible and scalable manner. These EPI aggregates break symmetry and axially elongate without external chemical stimulation. Inhibition of WNT signaling in early stages of development leads to the formation of gastruloids with anterior neural tissues. Thus, we provide a new tool to study the development of the mouse after implantation in vitro, especially the formation of anterior neural regions., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Gastrulation consists in the dramatic reorganisation of the epiblast, a one-cell thick epithelial sheet, into a multilayered embryo. In chick, the formation of the internal layers requires the generation of a macroscopic convection-like flow, which involves up to 50,000 epithelial cells in the epiblast. These cell movements locate the mesendoderm precursors into the midline of the epiblast to form the primitive streak. There they acquire a mesenchymal phenotype, ingress into the embryo and migrate outward to populate the inner embryonic layers. This review covers what is currently understood about how cell behaviours ultimately cause these morphogenetic events and how they are regulated. We discuss 1) how the biochemical patterning of the embryo before gastrulation creates compartments of differential cell behaviours, 2) how the global epithelial flows arise from the coordinated actions of individual cells, 3) how the cells delaminate individually from the epiblast during the ingression, and 4) how cells move after the ingression following stereotypical migration routes. We conclude by exploring new technical advances that will facilitate future research in the chick model system., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The Brachyury gene encodes a transcription factor that is conserved across all animals. In non-chordate metazoans, brachyury is primarily expressed in ectoderm regions that are added to the endodermal gut during development, and often form a ring around the site of endoderm internalization in the gastrula, the blastopore. In chordates, this brachyury ring is conserved, but the gene has taken on a new role in the formation of the mesoderm. In this phylum, a novel type of mesoderm that develops into notochord and somites has been added to the ancestral lateral plate mesoderm. Brachyury contributes to a shift in cell fate from neural ectoderm to posterior notochord and somites during a major lineage segregation event that in Xenopus and in the zebrafish takes place in the early gastrula. In the absence of this brachyury function, impaired formation of posterior mesoderm indirectly affects the gastrulation movements of peak involution and convergent extension. These movements are confined to specific regions and stages, leaving open the question why brachyury expression in an extensive, coherent ring, before, during and after gastrulation, is conserved in the two species whose gastrulation modes differ considerably, and also in many other metazoan gastrulae of diverse structure., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 mouse embryonic development a mass of pluripotent epiblast tissue is transformed during gastrulation to generate the three definitive germ layers: endoderm, mesoderm, and ectoderm. During gastrulation, a spatiotemporally controlled sequence of events results in the generation of organ progenitors and positions them in a stereotypical fashion throughout the embryo. Key to the correct specification and differentiation of these cell fates is the establishment of an axial coordinate system along with the integration of multiple signals by individual epiblast cells to produce distinct outcomes. These signaling domains evolve as the anterior-posterior axis is established and the embryo grows in size. Gastrulation is initiated at the posteriorly positioned primitive streak, from which nascent mesoderm and endoderm progenitors ingress and begin to diversify. Advances in technology have facilitated the elaboration of landmark findings that originally described the epiblast fate map and signaling pathways required to execute those fates. Here we will discuss the current state of the field and reflect on how our understanding has shifted in recent years., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

Morphogenetic flows in developmental biology are characterized by the coordinated motion of thousands of cells that organize into tissues, naturally raising the question of how this collective organization arises. Using only the kinematics of tissue deformation, which naturally integrates local and global mechanisms along cell paths, we identify the dynamic morphoskeletons behind morphogenesis, i.e., the evolving centerpieces of multicellular trajectory patterns. These features are model- and parameter-free, frame-invariant, and robust to measurement errors and can be computed from unfiltered cell-velocity data. We reveal the spatial attractors and repellers of the embryo by quantifying its Lagrangian deformation, information that is inaccessible to simple trajectory inspection or Eulerian methods that are local and typically frame-dependent. Computing these dynamic morphoskeletons in wild-type and mutant chick and fly embryos, we find that they capture the early footprint of known morphogenetic features, reveal new ones, and quantitatively distinguish between different phenotypes., Competing Interests: The authors declare no competing interest.

Deciphering the key mechanisms of morphogenesis during embryonic development is crucial to understanding the guiding principles of the body plan and promote applications in biomedical research fields. Although several computational tissue reconstruction methods using cellular gene expression data have been proposed, those methods are insufficient with regard to arranging cells in their correct positions in tissues or organs unless spatial information is explicitly provided. Here, we report SPRESSO, a new in silico three-dimensional (3D) tissue reconstruction method using stochastic self-organizing map (stochastic-SOM) clustering, to estimate the spatial domains of cells in tissues or organs from only their gene expression profiles. With only five gene sets defined by Gene Ontology (GO), we successfully demonstrated the reconstruction of a four-domain structure of mid-gastrula mouse embryo (E7.0) with high reproducibility (success rate = 99%). Interestingly, the five GOs contain 20 genes, most of which are related to differentiation and morphogenesis, such as activin A receptor and Wnt family member genes. Further analysis indicated that Id2 is the most influential gene contributing to the reconstruction. SPRESSO may provide novel and better insights on the mechanisms of 3D structure formation of living tissues via informative genes playing a role as spatial discriminators.

Indirect evidence suggests that blastopore closure during gastrulation of anamniotes, including amphibians such as Xenopus laevis , depends on circumblastoporal convergence forces generated by the marginal zone (MZ), but direct evidence is lacking. We show that explanted MZs generate tensile convergence forces up to 1.5 μN during gastrulation and over 4 μN thereafter. These forces are generated by convergent thickening (CT) until the midgastrula and increasingly by convergent extension (CE) thereafter. Explants from ventralized embryos, which lack tissues expressing CE but close their blastopores, produce up to 2 μN of tensile force, showing that CT alone generates forces sufficient to close the blastopore. Uniaxial tensile stress relaxation assays show stiffening of mesodermal and ectodermal tissues around the onset of neurulation, potentially enhancing long-range transmission of convergence forces. These results illuminate the mechanobiology of early vertebrate morphogenic mechanisms, aid interpretation of phenotypes, and give insight into the evolution of blastopore closure mechanisms., Competing Interests: DS, EK, LD, RK No competing interests declared, (© 2018, Shook et al.)

Background: Gastrulation is a complex orchestration of movements by cells that are specified early in development. Until now, classical convergent extension was considered to be the main contributor to sea urchin archenteron extension, and the relative contributions of cell divisions were unknown. Active migration of cells along the axis of extension was also not considered as a major factor in invagination., Results: Cell transplantations plus live imaging were used to examine endoderm cell morphogenesis during gastrulation at high-resolution in the optically clear sea urchin embryo. The invagination sequence was imaged throughout gastrulation. One of the eight macromeres was replaced by a fluorescently labeled macromere at the 32 cell stage. At gastrulation those patches of fluorescent endoderm cell progeny initially about 4 cells wide, released a column of cells about 2 cells wide early in gastrulation and then often this column narrowed to one cell wide by the end of archenteron lengthening. The primary movement of the column of cells was in the direction of elongation of the archenteron with the narrowing (convergence) occurring as one of the two cells moved ahead of its neighbor. As the column narrowed, the labeled endoderm cells generally remained as a contiguous population of cells, rarely separated by intrusion of a lateral unlabeled cell. This longitudinal cell migration mechanism was assessed quantitatively and accounted for almost 90% of the elongation process. Much of the extension was the contribution of Veg2 endoderm with a minor contribution late in gastrulation by Veg1 endoderm cells. We also analyzed the contribution of cell divisions to elongation. Endoderm cells in Lytechinus variagatus were determined to go through approximately one cell doubling during gastrulation. That doubling occurs without a net increase in cell mass, but the question remained as to whether oriented divisions might contribute to archenteron elongation. We learned that indeed there was a biased orientation of cell divisions along the plane of archenteron elongation, but when the impact of that bias was analyzed quantitatively, it contributed a maximum 15% to the total elongation of the gut., Conclusions: The major driver of archenteron elongation in the sea urchin, Lytechinus variagatus, is directed movement of Veg2 endoderm cells as a narrowing column along the plane of elongation. The narrowing occurs as cells in the column converge as they migrate, so that the combination of migration and the angular convergence provide the major component of the lengthening. A minor contributor to elongation is oriented cell divisions that contribute to the lengthening but no more than about 15%., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cell-cell contact formation constitutes an essential step in evolution, leading to the differentiation of specialized cell types. However, remarkably little is known about whether and how the interplay between contact formation and fate specification affects development. Here, we identify a positive feedback loop between cell-cell contact duration, morphogen signaling, and mesendoderm cell-fate specification during zebrafish gastrulation. We show that long-lasting cell-cell contacts enhance the competence of prechordal plate (ppl) progenitor cells to respond to Nodal signaling, required for ppl cell-fate specification. We further show that Nodal signaling promotes ppl cell-cell contact duration, generating a positive feedback loop between ppl cell-cell contact duration and cell-fate specification. Finally, by combining mathematical modeling and experimentation, we show that this feedback determines whether anterior axial mesendoderm cells become ppl or, instead, turn into endoderm. Thus, the interdependent activities of cell-cell signaling and contact formation control fate diversification within the developing embryo., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Zebrafish is a model organism suitable for studying vertebrate development. We analyzed the N-glycan structures of zebrafish embryos and their alterations during zebrafish embryogenesis to obtain basic data for studying the roles of N-glycosylation. Multiple modes of high-performance liquid chromatography and multistage mass spectrometry were used for structural analysis of N-glycans. The N-glycans from deyolked embryos at 36 hours postfertilization, a mid-pharyngula stage, contained relatively higher amounts of complex- and hybrid-type glycans with LacNAc (Galβ1-4GlcNAc) and/or sialyl LacNAc without additional β1,4-Gal, which are commonly found in mammalian tissues, as well as abundant oligomannose-type glycans. Some of the complex- and hybrid-type glycans possessed various extended LacNAc structures, such as Galβ1-4LacNAc, LacNAc-repeat or unique (+/- dHex)-GalNAcα1-GlcNAcβ1-LacNAc. In contrast, the yolk of the embryo contains predominant oligomannose-type glycans and complex-type glycans with Galβ1-4(Siaα2-3)Galβ1-4(Fucα1-3)GlcNAc antennae. N-Glycan profiles obtained from deyolked embryos at different stages showed stage-dependent variation of complex- and hybrid-type glycans. At gastrula and early segmentation stages, complex- and hybrid-type glycans were minor components, and their antenna structures were mainly sialyl LacdiNAc (Siaα2-6GalNAcβ1-4GlcNAc). From the mid-segmentation to pharyngula stages, those with LacNAc and/or α2,6-sialyl LacNAc antenna structures increased remarkably, and those with α2,3-sialyl LacNAc antenna, core α1,6-Fuc and bisecting GlcNAc modifications increased gradually. These results suggest the presence of mechanisms for regulating the antenna structures of complex/hybrid N-glycan biosynthesis in the phylotypic stage of vertebrate development., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cell proliferation has generally been considered dispensable for anteroposterior extension of embryonic axis during vertebrate gastrulation. Signal transducer and activator of transcription 3 (Stat3), a conserved controller of cell proliferation, survival and regeneration, is associated with human scoliosis, cancer and Hyper IgE Syndrome. Zebrafish Stat3 was proposed to govern convergence and extension gastrulation movements in part by promoting Wnt/Planar Cell Polarity (PCP) signaling, a conserved regulator of mediolaterally polarized cell behaviors. Here, using zebrafish stat3 null mutants and pharmacological tools, we demonstrate that cell proliferation contributes to anteroposterior embryonic axis extension. Zebrafish embryos lacking maternal and zygotic Stat3 expression exhibit normal convergence movements and planar cell polarity signaling, but transient axis elongation defect due to insufficient number of cells resulting largely from reduced cell proliferation and increased apoptosis. Pharmacologic inhibition of cell proliferation during gastrulation phenocopied axis elongation defects. Stat3 regulates cell proliferation and axis extension in part via upregulation of Cdc25a expression during oogenesis. Accordingly, restoring Cdc25a expression in stat3 mutants partially suppressed cell proliferation and gastrulation defects. During later development, stat3 mutant zebrafish exhibit stunted growth, scoliosis, excessive inflammation, and fail to thrive, affording a genetic tool to study Stat3 function in vertebrate development, regeneration, and disease.

By the initial phase of gastrulation, Wnt pathway regulation mediates endomesoderm specification and establishes the animal-vegetal axis, thereby leading to proper gastrulation in starfish. To provide insight into the ancestral mechanism regulating deuterostome gastrulation, we identified the gene expression patterns of Wnt, Frizzled (Fz), and secreted frizzled-related protein (sFRP) family genes, which play a role in the initial stage of the Wnt pathway, in starfish Patiria (Asterina) pectinifera embryos using whole mount in situ hybridization. We identified ten Wnt, four Fz, and two sFRP paralogues. From the hatching blastula to the late gastrula stage, the majority of the Wnt genes and both Fz5/8 and sFRP1/5 were expressed in the posterior and anterior half of the embryo, respectively. Wnt8, Fz1, and Fz4 showed restricted expression in the lateral ectoderm. On the other hand, several genes were expressed de novo in the restricted domain of the archenteron at the late gastrula stage. These results suggest that the canonical and/or non-canonical Wnt pathway might implicate endomesoderm specification, anterior-posterior axis establishment, anterior-posterior patterning, and archenteron morphogenesis in the developmental context of starfish embryos. From comparison with the expression patterns observed in Patria miniata, we consider that the Wnt pathway is conserved among starfishes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In chordates, neural induction is the first step of a complex developmental process through which ectodermal cells acquire a neural identity. In ascidians, FGF-mediated neural induction occurs at the 32-cell stage in two blastomere pairs, precursors respectively of anterior and posterior neural tissue. We combined molecular embryology and cis-regulatory analysis to unveil in the ascidian Ciona intestinalis the remarkably simple proximal genetic network that controls posterior neural fate acquisition downstream of FGF. We report that the combined action of two direct FGF targets, the TGFβ factor Nodal, acting via Smad- and Fox-binding sites, and the transcription factor Otx suffices to trigger ascidian posterior neural tissue formation. Moreover, we found that this strategy is conserved in the distantly related ascidian Phallusia mammillata, in spite of extreme sequence divergence in the cis-regulatory sequences involved. Our results thus highlight that the modes of gene regulatory network evolution differ with the evolutionary scale considered. Within ascidians, developmental regulatory networks are remarkably robust to genome sequence divergence. Between ascidians and vertebrates, major fate determinants, such as Otx and Nodal, can be co-opted into different networks. Comparative developmental studies in ascidians with divergent genomes will thus uncover shared ascidian strategies, and contribute to a better understanding of the diversity of developmental strategies within chordates.

Cytokinesis terminates mitosis, resulting in separation of the two sister cells. Septins, a conserved family of GTP-binding cytoskeletal proteins, are an absolute requirement for cytokinesis in budding yeast. We demonstrate that septin-dependence of mammalian cytokinesis differs greatly between cell types: genetic loss of the pivotal septin subunit SEPT7 in vivo reveals that septins are indispensable for cytokinesis in fibroblasts, but expendable in cells of the hematopoietic system. SEPT7-deficient mouse embryos fail to gastrulate, and septin-deficient fibroblasts exhibit pleiotropic defects in the major cytokinetic machinery, including hyperacetylation/stabilization of microtubules and stalled midbody abscission, leading to constitutive multinucleation. We identified the microtubule depolymerizing protein stathmin as a key molecule aiding in septin-independent cytokinesis, demonstrated that stathmin supplementation is sufficient to override cytokinesis failure in SEPT7-null fibroblasts, and that knockdown of stathmin makes proliferation of a hematopoietic cell line sensitive to the septin inhibitor forchlorfenuron. Identification of septin-independent cytokinesis in the hematopoietic system could serve as a key to identify solid tumor-specific molecular targets for inhibition of cell proliferation.

We have previously shown that the cell sorting process of animal pole cells (AC) and vegetal pole cells (VC) from Xenopus gastrulae is considered to involve two steps: concentrification and polarization. In this study, we addressed the question of what specified the spatial relationship of the AC and VC clusters during the process. First, we examined the inhibitory or facilitatory treatment for myosin 2 activity during each of the two steps. The aggregates treated with Y27632 or blebbistatin during the concentrification step showed a cluster random arrangement, suggesting the prevention of the cell sorting by inhibition of myosin 2. Meanwhile, the treatment with a Rac1 inhibitor, NSC23766, during the same step resulted in promotion of the fusion of the AC clusters and the progression of the cell sorting, presumably by an indirect activation of myosin 2. On the other hand, the treatments with any of the three drugs during the polarization step showed that the two clusters did not appose, and their array remained concentric. Thus, the modulation of cell contraction might be indispensable to each of the two steps. Next, the activin/nodal TGF-β signaling was perturbed by using a specific activin receptor-like kinase inhibitor, SB431542. The results revealed a bimodal participation of the activin/nodal TGF-β signaling, i.e., suppressive and promotive effects on the concentrification and the polarization, respectively. Thus, the present in vitro system, which permits not only the cell contraction-mediated cell sorting but also the TGF-β-directed mesodermal induction such as cartilage formation, may fairly reflect the embryogenesis in vivo.

Although much has been learned about genetic networks, cell mechanics, and whole-embryo mechanics through experimental and computational studies, the challenge of connecting these separate bodies of knowledge into an integrated whole remains. Here, we offer a multiscale biochemical-mechanical framework from which such integration might proceed. We identify components of the framework for which quantitative descriptions are currently available, and use the framework to gain insight into convergent extension and gastrulation--crucial tissue movements that occur in early stage amphibian embryos.

We analyzed the notochord formation, formation of the prechordal plate, and patterning of anteroposterior regional specificity of the involuting and extending archenteron roof of a urodele, Cynops pyrrhogaster. The lower (LDMZ) and upper (UDMZ) domains of the dorsal marginal zone (DMZ) of the early gastrula involuted and formed two distinct domains: the anterior fore-notochordal endodermal roof and the posterior domain containing the prospective notochord. Cygsc is expressed in the LDMZ from the onset of gastrulation, and the Cygsc-expressing LDMZ planarly induces the notochord in the UDMZ at the early to mid gastrula stages. At the mid to late gastrula stages, part of the Cygsc-expressing LDMZ is confined to the prechordal plate. On the other hand, Cybra expression only begins at mid gastrula stage, coincident with notochord induction at this stage. Anteroposterior regional specificity of the neural plate was patterned by the posterior domain of the involuting archenteron roof containing the prospective notochord at the mid to late gastrula stages. Cynops gastrulation thus differs significantly from Xenopus gastrulation in that the regions of the DMZ are specified from the onset of gastrulation, while the equivalent state of specification does not occur in Cynops until the middle of gastrulation. Thus we propose that Cynops gastrulation is divided into two phases: a notochord induction phase in the early to mid gastrula, and a neural induction phase in the mid to late gastrula.

A major goal of contemporary studies of embryonic development is to understand large sets of regulatory changes that accompany the phenomenon of embryonic induction. The highly resolved sea urchin pregastrular endomesoderm-gene regulatory network (EM-GRN) provides a unique framework to study the global regulatory interactions underlying endomesoderm induction. Vegetal micromeres of the sea urchin embryo constitute a classic endomesoderm signaling center, whose potential to induce archenteron formation from presumptive ectoderm was demonstrated almost a century ago. In this work, we ectopically activate the primary mesenchyme cell-GRN (PMC-GRN) that operates in micromere progeny by misexpressing the micromere determinant Pmar1 and identify the responding EM-GRN that is induced in animal blastomeres. Using localized loss-of -function analyses in conjunction with expression of endo16, the molecular definition of micromere-dependent endomesoderm specification, we show that the TGFbeta cytokine, ActivinB, is an essential component of this induction in blastomeres that emit this signal, as well as in cells that respond to it. We report that normal pregastrular endomesoderm specification requires activation of the Pmar1-inducible subset of the EM-GRN by the same cytokine, strongly suggesting that early micromere-mediated endomesoderm specification, which regulates timely gastrulation in the sea urchin embryo, is also ActivinB dependent. This study unexpectedly uncovers the existence of an additional uncharacterized micromere signal to endomesoderm progenitors, significantly revising existing models. In one of the first network-level characterizations of an intercellular inductive phenomenon, we describe an important in vivo model of the requirement of ActivinB signaling in the earliest steps of embryonic endomesoderm progenitor specification., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Toxic effects of 1-methyl-3-octylimidazolium bromide ([C8mim]Br) on the early embryonic development of the frog Rana nigromaculata were evaluated. Frog embryos in different developmental stages (early cleavage, early gastrula, or neural plate) were exposed to 0, 45, 63, or 88.2 mg/L of the ionic liquid [C8mim]Br for 96 h. The 96-h median lethal concentration values at the early cleavage, early gastrula, and neural plate stages of development were 85.1, 43.4, and 42.4 mg/L, respectively. In embryos exposed to [C8mim]Br, the duration of embryo dechorionation was prolonged in the early cleavage and neural plate, but not the early gastrula, stages of development compared with control embryos. Embryos in the neural plate developmental stage were found to have the highest mortality rate following [C8mim]Br exposure. These results suggest that [C8mim]Br has toxic effects on the early embryonic development of the frog.

Importin-beta is an essential component of nuclear protein import, spindle formation and nuclear envelope assembly. Formerly, the function of the Drosophila Ketel gene, which encodes importin-beta and is essential for the survival to adulthood, seemed to be required only in the mitotically active cells. We report here that importin-beta function is required in every cell and that this protein possesses an exceptionally long life span. Mosaic analysis, using gynanders, indicated that zygotic function of the Ketel gene is essential in a large group of cells in the embryos. Expression of a UAS-Ketel transgene by different tissue specific Gal4 drivers on ketel(null)/- hemizygous background revealed the requirement of Ketel gene function in the ectoderm. Elimination of the Ketel gene function using a UAS-Ketel-RNAi transgene driven by different Gal4 drivers confirmed the indispensability of the Ketel gene in the ectoderm. Using GFP-tagged importin-beta (encoded by a ketel(GFP) allele) we revealed that the maternally provided GFP-importin-beta molecules persist up to larval life. The zygotic Ketel gene is expressed in every cell during early gastrulation. Although the gene is then turned off in the non-dividing cells, the produced importin-beta molecules persist long and carry out nuclear protein import throughout the subsequent stages of development. In the continuously dividing diploid cells, the Ketel gene is constitutively expressed to fulfill all three functions of importin-beta.