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PubMed2001

Transgenic Xenopus embryos reveal that anterior neural development requires continued suppression of BMP signaling after gastrulation.

In vertebrates, BMP signaling before gastrulation suppresses neural development. Later in development, BMP signaling specifies a dorsal and ventral fate in the forebrain and dorsal fate in the spinal cord. It is therefore possible that a change in the competence of the ectoderm to respond to BMP signaling occurs at some point in development. We report that exposure of the anterior neural plate to BMP4 before gastrulation causes suppression of all neural markers tested. To determine the effects of BMP4 after gastrulation, we misexpressed BMP4 using a Pax-6 promoter fragment in transgenic frog embryos and implanted beads soaked in BMP4 in the anterior neural plate. Suppression of most anterior neural markers was observed. We conclude that most neural genes continue to require suppression of BMP signaling into the neurula stages. Additionally, we report that BMP4 and BMP7 are abundantly expressed in the prechordal mesoderm of the neurula stage embryo. This poses the paradox of how the expression of most neural genes is maintained if they can be inhibited by BMP signaling. We show that at least one gene in the anterior neural plate suppresses the response of the ectoderm to BMP signaling. We propose that the suppressive effect of BMP signaling on the expression of neural genes coupled with localized suppressors of BMP signaling result in the fine-tuning of gene expression in the anterior neural plate.

Animals↗
PubMed2002

Xenopus Cdc42 regulates convergent extension movements during gastrulation through Wnt/Ca2+ signaling pathway.

Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, cell adhesion, cell-cycle progression, and transcription. We have isolated the Xenopus homolog of Rho GTPase Cdc42 and examined its potential role during gastrulation movements in early Xenopus embryos. XCdc42 is expressed in tissues undergoing extensive morphogenetic changes, such as the deep layers of involuting mesoderm and posterior neuroectoderm during gastrulation, and somitic mesoderm at neurula stages. Overexpression of either wild-type (WT) or dominant-negative (DN) XCdc42 interferes with convergent extension movements in intact embryos, activin-stimulated animal caps, and dorsal marginal zone explants. These effects occur without affecting mesodermal specification. Overexpression of WT or DN XCdc42 leads to the decrease and increase of cell adhesiveness of blastomeres, respectively, as demonstrated by the cell adhesion assay. In addition, when overexpressed, PKC-alpha, XWnt-5a, and Mfz-3 inhibit activin-induced convergent extension in animal cap explants. This inhibition can be rescued by coexpression of DN XCdc42, implying that XCdc42 acts downstream of the Wnt/Ca2+ signaling pathway involving PKC activation. XCdc42 also lies downstream of XWnt-5a in the regulation of Ca2+-dependent cell adhesion. Taken together, our results suggest that XCdc42 plays a role in the regulation of convergent extension movements during gastrulation through the protein kinase C-mediated Wnt/Ca2+ pathway.

Amino Acid Sequence↗
PubMed1983

Fibronectin in early amphibian embryos. Migrating mesodermal cells contact fibronectin established prior to gastrulation.

The three-dimensional organisation of fibronectin (FN) in early amphibian embryos (Ambystoma mexicanum, Pleurodeles waltlii) was studied with the use of antibodies against amphibian-FN. Immunofluorescence labelling was performed on whole-mount specimens. It was shown that before gastrulation an extensive extracellular network consisting of anastomosed FN-fibrils underlies the roof of the blastocoel prior to the migration of mesodermal cells. Initially, FN-fibrils develop radially on the inner surface of ectodermal cells (early blastula stage) and become confluent there to elaborate a fibrillar network. During the late blastula stage the entire surface of the blastocoel roof (presumptive ectoderm and mesoderm) is covered by the FN-rich extracellular matrix. As gastrulation proceeds, the migrating mesodermal cells interact with the FN-fibrillar network. The results suggest that the FN-containing extracellular matrix plays an important role in mediating mesodermal cell-substratum contacts in gastrulating embryos. No FN-fibrils were found in other parts of the embryo.

Ambystoma↗
PubMed1995

Fibronectin peptide DRVPHSRNSIT and fibronectin receptor peptide DLYYLMDL arrest gastrulation of Rana pipiens.

Gastrulation is characterized by dramatic cell migration which is thought to require the interaction of cell adhesion molecules with extracellular molecules. We have tested two novel peptides, a fibronectin peptide and a fibronectin receptor peptide, for their effects on gastrulation of the leopard frog Rana pipiens. The fibronectin peptide DRVPHSRNSIT corresponds to residues 1373-1383 of the cell-binding domain of fibronectin; the receptor peptide DLYYLMDL corresponds to residues 124-131 of beta 1 subunit of a variety of integrins including alpha 5 beta 1. Either of these peptides significantly inhibited gastrulation after being microinjected into mid-blastulae. These results indicate that these sequences may correspond to the ligand/receptor interaction sites of fibronectin and its receptor(s).

Amino Acid Sequence↗
PubMed2001

Evolutionary conservation, developmental expression, and genomic mapping of mammalian Twisted gastrulation.

The twisted gastrulation gene (tsg) encodes a secreted protein required for the correct specification of dorsal midline cell fate during gastrulation in Drosophila. We report that tsg homologs from human, mouse, zebrafish, and Xenopus share 72-98% identity at the amino acid level and retain all 24 cysteine residues from Drosophila. In contrast to Drosophila where tsg expression is limited to early embryos, expression is found throughout mouse and human development. In Drosophila, tsg acts in synergy with decapentaplegic (dpp), a member of the TGF-beta family of secreted proteins. The vertebrate orthologs of dpp, BMP-2 and -4, are crucial for gastrulation and neural induction, and aberrant signaling by BMPs and other TGF-beta family members results in developmental defects including holoprosencephaly (HPE). Interestingly, human TSG maps to the HPE4 locus on Chromosome 18p11.3, and our analysis places the gene within 5 Mbp of TG-interacting factor (TGIF).

Amino Acid Sequence↗
PubMed2004

Unusual cleavage and gastrulation in a freshwater nematode: developmental and phylogenetic implications.

Early embryogenesis in nematodes as seen in Caenorhabditis elegans and many other species of this phylum features several characteristic events. These include the visible presence of a germline from the very beginning generating different somatic lineages via asymmetric cleavages, the absence of a coeloblastula and a unique type of gastrulation with immigration of just two gut precursor cells. Here it is shown by using Nomarski optics that development of the freshwater nematode Tobrilus diversipapillatus differs from this pattern in two prominent aspects. (1) No asymmetric cleavages and no distinct cell lineages are generated; (2) in contrast to all other nematodes studied so far, a prominent coeloblastula is formed and gastrulation resembles the "classical" pattern found all over the animal kingdom. These developmental peculiarities are considered to be plesiomorphic and thus the order "Triplonchida", to which Tobrilus belongs, may occupy a phylogenetic position at the base of the nematode phylum. The findings reported here allow us to reject a number of conceivable correlations between the type of gastrulation and other developmental parameters.

Animals↗
PubMed1989

Local shifts in position and polarized motility drive cell rearrangement during sea urchin gastrulation.

This study examines the mechanisms of epithelial cell rearrangement during archenteron elongation in the sea urchin embryo using scanning electron microscopy, differential interference contrast videomicroscopy, cell marking, and fluorescently labeled chimaeric clones. Archenteron elongation involves two major processes: local shifts in position of cells in the archenteron wall and polarized motility of the cells as they rearrange. Fluorescently labeled chimaeric clones introduced into the archenteron of Lytechinus pictus are initially 4-5 cells wide; by the end of gastrulation the clones elongate and narrow, so that they are one cell wide in the narrowest region of the archenteron. The extent of clonal mixing indicates that cells in the archenteron change their relative positions by only 1-2 cell diameters during cell rearrangement. Cells at the blastopore rearrange concomitantly with cells in the archenteron, resulting in a 35% decrease in blastopore diameter. Endoderm cells undergo polarized, stage-specific changes in shape and motility as they rearrange; (1) they flatten markedly along their apical-basal axis throughout archenteron elongation; (2) just prior to the onset of cell rearrangement, basal surfaces of all cells in the archenteron extend long, polarized lamellipodial protrusions along the axis of extension of the archenteron; (3) as cell rearrangement begins, basal surfaces round up and the cells become isodiametric; (4) by the 3/4 gastrula stage the cells become stretched along the animal-vegetal axis, apparently due to filopodial traction, and finally (5) they continue to rearrange, returning to a less elongated shape by the end of gastrulation. Direct observation of gastrulation in the cidaroid Eucidaris tribuloides indicates that in this species cell rearrangement is accomplished by progressive circumferential intercalation of cells without upwardly directed filopodia. This intercalation is accompanied by explosive, apparently stochastic, cortical blebbing activity at the boundaries between cells, suggesting that in addition to whatever cell rearrangement may be generated by filopodial tension, such activity is an important component of the active rearrangement process.

Animals↗
PubMed1990

Target recognition by the archenteron during sea urchin gastrulation.

During sea urchin gastrulation filopodia are sent out by secondary mesenchyme cells (SMCs) at the tip of the archenteron in continual cycles of extension, attachment, and retraction. Eventually the archenteron ceases its elongation and its tip localizes to the animal pole region of the embryo (Gustafson and Kinnander, 1956, Exp. Cell Res. 11, 36-57; Dan and Okazaki, 1956, Biol. Bull. 110, 29-42). We have investigated the mechanisms and specificity of this localization by analyzing filopodial behavior and by experimental manipulation of the interaction of the archenteron with the animal pole region. When the tip of the archenteron nears the animal pole, some filopodia make contact with a well-defined locus within this region. Filopodia that make contact with the locus remain attached 20-50 times longer than attachments observed at any other site along the blastocoel wall. The SMCs bearing the long-lived filopodia eventually change their phenotype by flattening and spreading onto this region. Several lines of experimental evidence indicate that contact with the animal pole locus, or "target" region, is crucial for the change in phenotype of the SMCs: (1) the phenotypic change can be induced precociously by bringing the animal pole region within reach of the tip of the archenteron early in gastrulation. Precocious contact with other regions of the blastocoel wall does not induce a similar change. (2) The phenotypic change can be delayed by placing the animal pole out of reach late in gastrulation, resulting in artificial prolongation of exploratory behavior by filopodia. (3) Ectopic combinations of animal pole ectoderm and archenterons in fused multiple embryos and chimaeras result in attachment of archenterons to the nearest available target, and (4) freely migrating SMCs are observed to migrate randomly within the blastocoel, then stop at the animal pole and undergo the change in phenotype. Filopodia rapidly attach to the animal pole when the shape of early gastrulae is altered such that the animal pole is less than 35 microns from the tip of the archenteron, even though such attachments only occur in normal embryos at the 2/3-3/4 gastrula stage. Since it has previously been shown that the archenteron elongates autonomously to 2/3 of its final length (Hardin, 1988, Development 103, 317-324), it appears that autonomous extension of the archenteron is required to place filopodia close enough to the animal pole to allow them to interact with it.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

The Drosophila gastrulation gene concertina encodes a G alpha-like protein.

Gastrulation is a complex process requiring the coordination of cell shape changes and cell movements. In Drosophila, gastrulation begins immediately upon cellularization of the blastoderm stage embryo with the formation of the ventral furrow and posterior midgut. Cells that form both of these invaginations change their shape via apical constriction. Embryos from mothers homozygous for mutations in the concertina (cta) gene begin furrow formation by forming a zone of tightly apposed cells, constrict some cells, and then fail to constrict enough cells to form an organized groove. The cta gene has been cloned, and sequence analysis suggests that it encodes an alpha subunit of a G protein. G proteins have a role in cell-cell communication as mediators of signals between membrane-bound receptors and intracellular effectors. The phenotype of embryos from homozygous cta mothers suggests that the cta gene plays a role in a signal transduction pathway used during gastrulation.

Amino Acid Sequence↗
PubMed1995

Down-regulation of ornithine decarboxylase by an increased degradation of the enzyme during gastrulation of Xenopus laevis.

The present study was designed to analyze the regulation of the levels of the polyamines and their biosynthetic enzymes during embryonic development of Xenopus laevis. The activity of ornithine decarboxylase (ODC), a rate-controlling enzyme in polyamine biosynthesis, is elevated until, during gastrulation, there is a precipitous drop in activity. This is not attributable to a decrease in ODC mRNA content and polysome profiles reveal no apparent decrease in ODC message associated with polysomes. ODC synthesis seems to be maintained at a low, relatively constant rate until neurulation whereupon ribosome loading of ODC mRNA increases. During gastrulation the rate of ODC degradation increases dramatically, which can account for the decrease in ODC. S-Adenosylmethionine decarboxylase (AdoMetDC), another rate-controlling enzyme in polyamine biosynthesis, shows a low and constant activity from cleavage to neurulation. Subsequently, the AdoMetDC activity increases dramatically. The changes in AdoMetDC activity parallel the changes in AdoMetDC mRNA levels, suggesting a transcriptional control of AdoMetDC expression during this development period. The activities of ODC and AdoMetDC produce a steady increase in putrescine and spermidine content of the embryo. The spermine content also increases until gastrulation, but then decreases until the tailbud stage.

Adenosylmethionine Decarboxylase↗
PubMed1995

Expression of zebrafish GATA 3 (gta3) during gastrulation and neurulation suggests a role in the specification of cell fate.

In order to understand the role of the transcription factor GATA 3 in vertebrate development, we have examined its expression and some aspects of its regulation during gastrulation and neurulation in the zebrafish. The complete coding sequence of the cDNA encoding the zebrafish GATA 3 homologue, termed gta3, is described. Analysis of expression patterns by in situ hybridisation shows the gene to be expressed during gastrulation in the ventral region of the embryo which includes tissue fated to form the non-neural ectoderm. By the end of gastrulation, there is a clear border to the gta3 expression domain that is close to the edge of the neural plate. Subsequently, gta3 expresses in the pronephric duct and in defined regions of the central nervous system which include specific cells in each segment of the spinal cord and nuclei in the brain. Double labelling embryos with a probe for gta3 and antibodies which identify differentiated neurons suggest that gta3 is dynamically expressed during the early differentiation phase of a subset of neurons but not in the terminal phase. Analysis of gta3 expression in dorsalised embryos and in cyc and spt mutant embryos indicates that the neural expression of the gene is subject to control by signals from the mesoderm, including both the notochord and the somites, which influence the segmental organisation of expression in the spinal cord.

Amino Acid Sequence↗
PubMed1995

A novel gene expressed during zebrafish gastrulation identified by differential RNA display.

Vertebrate gastrulation is a dynamic period of development characterized by extensive cell migrations. This stage of development is likely to require the expression of a new genetic repertoire to initiate and direct these dramatic changes. The differential RNA display has been used to identify genes specifically expressed during the gastrula stage of a model vertebrate, the zebrafish. One of the genes isolated by the differential display technique has been sequenced and characterized for its spatial and temporal expression. This gene, called G12, is expressed during a narrow window of time during gastrulation and is restricted to a single cell type. At this time of development the zebrafish embryo consists of three cell types: the yolk cell, EVL cells and deep cells. Interestingly, both EVL and deep cells derive early in development from common progenitor cells but G12 expression is restricted only to the EVL lineage. Comparison of the amino acid sequence from this gene with the Genbank database indicates similarity to two previously reported mammalian genes. The similarity between these three genes suggests that they may serve a common function. The G12 gene is the first example of restricted gene expression in EVL cells of the zebrafish. The G12 gene should prove to be a useful model for the study of regulated gene expression during gastrulation.

Amino Acid Sequence↗
PubMed1996

Dominant negative expression of a cytoplasmically deleted mutant of XB/U-cadherin disturbs mesoderm migration during gastrulation in Xenopus laevis.

XB/U-cadherin is a maternal Xenopus cadherin which mediates interblastomere adhesion in early embryogenesis. In order to explore its role in gastrulation, we expressed a cytoplasmic deletion mutant of XB/U-cadherin (XB delta c38) under the control of the CMV promoter in Xenopus embryos. This truncated XB-cadherin fails to form complexes with catenins and does not mediate cell-cell aggregation as shown by transfection of mouse Ltk- cells. Injections of the deletion for XB/U-cadherin into the dorsal-marginal region of four cell stage embryos resulted in a dominant negative expression of the cadherin mutant after MBT. Two different phenotypes were observed in a dose dependent manner: high doses (125-250 pg DNA) led to severe distortions of the gastrulation movement. Involution of the mesoderm was impaired, posterior mesoderm migrated laterally around the blastopore and formed two bands of axial tissue. Low doses (up to 50 pg DNA) resulted in embryos of a posteriorized phenotype with disorganized neural structures. Both phenotypes could be rescued by coinjection of cDNA constructs containing wild-type XB/U-cadherin. Injections of constructs encoding a XB/U-cadherin protein truncated both in its extracellular and cytoplasmic domains yielded normal phenotypes. These results suggest that a proper function of XB/U-cadherin is essential for mesoderm movements during gastrulation.

Animals↗
PubMed1993

Genetic control of gastrulation in the mouse.

In the past, understanding of the process of gastrulation in the mouse has primarily been based on morphological analyses. Recently, a number of molecules have been implicated in mesoderm induction and axis formation in Xenopus, and several of these exhibit unique patterns of expression during mouse gastrulation. These gene-expression data, together with fate mapping, ectopic expression experiments and mutational analysis, will now facilitate studies on the functional aspects of gastrulation in the mouse.

Animals↗
PubMed2004

Conditional activation of RhoA suppresses the epithelial to mesenchymal transition at the primitive streak during mouse gastrulation.

Gastrulation is a pivotal event of mouse early embryogenesis. In telencephalin (TLCN)-Cre mice carrying the Cre recombinase gene inserted into the translational initiation site of the TLCN gene, Cre-mediated recombination took place at the postimplantation stage. To examine the role of RhoA signaling in early embryogenesis, we produced Rho36 mice carrying constitutively active RhoA(G14V) gene inducible by Cre recombinase and crossed with TLCN-Cre mice. In doubly transgenic embryos at the gastrulation stage, there appeared an abnormal bulge of cells protruded from the primitive streak region into the amniotic cavity. The bulged cell mass expressed the epiblast marker gene Oct3 and E-cadherin, but not the primitive streak marker gene T except for the basal portion. These results suggest that the conditional activation of RhoA signaling suppressed the epithelial to mesenchymal transition at the primitive streak during mouse gastrulation.

Allantois↗
PubMed2005

Wnt11 functions in gastrulation by controlling cell cohesion through Rab5c and E-cadherin.

Wnt11 plays a central role in tissue morphogenesis during vertebrate gastrulation, but the molecular and cellular mechanisms by which Wnt11 exerts its effects remain poorly understood. Here, we show that Wnt11 functions during zebrafish gastrulation by regulating the cohesion of mesodermal and endodermal (mesendodermal) progenitor cells. Importantly, we demonstrate that Wnt11 activity in this process is mediated by the GTPase Rab5, a key regulator of early endocytosis, as blocking Rab5c activity in wild-type embryos phenocopies slb/wnt11 mutants, and enhancing Rab5c activity in slb/wnt11 mutant embryos rescues the mutant phenotype. In addition, we find that Wnt11 and Rab5c control the endocytosis of E-cadherin and are required in mesendodermal cells for E-cadherin-mediated cell cohesion. Together, our results suggest that Wnt11 controls tissue morphogenesis by modulating E-cadherin-mediated cell cohesion through Rab5c, a novel mechanism of Wnt signaling in gastrulation.

Animals↗
PubMed2006

A role for GATA factors in Xenopus gastrulation movements.

Gastrulation movements in Xenopus laevis are becoming increasingly well characterised, however the molecular mechanisms involved are less clear. Active migration of the leading edge mesendoderm across the fibronectin-coated blastocoel roof is necessary for further development of tissues such as head mesoderm, heart, blood and liver. The zinc finger transcription factors GATA4 and GATA6 are expressed in this migratory tissue during gastrulation, but their role here is unknown. This study further characterises the expression of GATA4 and 6 during gastrulation, and investigates their function in migratory behaviour. Gain-of-function experiments with these GATA factors induce cell spreading, polarisation and migration in non-motile presumptive ectoderm cells. Expression of a dominant-interfering form of GATA6, which inhibits transactivation of GATA targets, severely impairs the ability of dorsal leading edge mesendoderm to spread and translocate on fibronectin. Mosaic inhibition of GATA activity indicates that GATA factors function cell autonomously to induce cell spreading and movement in dorsal mesendoderm. Knockdown of specific GATA factors using anti-sense morpholinos indicates that GATA4 and GATA6 both contribute to dorsal mesendoderm migration in vitro. GATA4 and GATA6 are known to be involved in cell-specification of mesoderm and endoderm-derived tissues, but this is the first description of an additional role for these factors in cell migration.

Activins↗
PubMed2025

Self-organization of mouse embryonic stem cells into reproducible pre-gastrulation embryo models via CRISPRa programming.

Embryonic stem cells (ESCs) can self-organize into structures with spatial and molecular similarities to natural embryos. During development, embryonic and extraembryonic cells differentiate through activation of endogenous regulatory elements while co-developing via cell-cell interactions. However, engineering regulatory elements to self-organize ESCs into embryo models remains underexplored. Here, we demonstrate that CRISPR activation (CRISPRa) of two regulatory elements near Gata6 and Cdx2 generates embryonic patterns resembling pre-gastrulation mouse embryos. Live single-cell imaging revealed that self-patterning occurs through orchestrated collective movement driven by cell-intrinsic fate induction. In 3D, CRISPRa-programmed embryo models (CPEMs) exhibit morphological and transcriptomic similarity to pre-gastrulation mouse embryos. CPEMs allow versatile perturbations, including dual Cdx2-Elf5 activation to enhance trophoblast differentiation and lineage-specific activation of laminin and matrix metalloproteinases, uncovering their roles in basement membrane remodeling and embryo model morphology. Our findings demonstrate that minimal intrinsic epigenome editing can self-organize ESCs into programmable pre-gastrulation embryo models with robust lineage-specific perturbation capabilities.

Animals↗