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We have undertaken the first detailed analysis of Rho GTPase function during vertebrate development by analyzing how RhoA and Rac1 control convergent extension of axial mesoderm during Xenopus gastrulation. Monitoring of a number of parameters in time-lapse recordings of mesoderm explants revealed that Rac and Rho have both distinct and overlapping roles in regulating the motility of axial mesoderm cells. The cell behaviors revealed by activated or inhibitory versions of these GTPases in native tissue were clearly distinct from those previously documented in cultured fibroblasts. The dynamic properties and polarity of protrusive activity, along with lamellipodia formation, were controlled by the two GTPases operating in a partially redundant manner, while Rho and Rac contributed separately to cell shape and filopodia formation. We propose that Rho and Rac operate in distinct signaling pathways that are integrated to control cell motility during convergent extension. 相似文献
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We previously identified the adaptor protein PACSIN2 as a negative regulator of ADAM13 proteolytic function. In Xenopus embryos, PACSIN2 is ubiquitously expressed, suggesting that PACSIN2 may control other proteins during development. To investigate this possibility, we studied PACSIN2 function during Xenopus gastrulation and in XTC cells. Our results show that PACSIN2 is localized to the plasma membrane via its coiled-coil domain. We also show that increased levels of PACSIN2 in embryos inhibit gastrulation, fibronectin (FN) fibrillogenesis and the ability of ectodermal cells to spread on a FN substrate. These effects require PACSIN2 coiled-coil domain and are not due to a reduction of FN or integrin expression and/or trafficking. The expression of a Mitochondria Anchored PACSIN2 (PACSIN2-MA) sequesters wild type PACSIN2 to mitochondria, and blocks gastrulation without interfering with cell spreading or FN fibrillogenesis but perturbs both epiboly and convergence/extension. In XTC cells, the over-expression of PACSIN2 but not PACSIN2-MA prevents the localization of integrin β1 to focal adhesions (FA) and filamin to stress fiber. PACSIN2-MA prevents filamin localization to membrane ruffles but not to stress fiber. We propose that PACSIN2 may regulate gastrulation by controlling the population of activated α5β1 integrin and cytoskeleton strength during cell movement. 相似文献
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Platelet-derived growth factor receptor (PDGFR) signaling is required for normal gastrulation in Xenopus laevis. Embryos deprived of PDGFR signaling develop with a range of gastrulation-specific defects including spina bifida, shortened anteroposterior axis, and reduced anterior structures. These defects arise because the involuting mesoderm fails to move appropriately. In this study, we determine that inhibition of PDGFR signaling causes prospective head mesoderm cells to appear in the blastocoel cavity at the onset of gastrulation, stage 10. These aberrant cells undergo apoptosis via the caspase 3 pathway at an embryonic checkpoint called the early gastrula transition (EGT). They are TUNEL-positive and have increased levels of caspase 3 activity compared to control embryos. Apoptotic death of these mesoderm cells can be prevented by co-injection of mRNA encoding Bcl-2 or by injection of either a general caspase inhibitor or a caspase 3-specific inhibitor. Prevention of cell death, however, is not sufficient to rescue gastrulation defects in these embryos. Based on these data, we propose that PDGFR signaling is necessary for survival of prospective head mesoderm cells, and also plays an essential role in the control of their cell movement during gastrulation. 相似文献
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We used cDNA microarray analysis to screen for FGF target genes in Xenopus embryos treated with the FGFR1 inhibitor SU5402, and identified neurotrophin receptor homolog (NRH) as an FGF target. Causing gain of NRH function by NRH mRNA or loss of NRH function using a Morpholino antisense-oligonucleotide (Mo) led to gastrulation defects without affecting mesoderm differentiation. Depletion of NRH by the Mo perturbed the polarization of cells in the dorsal marginal zone (DMZ), thereby inhibiting the intercalation of the cells during convergent extension as well as the filopodia formation on DMZ cells. Deletion analysis showed that the carboxyl-terminal region of NRH, which includes the "death domain," was necessary and sufficient to rescue gastrulation defects and to induce the protrusive cell morphology. Furthermore, we found that the FGF signal was both capable of inducing filopodia in animal cap cells, where they do not normally form, and necessary for filopodia formation in DMZ cells. Finally, we demonstrated that FGF required NRH function to induce normal DMZ cell morphology. This study is the first to identify an in vivo role for FGF in the regulation of cell morphology, and we have linked this function to the control of gastrulation cell movements via NRH. 相似文献
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Myristoylated alanine-rich C kinase substrate (MARCKS) is an actin-binding, membrane-associated protein expressed during Xenopus embryogenesis. We analyzed its function in cytoskeletal regulation during gastrulation. Here, we show that blockade of its function impaired morphogenetic movements, including convergent extension. MARCKS was required for control of cell morphology, motility, adhesion, protrusive activity, and cortical actin formation in embryonic cells. We also demonstrate that the noncanonical Wnt pathway promotes the formation of lamellipodia- and filopodia-like protrusions and that MARCKS is necessary for this activity. These findings show that MARCKS regulates the cortical actin formation that is requisite for dynamic morphogenetic movements. 相似文献
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Fibrillin-based human diseases such as Marfan syndrome and congenital contractural arachnodactyly implicate fibrillins in the function and homeostasis of multiple adult tissues. Fibrillins are also expressed in embryos, but no early developmental role has been described for these proteins. We use three independent methods to reveal a role for Xenopus fibrillin (XF) at gastrulation. First, expressing truncated forms of XF in the embryo leads to failure of gastrulation concomitant with a dominant-negative effect on native fibrillin fibril assembly. Expressing truncated XF also inhibits normal progression of the patterned, polarized cell motility that drives convergence and extension at gastrulation and perturbs directed extension in cultured explants of dorsal mesoderm. Second, injection of a synthetic peptide encoding a cell-binding domain of XF into midgastrula embryos causes acute failure of gastrulation associated with defective fibrillin fibril assembly. These injections also reveal a critical role for this peptide in the fibril assembly process. Third, morpholino-mediated knockdown of translation of XF in the embryo also perturbs normal gastrulation and directed extension. Together, these data show that native Xenopus fibrillin is essential for the process of directed convergent extension in presumptive notochord at gastrulation. 相似文献
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Boissel L Houssin N Chikh A Rynditch A Van Hove L Moreau J 《Developmental biology》2007,312(1):331-343
The transduction pathways that branch out of fibroblast growth factor signaling are essential for the induction of the mesoderm and the specification of the vertebrate body plan. One of these pathways is thought to control remodeling of the actin cytoskeleton through the Ral binding protein (RLIP also known as RalBP1), an effector of the small G protein Ral. RLIP contains a region of homology with the GTPase-activating protein (GAP) domain involved in the regulation of GTPases of the Rho family. We demonstrate here that the GAP domain of RLIP is responsible for the stability of the actin cytoskeleton in Xenopus laevis embryos. We also demonstrate that the complete N-terminal domain of RLIP containing the μ2 binding domain (μ2BD) and the GAP domain induces disruption of the actin cytoskeleton when targeted to the plasma membrane. Neither domain, however, has any effect on the actin cytoskeleton when individually targeted to the plasma membrane. We also determined that Cdc42-GDP, but neither Rac-GDP nor Rho-GDP, rescues the effect of expression of the membrane-localized Xenopus RLIP on the actin cytoskeleton. We show that the GAP domain of RLIP interacts in vivo with Cdc42-GTP and Cdc42-GDP. Finally, a single mutation (K244A) in the GAP sequence prevented embryos from gastrulating. These results demonstrate that to participate in the control of the actin cytoskeleton, RLIP needs its complete N-terminal region coding for the μ2BD and the GAP domain. We suggest that RLIP, by coordinating two complementary mechanisms, the endocytosis of clathrin-coated pits and the remodeling of cortical actin, participates in the gastrulation process. 相似文献
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In Xenopus gastrulation, the involuting mesodermal and non-involuting ectodermal cells remain separated from each other, undergoing convergent extension. Here, we show that Eph–ephrin signaling is crucial for the tissue separation and convergence during gastrulation. The loss of EphA4 function results in aberrant gastrulation movements, which are due to selective inhibition of tissue constriction and separation. At the cellular levels, knockdown of EphA4 impairs polarization and migratory activity of gastrulating cells but not specification of their fates. Importantly, rescue experiments demonstrate that EphA4 controls tissue separation via RhoA GTPase in parallel to Fz7 and PAPC signaling. In addition, we show that EphA4 and its putative ligand, ephrin-A1 are expressed in a complementary manner in the involuting mesodermal and non-involuting ectodermal layers of early gastrulae, respectively. Depletion of ephrin-A1 also abrogates tissue separation behaviors. Therefore, these results suggest that Eph receptor and its ephrin ligand might mediate repulsive interaction for tissue separation and convergence during early Xenopus gastrulation movements. 相似文献
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Shimizu T Yabe T Muraoka O Yonemura S Aramaki S Hatta K Bae YK Nojima H Hibi M 《Mechanisms of development》2005,122(6):747-763
E-cadherin is a member of the classical cadherin family and is known to be involved in cell-cell adhesion and the adhesion-dependent morphogenesis of various tissues. We isolated a zebrafish mutant (cdh1(rk3)) that has a mutation in the e-cadherin/cdh1 gene. The mutation rk3 is a hypomorphic allele, and the homozygous mutant embryos displayed variable phenotypes in gastrulation and tissue morphogenesis. The most severely affected embryos displayed epiboly delay, decreased convergence and extension movements, and the dissociation of cells from the embryos, resulting in early embryonic lethality. The less severely affected embryos survived through the pharyngula stage and showed flattened anterior neural tissue, abnormal positioning and morphology of the hatching gland, scattered trigeminal ganglia, and aberrant axon bundles from the trigeminal ganglia. Maternal-zygotic cdh1(rk3) embryos displayed epiboly arrest during gastrulation, in which the enveloping layer (EVL) and the yolk syncytial layer but not the deep cells (DC) completed epiboly. A similar phenotype was observed in embryos that received antisense morpholino oligonucleotides (cdh1MO) against E-cadherin, and in zebrafish epiboly mutants. Complementation analysis with the zebrafish epiboly mutant weg suggested that cdh1(rk3) is allelic to half baked/weg. Immunohistochemistry with an anti-beta-catenin antibody and electron microscopy revealed that adhesion between the DCs and the EVL was mostly disrupted but the adhesion between DCs was relatively unaffected in the MZcdh1(rk3) mutant and cdh1 morphant embryos. These data suggest that E-cadherin-mediated cell adhesion between the DC and EVL plays a role in the epiboly movement in zebrafish. 相似文献
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Fukui A Goto T Kitamoto J Homma M Asashima M 《Biochemical and biophysical research communications》2007,354(2):472-477
During frog gastrulation, mesendodermal cells become apposed to the blastocoel roof (BCR) by endoderm rotation, and migrate towards the animal pole. The leading edge of the mesendodermal cells (LEM) contributes to the directional migration of involuting marginal zone (IMZ) cells, but the molecular mechanism of this process is not well understood. Here we show that CXCR4/SDF-1 signaling mediates the directional movement of the LEM in Xenopus embryos. Expression of xCXCR4 was detected in the IMZ, and was complemented by xSDF-1alpha expression in the inner surface of the BCR. Over-expression of xCXCR4 and xSDF-1alpha caused gastrulation defects. An xCXCR4 N-terminus deletion construct and xSDF-1alpha-MO also inhibited gastrulation. Furthermore, explants of LEM migrate towards the dorsal BCR in the presence of xSDF-1alpha, and altered xCXCR4 expression in the LEM inhibited LEM migration. These results suggest that CXCR4/SDF-1 signaling is necessary for the migrations of massive numbers of cells during gastrulation. 相似文献
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The product of the Drosophila gene tribbles inhibits cell division in the ventral furrow of the embryo and thereby allows the normal prosecution of gastrulation. Cell division is also absent in involuting dorsal mesoderm during gastrulation in Xenopus, and to ask whether the two species employ similar mechanisms to coordinate morphogenesis and the cell cycle, we isolated a putative Xenopus homologue of tribbles which we call Xtrb2. Extensive cDNA cloning identified long and short forms of Xtrb2, termed Xtrb2-L and Xtrb2-S, respectively. Xtrb2 is expressed maternally and in mesoderm and ectoderm at blastula and gastrula stages. Later, it is expressed in dorsal neural tube, eyes, and cephalic neural crest. Time-lapse imaging of GFP-tagged Xtrb2-L suggests that during cell division, it is associated with mitotic spindles. Knockdown of Xtrb2 by antisense morpholino oligonucleotides (MOs) disrupted synchronous cell divisions during blastula stages, apparently as a result of delayed progression through mitosis and cytokinesis. At later stages, tissues expressing the highest levels of Xtrb2 were most markedly affected by morpholino knockdown, with perturbation of neural crest and eye development. 相似文献
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Protease nexin-1 (PN-1)/glia-derived nexin (GDN) is a member of the Serpin (serine proteinase inhibitor) family, and can inhibit thrombin, plasmin, and plasminogen activators. PN-1 has been shown to be a neuroprotective factor in a number of assay systems, and this activity has been assumed to be a function of its protease inhibitory function. Here, we report cloning and characterization of a Xenopus orthologue of PN-1 (xPN-1). xPN-1 was isolated in a functional screen of an egg cDNA library for factors that modify early axial patterning. xPN-1 is expressed maternally through late tadpole stages, and is expressed preferentially in the notochord, the pharyngeal endoderm, the otic vesicle, and the ventral region of the brain in tailbud embryos. Over-expression of xPN-1 causes defective gastrulation, inhibits convergent extension movements in activin induced animal caps, and inhibits expression of a distinct subset of activin induced mesendodermal markers. Interestingly, expression of point or deletion mutation of the Reactive Center Loop of xPN1,which is essential for the protease inhibitory activity of all serpins, had effects on Xenopus development indistinguishable from those of wild type xPN-1. These observations suggest the possibility that xPN-1 has a novel activity in addition to its established function as an inhibitor of serine proteases. 相似文献
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Activin-like signaling plays an important role in early embryogenesis. Activin A, a TGF-beta family protein, induces mesodermal/endodermal tissues in animal cap assays. In a screen for genes expressed early after treatment with activin A, we isolated a novel gene, denoted as BENI (Brachyury Expression Nuclear Inhibitor). The BENI protein has a conserved domain at the N-terminus that contains a nuclear localization signal (NLS), and two other NLSs in the C-terminal domain. BENI mRNA was localized to the animal hemisphere at the gastrula stages and to ectoderm except for neural regions at stage 17; expression persisted until the tadpole stage. The overexpression of BENI caused gastrulation defects and inhibition of elongation of activin-treated animal caps with reduction of Xbra expression. Moreover, whole-mount in situ hybridization revealed reduced expression of Xbra in BENI mRNA-injected regions of gastrula embryos. Functional knockdown of BENI using an antisense morpholino oligonucleotide also resulted in an abnormal phenotype of embryos curling to the dorsal side, and excessive elongation of activin-treated animal caps without altered expression of mesodermal markers. These results suggested that BENI expression is regulated by activin-like signaling, and that this regulation is crucial for Xbra expression. 相似文献
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In Xenopus, the pronephros is the functional larval kidney and consists of two identifiable components; the glomus, the pronephric tubules, which can be divided into four separate segments, based on marker gene expression. The simplicity of this organ, coupled with the fact that it displays the same basic organization and function as more complex mesonephros and metanephros, makes this an attractive model to study vertebrate kidney formation. In this study, we have performed a functional screen specifically to identify genes involved in pronephros development in Xenopus. Gain-of-function screens are performed by injecting mRNA pools made from a non-redundant X. tropicalis full-length plasmid cDNA library into X. laevis eggs, followed by sib-selection to identify the single clone that caused abnormal phenotypes in the pronephros. Out of 768 egg and gastrula stage cDNA clones, 31 genes, approximately 4% of the screened clones, affected pronephric marker expression examined by whole mount in situ hybridization or antibody staining. Most of the positive clones had clear expression patterns in pronephros and predicted/established functions highly likely to be involved in developmental processes. In order to carry out a more detailed study, we selected Sox7, Cpeb3, P53csv, Mecr and Dnajc15, which had highly specific expression patterns in the pronephric region. The over-expression of these five selected clones indicated that they caused pronephric abnormalities with different temporal and spatial effects. These results suggest that our strategy to identify novel genes involved in pronephros development was highly successful, and that this strategy is effective for the identification of novel genes involved in late developmental events. 相似文献