Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
Ras proteins mediate biological responses through various effectors and play a key role in relaying the Fibroblast Growth Factor (FGF) mesoderm induction signal during embryogenesis of the frog, Xenopus laevis. One Ras effector pathway involves the activation of the small G protein Ral. In the present study, we have investigated the role of key components in the Ral branch of FGF and Ras signalling during early Xenopus development. Treatment of animal caps with bFGF, which converts prospective ectoderm to mesoderm, activates Xral. The Ras mutant 12V37G, which can bind to Ral-GDS but not Raf, also activates Xral as well as causing developmental defects and cortical F-actin disassembly. A similar phenotype is induced by Ral-GDS itself. FGF-induced expression of several signature mesodermal genes, by contrast, is independent of Xral signalling. This and other data suggest that the RalB branch of Ras and FGF signalling regulates the actin cytoskeleton and morphogenesis in a transcriptionally independent manner. We also find Xral to be specifically activated in the marginal zone of Xenopus embryos, and find that disruption of the Ral pathway in this region prevents closure of the blastopore during gastrulation. We conclude that Ral signalling is autonomously required by mesodermal cells to effect essential morphogenetic changes during Xenopus gastrulation.
Fig.1. FGF activates the Xral protein. Animal caps explanted from post-MBT embryos (stage 9) were cultured for 4 hours in the presence (+) or absence (–) of 100 ng/ml bFGF (A) and analysed by RT-PCR for Xbra expression (B) or by immunoblotting to detect the GTP form of Xral (C). Active Ral-GTP was affinity purified from lysates of 15 animal caps (C) using the Ral-binding domain of RalBP1, and detected with anti-Ral antibodies. The result is representative from two separate experiments.
Fig.3. Morphogenetic perturbations induced by the Ral-GDS binding, Raf non-binding, Ras 12V37G. (A) Phenotypic effects at the blastula (stage 8) and neural plate (stage 14) stages resulting from Ras mutant mRNA injections. mRNAs encoding Ras 12V35S and 12V37G (500 pg/blastomere) were injected into the animal pole of each blastomere in two-cell embryos. The arrow indicates patches of abnormal cells in embryos injected with Ras 12V37G. Embryos co-injected with Ras 12V37G and XralB S28N were developmentally arrested during gastrulation but did not display necrotic cells. (B) Analysis of the cortical actin cytoskeleton in embryos injected with either 12V35S or 12V37G, and the rescue effect of XralB S28N on Ras 12V37G. Embryos were injected with Ras 12V35S or Ras 12V37G mRNA (500 pg/blastomere), or co-injected with Ras 12V37G (500 pg/blastomere) and XralB S28N (3 ng/blastomere), respectively. The arrowhead shows the reconstituted cortical actin cytoskeleton in embryos co-injected with Ras 12V37G and RalB S28N mRNAs. The actin cytoskeleton of animal caps was analysed at the MBT stage. Scale bars: 50 μm; confocal optical sections are 1 μm.
Fig.4. Onset of Ras 12V37G induced actin disruption after the midblastula transition. (A,C,E) Uninjected control embryos, (B,D,F) embryos injected in the animal hemisphere with Ras 12V37G mRNA (500 pg/blastomeres). Cortical actin was analysed at the 500-cell stage (A-B), the 2000-cell stage (C-D), and the MBT (E-F).
Fig.5. Morphogenetic perturbations and Xral activation induced by Ral-GDS. (A) Phenotypic effects of Ral-GDS mRNA and rescue by the XralB S28N mutant. Embryos either injected with either Ral-GDS mRNA (1.5 ng/blastomere) or co-injected with XralB S28N mRNA (4 ng/blastomere) in the animal pole of each blastomere of two-cell stage embryos. In embryos coinjected with XralB S28N, the arrows indicate the ectodermal roll resulting from to the incomplete closure of the blastopore at the neurula stage. (B) Analysis of cortical actin cytoskeleton of embryos injected with Ral-GDS and rescue effect of XralB S28N. Embryos were injected with Ral-GDS mRNA (1.5 ng/blastomere) alone or in combination with XralB S28N (4 ng/blastomere each) mRNAs. The animal cap actin cytoskeleton was analysed after the MBT stage. The arrows indicate the reconstituted cortical actin cytoskeleton in embryos co-injected with Ral-GDS and RalB S28N mRNAs. Scale bars: 50 μm and confocal optical sections are 1 μm. (C) Xral activation was analysed by pull-down from embryos at 128/256 cell stage as described in the Materials and Methods and Fig. 2. Precipitated Ral-GTP and total Ral protein from whole-embryo lysate were detected after immunoblotting with specific antibodies.
Fig.7. Inhibition of Ral signalling in prospective mesoderm causes gastrulation defects. Effect of XralB S28N on early development. Embryos were co-injected in each blastomere of 4-cell stage embryos with XralB S28N (500 pg/blastomere), and ß-Galactosidase (500 pg/blastomere) RNAs, in the animal apical hemisphere (A), in the marginal zone (B) or in the bottom of the vegetal hemisphere (C). (D) Effect of the Ral binding domain of RLIP on early development. Embryos at the 4-cell stage were injected in the marginal zone with 500 pg/blastomere of mRNA encoding the Ral binding domain of RLIP (RalBD). These embryos remained blocked during gastrulation, even when control embryos had reached stage 22. (E) Embryos injected in the marginal zone with mRNA encoding wild-type XralB (4× 750 pg). The site of RNA expression was monitored by detection of co-injected β-galactosidase expression. (2) and (4) show embryos corresponding to sibling controls at stage 17. Embryos had X-gal-stained cells in the marginal zone. Arrows in Band D indicate the ring corresponding to the open blastopore. (F) Rescue of XralB S28N by coexpression with wild-type XralB. All embryos were microinjected in marginal zone at 4-cell stage with XralB S28N (4× 750 pg) mRNA or co-injected with wild-type XralB (4× 1.5 ng) mRNA. Vegetal view of embryos all at the same time after injection. (G) Protein expressed in embryos injected with XralB S28N and wild-type XralB mRNAs were controlled by western blot.
