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First published online October 22, 2003
doi: 10.1242/10.1242/jcs.00763

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Control of embryonic Xenopus morphogenesis by a Ral-GDS/Xral branch of the Ras signalling pathway

Institut Jacques Monod, CNRS, Universités Paris VI et Paris VII, Mécanismes Moléculaires du Développement, 2 Place Jussieu, 75251 Paris cedex 05, France

View larger version (80K): [in a new window]   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.

View larger version (37K): [in a new window]   Fig. 2. Activation of endogenous Xral by Ras 12V37G. (A) Protein from embryos injected with 2x 500 pg of Ras 12V35S, Ras 12V37G or Ras 12V37G in combination with 3 ng of XralB S28N, were extracted (128-256 cell stage), the Ral-GTP was immunoprecipitated with RalBD-conjugated glutathion-sepharose, and total Ral protein in whole-embryo lysates were detected subsequent to SDS-PAGE by immunoblotting with specific antibodies, as described in the Materials and Methods. The signal from each Ral-GTP band from experiments was quantified by densitometry and analysed by Image-Quant. The values express the ratio of the immunoprecipitated Ral-GTP signal/total Ral protein signal. (B) Animals caps explanted from embryos injected either with Ras 17N or Rap1A (2x 500 pg) mRNAs at 2-cell stage was analysed for the activated form of Xral as described in Fig. 1.

View larger version (79K): [in a new window]   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.

View larger version (136K): [in a new window]   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).

View larger version (79K): [in a new window]   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.

View larger version (43K): [in a new window]   Fig. 6. Activation of Xral in the marginal zone of Xenopus embryos. (A,C) Experimental scheme showing animal cap (AN), marginal zone (MZ), endoderm (EN) and vegetative pole (VG) domains that were dissected (A) or injected with wild-type XralB mRNA (4x 500 pg) (C). Protein from explants (B) or whole, injected embryos (D) were extracted and analysed for RalB-GTP content by pull-down, as described in the Materials and Methods and in Fig. 1.

View larger version (78K): [in a new window]   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 (4x 750 pg). The site of RNA expression was monitored by detection of co-injected ß-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 (4x 750 pg) mRNA or co-injected with wild-type XralB (4x 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.

View larger version (105K): [in a new window]   Fig. 8. bFGF-induced expression of key mesoderm genes is independent of Ral signalling. RT-PCR analysis of mRNA extracted from animal caps cultured until end of gastrulation. Embryos at the four-cell stage were injected in the animal hemisphere of each blastomere with either 4x 750 pg of XralB S28N or Raf KD RNAs. Animal caps were dissected at the midblastula stage and cultured, with bFGF (+) (100 ng/ml) or without (–) bFGF, until the siblings embryos reached the gastrulation (stage 12).

View larger version (14K): [in a new window]   Fig. 9. Ras signalling is mediated by three independent effector proteins during Xenopus mesoderm induction and gastrulation. Ras activation leads to signalling through the Raf/MAP kinase and PI 3-kinase (PI3K) pathways to activate gene expression and through the Ral-GDS/Ral/RLIP pathway to regulate assembly and disassembly of the actin cytoskeleton in marginal-zone-derived cells during gastrulation.

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