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.