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Fig. 4. Inhibition of Xgly4 translation blocks gastrulation movements. (A) Western blot analysis of Xgly4 protein tagged with a Flag epitope at the C terminus using an anti-Flag antibody. Translation of the injected globin-Flag (50 pg) and Xgly4-Flag (500 pg) mRNA lacking the 5′ UTR sequence was not inhibited by the morpholino oligonucleotide Xgly4Mo (41 ng), directed against the 5′ untranslated region and the first methionine region of Xgly4 mRNA. This finding indicates that Xgly4Mo does not inhibit translation nonspecifically. (B) Embryos injected with Xgly4Mo (41 ng) and Xgly4 mRNA (1 ng) at stage 35/36. Xgly4 morpholino oligonucleotide, which was injected into dorsal region, blocked gastrulation movements. (C) In situ hybridization for Xvent1 at stage 11, XmyoD at stage 11, Xwnt11 at stage 11, Xbra at stage 11, Xen2 at stage 13 and XmyoD at stage 25 in uninjected embryos (left) and embryos injected with XglyMo (41ng; right). (D) Injection of Xgly4 mRNA rescues the phenotypes of the knypek (kny) mutant. Embryos from crossing of knym119 heterozygotes were injected with 20 pg Xgly4 RNA at one-cell stage and scored at pharyngula stage. Twenty-five percent of uninjected embryos showed the kny mutant phenotype (n=64) (Topczewski et al., 2001), whereas none of injected embryos showed the phenotype. Rather, injected embryos were indistinguishable from wild-type embryos except that they occasionally exhibited a slightly curly tip of the tail (n=112). This result suggests that Xgly4 is a functional homolog of the zebrafish kny gene. |
