| |
FIGURE 2. Skip inhibits neural crest development. A, Xenopus skip expression at the indicated development stages was analyzed by RT-PCR. −RT, minus reverse transcription. Histone H4 was used for normalization. B, expression profile of skip in Xenopus embryos was shown by whole mount in situ hybridization. st, stage. C, Skip inhibits β-catenin signaling in a dose-dependent manner in Xenopus embryos. 4-Cell-stage embryos were injected equatorially with 250 pg of β-catenin and/or 50, 250, or 500 pg of skip mRNA, together with a TOPflash reporter plasmid, in each blastomere as indicated. Embryos were gathered at stage 11.5 and analyzed by the Dual-Luciferase assay system. D, skip mRNA injection causes inhibition of slug expression. 2-Cell-stage embryos were injected in one blasteomere with control (Con) or skip mRNA (2 ng) and fixed at the early neurula stage for whole mount in situ hybridization analysis with slug, Sox3, and engrailed2 (en2) probes. lacZ mRNA (0.5 ng) was co-injected as a lineage tracer. Light blue, lacZ staining; dark blue and brown, hybridization signal. E, Skip blocks slug induction by Wnt signaling in Xenopus animal caps. 100 pg of noggin, 50 pg of wnt8, and 1 ng of skip mRNA were injected animally into each blastomere at the 4-cell-stage as indicated. At stage 8, animal caps were dissected, cultured until stage 14, and analyzed for expressions of slug, NCAM, Xbra, and H4 by RT-PCR. H4 was used as the loading control, and Xbra was used to confirm that the animal caps were without mesoderm contamination. −RT, minus reverse transcription; WE, whole embryo.
This research was originally published in The Journal of Biological Chemistry. Wang et al “Xenopus skip modulates Wnt/beta-catenin signaling and functions in neural crest induction.” April 2, 2010; 285 (14):10890-10901. © The American Society for Biochemistry and Molecular Biology. Reproduced with permission. |
