Fig. 4. The response of Grsf1 and Fragilis2 to Wnt signaling and shRNA silencing. (A) Co-culture of ES cells on lacZ and Wnt1-expressing fibroblasts induces Grsf1 endogenous mRNA expression in Wnt1-treated ES cells, but not Fragilis2 expression. Gene-specific expression was normalized to Gapdh and the fold change was calculated. Results of three independent co-culture experiments are shown. Whole-mount in situ hybridization of wild-type and ß-catenin mutants (B,C), and of wild-type and shRNA-silenced embryos (E,F) with Grsf1 and Fragilis2 probes at late-gastrulation stage. All embryos are depicted in a lateral view, anterior to the left. Grsf1 and Fragilis2 are strongly downregulated in the primitive streak of ß-catenin mutant embryos (B,C) and knock-down embryos (E,F). Arrow in C indicates remaining Fragilis2 expression at the base of the allantois. (D) Northern blot analysis for Grsf1 and Fragilis2 for eight different shRNA ES-cell lines (1-8) and wild-type ES cells. 28S, 18S and 5.8S ribosomal RNAs are indicated as molecular length markers, and the ethidium bromide (EtBr)-stained agarose gels served as loading control.