Fig. 7

Casanova activity is negatively regulated by the FGF-ERK signalling pathway. Expression of sox17 (blue) in embryos at 30% epiboly (A,B) or 50% epiboly (C-H). In C-E, FLDX used as lineage marker was revealed by immunochemistry (red labelling). Embryos injected at the 16-cell stage with RNA encoding Casanova-WT (A) or Casanova-S47A (B), each at 10 ng/µl (see also Fig. S2 in the supplementary material). Mutation of the putative phosphorylation site slightly increases Casanova activity. Co-injection with fgf8 RNA at 10 ng/µl (C), CA-Ras RNA at 5 ng/µl (E) or ERK* RNA at 50 ng/µl (G) inhibits the ability of Casanova-WT to induce sox17 (see Fig. S3 in the supplementary material). However Casanova-S47A is insensitive to Fgf8 (D), CA-Ras (F) or ERK* (H) overexpression. (I) RT-PCR analysis from three embryos injected with cas RNA at 10 ng/µl alone or together with ERK2* RNA at 50 ng/µl. (J) Differential activity of Casanova WT and Casanova S47A, based on the percentage of embryos ectopically expressing sox17 (see Table 4). The activity of exogenous Casanova-WT was compared with the activity of Casanova-S47A, treated or not with MAPK, FGF inhibitors or an activated form of Erk2.