Fig. 8. Molecular analysis of the differentiated animal cap phenotypes induced by HyBra proteins. Marker analysis by double (A,C,E,G,I-L) or single (B,D,F,H) RNA in situ hybridisation; probes are listed within panels, black letters indicate BM-Purple stain, red letters indicate Fast-Red stain (n3 independent repeats). (A,B) GFP-injected control explants showed no staining of tested marker genes. Injections of Xbra (C) and HyBra1 (E) induced muscle actin, but not xcg-1 expression, whereas HyBra2 (G) triggers the converse gene expression pattern. Furthermore, mRNA of the differentiated neuronal marker ß-tubulin was often present in HyBra2- (H), but never in HyBra1- (F), and only rarely in Xbra-injected explants (D). (I-L) Double in situ staining of animal caps injected with the Hybra2/Xbra chimerae. (I,J) H2TXA-injected animal caps show no xcg-1, ß-tubulin induction and very limited muscle actin expression. (K,L) By contrast, XTH2A-injected caps clearly show strong induction of xcg-1 and ß-tubulin, but no muscle actin mRNA. (M) Statistical overview of induced markers (xcg-1, muscle actin, ß-tubulin) by injection of particular mRNAs (2-6 independent single and/or double in situ hybridisation experiments).