Fig. 7. Secondary axis and Wnt/ß-catenin pathway activation by Xcyr61. (A-F) Xcyr61 mRNA (500 pg) was injected into ventral cells of Xenopus embryos at the four-cell stage. (A) Partial secondary axis induced by Xcyr61 (arrow). (B) Complete secondary axis, with secondary head and notochord, revealed by MZ15 antibody staining. (C-F) Dorsalisation caused by Xcyr61, in decreasing order of severity. Muscle is visualised by a cardiac actin probe. Arrows indicate the effects of Xcyr61. (C) Partial secondary axis, (D) secondary muscle outgrowth, (E) split somite and (F) isolated muscle cells at ectopic location. (G) Xcyr61 induces expression of Siamois, a direct target of Wnt/ß-catenin signalling, in ventral marginal zone tissue. Siamois is expressed at high levels in dorsal marginal zone tissue and is also induced in ventral marginal zone cells by Xwnt8. Ornithine decarboxylase acts as a loading control. (H-K) Xcyr61, and a deletion comprising only domains 1 and 2 (IGFBP and VWC) of the protein, causes dorsalisation of ventral marginal zone tissue. Ventral marginal zone tissue was dissected from embryos that had previously been injected with the indicated Xcyr61 constructs. They were allowed to develop to the equivalent of stage 32 when they were assayed for expression of cardiac actin. A control dorsal marginal zone explant is included for comparison. (L) Like Dishevelled (Dsh), Xcyr61 can activate of the TOPFLASH reporter, albeit weakly. Activity resides in the IGFBP domain (domain 1).