Fig. 3. In vitro analysis of Cv-2 activity. (A) Western blot analysis and co-immunoprecipitation reveals the direct interaction of V5-tagged Cv-2 protein with recombinant human BMP4 protein. V5-tagged chordin is used as a positive control for BMP binding. (B-E) Effect of Cv-2 injection on axis formation in Xenopus at stage 14 (B) and stage 27 (C-E). Ventral injections of Cv-2 RNA induce a partial secondary axis (arrows; B,C,E). Transverse section through the trunk of a stage 27 embryo injected ventrally with Cv-2 confirms the presence of an ectopic secondary neural epithelium (arrow, D). (F,G) RT-PCR analyses of Xenopus animal cap explants. (F) Xenopus Noggin mRNA induces expression of the general neural marker NCAM and anterior neural marker BF1. Increasing amounts of Cv-2 mRNA do not induce expression of either neural marker. (G) Co-injection of 2 ng Cv-2 RNA plus 0.1 ng noggin RNA increases the induction of BF-1 when compared with injection of 0.1 ng noggin RNA alone. The mesodermal marker muscle actin is used to confirm the absence of mesoderm in the explants and elongation factor-1 alpha (EF-1) is used to assess the relative amounts of recovered mRNA.