Proteolytic cleavage converts Cvl2 from an anti- to a pro-Bmp factor. (A) Western blot with anti-Myc antibody after SDS-PAGE with embryonic protein extracts of cvl2-myc mRNA-injected embryos. Under reducing conditions [+mercaptoethanol (ME)], full-length Cvl2 and a C-terminal cleavage fragment are detectable. Under non-reducing conditions (without ME), the cleavage products remain associated, and only the high molecular weight band is detectable. (B) Illustration of Cvl2 proteins encoded by the different mRNAs. The protein encoded by cvl2-WT is either cleaved, with the two fragments remaining associated via disulfide bounds of thus far unidentified residues (top), or uncleaved, resembling Cvl2-CM (lower panel). (C) Western Blot showing that Cvl2-CM is not cleaved in embryos, and that Cvl2-N is readily synthesized. (D-G) Lateral views of 32 hpf embryos; (H-O) animal views, dorsal rightwards, after in situ hybridization at 80% epiboly for the ventral marker eve1 (H-K) or the dorsal marker chordin (chd; L-O) (Schulte-Merker et al., 1997). Injection of cvl2-wt mRNA leads to both weakly ventralized (V1 in D and right embryos in H,L) and weakly (C1 in D) or moderately dorsalized embryos (C2 in D and left embryos in H,L). Injection of cvl2-CM mRNA causes strong dorsalization (F,J,N), while cvl2-N mRNA causes ventralization (G,K,O). Arrows in I,K,M,O indicate borders of the eve1 or chd expression domains. Ratios of obtained phenotypes were: (D-G) see Table 1; (H) dorsalization, 17/44; wild type, 22/44; mild ventralization, 5/44; (J) 52/55; (K) 21/43; (L) dorsalization, 21/56; wild type, 29/56; mild ventralization 6/56; (N) 45/48; (O) 21/36.
|
