Fig. 5

Vg1 and Nodal form heterodimers, and are only active when co-expressed.

(A) Anti-Flag reducing immunoblot (IB) of anti-HA immunoprecipitates (IP) from lysates of Mvg1 embryos injected with 50 pg of cyc-HA, sqt-HA and/or 50 pg of vg1-Flag mRNA. Black arrowhead indicates full-length Vg1-Flag; open arrowhead indicates cleaved Vg1-Flag. See also Figure 5—figure supplements 1A and B. (B) Anti-Flag reducing and non-reducing immunoblots of WT and Mvg1 embryos injected with 50 pg of vg1-Flag, cyc-Flag or bmp7a-Flag, collected at 50% epiboly. Under reducing conditions the proteins migrated at sizes consistent with the theoretical molecular weights for full-length monomers: Vg1-Flag – 42 kDa; Cyc-Flag – 58 kDa; Bmp7a-Flag – 50 kDa. Open arrowhead indicates where mature Bmp7a-Flag homodimers are expected to migrate as two species under non-reducing conditions (Little and Mullins, 2009). For an annotated gel, see Figure 5—figure supplement 1C. (C) Anti-Flag reducing immunoblot of anti-HA IP from lysates of Mvg1 embryos injected with 50 pg of sqt-HA and vg1-Flag mRNAs or vg1-HA and vg1-Flag mRNAs. Black arrowhead indicates full-length Vg1-Flag; open arrowhead indicates cleaved Vg1-Flag. (D) Transplantation of cells from donor embryos injected with cyc, vg1 or cyc and vg1 mRNAs into host embryos for analysis of Nodal target gene lft1 expression at 50% epiboly. mRNAs were co-injected with sfGFP mRNA to verify successful transplantation using DAB staining (Figure 5—figure supplement 2A). (E) Injection of vg1 mRNA into the yolk syncytial layer (YSL) of Mvg1 mutants, shown at 32 hpf. For Nodal target gene expression in vg1 mRNA YSL-injected embryos see Figure 5—figure supplement 2B. vg1 was co-injected with a fluorescent dextran to verify YSL localization (Figure 5—figure supplement 2C).