Fig. 1. Vg1 is required for initiation of Smad2 phosphorylation and head induction. (A) Real-time RT-PCR analysis of oocytes and gastrula-stage embryos shows that maternal Vg1 mRNA is efficiently depleted by the Vg1A oligo (4 ng oligo, 12% of control levels; 6 ng oligo, 5% of control levels) and that no zygotic transcription of Vg1 is detected during gastrulation. (B) Vg1 protein is depleted in a dose-dependent manner by the Vg1A oligo. Oocytes injected with 45 ng of morpholino (Vg1MO; 50% of control levels), and 4 ng or 6 ng of antisense oligo (Vg1A; 70% and 58% of control levels in oocytes, and 61% and 45% of controls at stage 10, respectively) have reduced levels of Vg1 protein. -Tub, -tubulin. (C) Vg1-depleted embryos show a delay in gastrulation in a dose-dependent manner. Whole-mount in situ hybridization with probes specific for cerberus (cerb) and chordin (chd) shows that the expression of cerb and chd is reduced at mid-gastrula stage in a dose-dependent manner. In histological sections of tailbud stages, Vg1-depleted embryos show an absence of notochord and fusion of somites in the midline (arrow). (D) Real-time RT-PCR analysis of stage 10 embryos shows that expression of the ß-catenin/Xtcf3 target genes Xnr3 and siamois is unaffected by Vg1 depletion. (E) Vg1 depletion reduces the phosphorylation of Smad2 (arrow; 34% of control level at stage 9.5) and increases the phosphorylation of Smad1 (arrowhead; 140% of controls at stage 10.5), as analyzed by western blots. (F) Mesoderm-induction activity of Vg1-depleted vegetal masses (Vg1-), from 6 ng Vg1A-injected oocytes, is decreased compared with controls (wt), as determined by Nieuwkoop assays. Real-time RT-PCR analysis shows that Vg1 depletion reduces the induction of Xbra, Fgf8 and chordin in wild-type animal caps. One whole embryo at stage 11 (WE) was used for quantification. (G) Real-time RT-PCR analysis of embryos during gastrula stages shows that Vg1 depletion causes a downregulation of dorsally expressed cerberus, chordin, noggin and dickkopf (dkk), and an upregulation of dorsal marker Xnr1 and ventral marker sizzled, without affecting the levels of Xsox17 and Bmp4.