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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. α-Tub,α -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 β-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 Xsox17α and Bmp4. Image published in: Birsoy B et al. (2006) Copyright © 2006. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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