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Fig. 2. Active Vg1(S) allele rescues the Vg1 depletion phenotype. (A)
Alignment of the N-terminal 39 amino acids of the sequences of Vg1 homologs
from different Xenopus species. X. laevis, Xl Vg1(S)
AY838794 and Xl Vg1(P) BC090232; X. tropicalis, Xt Vg1 AL849026;
X. borealis, Xbo Vg1 AF041844. Asterisk indicates serine (S) versus
proline (P) residues at position 20. Alignment of antisense oligo (Vg1A)
sequence with Xl Vg1(S) and Xl Vg1(P) shows that it recognizes both forms.
(B) A comparison of the profiles of Vg1(P) and Vg1(S) in western blots
of oocytes and embryos (stage 10) injected with 300 pg of Vg1(P) or
Vg1(S) mRNA and probed with the anti-Vg1 antibody D5, showing that
the serine allele of Vg1 is more efficiently translated and processed than the
proline allele.  -Tub, -tubulin. (C) Vg1 depletion (Vg1-)
causes a gastrulation delay, which is partially rescued by the re-introduction
of Vg1(S) mRNA (200 pg) into Vg1-depleted oocytes. Un,
uninjected; Vg1-, 6 ng Vg1A injected. At mid-gastrulation, Smad2
phosphorylation is also partially rescued by Vg1(S) mRNA.
-Tub, -tubulin. (D) Vg1 depletion causes axial defects at
tailbud stages and this phenotype can be partially rescued by Vg1(S)
mRNA (200 pg). Un, uninjected; Vg1-, 6ng Vg1A injected. (E,F) Real-time
RT-PCR shows that dorsal [chordin, cerberus, noggin and
dickkopf (dkk)] and ventral marker expression
(sizzled) can be partially rescued by Vg1(S) mRNA
(200 pg). (G) Vegetal injection of Vg1 (200 pg) mRNA into
fertilized eggs causes the upregulation of Xnr1 and Fgf8 to
levels similar to those observed with Xnr5 (40 pg), and upregulation
of Xsox17 and chordin to a lesser extent, during
gastrulation. (H) Vg1 mRNA and protein are more abundant
dorsally than ventrally at the 32-cell stage. Real-time RT-PCR analysis of
wild-type embryos hemisected into dorsal and ventral halves at the 32-cell
stage indicates that Vg1 mRNA is enriched in the dorsal halves while
levels of VegT mRNA are equal. Western blot analysis of the dorsal
and ventral halves at the 32-cell stage from the same experiment shows that
Vg1 protein is more abundant in the dorsal halves (88% of control level) than
in the ventral halves (64% of control level). mRNA from two whole embryos
(WE), four dorsal (D) and four ventral (V) wild-type half embryos was used in
the RT-PCR analysis. Four whole embryos (WE), eight dorsal (D) or eight
ventral (V) wild-type half embryos were used for western blot analysis. The
results were repeated in three separate experiments and a representative set
is shown. -Tubulin (-Tub) was used as a loading control.
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