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Fig. 1. Wnt-mediated neural crest induction depends on Sox9 function. (A) Whole-mount in situ hybridization of embryos injected in one blastomere at the
two-cell stage with 100 pg of Wnt-1 or Wnt-3A plasmid DNA and 10 ng of Sox9 morpholino antisense (+Sox9-AS) or a control morpholino (+Co-AS).
Slug and Snail expansion mediated by Wnt-1 or Wnt-3A overexpression is blocked in Sox9-depleted embryos (+Sox9-AS). RNA encoding the lineage
tracer h-galactosidase was coinjected to identify the injected side (right side in all panels, arrows). Embryos are viewed from the dorsal side, anterior to the
top. (B) Quantification of Slug and Snail in situ hybridization results. The numbers at the top of each column indicate the number of cases analyzed. (C)
Real-time RT�PCR of control and Sox9-depleted animal explants isolated from embryos injected with a combination of noggin and Wnt-3A mRNA. The
relative expression levels of Snail, Slug, and Sox9 are displayed as histograms. In Sox9-depleted animal explants, Wnt-3A/noggin-mediated induction of
Slug and Sox9 is blocked, while Snail remains largely unaffected. Explants were harvested and analyzed at equivalent stage 17. In each experiment, EF1a is
used as a loading control (data not shown), and each result is normalized to the expression of EF1a. Values (n = 4) are presented as mean F SEM. *P b
0.05 versus noggin+Wnt-3A + CoAS. Single injection of Sox9-AS or Co-AS had no effect on the expression of these genes (not shown). |
