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Fig. 2. Regulation of Sox9 and Snail expression by Slug. (A) Whole-mount in situ hybridization of embryos injected in one blastomere at the two-cell stage
with 1�2 ng of DSlug mRNA. Slug (74% of the embryos, n = 62) and Sox9 (52% of the embryos, n = 87) expressions are reduced, while Snail expression
domain was either unaffected or expanded (31% and 52% of the embryos, respectively; n = 63). RNA encoding the lineage tracer h-galactosidase was
coinjected to identify the injected side (left side in all panels, arrows). Embryos are viewed from the dorsal side, anterior to the top. (B) Real-time RT�PCR of
control and DSlug-injected 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 the DSlug-injected animal explants, Wnt-3A/noggin-mediated induction of Slug and Sox9 is blocked.
While Snail remains 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 = 3) are presented as mean F SEM. *P b 0.05 versus noggin+Wnt-3A; **P b 0.1
versus noggin+Wnt-3A. Single injection of DSlug had no effect on the expression of these genes (not shown). |
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Fig. 3. Sox9 functions as a transcriptional activator during neural crest formation. (A) Schematic representation of the fusion constructs. The HMG box of Sox9
(blue) was fused in frame to either the repressor domain of engrailed (Sox9-Eng, orange) or the activation domain of VP16 (Sox9-VP16, green). (B) By
Western blot analysis using a pan-Sox antibody a single product is recognized in in vitro transcription/translation reactions directed by Sox9 (lane 1), Sox9-Eng
(lane 2), and Sox9-VP16 (lane 3) cDNAs. (C) Whole-mount in situ hybridization of embryos injected in one blastomere at the two-cell stage with 10 ng of
Sox9-AS or 400 pg of mRNA encoding either Sox9-Eng or Sox-VP16. Embryos were analyzed for expression of neural crest (Slug, Snail, and Sox10) and
neural plate (Sox2) markers at stage 15/17. Sox9-Eng construct blocks expression of the neural crest markers Slug, Snail, and Sox10 and expands the neural
plate-specific gene Sox2, thereby mimicking the phenotype of Sox9-depleted embryos (Sox9-AS). RNA encoding the lineage tracer h-galactosidase was
coinjected to identify the injected side (red staining, left side in all panels, arrows). Embryos are viewed from the dorsal side, anterior to the top. In each panel,
the number of embryos analyzed is indicated in the lower right corner (n=), and the percentage of embryos exhibiting the phenotype shown is indicated in the
upper right corner. |
