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Fig. 4. Sox9 is required for neural crest induction. (A) Schematic representation of the hormone-inducible inhibitory mutant Sox9 construct. Sox9 lacking the
transactivation domain (Sox9DC) is fused to the ligand-binding domain of human glucocorticoid receptor (hGR, purple). (B) Experimental timeline. Two-cell
stage embryos are injected in one blastomere with 1 ng of Sox9DC-GR mRNA. Embryos are subsequently incubated with dexamethasone (+DEX) at different
time point during development (stages 6, 8, 10, 12, or 14), and fixed at stage 17 for detection of Slug, Pax3, or AP2 by whole-mount in situ hybridization. (C)
Whole-mount in situ hybridization of embryos injected in one blastomere at the two-cell stage with 1 ng of Sox9DC-GR mRNA and treated with
dexamethasone (+DEX) at the gastrula (stage 10) or neurula (stage 14) stages. Embryos were analyzed for expression of three early neural crest markers (Slug,
Pax3, and AP2) at stage 15/17. Activation of Sox9 inhibitory mutant (Sox9DC-GR) before stage 14 blocks formation of neural crest progenitors. 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. (D) Quantification of Slug, Pax3, and AP2 in situ hybridization results. The numbers at the top of each bar indicate the
number of cases analyzed. Slug (red bars) and Pax3 (blue bars) expressions were analyzed after treatment with dexamethasone at five time points, stages 6, 8,
10, 12, and 14. AP2 expression (green bars) was analyzed after treatment with dexamethasone at three time points, stages 6, 10, and 14. |
