Fig. 6. Sox9 is required to specify the otic placode. (A) Experimental procedure. Embryos at the two-cell stage are injected in one blastomere with 1 ng of Sox9C-GR mRNA. Embryos are subsequently incubated with dexamethasone (+Dex) at different time points during development (stage 6, 11 and 15), and fixed at stage 22 for detection of Pax8 and Tbx2 by whole-mount in situ hybridization. (B) Addition of dexamethasone at the blastula (stage 6) and gastrula (stage 11) stages results in a severe reduction of Pax8 and Tbx2 expression domain in the otic placode. However, Pax8 and Tbx2 otic expression remain unaffected in embryos treated with dexamethasone at stage 15 (arrows). Upper panels are dorsal views, anterior towards the top. Lower panels are anterior views. RNA encoding the lineage tracer ß-galactosidase was co-injected to identify the injected side (red staining). In all panels, embryos are injected on the left side. (C) Quantification of the in situ hybridization results. The number of cases analyzed for each time point is indicated.