Fig. 5. Dlx activity is required before the end of gastrulation to position the neural plate border and specify adjacent cell fates. Embryos were injected unilaterally with either VP16-Dlx3hd-GR or EnR-Dlx3hd-GR mRNAs as above and dexamethasone was then added at various times to examine the temporal requirements for Dlx activity. Embryos were assayed for Xsox2 (stage 15) and Xslug (stage 17) by in situ hybridization (blue stain). ß-galactosidase staining (magenta stain) marks the progeny of the injected blastomeres. In all cases, control embryos injected with the fusion protein constructs but cultured without dexamethasone remained unaffected (not shown). (A-D) Addition of dexamethasone at stage 6 expanded Xsox2 (A) or outwardly shifted Xslug (C) in embryos expressing EnR-Dlx3dh and ablated both markers in embryos expressing VP16-Dlx3hd (arrows in B and D). (E,F) In contrast, addition of dexamethasone at stage 11.5-12 to either EnR-Dlx3hd- or VP16-Dlx3hd-injected embryos did not affect Xsox2. (G) Similarly, stage 11.5-12 dexamethasone addition to EnR-Dlx3hd-injected embryos caused no change in Xslug. (H) However, stage 11.5-12 dexamethasone addition to VP16-Dlx3hd-injected embryos ablated Xslug, indicating that the neural crest remains sensitive to Dlx activity after gastrulation. (I) The time course of the dexamethasone effect on EnR-Dlx3hd-expressing embryos suggests that endogenous Dlx activity affects neural crest patterning before the end of gastrulation.