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Fig. 6. Sox10 expands Slug and induces ectopic Trp2-expressing cells and melanocytes at the gastrula stage. (A) Ectopic Slug expression in embryos injected with Sox10-GR at the two-cell stage and treated with dexamethasone at stage 6 or stage 10 (left panels). Slug expression on the uninjected side is shown for comparison (right panels). (B) Ectopic Trp-2 expression in embryos injected with Sox10-GR at the two-cell stage and treated with dexamethasone at stage 10 (upper left panel). Trp-2 expression is unaffected in the neural crest lineage of embryos treated with dexamethasone at stage 13 (lower left panel). When targeted in the head region, Sox10-GR blocks Trp-2 expression in the pigmented epithelium of the retina (arrow, left panel). Trp-2 expression on the uninjected side is presented for comparison (right panels). Lateral view, anterior is to the left (left panels) or to the right (right panels). RNA encoding the lineage tracer beta-galactosidase was coinjected to identify the injected side (red staining). (C) Quantification of the in situ hybridization results. The number of cases analyzed for each time point (+Dex) is indicated in parentheses. (D) Western blot analysis. Detection of Sox10-GR protein in extracts from Sox10-GR-injected embryos (+Sox10-GR) collected at different stages after injection at the two-cell stage. The fusion protein is expressed at similar levels at all stages (st 10–22). This Sox10 antibody does not allow detection of endogenous Sox10 protein (first lane). Tubulin is presented as a loading control. (E) Development of ectopic melanocytes in Sox10-GR-injected embryos. Embryos injected with Sox10-GR at the two-cell stage and treated with dexamathasone at stage 10 (upper panel, +Dex st 10) displays ectopic formation of pigment cells in the head (white arrow) and the trunk (black arrows) regions. The pattern of pigment cells is unaffected in sibling embryos treated with dexamethasone at stage 16 (lower panel, +Dex st 16). (F) Induction of differentiated melanocytes in animal explants at equivalent stage 40. Animal explants isolated at the blastula stage from embryo injected with 2 ng of Sox10-GR mRNA at the two-cell stage were treated with dexamethasone at stage 9 (+Dex st. 9) or at stage 16 (+Dex st. 16). Induction of melanocytes is only observed in animal explants treated by dexamethasone at stage 9. |
