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Fig. 7. RhoV overexpression expands the neural crest territory. (A) In situ hybridization analysis of neurula stage embryos injected at two-cell stage with 100 ng of wt-RhoV mRNA and the β-galactosidase mRNA lineage tracer. Sox9 (a), Sox10 (b), Slug (c), Snail (d) and Sox2 (e) analysis on early neurula stage embryos. Twist (f, g) analysis on late neurula stage embryos. At early neurula stage, the ectopic expression of wt-RhoV enlarges the domain stained by all neural crest markers and reduces the domain stained by the neural plate marker Sox2 as indicated by black arrows. At late neurula stage, forced expression of RhoV led to an increase in Twist signal on the injected side (g) compared to the control side (f). (B) Expression of wt-RhoV does not affect cell proliferation. Two-cell stage embryos were co-injected in one cell with 100 ng of wt-RhoV and the β-galactosidase mRNA lineage tracer (red staining). At early neurula stage, injected embryos were processed either for Sox9 in situ hybridization (a) or for anti-phospho-histone H3 staining by immunochemistry (b). β-galactosidase activity was revealed using Red-Gal (a) or X-Gal (b). A magnified view of the area boxed in panel b is shown in panel c. No significant difference was observed in the number of mitotic cells in either side of the embryo. (C) The expansion of the neural crest territory induced by RhoV does not require cell proliferation. Two-cell stage embryos were co-injected in one cell with 100 ng of wt-RhoV mRNA and β-galactosidase mRNA as a tracer. At stage 11, control (a, b) and injected embryos (c, d) were treated (b, d) or not (a, c) with HUA and fixed at stage 15. Injected embryos were processed for Sox9 in situ hybridization and control embryos for anti-phospho-histone staining H3. Sox9 expansion (c) is not affected by HUA treatment (d), whereas it induced a 90% reduction in the number of mitotic cells (compare panel b with a). |
