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Fig. 5. FoxD3 is required for neural crest development both in vivo and in animal caps. (A-C) Injection of FoxD3delN mRNA (50 pg) into two right blastomeres at the 8-cell stage suppressed Slug (A), and endogenous FoxD3 (detected with 3′UTR probe) (B). Sox2 was induced in the expected neural crest region (shown by an arrow in C) at stage 16. (D-O) Co-injection of FoxD3delN and Slug rescues the expression of neural crest markers. Injection of Slug mRNA (100 pg) into two right blastomeres at 8-cell stage moderately expands the expression of endogenous Slug (detected with 3′UTR probe) (D), FoxD3 (G) and Twist (J), but not Sox2 (M). Injection of FoxD3delN suppressed expression of the neural crest markers (E,H,K), while co-injection of FoxD3delN and Slug rescued their expression (F,I,L). Expansion of Sox2 caused by FoxD3delN was suppressed by coinjecting Slug (N,O). (P) Animal caps were prepared from embryos injected with Slug (50 pg) + Wnt3a (50 pg; lane 3), Slug (50 pg) + Wnt3a (50 pg) + FoxD3delN (100 pg; lane 4), and Slug (50 pg) + Wnt3a (50 pg) + XBF2delN (100 pg; lane 5) mRNAs. They were harvested at stage17 equivalent and analyzed by RT-PCR. |
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Fig. 3. Overexpression of FoxD3 promotes neural crest differentiation as well as neuronal differentiation in vivo and in vitro. (A-D) FoxD3 mRNA (100pg) was injected into the left animal blastomeres at the 8-cell stage. Embryos were harvested at stage 23 and subjected to whole-mount in situ hybridization with (A) Slug, (B) FoxD3, (C) Ets-1, and (D) Sox2 probes. Ectopic expression is shown by arrows. (E-G) Overexpression of FoxD3 in animal cap explants. Animal caps were prepared from embryos injected with (E) control mRNA (25 pg) or (F) FoxD3 mRNA (25 pg), and harvested when siblings (E inset) reached stage 40. (G) RT-PCR analysis. Animal caps injected with control or FoxD3 mRNA were harvested at stage 17 equivalent. (H) Overexpression of XBF2 in animal cap explants. Animal caps injected with control (25 pg) or XBF2 (25 pg) mRNA were analyzed as in G. |
