Fig. 6. Differential requirement of FoxD3 and Slug in neural crest development. (A) A truncated mutant that encodes only the DNA-binding domain of Slug (bottom; SlugBD, labeled dn Slug). (B) Injection of SlugBD mRNA (100 pg) into two right blastomeres at 8-cell stage suppressed endogenous Slug expression (detected with 3'UTR probe) at stage 15 on the injected side. (C,D) Slug expression was suppressed in animal caps by the co-injection of 100 pg of SlugBD mRNA with Slug (50 pg) and Wnt3a (50 pg) mRNAs at stage 17 (C) (animal caps injected with Slug (50 pg) and Wnt3a (50 pg); C inset). This suppression was reversed by increasing wild-type Slug mRNA to 200 pg (D). (E) Effects of SlugBD on neural crest markers in FoxD3-injected animal caps. Control mRNA, FoxD3 mRNA (25 pg), or combination of FoxD3 (25 pg) and SlugBD (100 pg) mRNAs were injected into animal blastomeres in 8-cell stage embryos. Animal caps were prepared at stage 10.5 and harvested at stage 21 for RT-PCR analysis. (F) Animal caps were prepared from embryos injected with Zic-r1 (100 pg; lane 3), Zic-r1 + FoxD3delN (100 pg; lane 4), Zic-r1 + SlugBD (100 pg; lane 5), Chd (50 pg) + Wnt3a (50 pg; lane 6), Chd + Wnt3a + FoxD3delN (lane 7), or Chd + Wnt3a + SlugBD (lane 8) mRNAs. Animal caps were harvested at stage 14 for RT-PCR analysis. (G) Animal caps were prepared at stage 10.5 from embryos injected with control (100 pg; lanes 2 and 6), Slug (50 pg; lanes 3 and 7), FoxD3 (25 pg; lanes 4 and 8), or FoxD3 + Slug (lanes 5 and 9) mRNAs, with (lanes 6-9) or without (lanes 2-5) Chordin (50 pg) mRNA.