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Fox factors (winged-helix transcription factors) play important roles in early embryonic patterning. We show here that FoxD3 (Forkhead 6) regulates neural crest determination in Xenopus embryos. Expression of FoxD3 in the presumptive neural crest region starts at the late gastrula stage in a manner similar to that of Slug, and overlaps with that of Zic-r1. When overexpressed in the embryo and in ectodermal explants, FoxD3 induces expression of neural crest markers. Attenuation of FoxD3-related signaling by a dominant-negative FoxD3 construct (FoxD3delN) inhibits neural crest differentiation in vivo without suppressing the CNS marker Sox2. Interestingly, these loss-of-function phenotypes are reversed by coinjecting SLUG: In animal cap explants, neural crest differentiation induced by Slug and Wnt3a is also inhibited by FoxD3delN but not by a dominant-negative form of XBF2. Loss-of-function studies using dominant-negative forms of FoxD3 and Slug indicate that Slug induction by Zic factors requires FoxD3-related signaling, and that FoxD3 and Slug have different requirements in inducing downstream neural crest markers. These data demonstrate that FoxD3 (or its closely related factor) is an essential upstream regulator of neural crest determination.
Fig. 1. Expression of FoxD3 in early Xenopus embryos and comparison to that of Slug. (A-H) Spatial and temporal expression of FoxD3 (A,C,E,G) and Slug (B,D,F,H) analyzed by whole-mount in situ hybridization. (A,B) Early gastrula stage 11 (vegetal view), arrowhead indicates dorsal lip; (C,D) mid-gastrula stage 12; (E,F) late gastrula stage 12.5; and (G,H) mid-neurula stage 16. (I-K) Double-labeled in situ hybridization. (I) Sox2 (light blue) and FoxD3 (indigo), (J) Sox2 (light blue) and Slug (indigo), (K) Zic-r1 (light blue) and FoxD3 (indigo). (C-K) Dorsal views. (L) Histological analysis of FoxD3 distribution at mid-neurula stage. np, neural plate.
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.
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.