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Requirement of FoxD3-class signaling for neural crest determination in Xenopus

Noriaki Sasai1,2, Kenji Mizuseki1 and Yoshiki Sasai1,3,*

1 Department of Medical Embryology and Neurobiology, and
2 Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Sakyo, Kyoto 606-8507, Japan
3 Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan



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  		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.

 


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  		Fig. 2. Regulation of FoxD3 expression in animal cap assay. Animal caps were prepared from stage 10.5 embryos that had been injected with (A,B) control mRNA (100 pg), (C,D) Chd mRNA (50 pg), (E,F) Wnt3a mRNA (50 pg), (G,H) Chd and Wnt3a mRNAs, (I,J) Slug mRNA (50 pg), (K,L) Slug and Wnt3a mRNAs, and (M,N) Zic-r1 mRNA (100 pg). The animal caps were harvested at stage 17 and analyzed with FoxD3 probe (A,C,E,G,I,K,M) or Slug 3'UTR probe (B,D,F,H,J,L,N).

 


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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.

 


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  		Fig. 4. Generation of a dominant-negative FoxD3 construct. (A) A dominant-negative FoxD3 construct, FoxD3delN. Amino acid residue numbers are indicated above. NLS (grey box), SV40 nuclear localization signal; black box, DNA-binding domain. (B) Animal caps were prepared from embryos injected with control RNA (150 pg), FoxD3 (25 pg; lane 3), FoxD3 (25 pg) + FoxD3delN (50 pg) (lane 4), FoxD3 (100 pg) + FoxD3delN (50 pg) (lane 5), XBF2 (25 pg) + FoxD3delN (50 pg) (lane 6), or XBF2 (25 pg) + FoxD3delN (100 pg) (lane 7) mRNAs. As a specificity control, XBF2delN was also constructed (see Materials and Methods). Animal caps were prepared from embryos injected with XBF2 (25 pg) (lane 8), XBF2 (25 pg) + XBF2delN (50 pg) (lane 9), XBF2 (100 pg) + XBF2delN (50 pg) (lane 10), FoxD3 (25 pg) + XBF2delN (50 pg) (lane 11), or FoxD3 (25 pg) + XBF2delN (100 pg) (lane 12) mRNAs. They were harvested at stage17 equivalent and analyzed by RT-PCR.

 


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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. 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.

 


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  		Fig. 7. FoxD3 and XBF2 show distinct transcriptional regulation in Slug induction. (A) FoxD3 is a transcriptional repressor. Animal caps were prepared from embryos injected with FoxD3 (25 pg; lane 3), FoxD3 (25 pg) + FoxD3-VP16 (50 pg; lane 4), FoxD3 (75 pg) + FoxD3-VP16 (50 pg; lane 5), FoxD3 (25 pg) + FoxA4-VP16 (100 pg; lane 6), and FoxD3-EnR (50 pg; lane 7) mRNAs. Animal caps were harvested at stage 17 and analyzed by RT-PCR. (B-P) Animal caps were prepared from embryos injected with control (50 pg; B-D), FoxA4 (50 pg; E-G), FoxD3 (25 pg; H-J), XBF2 (50 pg; K-M) or dnBMPR (200 pg; N-P) mRNAs, and harvested at stage 17 equivalent. Animal caps were analyzed by in situ hybridization with BMP4 (B,E,H,K,N), Sox2 (C,F,I,L,O), and Slug (D,G,J,M,P) probes. Injection of FoxA4, FoxD3, XBF2 and dnBMPR mRNA suppressed BMP expression and induced Sox2 expression. In contrast, FoxD3 induced Slug expression in the animal cap while neither FoxA4, XBF2 nor dnBMPR did.

 

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© The Company of Biologists Ltd 2001