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First published online 20 April 2005
doi: 10.1242/dev.01823

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Neural crest determination by co-activation of Pax3 and Zic1 genes in Xenopus ectoderm

¹ Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan
² Department of Medical Embryology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan

View larger version (83K): [in a new window]   Fig. 1. Regulation of Pax3, Zic1 and Foxd3 expression in early Xenopus embryos. (A-C) The expression of Pax3 (A), Zic1 (B) and Foxd3 (C) at the early neurula stage as analyzed by whole-mount in situ hybridization. (D-F) Double-labeled in situ hybridization at the neurula stage (stage 15). (D) Pax3 (light blue) and Foxd3 (magenta); (E) Zic1 (light blue) and Foxd3 (magenta); (F) Pax3 (magenta) and Zic1 (light blue). (G-N) Effects of BMP and Wnt signals on Pax3, Zic1 and Foxd3 expression. Synthetic mRNAs of CA-BMPR (50 pg/cell; G-I), dnBMPR (200 pg/cell; J-L) or Wnt3a (10 pg of DNA/cell; M,N) were injected into two left animal blastomeres at the eight-cell stage. Whole-mount in situ hybridization was performed with Pax3 (G,J,M), Zic1 (H,K) and FoxD3 (I,L,N) probes. Arrows in J-N indicate ectopic expression of each marker gene; arrowheads in F indicate the Pac3+/Zic1+ area.

View larger version (72K): [in a new window]   Fig. 2. Overexpression of Pax3 and Zic1 induces ectopic expression of neural crest markers. Synthetic mRNA was injected into two left animal blastomeres (A-L) or into the ventral animal blastomeres (M-P) at the eight-cell stage. Embryos were harvested at stage 15 and subjected to in situ hybridization with Foxd3 (A,D,G-M,O,P), Slug (B,E,N), Zic1 (C) and Pax3 (F) probes. Pax3 mRNA injection (50 pg/cell; A-C), Zic1 mRNA injection (50 pg/cell; D-F), Pax3-GR mRNA injection (100 pg/cell; G-I), Zic1-GR mRNA injection (100 pg/cell; J-L), and injection of both Pax3 and Zic1 mRNAs (50 pg/cell each; E-H) with lacZ mRNA (200 pg/cell). Dexamethasone (Dex; 10 µM) was added to culture solution at stage 10.5 (H,K) or stage 12 (I,L). Arrows in A,B,H,K,M and N indicate ectopic expression of each marker gene.

View larger version (71K): [in a new window]   Fig. 3. Both Pax3 and Zic1 are required for neural crest determination in vivo. Pax3-MO (20 ng/cell; A-F), Zic1-MO (20 ng/cell; G-L), Foxd3-MO (20 ng/cell; M-O), Msx1-MO (50 ng/cell; P-R) or dnBMPR (200 pg/cell; S-U) were injected unilaterally into the animal blastomeres of eight-cell-stage embryos. Foxd3 expression suppressed by Pax3-MO (A) was rescued by Pax3 mRNA (C), but not by Zic1 nor Msx1 (E,F). Zic1-MO-induced suppression of Foxd3 (G) was reversed by Zic1 mRNA (I), but not by Pax3 nor Msx1 (K,L). Msx1-MO suppresses the expression of Foxd3 but not of Pax3 and Zic1 (P-R). Foxd3 expression, upregulated by dnBMPR (S), was suppressed by Pax3-MO and Zic1-MO (T,U). Whole-mount in situ hybridization with Foxd3 (A,C,E-G,I,K,L,P,S-U), Slug (B,H,M), Pax3 (J,N,Q) and Zic1 (D,O,R) probes.

View larger version (38K): [in a new window]   Fig. 4. Pax3 and Zic1 promote neural crest differentiation in Wnt-treated animal cap ectoderm. All animal blastomeres of eight-cell-stage embryos were injected with: Pax3 (50 pg/cell; A,R,S); Pax3 and Wnt3a (50 pg of each/cell; B,C); Zic1 (50 pg/cell; E); Zic1 and Wnt3a (50 pg of each/cell; F,G); Pax3 (50 pg/cell), Wnt3a (50 pg/cell) and Zic1-MO (10 ng/cell; D); Zic1 (50 pg/cell), Wnt3a (50 pg/cell) and Pax3-MO (10 ng/cell; H); Wnt3a (50 pg/cell; inset of B); Pax3, Wnt3a and Bmp4 (50 pg of each /cell; I,J); Zic1, Wnt3a and Bmp4 (50 pg of each/cell; K,L); Pax3, Zic1 and Wnt3a (50 pg of each/cell; M); Pax3, Zic1, Wnt3a and Bmp4 (50 pg of each/cell; N); Chd (50 pg/cell; O); Chd and Bmp4 (50 pg of each/cell; P); or Pax3 (50 pg/cell) and Zic1-MO (10 ng/cell; S). Bmp4 injection was performed by using plasmid DNA (pCS2-Bmp4). Animal cap explants were prepared at stage 9 and harvested at stage 15 for in situ hybridization with Foxd3 (A,B,D-F,H,I,K,M,N), Zic1 (C,J), Pax3 (G,L) or nrp (O,P) probes. (Q) Scheme of the procedure of the animal cap dissociation experiments. (R,S) Animal cap cells were dissociated at stage 9 and harvested for RT-PCR when the siblings reached stage 15. Wnt3a proteins were added at the stage indicated above.

View larger version (80K): [in a new window]   Fig. 5. Co-activation of Pax3 and Zic1 in concert with Wnt signaling is essential for neural crest determination of the ectoderm in vivo. (A-F) Synthetic mRNA was injected into a ventral animal blastomere at the eight-cell stage. Embryos were harvested at stage 15 for in situ hybridization with Foxd3 (A,C,E) or Slug (B,D,F) probes. A higher magnification view is shown in the right half of each panel. Pax3 and Zic1 mRNA injection with lacZ mRNA (A-F; ß-gal activity was visualized by incubating with Red-Gal), ß-catenin-MO (Gene Tools; 10 ng/cell; C-F), and ß-catenin (50 pg of DNA/cell; E,F).

View larger version (25K): [in a new window]   Fig. 6. Working model for neural crest determination by combined functions of Pax3, Zic1 and Wnt.

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