Sato T et al. (2005), Neural crest determination by co-activation of ...

XB-ART-34624

Development 2005 May 01;13210:2355-63. doi: 10.1242/dev.01823.

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

Sato T , Sasai N , Sasai Y .

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A number of regulatory genes have been implicated in neural crest development. However, the molecular mechanism of how neural crest determination is initiated in the exact ectodermal location still remains elusive. Here, we show that the cooperative function of Pax3 and Zic1 determines the neural crest fate in the amphibian ectoderm. Pax3 and Zic1 are expressed in an overlapping manner in the presumptive neural crest area of the Xenopus gastrula, even prior to the onset of the expression of the early bona fide neural crest marker genes Foxd3 and Slug. Misexpression of both Pax3 and Zic1 together efficiently induces ectopic neural crest differentiation in the ventral ectoderm, whereas overexpression of either one of them only expands the expression of neural crest markers within the dorsolateral ectoderm. The induction of neural crest differentiation by Pax3 and Zic1 requires Wnt signaling. Loss-of-function studies in vivo and in the animal cap show that co-presence of Pax3 and Zic1 is essential for the initiation of neural crest differentiation. Thus, co-activation of Pax3 and Zic1, in concert with Wnt, plays a decisive role for early neural crest determination in the correct place of the Xenopus ectoderm.

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Species referenced: Xenopus
Genes referenced: bmp4 chrd foxd3 gal.2 msx1 nrp1 pax3 snai2 wnt3a zic1
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	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.
	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.
	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.
	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; β-gal activity was visualized by incubating with Red-Gal), β-catenin-MO (Gene Tools; 10 ng/cell; C-F), and β-catenin (50 pg of DNA/cell; E,F).
	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.