Abu-Elmagd M et al. (2006), Frizzled7 mediates canonical Wnt signaling in n...

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Dev Biol 2006 Oct 01;2981:285-98. doi: 10.1016/j.ydbio.2006.06.037.

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Frizzled7 mediates canonical Wnt signaling in neural crest induction.

Abu-Elmagd M , Garcia-Morales C , Wheeler GN .

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The neural crest is a multipotent cell population that migrates from the dorsal edge of the neural tube to various parts of the embryo where it differentiates into a remarkable variety of different cell types. Initial induction of neural crest is mediated by a combination of BMP, Wnt, FGF, Retinoic acid and Notch/Delta signaling. The two-signal model for neural crest induction suggests that BMP signaling induces the competence to become neural crest. The second signal involves Wnt acting through the canonical pathway and leads to expression of neural crest markers such as slug. Wnt signals from the neural plate, non-neural ectoderm and paraxial mesoderm have all been suggested to play a role in neural crest induction. We show that Xenopus frizzled7 (Xfz7) is expressed in the dorsal ectoderm including early neural crest progenitors and is a key mediator of the Wnt inductive signal. We demonstrate that Xfz7 expression is induced in response to a BMP antagonist, noggin, and that Xfz7 can induce neural crest specific genes in noggin-treated ectodermal explants (animal caps). Morpholino-mediated or dominant negative inhibition of Xfz7 inhibits Wnt induced Xslug expression in the animal cap assay and in the whole embryo leading to a loss of neural crest derived pigment cells. Full-length Xfz7 rescues the morpholino-induced phenotype, as does activated beta-catenin, suggesting that Xfz7 is signaling through the canonical pathway. We therefore demonstrate that Xfz7 is regulated by BMP antagonism and is required for neural crest induction by Wnt in the developing vertebrate embryo.

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Species referenced: Xenopus laevis
Genes referenced: foxd3 fzd3 fzd7 msx1 nog notch1 pax3 snai1 snai2 sox10 sox2 sox9 twist1 wnt1 wnt3a wnt7b wnt8a
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	Fig. 1. Xfz7 rather than Xfz3 is expressed in the early neural crest. (A) Embryos were harvested at the stages indicated and expression of Xfz7, Xfz3, Slug and Pax3 was assessed by RT-PCR. Histone H4 was used as a loading control. (B–E) Double label in situ expression of Xfz7 and Xfz3 with Xslug at early neurula (stage 13, B and D) and late neurula stage (stage 18, Ci and Ei). Xfz7 and Xfz3 (red) and Slug (purple). Cii and Eii are frozen sections cut at the level shown in panels Ci and Ei. (F and G) Wholemount in situ expression of panels F slug and G Xfz3 on stage 13 embryos.
	Fig. 4. Analysis of Xfz7 function in neural crest formation in whole embryos. Embryos were injected with RNA or MO at the 2-cell (A–F), 8-cell (G, H, J–L) or 16-cell stage (I) into the animal pole region of one blastomere except for (G) where the embryo was injected twice into the DMZ. RNA's or MO's used were: (A) Xfz7 full-length (1 ng), (B) Xfz7CRD (1 ng), (C) Xfz7MO (35 ng), (D) Xfz3 full-length (1 ng), (E) Xfz3CRD (1 ng), (F) Xfz10MO (50 ng), (G) Xfz7MO (60 ng), (H) Xfz7CRD (500 pg). (I) Xfz7MO (40 ng) into the D1.2 blastomere, (J) CMO (35 ng), (K) Xfz7MO (35 ng), (L) Xfz7MO (35 ng). All injections contained the lineage tracer β-galactosidase (250 pg). Thus, the red nuclear β-galactosidase staining indicates the injected side. The dark purple indicates Xslug expression except (L) where it is sox2 expression. All embryos are stage 18 except for panels H, J–L which are stage 13. All embryos are dorsal views with anterior to the top.
	Fig. 5. Loss of Xfz7 function leads to downregulation of neural crest gene expression in whole embryos and expansion of neural gene expression. Embryos were injected at the 2-cell stage (except K, 8 cell stage) with RNA in the animal pole of one blastomere. MOs or RNA' used were standard control MO (60 ng) (A, D, G, J, M, P), Xfz7MO (60 ng except (K) 40 ng) (B, E, H, N, Q) and Xfz7CRD (1 ng) (C, F, I, L, O, R). All injections contained the lineage tracer β-galactosidase (250 pg). Thus, the red nuclear β-galactosidase staining indicates the injected side. The dark purple indicates the expression of FoxD3 (A–C), Sox10 (D–F), Sox2 (G–I), Sox9 (J–L), Snail (M–O) and Twist (P–R). All embryos are dorsal views with anterior to the top. The dashed line in panels H and I indicates the embryonic midline.
	foxd3 (forkhead box D3) gene expression in a Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior left.
	sox10 (SRY (sex determining region Y)-box 10) gene expression in a Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 17, dorsal view, anterior left.
	fzd3 (frizzled 3) gene expression in Xenopus laevis embryo, NF stage 13, assayed via in situ hybridization, dorsal view, anterior up.
	Fig. 2. Xfz7 is important for convergent extension movements. (A) injection of 250 pg of Xfz7 CRD leads to boat shape embryos typical of defects in convergent extension movements. (B) Co-injection of 250 pg Xfz7 CRD and 250 pg Xfz7. Full-length leads to normal looking embryos. (C and D) Injection of increasing concentrations of Xfz7 leads to increasing convergent extension defects. (E) Diagram showing the binding sites for MO1, 2 and 3 to Xfz7A (Wheeler and Hoppler, 1999) and Xfz7B (Medina et al., 2000 and Sumanas et al., 2000). (F and G) mRNA and protein were prepared from Xfz7 plasmid using an in vitro transcription translation system. 35S-labeled translation products were analyzed on SDSPAGE gels. (F) Translation of Xfz7 RNA (lane 1) was decreased in the presence of MO2 (lane 3) but not by MO1 (lane 2), MO3 (lane 4) and control MO (lane 5). The results shown are representative for at least 3 independent experiments. (G) Translation of Xfz7 RNA was decreased as the amount of MO2 was increased. Lane 1, control MO, lanes 2–5, 20, 40, 60 and 80 ng MO2, lane 6, Xfz7 on its own.
	Fig. 3. Xfz7 induces neural crest markers in animal caps. (A) Xfz7 induces the same neural crest markers as Xwnt1 and Xfz3 shown by RT-PCR analysis of animal caps expressing noggin (500 pg) alone (lane 1) or with Xwnt1 (100 pg, lane 2), Xfz3 (1 ng, lane 3) or Xfz7 (500 pg, lane 4). (B) 500 pg and 750 pg of noggin injected into animal caps induces expression of Sox2, Xfz7 and Xfz3 but not Xslug, Wnt1, Wnt8 and Xbra1 (lanes 1 and 2) compared with the non-injected control (Ni, lane 3). WE in lane 4 stands for whole embryo. (C) RT-PCR analysis of slug expression is shown for animal caps injected with noggin (500 pg) alone (lane 1) or with increasing amounts of Xfz7 250 pg (lane 2), 500 pg (lane 3), 1 ng (lane 4). ‘Ni’ indicates animal cap from uninjected embryo and ‘WE’ indicates RNA made from whole embryos at stage 19.
	Fig. 6. Downregulation of Xfz7 expression leads to the loss of melanocytes in whole embryos. (A and B) Control embryos. The arrows show the neural crest derived melanocytes concentrated in the trunk. (C and D) Injection of Xfz7 MO (60 ng) into the DMZ of a 4-cell stage embryo results in decreased melanocytes (arrow) and morphogenetic defects. (E–F) Injection of Xfz7 MO (60 ng) into the animal pole region of both blastomeres of a 2-cell stage embryo results in absent melanocytes (arrow).
	Fig. 7. Xfz7 MO-mediated downregulation of neural crest induction can be rescued by Xfz7 and β-catenin. Embryos were injected at the 2-cell stage with MO and RNA in the animal pole of one blastomere. The MOs used were Xfz7MO (A and B) and Co MO (A). RNAs used were Xfz7 SDM (A) and constitutively active β-catenin (B). (A) Xfz7 MO on its own gives 22% embryos showing normal slug expression (n = 27). Xfz7 MO plus 50 pg Xfz7SDM gives 86% normal slug expression (n = 29). 50 ng Co MO plus 1 ng Xfz7 SDM results in 90% of embryos having normal slug expression (n = 27). (B) 35 ng Xfz7MO gives 16% of embryos showing normal slug expression (n = 29). Xfz7MO plus 50 pg β-catenin and 100 pg β-catenin gives embryos showing increasingly normal slug expression (67% and 88%, respectively, n = 29 and 34). 500 pg β-catenin on its own gives 94% of embryos with normal or increased slug expression (n = 29). All injections contained the lineage tracer β-galactosidase (250 pg).
	Fig. 8. Xfz7 can signal neural crest induction via a number of different Wnts. In the presence of 500 pg noggin, 100 pg of Xwnt1 (lane 1), Xwnt7b (lane 3) and Xwnt8 (lane 5), all induce slug expression. When 40 ng of Xfz7MO is co-injected, the slug signal is decreased (lanes 2, 4 and 6). ‘Ni’ indicates animal cap from uninjected embryo and ‘WE’ indicates RNA made from whole embryos at stage 19.