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Wnts are a large family of secreted molecules implicated in numerous developmental processes. Frizzled proteins are likely receptors for Wnts and are required for Wnt signaling in invertebrates. A large number of vertebrate frizzled genes have also been identified, but their roles in mediating specific responses to endogenous Wnts have not been well defined. Using a functional assay in Xenopus, we have performed a large screen to identify potential interactions between Wnts and frizzleds. We find that signaling by Xwnt1, but not other Wnts, can be specifically enhanced by frizzled 3 (Xfz3). As both Xfz3 and Xwnt1 are highly localized to dorsal neural tissues that give rise to neural crest, we examined whether Xfz3 mediates Xwnt1 signaling in the formation of neural crest. Xfz3 specifically induces neural crest in ectodermal explants and in embryos, similar to Xwnt1, and at lower levels of expression, synergizes with Xwnt1 in neural crest induction. Furthermore, loss of Xfz3 function, either by depletion with a Xfz3-directed morpholino antisense oligonucleotide or by expression of an inhibitory form of Xfz3 (Nfz3), prevents Xwnt1-dependent neural crest induction in ectodermal explants and blocks neural crest formation in whole embryos. These results show that Xfz3 is required for Xwnt1 signaling in the formation of the neural crest in the developing vertebrate embryo.
Fig. 2. Xfz3 and Xwnt1 are co-expressed in developing neural tissues. (A) Whole-mount in situ analysis of Xfz3 and Xwnt1 in neurula stage embryos (top, stage 18) and early tadpoles (bottom, stage 25). (B) Embryos were harvested at stages indicated and expression of dorsal neural markers was assessed by RT-PCR. Fgf receptor (FGFR1) was used as a loading control.
Fig. 7. Analysis of Xfz3 activity in whole embryos. Albino embryos were injected at the two cell stage with RNA into the animal pole region of either the left or rightblastomere. RNAs used were Xfz3 (0.75-3.0 ng), Nfz3 (0.75-3.0 ng), Xwnt1 (10 pg) and the lineage tracer β-galactosidase (200 pg). The red nuclear β-galactosidase staining indicates the side injected. The dark purple indicates Xslug expression. All panels are dorsal views with anterior to the left; the view of Xfz3 is slightly oblique with the arrow indicating ectopic Xslug on the injected ventrallateral side of the embryo.
Fig. 8. Xfz3 is required for neural crest formation. (A) Xfz3-directed antisense morpholino oligonucleotide blocks translation of injected Xfz3 mRNA. Xfz3 mRNA was injected into one cell embryos together with Xfz3 morpholino at the doses indicated. Embryos were harvested at stage 10 and analyzed by western blot with Xfz3 antibodies. (B) Xfz3 mRNA was injected together with control (Con), Kermit (Ker) or Xfz3 morpholinos (2 ng each) or with no oligo (-) and stage 10 embryos were western blotted for Xfz3 as above. (C) RT-PCR analysis of Xslug expression (as in previous figures) in animal caps expressing chordin and Xwnt1, co-injected with either Xfz3 morpholino (FM; 4 ng) or control morpholino (CM; 4 ng) and harvested at stage 18. Xwnt1-dependent induction of Xslug was rescued by co-injection of an Xfz3 mRNA (F3) lacking the morpholino target sequence; this dose of F3 mRNA (10pg) caused minimal induction of Xslug in the absence of Xwnt1. (D-F) 2 ng of the control morpholino (D) or Xfz3 morpholino (E,F) were co-injected with mRNA for nuclear β-galactosidase into a single dorsal-lateral (B2) blastomere at the 32-cell stage. For rescue of Xfz3 depletion, mouse Xfz3 mRNA (0.4 ng) was co-injected with Xfz3 morpholino (F). Embryos were fixed at stage 18 and β-galactosidase activity was measured in situ (magenta) followed by whole-mount in situ hybridization for Xslug.
