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A role for frizzled 3 in neural crest development

¹ Cell and Molecular Biology Graduate Group (School of Medicine), University of Pennsylvania, Philadelphia, PA 19104, USA
² Department of Medicine (School of Medicine), University of Pennsylvania, Philadelphia, PA 19104, USA
³ Department of Animal Biology (School of Veterinary Medicine), University of Pennsylvania, Philadelphia, PA 19104, USA
⁴ Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA 19104, USA

View larger version (34K): [in a new window]   Fig. 1. Xwnt1 and Xfz3 synergize in the dorsal axis induction assay. (A) Xenopus ectopic axis assay showing synergy between Wnts and frizzleds. RNAs encoding frizzleds (Xfz) and Wnts were injected at low concentrations either alone or in combination into a ventral blastomere of a four- to eight-cell embryo. Secondary axes (indicated by arrows) were scored at the late neurula stage. (B) Summary of screen to identify synergistic Wnt/frizzled combinations. Percent of embryos with ectopic axes is plotted with respect to RNAs injected. The black represents complete axes with cement gland and at least one eye at stage 30. The white represents partial axes. n=61 per combination. Doses of RNA used: Xfz2 (1 ng), Xfz3 (100 pg), Xfz7 (500 pg), Xfz8 (20 pg), mouse Wnt1 (1 pg), Xwnt2b (2.5 pg), Xwnt3A (0.5 pg), Xwnt4 (1 ng), Xwnt5A (200 pg), Xwnt7b (0.1 pg), Xwnt8 (25 pg) and Xwnt11 (500 pg). Synergy in the axis duplication assay was similar for Xenopus and mouse Xwnt1 and frizzled 3 (data not shown).

View larger version (64K): [in a new window]   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.

View larger version (85K): [in a new window]   Fig. 3. Xfz3 induces neural crest in animal caps. (A) Xfz3 induces neural crest in a dose-dependent manner. RT-PCR was performed on animal caps ectopically expressing Xfz3 (10 pg, 100 pg, 1 ng) or Xwnt1 (1 pg, 10 pg, 100 pg), either alone or with chordin (1 ng). Stage 23 embryos were used as a positive control. -RT, without reverse transcriptase; EF1 is a loading control. (B) Xfz3 induces the same neural crest markers as Xwnt1 and Xwnt8, shown by RT-PCR analysis of animal caps expressing chordin (1 ng) alone or with Xfz3 (1 ng), Xwnt1 (0.1 ng) or Xwnt8 (0.1 ng). (C) Xfz3 induces migratory melanocytes in animal caps. Albino eggs were fertilized with pigmented male sperm and injected with RNAs for chordin (1 ng) alone or with Xfz3 (100 pg), Xwnt1 (10 pg) or Xfz3/CS2 (50 pg) as indicated. Punctate pigmented spots are migratory melanocytes. ‘uninj’ indicates animal cap from uninjected embryo (A-C). ‘embryo’ in C (upper right) indicates close-up view of melanocytes in uninjected embryo.

View larger version (36K): [in a new window]   Fig. 4. Xfz3 and Xwnt1 induce Xslug at similar times in early neurula stage explants, in parallel with onset of Xslug expression in whole embryos. Animal caps expressing chordin, chordin plus Xfz3, or chordin plus Xwnt1, as well as control animal caps and sibling embryos, were harvested at the stages indicated and processed for RT-PCR analysis as in Fig. 3.

View larger version (54K): [in a new window]   Fig. 5. Interaction between Xwnt1 and Xfz3 in neural crest induction. (A) Xwnt1 synergizes with Xfz3 to induce Xslug. RT-PCR analysis of Xslug expression in animal caps expressing chordin (1 ng) alone, with Xfz3 (10 pg), Xwnt1 (1 pg) or Xwnt8 (1 pg) or combinations thereof. Shown below each lane are the Xslug expression levels, normalized to EF1, relative to the chordin alone sample (defined as 1). (B) An inhibitory form of Xfz3 encoding the CRD (Nfz3) inhibits neural crest formation and this is overcome by increasing Xwnt1. RT-PCR analysis of Xslug expression in animal caps expressing chordin (1 ng) alone, with Xwnt1 (10 pg, 30 pg, 100 pg), with Nfz3 (4 ng) or combinations thereof. Controls are as in Fig. 3. (C) Activity of Xfz3 in neural crest induction requires the CRD (putative ligand binding domain). Co-expression of Xfz3 with Nxfz3 blocks induction of Xslug by Xfz3 and a construct lacking the CRD (Xfz3N) is not active in neural crest induction. (All samples were harvested at stage 23.)

View larger version (61K): [in a new window]   Fig. 6. (A) Xfz3 specifically induces neural crest. RT-PCR analysis of Xslug expression is shown for animal caps expressing chordin (1 ng) alone or with Xfz2 (0.1 ng, 1 ng), Xfz3 (0.1 ng, 1 ng), Xfz7 (0.1 ng, 1 ng), Xfz8 (0.1 ng, 1 ng) and Xwnt1 (0.1 ng). (B) Dendrogram analysis of Drosophila, mouse and Xenopus frizzleds. (C) Nxfz3, but not Nxfz2 or Nxfz7, inhibits Xwnt1 induction of Xslug. RT-PCR analysis of Xslug expression in animal caps expressing chordin (1 ng) alone, Xwnt1 and Nxfz3, 2 or 7 (0.1 and 1.0 ng).

View larger version (101K): [in a new window]   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 right blastomere. RNAs used were Xfz3 (0.75-3.0 ng), Nfz3 (0.75-3.0 ng), Xwnt1 (10 pg) and the lineage tracer ß-galactosidase (200 pg). The red nuclear ß-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 ventral lateral side of the embryo.

View larger version (65K): [in a new window]   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 ß-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 ß-galactosidase activity was measured in situ (magenta) followed by whole-mount in situ hybridization for Xslug.