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The USP46 complex regulates Wnt signaling in Xenopus and zebrafish. A, B The USP46 complex induces secondary axis formation and Wnt target gene expression in Xenopus embryos. AXenopus embryos were injected ventrally at the 4-cell stage with Xwnt8 mRNA (10 pg), individual USP46 members (1 ng), or a 1:1:1 mixture (0.33 ng each). The percentage of axis duplication is shown with absolute numbers on the top of each bar and representative images. p-values for injection of individual components versus the complex and for injected embryos compared to wildtype is <0.0001. Scale bar = 200 mm. B RT-PCR show induction of Xenopus Wnt target genes, Xnr3 and Chordin. Expression is shown as a ratio of Ornithine decarboxylase (Odc) normalized to Xwnt8 injected animal caps. Control is un-injected. p-values compare injections of individual components versus the USP46 complex. C, D Knockdown of the USP46 complex in zebrafish induces a cyclopic phenotype and reduction of Wnt target gene transcripts, which are rescued by co-injecting homologous human mRNAs. C Embryos were injected at the single-cell stage with Morpholino oligonucleotides (MO, 3 ng) and rescued with mRNAs (0.8 ng). The percentage of cyclopic embryos is shown with absolute numbers on the top of each bar. Arrows show developing eyes. p-values compare MO-injected versus rescue. p-values for all injected embryos compared to wildtype is <0.0001. Scale bar = 200 mm. D mRNAs were isolated, and Lef1 and CyclinD1 levels were quantified by RT-PCR. Gene expression is graphed as a ratio to β-actin control and normalized to un-injected embryos. p-values shown compare MO-injected versus un-injected embryos and morpholino-injected versus corresponding rescued embryos. B, D Graphs show mean ± SEM, n = 3 independent pools of embryos. Significance was analyzed by two-tailed Student’s t test. Source data are provided as a Source data file.
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