Fig. 2

Waif1a Inhibits Wnt/β-Catenin Signaling (A) Classes of phenotypes induced by wnt8 overexpression: class 1, small eyes (arrow); class 2, no eyes (arrow); class 3, reduced forebrain and midbrain (arrowheads); class 4, loss of notochord indicative of enhancement of zygotic, organizer-restricting Wnt signaling; and class 5, hyperdorsalized, indicative of activation of maternal, organizer-inducing Wnt signaling. (B) Distribution of phenotypes in embryos injected with wnt8 (8 pg) plus waif1a RNA (60 pg) or equimolar amounts of GFP control RNA. ^***p < 0.001, chi-square test. (C) Dorsoventral patterning phenotypes indicated by loss or reduction of cement glands (arrowheads) and quantified using the DAI of Xenopus embryos injected on the dorsal side with waif1a RNA (200 pg), axin2 (conductin, 500 pg), xdkk1 (100 pg), lrp6ΔN (60 pg), and xnr3 (200 pg). ^*p < 0.05, Student′s t test. (D) pBAR reporter activity (relative to IL-4R-GFP control) in HEK293T cells transfected with the indicated constructs. ^***p < 0.001, Student′s t test. (E) Phenotypic classes as defined in (A) of embryos injected with 8 pg wnt8 RNA, 4 ng waif1a MO1, and 32 pg of MO-insensitive waif1a RNA or equimolar control reagents. ^***p < 0.001, chi-square-test. (F) Waif1/5T4 knockdown in HEK293T cells enhances pBAR activation in response to Wnt3a conditioned media. ^**p < 0.01 and ^***p < 0.001, Student′s t test. (G) Waif1/5T4 knockout MEFs show increased pBAR activation in response to purified Wnt3a (100 ng/μl). ^*p < 0.05, two-way ANOVA with Bonferroni′s post hoc test. (H) Whole-mount in situ hybridization reveals upregulation of sp5l (15 of 16 embryos), cdx4 (15 of 15), and vent (15 of 16) at the 90% epiboly stage in embryos injected with10 ng waif1a MO1. Note that staining reactions were stopped before the normal wild-type expression in control embryos became visible. See also Figure S2.