Fig. 4. Maternal Wnt5a is essential for both canonical and non-canonical Wnt signaling. (A) Control uninjected and sibling, Wnt5a antisense oligo-injected oocytes were assayed by real-time RT-PCR for the relative expression of Wnt5a, Wnt11, β-catenin and Dishevelled 2 (Dvl2) mRNA. (B) Real-time RT-PCR assay of the relative expression of Wnt5a in uninjected and Wnt5a-depleted oocytes at blastulae (stage 9) and gastrulae (stages 10, 12). (C) Embryos derived from sibling control and Wnt5a-depleted oocytes at the early tailbud stage. This phenotype was seen in four experiments in a total of 99% of cases (102/104); controls were normal in 99% of cases (91/92). (D) The phenotype of Wnt5a depleted (Wnt5a^-) embryos was partially rescued by the reintroduction of 25 pg Xenopus Wnt5a mRNA (Wnt5a^-+Wnt5a mRNA) before fertilization. Wnt5a-depleted early tailbud stage embryos (5/6) had the ventralized phenotype shown compared with 0/6 Wnt5a^-+25 pg Wnt5a mRNA embryos and 0/10 Wnt5a^-+100 pg Wnt5a mRNA embryos; 11/11 controls were normal. The experiment was repeated with a similar result. (E) The relative expression levels of Xnr3 and siamois in control, Wnt5a-depleted and Wnt5a-depleted+100 pg Xenopus Wnt5a mRNA-injected embryos assayed by real-time RT-PCR at the early gastrula stage. (F) TOPflash reporter activation in control and Wnt5a-depleted embryos frozen at the late blastula stage. (G) TOPflash reporter activation in Wnt5a mRNA overexpressing embryos (100 pg), β-catenin-depleted embryos and sibling embryos injected with both Wnt5a mRNA and β-catenin MO. (H) Western blot of p-JNK-1 protein in control, Dkk1-depleted, Wnt11-depleted, Wnt5a-depleted, Dkk1/Wnt11- and Dkk1/Wnt5a-depleted late blastulae. Quantitation is shown below. P<0.01, Student's t-test. (I) The relative expression levels of Xnr5 and chordin in control, Dkk1-depleted, Wnt11-depleted and Dkk1/Wnt11-depleted embryos assayed by real-time RT-PCR at the early gastrula stage. (J) The relative expression levels of Xnr5 and chordin in control, Dkk1-depleted, Wnt5a-deleted and Dkk1/Wnt5a-depleted embryos assayed by real-time RT-PCR at the early gastrula stage. (K,L) Batches of two whole and four dorsal- or ventral-half, 32-cell stage control and Wnt5a-depleted embryos assayed by real-time RT-PCR for the relative expression of Wnt11 mRNA (K) and Wnt5a mRNA (L).