Fig. 3. Maternal Dkk1 inhibits non-canonical Wnt signaling. (A) Embryos derived from sibling control, β-catenin-depleted (β-catMO), Dkk1-depleted and Dkk1/β-catenin-depleted oocytes at the early gastrula stage. The Dkk1-depleted abnormal shape change was seen in 22/26 cases, and was present in 0/25 β-catenin-depleted, 18/21 Dkk/β-catenin-depleted cases and 2/24 controls. The experiment was repeated with similar results. (B) Embryos derived from sibling control, Dkk1-depleted, β-catenin-depleted and Dkk/β-catenin-depleted oocytes at the early tailbud stage. 18/20 Dkk1-depleted embryos had the phenotype shown, whereas β-catenin-depleted embryos were ventralized (20/20, 10 ng MO; 18/18, 20 ng MO). Dkk/β-catenin-depleted embryos were also ventralized (19/20 Dkk^-/10ng MO; 17/17 Dkk^-/20ng MO) and 24/24 controls were normal. (C) The relative expression levels of Xnr5 in control, Dkk1-depleted, β-catenin-depleted and Dkk1/β-catenin-depleted embryos assayed by real-time RT-PCR at the early gastrula stage. (D) Equatorial zones from control, Dkk1-depleted, β-catenin-depleted and Dkk1/β-catenin-depleted dissected at the mid-blastula stage and cultured until the late neurula stage. Dkk1-depleted (9/9) and Dkk1/β-catenin-depleted explants (10/11 Dkk^-/10 ng MO; 11/11 Dkk^-/20 ng MO) were hyper-elongated compared with control, whereas β-catenin-depleted embryos were round (8/8 with 10 ng MO; 9/9 with 20 ng MO). The experiment was repeated with similar results. (E) Western blot of p-JNK-1 protein in control and Dkk1-depleted sibling at late blastulae (stage 9.5) and early gastrulae (stage 10.5), hemisected into dorsal (dor) and ventral (ven) halves before freezing. A total JNK antibody was used as loading control. (F) Western blot of p-JNK-1 protein in control and Dkk1-depleted vegetal masses compared with equatorial explants. Ten vegetal masses and five equators were compared. Quantitation is shown on the right. P<0.01, Student's t-test. (G) Western blot of p-JNK-1 protein in control, β-catenin-depleted, Dkk1-depleted and Dkk1/β-catenin-depleted (Dkk^-/β-catMO) late blastulae. Quantitation is shown on the right. P<0.03, Student's t-test. (H) Equatorial zones from control, Dkk-depleted and JNK inhibitor SP600125-treated equatorial zones dissected at the mid-blastula stage and cultured until the late neurula stage. 0/10 uninjected, 10/10 Dkk^- and 1/10 Dkk^-/SP600125-treated explants had the Dkk1-depleted shape. The experiment was repeated with similar results.