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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.
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