Fig. 8

Fig. 8

Waif1a Activates Noncanonical Wnt Signaling by a Dkk1-Dependent Mechanism (A) Overexpression of zebrafish waif1a RNA (200 pg) inhibits CE movements of Xenopus Keller explants. noninj., noninjected. (B) Fraction of elongated explants: waif1a, 200 pg; waif1c, 200 pg; dnRhoA, 50 pg; dnRac1, 50 pg; DshΔDEP, 500 pg; frizzled 7 MO, 1.6 pmol; frizzled 8 MO, 1.6 pmol; xnr3, 200 pg; lrp6, 60 pg. ^*p < 0.05, Student′s t test. The average of three independent experiments is shown. (C) Western blot of Xenopus embryo extracts at stage 10.5 blotted for endogenous phospho-JNK and total JNK. xwnt5a, 100 pg; xwaif1a.2, 50 pg; xdkk1, 100 pg; xwaif1a.2 MO, 1.6 pmol; xdkk1 MO, 1.6 pmol. (D) dnRhoA (50 pg) does not, but xnr3 (200 pg) can, fully rescue zebrafish waif1a (200 pg)-induced ventralization of Xenopus embryos quantified using the DAI. ^*p < 0.05, Student′s t test. (E) Fraction of elongated explants. dkk1 MO low, 0.8 pmol; high, 1.6 pmol. The average of three independent experiments is shown. ^*p < 0.05, Student′s t test. (F) CE defects in zebrafish embryos coinjected with waif1a MO1 and dkk1 MO (52 of 61 embryos) shown by compressed myoD-positive somites (arrowheads) and widened krox20 and pax2.1-positive neural expression domains (brackets) at the 15 somite stage. waif1a MO1, 2 ng; control MO, 2 ng; dkk1 MO, 1 ng. (G) Model of Waif1a function as modifier of Wnt signaling pathways. waif1a expression is directly regulated by β-catenin signaling during gastrulation and acts as a cell-autonomous feedback inhibitor of the pathway in Wnt-receiving cells. Waif1 interacts with LRP6 and interferes with Wnt-dependent internalization of LRP6, without affecting LRP/Frizzled interaction or LRP6 phosphorylation. See also Figure S8.