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Wnt signaling in development and adult tissue homeostasis requires tight regulation to prevent patterning abnormalities and tumor formation. Here, we show that the maternal Wnt antagonist Dkk1 downregulates both the canonical and non-canonical signaling that are required for the correct establishment of the axes of the Xenopus embryo. We find that the target Wnts of Dkk activity are maternal Wnt5a and Wnt11, and that both Wnts are essential for canonical and non-canonical signaling. We determine that Wnt5a and Wnt11 form a previously unrecognized complex. This work suggests a new aspect of Wnt signaling: two Wnts acting in a complex together to regulate embryonic patterning.
Fig. 2. Maternal Dkk1 inhibits canonical Wnt signaling. (A) Embryos derived from sibling control (uninj.) and Dkk1-depleted oocytes (5, 7.5 and 10 ng oligo injected) at the early tailbud stage. This phenotype was seen in seven experiments in a total of 85% of cases (124/165). (B) The phenotype of Dkk1-depleted (Dkk-) embryos was partially rescued by the reintroduction of 20 pg human Dkk1 mRNA (Dkk-+mRNA) before fertilization. Here, Dkk1-depleted embryos (14/17) had the elongated phenotype shown compared with 4/14 for Dkk1-+mRNA and 0/24 uninjected; 20 pg human Dkk1 mRNA alone (Dkk mRNA) caused enlargement of head structures (16/18). The experiment was repeated with a similar result. (C) The relative expression levels of Xnr3 and Xnr5 in control (uninj.), in Dkk1 depleted (Dkk-), in 20 pg human Dkk1 mRNA (Dkk mRNA) and in Dkk1 depleted+20 pg human Dkk1 mRNA injected (Dkk-+mRNA) embryos assayed by real-time RT-PCR at the late blastula stage; siblings of those shown in B. (D) TOPflash reporter activation after injection into two dorsal cells of four-cell stage control embryos compared with sibling Dkk1-depleted embryos frozen at the eight-cell, mid-(stage 8), late blastula (stages 9, 9.5) and early gastrula stages (stages 10, 10.5). (E) Western blot of total β-catenin protein in control and Dkk1-depleted sibling early blastulae (stage 7), usingα -tubulin as a loading control. Quantitation is shown on the right. (F,G) In situ hybridization of sibling control and Dkk1-depleted early gastrulae (Xnr5, F; chordin, G). (H) Western blot of phospho-Smad2 and -Smad1 proteins in control and Dkk1-depleted sibling at late blastulae (stage 9.5) and early gastrulae (stage 10.5). Before freezing, embryos were hemisected into batches of four dorsal (dor) and four ventral (ven) halves.
Fig. S1. Dkk-1 depletion affects Xnr3 expression pattern. Whole-mount in situ hybridization for Xnr3 mRNA in uninjected and Dkk1-depleted early gastrulae (stage 10). The Xnr3 expression territory is narrowed in the left-right axis and expanded along animal-vegetal axis. The dorsal lip is marked with a white arrowhead.
Fig. S4. Dkk1 depletion induces elongation movement in both dorsal and ventral explant. For dorsal and ventral explants, the embryos were marked on the dorsal side at the four-cell stage with Nile Blue, and the equatorial zone was dissected and cut into dorsal and ventral halves at the late blastula stage.
Fig. S5. The Dkk1 depletion phenotype depends on both Wnt5a and Wnt11. (A) The phenotypes of control, Wnt5a-depleted, Dkk1-depleted and Wnt5a/Dkk1-depleted embryos at the neurula stage. Wnt5a/Dkk1-depleted embryos have the same ventralized phenotype as Wnt5a-depleted embryos. (B) The phenotypes of control, Wnt11-depleted, Dkk1-depleted and Wnt11/Dkk1-depleted embryos at the mid-gastrula stage. Wnt11/Dkk1-depleted embryos have the same phenotype as Wnt11-depleted embryos.