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Dev Cell
2011 Dec 13;216:1129-43. doi: 10.1016/j.devcel.2011.10.015.
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Waif1/5T4 inhibits Wnt/β-catenin signaling and activates noncanonical Wnt pathways by modifying LRP6 subcellular localization.
Kagermeier-Schenk B, Wehner D, Ozhan-Kizil G, Yamamoto H, Li J, Kirchner K, Hoffmann C, Stern P, Kikuchi A, Schambony A, Weidinger G.
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Wnt proteins can activate distinct signaling pathways, but little is known about the mechanisms regulating pathway selection. Here we show that the metastasis-associated transmembrane protein Wnt-activated inhibitory factor 1 (Waif1/5T4) interferes with Wnt/β-catenin signaling and concomitantly activates noncanonical Wnt pathways. Waif1 inhibits β-catenin signaling in zebrafish and Xenopus embryos as well as in mammalian cells, and zebrafish waif1a acts as a direct feedback inhibitor of wnt8-mediated mesoderm and neuroectoderm patterning during zebrafish gastrulation. Waif1a binds to the Wnt coreceptor LRP6 and inhibits Wnt-induced LRP6 internalization into endocytic vesicles, a process that is required for pathway activation. Thus, Waif1a modifies Wnt/β-catenin signaling by regulating LRP6 subcellular localization. In addition, Waif1a enhances β-catenin-independent Wnt signaling in zebrafish embryos and Xenopus explants by promoting a noncanonical function of Dickkopf1. These results suggest that Waif1 modulates pathway selection in Wnt-receiving cells.
Figure 2. Waif1a Inhibits Wnt/b-Catenin Signaling (A) Classes of phenotypes induced by wnt8 overex- pression: class 1, small eyes (arrow); class 2, no eyes (arrow); class 3, reduced forebrain and midbrain (arrow- heads); class 4, loss of notochord indicative of enhance- ment of zygotic, organizer-restricting Wnt signaling; and class 5, hyperdorsalized, indicative of activation of maternal, organizer-inducing Wnt signaling.
(B) Distribution of phenotypes in embryos injected with wnt8 (8 pg) plus waif1a RNA (60 pg) or equimolar amounts of GFP control RNA. ***p < 0.001, chi-square test. (C) Dorsoventral patterning phenotypes indicated by loss or reduction of cement glands (arrowheads) and quanti- fied using the DAI of Xenopus embryos injected on the dorsal side with waif1a RNA (200 pg), axin2 (conductin, 500 pg), xdkk1 (100 pg), lrp6DN (60 pg), and xnr3 (200 pg). *p < 0.05, Student t test.
(D) pBAR reporter activity (relative to IL-4R-GFP control) in HEK293T cells transfected with the indicated constructs. ***p < 0.001, Student t test. (E) Phenotypic classes as defined in (A) of embryos injected with 8 pg wnt8 RNA, 4 ng waif1a MO1, and 32 pg of MO-insensitive waif1a RNA or equimolar control re- agents. ***p < 0.001, chi-square-test.
(F) Waif1/5T4 knockdown in HEK293T cells enhances pBAR activation in response to Wnt3a conditioned media. **p < 0.01 and ***p < 0.001, Student t test. (G) Waif1/5T4 knockout MEFs show increased pBAR activation in response to purified Wnt3a (100 ng/ml). *p < 0.05, two-way ANOVA with Bonferroni post hoc test. (H) Whole-mount in situ hybridization reveals upregula- tion of sp5l (15 of 16 embryos), cdx4 (15 of 15), and vent (15 of 16) at the 90% epiboly stage in embryos injected with10 ng waif1a MO1. Note that staining reactions were stopped before the normal wild-type expression in control embryos became visible.
See also Figure S2.
Figure 3. waif1a Inhibits wnt8 Function in Mesoderm and Neuroec- toderm Patterning (A) Reduction of the forebrain marker otx2 and expansion of the posterior neural marker hoxb1b at the 100% epiboly stage in embryos injected with 10 ng waif1a MO1 or MO2 (23 of 28 embryos MO1, 21 of 24 MO2).
(B) Changes in size of the telencephalic foxg1a expression domain at the one somite stage (bracket) in embryos injected with 2 ng each of wnt8.1 and wnt8.2 MOs (20 of 21 embryos), 160 pg waif1a RNA (36 of 36), 0.4 pg wnt8 RNA (22 of 26), and 4 ng of waif1a MO1 (16 of 25) without or with 2 pg axin1 RNA (17 of 23). (C) Changes in the size of the dorsal gsc expression domain at the 50% epiboly stage (arrowheads) in embryos injected with 2 ng each of wnt8.1 and wnt8.2 MOs (41 of 42 embryos), 150 pg waif1a RNA (35 of 44), and 10 ng waif1a MO1 (20 of 21 embryos), relative to embryos injected with equimolar amounts of luciferase control RNA plus control MO and in hsp70l:Wnt8-GFP transgenic embryos heat shocked at 30% epiboly (46 of 46 embryos). (D) Head regions with eyes (arrows) and midbrain-hindbrain boundary (arrowheads) indicated at 24 hpf of embryos injected with 10 ng waif1a MO1 and 0.2 ng standard control MO, or 0.2 ng tcf3a MO plus 10 ng control MO. (E) Quantification of the tcf3a and waif1a knockdown synergy. Embryos were classified using the criteria introduced in Figure 2A, ***p < 0.001, chi-square test.
See also Figure S3.
Figure S1, related to Figure 1. Waif phylogeny and expression during zebrafish and Xenopus embryogenesis and creation of a transgenic zebrafish line expressing Axin1-YFP after heat- shock. Data characterizing a newly created hs:axin1YFP transgenic fish line are presented, as well as, the primary sequence conservation of waif family members, the phylogenetic relationship of zebrafish and mammalian Waif proteins, the expression pattern of zebrafish waif1a and Xenopus waif1a.2 in wildtype embryos and data showing that waif1a expression is regulated by wnt8 in zebrafish embryos.
tpbg (trophoblast glycoprotein ) gene expression in Xenopus laevis embryos, NF stage 24, as assayed by in situ hybridization. Lateral view: anteriorleft, dorsal up.
