The Wnt coreceptor Ryk regulates Wnt/planar cell polarity by modulating the degradation of the core planar cell polarity component Vangl2

Abstract

The Wnt signaling pathways control many critical developmental and adult physiological processes. In vertebrates, one fundamentally important function of Wnts is to provide directional information by regulating the evolutionarily conserved planar cell polarity (PCP) pathway during embryonic morphogenesis. However, despite the critical roles of Wnts and PCP in vertebrate development and disease, little is known about the molecular mechanisms underlying Wnt regulation of PCP. Here, we have found that the receptor-like tyrosine kinase (Ryk), a Wnt5a-binding protein required in axon guidance, regulates PCP signaling. We show that Ryk interacts with Vangl2 genetically and biochemically, and such interaction is potentiated by Wnt5a. Loss of Ryk in a Vangl2(+/-) background results in classic PCP defects, including open neural tube, misalignment of sensory hair cells in the inner ear, and shortened long bones in the limbs. Complete loss of both Ryk and Vangl2 results in more severe phenotypes that resemble the Wnt5a(-/-) mutant in many aspects such as shortened anterior-posterior body axis, limb, and frontonasal process. Our data identify the Wnt5a-binding protein Ryk as a general regulator of the mammalian Wnt/PCP signaling pathway. We show that Ryk transduces Wnt5a signaling by forming a complex with Vangl2 and that Ryk regulates PCP by at least in part promoting Vangl2 stability. As human mutations in WNT5A and VANGL2 are found to cause Robinow syndrome and neural tube defects, respectively, our results further suggest that human mutations in RYK may also be involved in these diseases.

FIGURE 1.

Genetic interaction of Ryk and Vangl2. Lateral views of E18.5 mouse embryos (top row) and their skeletal preparations (bottom row). The Vangl2^−/− embryo showed a complete open neural tube and shortened frontonasal process, limbs, and anterior-posterior body axis. The Vangl2^−/− phenotype was not enhanced by heterozygous loss of Ryk. However, the Ryk^−/−;Vangl2^+/− embryo phenocopied the Vangl2^−/− embryos. The Ryk^−/−;Vangl2^−/− embryo resembled the Wnt5a^−/− embryo and was more severe phenotypically compared with the Vangl2^−/− embryo.

FIGURE 2.

Severe length reduction in the Ryk^−/−;Vangl2^−/− humerus. A–F, Skeletal preparation of the forelimbs of the indicated genotypes at E18.5. Bone length was only mildly reduced in the Vangl2^−/− (C) and Ryk^−/−(B) mutant, and a more severe reduction in limb elongation was found in the Ryk^−/−;Vangl2^+/− limb (D). Reduction in humerus length in Ryk^−/−;Vangl2^−/− embryos (E) was very severe and similar to that in the Wnt5a^−/− limb (F). A′–F′, enlarged views of the anterior paws shown in A–F. One phalange in the Ryk^−/−;Vangl2^−/− embryo was missing (E′). Distal elements were completely missing in the Wnt5a^−/− limb (F′). G–J, boxed rib regions of E18.5 embryos are shown at higher magnification. G, normal development of the rib cage in the Ryk^+/−;Vangl2^+/− embryo. H, single bifurcation in the Vangl2^−/− rib cage. I, reduced number of ribs accompanied by severe bifurcation in the Ryk^−/−;Vangl2^−/− embryo. J, bifurcation of a subset of the ribs in the Wnt5a^−/− embryo. G′–J′, ventral views of sternum in embryos shown in G–H. The sternum in the Ryk^−/−;Vangl2^−/− (I′) and the Wnt5a^−/− (J′) embryos were closed incompletely (arrows).

FIGURE 3.

Misalignment of sensory hair cells in the Ryk and Vangl2 mutant cochleae. A–E, projection of a z-stack of confocal sections of sensory hair cells of E18.5 cochleae stained for acetylated tubulin (green) and actin (red). F–I, quantification of hair cell alignment. Number of hair cells in relation to their deviation from correct alignment is displayed. Normal alignment of hair cells in Ryk^+/−;Vangl2^+/− (A) and Ryk^−/− (B) inner ears. C, loss of orientation in the Vangl2^−/− cochlea. D, in the Ryk^−/−;Vangl2^+/− cochlea, the orientation of the hair cells was impaired, most severely in the outer hair cell row 3 (I, p < 0.0001, two-tailed Student's t test against Ryk^+/−;Vangl2^+/−) and to a minor degree in the remaining hair cell rows (F and G). E and I, misalignment of hair cells was similar in the Ryk^−/−;Vangl2^−/− cochlea compared with Vangl2^−/−. One and up to two additional rows of hair cells were detected in the middle but not the proximal cochlea of Ryk^−/−;Vangl2^+/− and Ryk^−/−;Vangl2^−/− embryos, respectively (marked by arrows in E and F). IHC, inner hair cell; OHC, outer hair cell.

FIGURE 4.

Ryk and Vangl2 interaction is enhanced by Wnt5. A and B, co-immunoprecipitation of Ryk and Vangl2 expressed in the HEK 293T cells. Wnt5a enhanced Ryk and Vangl2 interaction. The ratio of co-immunoprecipitated Vangl2 to Ryk was calculated and compared. The results of four independent experiments were analyzed using the Student's t test and shown on the right.

FIGURE 5.

Phosphorylation of Vangl2 is not induced by Ryk. A, in transfected CHO cells, unphosphorylated (lower arrow) and basal phosphorylated (middle arrow) Vangl2 was detected. In the presence of Wnt5a, phosphorylation of Vangl2 (upper arrows) was induced by Ror2 but not by Ryk. B, phospho-specific Vangl2 (pVangl2) antibody that only recognizes phosphorylated founder sites of Vangl2, Ser-82 and Ser-84 (3), showed a mobility shift of Vangl2 (upper arrow) only in the presence of Wnt5a and Ror2 but not Ryk.

FIGURE 6.

Ryk increases the stability of Vangl2. A, in the E9.5 whole embryo lysates, Vangl2 protein levels in the Ryk^−/−;Vangl2^+/− embryos were decreased compared with those in the Ryk^+/+;Vangl2^+/− littermate. B, decrease of Vangl2 protein levels in the Ryk^−/− MEF cells compared with wild type MEF cells. The ratio of Vangl2 to actin protein levels was calculated and compared between Ryk^−/− and wild type cells. The results of three independent experiments were analyzed using Student's t test and shown on the right. C, dose-dependent increase in levels of Vangl2 by Ryk or the lysosome inhibitor bafilomycin A1 (baf A1, 400 nm). Increasing amounts of Ryk DNA in transfection resulted in higher levels of Vangl2 protein. The ratio of Vangl2 to actin protein levels was calculated and compared. The results of four independent experiments were analyzed using Student's t test and shown on the right. D, phospho-deficient Vangl2 (ΔpVangl2), lacking all Ser and Thr phosphorylation sites (3), was stabilized by Ryk. The ratio of ΔpVangl2 to actin protein levels was calculated and compared. The results of four independent experiments were analyzed using Student's t test. E, Vangl2 degradation was analyzed using the protein synthesis inhibitor cycloheximide. In the presence of Ryk, the degradation of Vangl2 is reduced.

FIGURE 7.

Wnt5a regulates Vangl2 stability. A, Vangl2 protein levels were decreased in the E13.5 embryonic limb lysate of the Wnt5a^−/− mutants compared with control littermates. The ratio between Vangl2 and Actin was calculated, and the results of four independent experiments were analyzed using Student's t test. B, the levels of Vangl2 were increased in CHO cells that stably express Vangl2 in the presence of Wnt5a-conditioned medium for 12 h. The ratio betweeenVangl2 and actin was calculated, and the results of four independent experiments were analyzed using Student's t test. C, Wnt5a-conditioned medium increased the levels of Vangl2 in wild type MEF cells but not in Ryk^−/− MEF cells. Cells were incubated for 12 h. D, in the Vangl2-expressing CHO cells, Wnt5a expression increased the levels of Vangl2 similar to Ryk expression or the treatment with lysosome inhibitor bafilomycin A1 (baf A1, 400 nm). Co-expression of Ryk and Wnt5a further increased the stability of Vangl2. The ratio of Vangl2 to actin protein levels was calculated and compared. The results of four independent experiments were analyzed using Student's t test and shown on the right.