Seo HS et al. (2026), Prickle and Ror modulate Dishevelled-Vangl inte...

XB-ART-61829

Elife 2026 Apr 30;12. doi: 10.7554/eLife.91199.

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Prickle and Ror modulate Dishevelled-Vangl interaction to regulate non-canonical Wnt signaling during convergent extension in Xenopus.

Seo HS, Yu D, Popov IK, Tao J, Angermeier AR, Yang F, Marchetto S, Borg JP, Sha B, Axelrod JD, Chang C, Wang J.

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Convergent extension (CE) is a fundamental morphogenetic process where oriented cell behaviors lead to polarized extension of diverse tissues. In vertebrates, regulation of CE requires both non-canonical Wnt, its co-receptor Ror, and several 'core members' of the planar cell polarity (PCP) pathway. PCP was originally identified as a mechanism to coordinate the cellular polarity in the plane of static epithelium, where core proteins Frizzled (Fz)/Dishevelled (Dvl) and Van Gogh-like (Vangl)/Prickle (Pk) partition to opposing cell cortex. But how core PCP proteins interact with each other to mediate non-canonical Wnt/Ror signaling during CE is not clear. We found previously that during CE, Vangl cell-autonomously recruits Dvl to the plasma membrane and keeps Dvl inactive. In this study, we show that non-canonical Wnt induces Dvl to transition from Vangl to Fz in Xenopus embryos. Pk inhibits the transition and functionally synergizes with Vangl to suppress Dvl during CE. Conversely, Ror is required for the transition and functionally antagonizes Vangl. Biochemically, Vangl interacts directly with both Ror and Dvl. Ror and Dvl do not bind directly but can be co-fractionated with Vangl. Collectively, we propose that Pk assists Vangl to function as an unconventional adaptor that brings Dvl and Ror into a complex to serve two functions: (1) simultaneously preventing both Dvl and Ror from ectopically activating non-canonical Wnt signaling; and (2) relaying Dvl to Fz for signaling activation upon non-canonical Wnt-induced dimerization of Fz and Ror.

???displayArticle.pubmedLink??? 42059436
???displayArticle.pmcLink??? PMC13132548
???displayArticle.link??? Elife
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Species referenced: Xenopus laevis
Genes referenced: dvl2 fzd7 prickle1 ror2 vangl2 wnt11
GO keywords: non-canonical Wnt signaling pathway [+]
???displayArticle.antibodies??? Dvl2 Ab3 FLAG Ab3 GFP Ab7 Myc Ab23 Ror2 Ab5
???displayArticle.morpholinos??? prickle1 MO1 ror2 MO2 vangl2 MO2

???displayArticle.disOnts??? Robinow syndrome [+]

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	Figure 1. Prickle (Pk) synergizes with Vangl to suppress Dishevelled (Dvl) during convergent extension (CE). Injecting 0.1 ng mouse Vangl2 mRNA (mVangl2) into the dorsal marginal zone (DMZ) results in moderate CE defects and reduction of the length- to- width ratio (LWR), and the phenotypes are significantly enhanced by co- injecting a small dose of Xpk (a, a’) or mPk2 (b, b’) mRNA that causes minimal or no CE defect per se. On the other hand, higher dose mVangl2 (0.15 ng) induced more severe CE defects that can be rescued by knocking down endogenous Pk using XpkMO (c, c’); whereas, knockdown of endogenous vangl2 (XVMO) induced CE defects that can be rescued by moderate mPk2 overexpression (d, d’). Conversely, 0.5 ng mouse Dvl2 injection- induced CE defects can be dose- dependently rescued by mPk2 (e, e’) or Xpk (f, f’) co- overexpression; and mVangl2- mPk2 co- overexpression- induced severe CE defect can be rescued by co- injecting Dvl2 (g, g’). Similarly, in activin- induced animal cap elongation assay, mVangl2 synergizes with Xpk to induce strong CE defect, which can be rescued significantly by co- injection of mouse Dvl2 (h, h’). CE phenotype was determined by quantifying the length- to- width ratio (LWR) of the embryos or animal cap explants in each group (a, b, c, d, e, f, g, h). Experiments were repeated three times, and the total number of embryos or explants analyzed is indicated below each panel in (a)-(h). Data are presented as box plots in (a’), (b’), (c’), (d’), (e’), (f’), (g’) and (h’), with the whiskers indicating the minima and maxima, the center lines representing median, the box upper and lower bounds representing 75th and 25th percentile, respectively. Two- tailed, unpaired T- test was used to compare the LWR of different groups, and the p values are indicated in (a’)-(h’) between different groups.
	Figure 1—figure supplement 1. Prickle (Pk) knockdown or overexpression induces convergent extension (CE) defects. Dorsal marginal zone (DMZ) injection to over-express (a, a’) or morpholino knockdown (b, b’) of Xpk can dose-dependently block CE, resulting in shortened body axes and reduced LWR. In the animal cap explant, XpkMO knockdown can also block activin-induced CE (c, c’), but the defect can be rescued by co-injecting RNA encoding mouse Pk2 (d, d’). CE phenotype in (a) and (b) was determined by quantifying the length-to-width ratio (LWR) of the embryos in each group. Experiments were repeated three times, and the total number of embryos analyzed is indicated below each panel in (a) and (b). Quantification of the length of the animal cap explants are in (c’) and (d’). Data are presented as box plots in (a’), (b’), (c’), and (d’), with the whiskers indicating the minima and maxima, the center lines representing median, the box upper and lower bounds representing 75th and 25th percentiles, respectively. Two-tailed, unpaired t-test was used to compare the LWR of different groups, and the p values are indicated in (a’)-(d’) between different groups.
	Figure 2. Vangl2 RH variant diminishes membrane recruitment of Prickle (Pk) and reduces functional synergy with Pk. Schematic illustration showing the structure of Vangl2 and location of the R177H variant at the intracellular loop region between transmembrane domains 2 and 3 (a). When expressed in Xenopus animal cap cells, EGFP-Vangl2 RH displays plasma membrane localization indistinguishable from wild-type EGFP-Vangl2 (b, b’). Immunostaining shows that flag-XPk displays diffuse cytoplasmic localization with some membrane enrichment when expressed alone (c, c’). The plasma membrane localization of flag-XPk is enhanced significantly by co-expression of wild-type Vangl2 (d, d’), but only modestly by Vangl2 RH variant (e, e’). (f) Quantification of the ratio of plasma membrane vs. cytoplasmic flag-XPk signal intensity in (c), (d), and (e). Co-IP and western blot show that the R177H mutation does not alter Vangl2 protein level but reduces binding to flag-XPk (g, n=3 biological repeats). Functionally, co-injection of 0.05 ng Vangl2 and 0.5 ng Xpk can strongly synergize to disrupt convergent extension (CE), but the synergy is significantly reduced when Vangl2 RH variant mRNA is co-injected with Xpk (h). The CE phenotype was determined by quantifying the length-to-width ratio (LWR) of the embryos in each group in (h). Experiments were repeated three times, and the total number of embryos analyzed is indicated below each panel in (h). Data are presented as box plots in (i), with the whiskers indicating the minima and maxima, the center lines representing median, the box upper and lower bounds representing 75th and 25th percentiles, respectively. Two-tailed, unpaired t-test was used to compare the LWR of different groups, and the p values are indicated between different groups. Scale bars represent 30 μm in b-e; 10 μm in c’, d’, e’.
	Figure 2—figure supplement 1. Prickle (Pk), Vangl2 and Dvl2 do not cross- regulate each other’s protein stability in Xenopus embryos during convergent extension (CE). Prickle (Pk), Vangl2 and Dvl2 do not cross-regulate each other’s protein stability in Xenopus embryos during convergent extension (CE). EGFP-Vangl2, flag-Dvl2, and GFP-mPk2 mRNA were injected into the dorsal marginal zone (DMZ), either alone or together, at 4 cell stage, and embryos were harvested at stages 10.5–11 for western blot. Co-overexpressing Vangl2 with mPk2, or Dvl2 with mPk2, does not significantly alter each other’s protein level (a). Similarly, co-overexpressing Flag-XPk with EGFP-Vangl2 in activin-treated animal cap explant also does not alter each other’s protein level (b). Knocking down endogenous Xvangl2 does not alter the protein level of injected Flag-XPk either (c). Interestingly, however, co-transfecting Vangl2 with XPk into 293T cells reduced Xenopus Pk1 (XPk) protein level (d).
	Figure 2—figure supplement 2. Vangl2 enhances Pk2 plasma membrane localization in animal cap cells. EGFP-tagged mPk2 displays uniform cytoplasmic distribution with weak enrichment at the plasma membrane when expressed in animal cap cells (a, c, and enlarged view in c). Plasma membrane enrichment of mPk2 is enhanced by co-injection of mVangl2 (b), but eliminated by morpholino knockdown of endogenous XVangl2 (d). Injecting a small amount of mVangl2 (0.05 ng) could restore mPk2 plasma membrane enrichment in XVangl2 morphant animal cap explants (e). Scale bars represent 30 μm.
	Figure 2—figure supplement 3. Vangl2 enhances Pk2 plasma membrane localization in dorsal marginal zone (DMZ) cells. EGFP-mPk2 displayed both diffused cytoplasmic distribution and enrichment at the plasma membrane when injected into the DMZ (a-a”). Plasma membrane enrichment of mPk2 is enhanced by co-injection of 0.05 ng mVangl2 (b-b”), but diminished by knockdown of endogenous XVangl2 with 30 ng morpholino (c-c”). Co-injecting 0.05 ng mVangl2 mRNA can rescue mPk2 plasma membrane enrichment in XVangl2 morphants (d-d"). EGFP-mPk2 membrane enrichment was measured by calculating the ‘normalized EGFP-mPk2 membrane/ cytoplasm ratio’ in (e). For this calculation, membrane mCherry (mem-mCh) was co-injected in each experiment to mark the plasma membrane, and mCh signal was used to segment the plasma membrane from the cytoplasm in ImageJ. The average signal intensities of EGFP-mPk2 and mem-mCh on the plasma membrane and in cytoplasm were then measured using ImageJ. The membrane-to-cytoplasm signal intensity ratio of EGFP-mPk2 is divided by that of mem-mCh to calculate the ‘normalized EGFP-mPk2 membrane/ cytoplasm ratio.’ Each experiment was repeated with three separate injections on different days, and at least three embryos were injected each time for imaging analyses. An unpaired t-test was used to compare the ‘normalized mPk2 membrane/ cytoplasm ratio’ of different groups, and the p values are indicated between different groups. Scale bars represent 30 μm.
	Figure 2—figure supplement 4. Vangl2 R177H variant does not alter Vangl2-Dvl2 interaction. Dvl2-EGFP displays uniform cytoplasmic distribution when expressed alone in Xenopus animal cap cells (a, a’), but co-expression with either wild-type Vangl2 (b, b’) or Vangl2 R177H variant (c, c’) recruits Dvl2-EGFP to the plasma membrane. (d) Quantification of the ratio of plasma membrane vs. cytoplasmic Dvl2-EGFP signal intensity in (a), (b), and (c). Co-IP and western blot did not reveal significant impact of R177H on either Vangl2 protein level or binding to Dvl2 (e). Scale bars represent 30 μm in a-c; 6 μm in a’-c’.
	Figure 2—figure supplement 5. R177H mutation diminishes Vangl2 activity in convergent extension (CE). When injected into the dorsal marginal zone (DMZ), 0.2 ng, and 0.1 ng wild-type mouse Vangl2 mRNA cause severe and moderate CE defect, respectively. Vangl2 R177H mRNA, however, causes significantly milder CE defect (a). Fz7 and Dvl2 co-injection caused severe CE defects that can be rescued by 0.1 wild-type Vangl2, but not by Vangl2 R177H (b). CE phenotype in (a) and (b) was determined by quantifying the length-to-width ratio (LWR) of the embryos in each group. Experiments were repeated three times, and the total number of embryos analyzed is indicated below each panel in (a) and (b). Data are presented as box plots in (a’) and (b’), with the whiskers indicating the minima and maxima, the center lines representing median, the box upper and lower bounds representing 75th and 25th percentile, respectively. Two-tailed, unpaired t-test was used to compare the length-to-width ratio (LWR) of different groups, and the p values are indicated in (a’) and (b’) between different groups.
	Figure 3. Prickle (Pk) synergizes with Vangl2 to inhibit Wnt11-induced formation of Dishevelled (Dvl) patches. In animal cap explants, 0.25 ng Wnt11 injection with 0.5 ng of mCh-tagged mouse Dvl2 (Dvl2-mCh) and membrane-GFP (mGFP) induces formation of distinct Dvl2 patches at the cell-cell contact (a-c’). Co-injection indicates that these Dvl2 patches completely overlap with Fz7-EGFP (d-f’). In contrast, Vangl2, when expressed at moderate levels (0.1 ng), is distributed more broadly along the plasma membrane (g–i), but also displays enrichment immediately outside and at the edge of Dvl2 patches (g’-I’, arrowheads and arrows, respectively). A high level of Vangl2 injection (0.5 ng) inhibits Wnt11-induced Dvl2 patch formation and makes Dvl2 more evenly distributed with Vangl2 (j-l’). The same effect can also be achieved by co-expressing Pk with moderate levels of Vangl2 (m-o’). (p–t) Measurement of the relative intensity of Dvl2 along the patches with either membrane GFP, Fz7, Vangl2 at moderate (0.1 ng) and high (0.5 ng) levels, or Vangl2 (0.1 ng) with Xpk (0.5 ng) co-injection. Scale bars represent 15 μm in a-o; 4 μm in a’-o’.
	Figure 3—figure supplement 1. Overexpression of Prickle (Pk) alone cannot effectively disrupt Dvl-Fz interaction in Xenopus. In both animal cap and dorsal marginal zone (DMZ) cells, Fz7 can recruit Dvl2 from the cytoplasm to the plasma membrane (compare b to a, and e to d). Overexpression of Pk2 does not inhibit Fz7-mediated plasma membrane recruitment of Dvl2 (c, f). Scale bars represent 30 μm.
	Figure 3—figure supplement 2. Prickle (Pk) inhibits Wnt11-induced dissociation of Dvl2 from Vangl2. (a) In dorsal marginal zone (DMZ) explant, binding between flag-Dvl2 and EGFP-Vangl2 is reduced by co-injection of Wnt11. But overexpression of Pk2 can prevent Wnt11-induced dissociation of Dvl2 and Vangl2. (b) Quantification of Vangl2 co-IP over pulled down Flag-Dvl2 from three separate experiments.
	Figure 3—figure supplement 3. Prickle (Pk) synergizes with Vangl2 to inhibit Wnt11-induced formation of Dishevelled (Dvl) patches in dorsal marginal zone (DMZ) explants. In DMZ explants, 0.1 ng of EGFP-tagged mouse Dvl2 (Dvl2-EGFP) and 0.2 ng of mScarletI-tagged mouse Dvl2 (Dvl2-mSc) are separately injected into two adjacent blastomeres with Wnt11. Wnt11-induced Dvl2-mSc and Dvl2-EGFP patches are both observed, and they are aligned along the border of adjacent cells (a-c, and an enlarged view of the boxed area in a’-c’). Co-injection of Dvl2-mSc with Fz7-EGFP indicates complete overlap between Dvl2 and Fz7 in the patches (d-f’). In contrast, Vangl2, when expressed at moderate levels (0.1 ng), is distributed more broadly along the plasma membrane (g–i), but also displays enrichment immediately outside and at the edge of Dvl2 patches (g’-I’, arrowheads and arrows, respectively). A high level of Vangl2 injection (0.5 ng) perturbs Wnt11-induced Dvl2 patch formation and makes Dvl2 more evenly distributed with Vangl2 (j-l’). The same effect can also be achieved by co-expressing Pk with moderate levels of Vangl2 (m-o’). (p–t) Measurement of the relative intensity of Dvl2-mSc along the patches with either Dvl2-EGFP, Fz7, Vangl2 at moderate (0.1 ng) and high (0.5 ng) levels, or Vangl2 (0.1 ng), and XPk (0.5 ng) co-injection. Scale bars represent 15 μm in a-o; 4 μm in a’-o’.
	Figure 3—figure supplement 4. Vangl2 forms rings to encircle Wnt11-induced Dvl2 patches on the plasma membrane. A moderate level of EGFP-Vangl2 (0.1 ng) was co-injected with Wnt11 and mCherry -tagged Dvl2. 3D reconstruction of confocal images shows that under this condition, all Dvl2 proteins on the plasma membrane formed discrete patches at the apical cell-cell junctions (a), while Vangl2 was distributed more diffusely on the plasma membrane (b) with enrichment overlapping with Dvl2 patches (c, white arrows). Enlarged views (a’-c’) show that enriched Vangl2 forms rings that encircle Dvl2 patches. Scale bars represent 30 μm in a-c; 4 μm in a’-c’.
	Figure 4. Prickle (Pk) helps Vangl2 to inhibit Wnt11-induced clustering of Fz7-Dvl2 complexes. In animal cap explants, Wnt11 induces formation of overlapping Fz7-EGFP and Dvl2-mCh patches at the cell-cell contact (a-c’). These patches are not affected by over-expressing moderate levels of Vangl2 (d-f’, 0.1 ng mRNA) or XPk (g-i’, 0.5 ng mRNA) individually. Vangl2 and XPk co-expression, however, not only disrupts Dvl2-mCh patches but also disperses Fz7-EGFP patches into small puncta (j–l). Enlarged views revealed that some of the Fz7-EGFP puncta are on the plasma membrane and remain co-localized with Dvl2-mCh, while the others are located in the cytoplasm near the plasma membrane (k’’, l’, arrows) and contain only Fz7 but not Dvl2 (compare arrows in j’ to k’). Scale bars represent 30 μm in a-l; 10 μm in a’-l’.
	Figure 4—figure supplement 1. Prickle (Pk) helps Vangl2 to inhibit Wnt11-induced clustering of Fz7-Dvl2 complexes in dorsal marginal zone (DMZ) explants. In DMZ explants, Wnt11 induces formation of overlapping Fz7-EGFP and Dvl2-mScarletI (Dvl2-mSc) patches at the cell-cell contact (a-c’). These patches are not affected by co-injecting moderate amounts of Vangl2 (d-f’, 0.1 ng mRNA) or XPk (g-i’, 0.5 ng mRNA) individually. Vangl2 and Pk co-expression, however, not only diminished Dvl2 patches but also dispersed Fz7 into small puncta (j–l). Enlarged views revealed that some of the Fz7 puncta are on the plasma membrane and remain co-localized with Dvl2, while the others are located in the cytoplasm near the plasma membrane (k’’, l’, arrows) and contain only Fz7 but not Dvl2 (compare arrows in j’ to k’). Scale bars represent 30 μm in a-l; 10 μm in a’-l’.
	Figure 4—figure supplement 2. Prickle (Pk)/Vangl2 co-expression induces Fz7 endocytosis. Xenopus explants were incubated with FM4-64FX, a fluorescent dye that is membrane impermeable and can only be internalized through endocytosis. In explants in which Fz7-GFP was co-injected only with Wnt11, Fz7 primarily formed patches on the cell cortex. Most of the internalized FM4-64FX puncta were devoid of Fz7, and the few Fz7 puncta were devoid of FM4-64FX (arrowheads in a-c, and enlarged views a’-c’). In contrast, in explants in which Fz7-GFP and Wnt11 were co-injected with Vangl2/Pk, the majority of the Fz7-GFP puncta are positive for FM4-64FX (arrows in d-f and enlarged views d’-f’), indicating that they were largely formed through endocytosis. Scale bars represent 15 μm in a-f; 4 μm in a’-f’.
	Figure 4—figure supplement 3. Mitochondria sequestration of endogenous Dishevelled (Dvl) induces Fz7 endocytosis. Wnt11 induces formation of Fz7 patches along the plasma membrane (a). Overexpression of DshMA, a mitochondria-tethered form of Dvl that can sequester endogenous Dvl/Dsh away from the plasma membrane, inhibits Fz7 patch formation, reduces membrane level of Fz7, and results in Fz7 cytoplasmic puncta (b). Scale bars represent 15 μm.
	Figure 5. Vangl2 R177H variant fails to synergize with Prickle (Pk) to inhibit Fz patch formation and down-regulate Fz stability at the plasma membrane. Wnt11-induced formation of Fz7-GFP patches on the plasma membrane (a, a’) was not affected by moderate expression either of wild-type Vangl2 (b, b’) or Vangl2 R177H variant (c, c’) alone. Xpk co-injection synergized with wild-type Vangl2 to diminish Fz7 patch formation and induce cytoplasmic Fz7 puncta (d, d’), but the synergy was not observed with Vangl2 R177H (e, e’). Scale bars represent 30 μm in a-e; 5 μm in a’-e’.
	Figure 6. Prickle (Pk) synergizes with Vangl2 to prevent Frizzled (Fz)-induced phosphorylation of Dishevelled (Dvl). In Xenopus extract, dorsal marginal zone (DMZ)-injected Dvl2 migrates as two bands, with the slower migrating band increasing in intensity from stage 10–12 as convergent extension (CE) starts and progresses during gastrulation (a). The slower migrating form of Dvl2 is increased by Fz7 co-injection but eliminated by phosphatase treatment (b). High-level Vangl2 injection can reduce the phosphorylated form of Dvl2, while Fz7 can counter Vangl2’s effect to increase Dvl2 phosphorylation (c). Co-IP experiment indicates that only the faster migrating, presumably unphosphorylated form of Dvl2 can be co-immunoprecipitated by Vangl2 (d). High-level Xpk (1 ng) or moderate Vangl2 (0.25 ng) cannot significantly reduce phosphorylated form of Dvl2 when injected individually, but their co-injection can suppress Dvl2 phosphorylation (e). (f) Quantification of the ratio between the slow-migrating/phosphorylated and fast-migrating/unphosphorylated forms of Dvl2 in (e), n=3 biological repeats.
	Figure 7. Ror2 is an obligatory component of the Frizzled (Fz)/Dishevelled (Dvl) cluster complex induced by Wnt11. EGFP-tagged Xenopus Ror2 (0.1 ng mRNA injection) is distributed homogeneously on the plasma membrane when injected with membrane-mCherry mem-mCh (a–c), but can be induced to form distinct patches upon Wnt11 co-injection (d-f, 0.25 ng). With Dvl2-mCh co-injection (0.5 ng), the Ror2 patches show overlap with Dvl2 patches (g–i). Unlike Dvl2, however, Ror2 additionally displays broad distribution along the entire cell cortex (compare g to h, and also see d). The enlarged views show that both Ror2 and Dvl2 accumulate at the center of the patches (red arrowhead), but Ror2 patches are slightly longer and extend beyond the border of Dvl2 patches (green arrowheads in g’-i’). (j) Measurement of the relative intensity of Dvl2-mCh along the patches with Ror2-EGFP (upper panel), and quantification of the ratio between Ror2 and Dvl2 intensity along the patches (bottom panel). Wnt11 induces Dvl2-mCh patch formation on the cell cortex (k) and is blocked by co-injecting 25 ng Xror2 morpholino (Xror2-MO) (l). Scale bars represent 15 μm in a-i; 4 μm in g’-i’; 30 μm in k-l.
	Figure 7—figure supplement 1. Ror2 co-overexpression rescues Vangl2 overexpression-induced convergent extension (CE) defects. Dorsal marginal zone (DMZ) injection of 0.2 ng of Vangl2 mRNA induces severe CE defects, which can be rescued by co-injecting 0.05 or 0.1 ng of Xror2 mRNA (a). CE phenotype in (a) was determined by quantifying the length-to-width ratio (LWR) of the embryos in each group. Experiments were repeated three times, and the total number of embryos analyzed is indicated below each panel in (a). Data are presented as box plots in (b), with the whiskers indicating the minima and maxima, the center lines representing median, the box upper and lower bounds representing 75th and 25th percentiles, respectively. Two-tailed, unpaired t-test was used to compare the LWR of different groups, and the p values are indicated in (b) between different groups.
	Figure 7—figure supplement 2. In Xenopus embryo extract, the co-IP experiment shows that binding between Dvl2-EGFP and Myc-Vangl2 is reduced by Wnt11 (compare lanes 2 and 3). Knocking down endogenous XRor2 with morpholino (Xror2 MO) can prevent Wnt11-induced dissociation of Dvl2 and Vangl2 (compare lanes 4 and 5) (a). (a’) Quantification of Dvl2-EGFP co-IP against Myc-Vangl2 pull down (n=3). Note that Dvl2-Vangl co-IP is reduced with XRor2 knockdown, either with or without Wnt11 co-injection. (b) Co-IP experiment shows that Ror2-Flag can be pulled down by co-injected Myc-Vangl2 but not Myc-Dvl2. (c) Fluorescence-detection size exclusion chromatography (FSEC) with protein extract from Xenopus embryos injected with Xror2-EGFP, HA-Vangl2, and Flag-Dvl2. A peak of EGFP signal is detected around fraction 15 (lower panel). Western blot analyses show co-fractionation of Ror2-EGFP, HA-Vangl2, and Flag-Dvl2 in fractions 14 (approximate molecular weight 773–1717 kD), 15 (~348–773 kD), and 16 (~166–348 kD).
	Figure 7—figure supplement 3. XRor2 knockdown does not alter plasma membrane recruitment of Dvl2 by Vangl2. (a) Scheme of making chimeric embryos to examine Dvl2 plasma membrane recruitment by Vangl2 with or without Ror2 knockdown. Two cell stage embryos receive the first injection into the animal side of both blastomeres, with mRNA encoding Dvl2-mScarlet-I and mem-miRFP670. The embryos are cultured to the 32 cell stage and receive a second injection into a single animal pole blastomere with mRNA encoding EGFP-Vangl2 or EGFP-Vangl2 with Ror2 MO. The embryos are cultured to stage 8, and the animal caps are dissected for imaging. (b) Dvl2 plasma membrane recruitment by Vangl2 is measured by the average plasma membrane-to-cytoplasm intensity ratio of Dvl2 in cells co-expressing Dvl2 and Vangl2, normalized by that in cells expressing Dvl2 alone. (c–f) Image of animal cap explant from embryos injected with EGFP-Vangl2 at 32 cell stage. (g–j) Image of animal cap explant from embryos injected with EGFP-Vangl2 and Ror2 MO at 32 cell stage. Scale bars represent 30 μm.
	Figure 7—figure supplement 4. Ror2 overexpression alone cannot recruit Dvl2 to the plasma membrane. (a) Scheme of making chimeric embryos to examine Dvl2 plasma membrane recruitment by Ror2. 2 cell stage embryos receive the first injection into the animal side of both blastomeres, with mRNA encoding Dvl2-mScarlet-I and mem-miRFP670, a plasma membrane marker. The embryos are cultured to the 32 cell stage and receive a second injection into a single animal pole blastomere with mRNA encoding either mem-EGFP or varying levels of Ror2-EGFP. The embryos are cultured to stage 8, and the animal caps are dissected for imaging. (b) Dvl2 plasma membrane localization is measured by the average plasma membrane-to-cytoplasm intensity ratio of Dvl2 in cells co-expressing Dvl2 and Ror2 (or mem-EGFP control), normalized by that in cells expressing Dvl2 alone. (c–f) Image of animal cap explant from embryos injected with mem-EGFP control at 32 cell stage. (g–r) Image of animal cap explant from embryos injected with 0.5 ng (g–j), 1 ng (k–n), or 1.5 ng (o–r) of Ror2-EGFP mRNA at 32 cell stage. Scale bars represent 30 μm.
	Figure 8. Vangl2/Prickle (Pk) exerts bimodal regulation of Ror2 in non-canonical Wnt signaling. (a–c) In animal cap explants, Wnt11 induces co-injected XRor2-tdTomato and Fz7-EGFP to co-cluster into patches on the cell cortex. Overexpression of a moderate level of Vangl2 (d-f, 0.1 ng) or Pk (g-I, 0.5 ng) individually does not perturb co-clustering of Ror2 with Fz7 into patches in response to Wnt11, but their co-overexpression (j–l) diminishes Ror2-Fz7 patches into small puncta and causes Fz7 to form cytoplasmic puncta, whereas Ror2 remains on the plasma membrane (compare arrows in j’-l’). a’-l’ are enlarged views of a-l, respectively. Conversely, partial knockdown of endogenous XVangl2 with a moderate level of XVMO (14 ng) also diminished Ror2/Fz7 patch formation in response to Wnt11 (m–o’) with simultaneous formation of intracellular puncta around the plasma membrane which contain Fz7 but not Ror2 (m’–o’), arrows. Scale bars represent 30 μm in a-o; 4 μm in a’-o’.
	Figure 8—figure supplement 1. Prickle (Pk) helps Vangl2 to inhibit Wnt11-induced clustering of Fz7 and Ror2. (a–c) In dorsal marginal zone (DMZ) explants, Wnt11 induces co-injected XRor2-tdTomato and Fz7-EGFP to co-cluster into patches on the cell cortex. Overexpression of a moderate level of Vangl2 (d-f, 0.1 ng) or Pk (g-I, 0.5 ng) individually does not perturb co-clustering of Ror2 with Fz7 into patches in response to Wnt11, but their co-overexpression (j–l) diminishes Ror2-Fz7 patches into puncta and causes Fz7 to form cytoplasmic puncta, whereas Ror2 remained on the plasma membrane (compare arrows in j’-l’). a’-l’ are enlarged views of a-l, respectively. Scale bars represent 30 μm in a-l; 4 μm in a’-l’.
	Figure 9. An integrated model for non-canonical Wnt signaling regulation during convergent extension (CE). (a, a’) In the absence of non-canonical Wnt, Pk helps Van Gogh-like (Vangl) to act as an adaptor that brings together Dishevelled (Dvl) and Ror, through either simultaneous binding of both Dvl and Ror to a single Vangl (a) or oligomerization of Vangl proteins bound separately to Dvl and Ror (a’), and keeps both Dvl and Ror inactive to prevent ectopic non-canonical Wnt signaling. (b, b’) Non-canonical Wnt initiates signaling by triggering Frizzled (Fz)-Ror heterodimerization, and in turn, the complexes consisting of Ror/Vangl/Dvl are brought close to Fz to deliver Dvl for activation of downstream targets. (c, c’) Non-canonical Wnt also induces other events, such as Dvl phosphorylation to facilitate Dvl dissociation from Vangl and transition to Fz in a spatially and temporally controlled manner.