Ossipova O , Chu CW , Fillatre J , Brott BK , Itoh K , Sokol SY .

R01 NS040972 NINDS NIH HHS , R21 HD078874 NICHD NIH HHS , R56 NS040972 NINDS NIH HHS , NS40972 NINDS NIH HHS

Xla Wt + dvl2{del_1236-2208} (fig.6.h)
Xla Wt + kif13b MO (fig.4.b,c)
Xla Wt + prickle3 MO (fig.3.b)
Xla Wt + prickle3 MO (fig.S2.e)
Xla Wt + prickle3{del} (fig.S2.c)
Xla Wt + prickle3{del} (fig.S3.b)
Xla Wt + prickle3{del} (fig.S3.d)
Xla Wt + prickle3{del} (fig.S3.e)
Xla Wt + vangl2 MO (fig.1.d, f, i)
Xla Wt + vangl2 MO (fig.6.b)
Xla Wt + vangl2 MO (fig.6.c-d)
Xla Wt + vangl2 MO (fig.6.e)
Xla Wt + vangl2 MO (fig.S1.b)
Xla Wt + vangl2 MO (fig.S1.d)
Xla Wt + {dn}dvl2 (fig.3.e)
Xla Wt + {dn}dvl2 (fig.6.g)
Xla Wt + {dn}dvl2 (fig.S3.a)
Xla Wt + {dn}dvl2 (fig.S3.d)

	Fig. 1. Vangl2 is required for the migration of skin multiciliated cells. (A, B) The Vangl2/Pk complex has a polarized apical distribution in MCC precursors. Four-cell embryos were injected with RNAs encoding CFP-Vangl2 (0.1 ng) and Pk (0.05–0.1 ng), embryos were fixed at stage 18 and immunostained with anti-GFP (A) or anti-GFP and anti-centrin antibodies (B). Arrows point to apically polarized Vangl2. Scale bar in A is 20 µm, bar in B, 10 µm, also applies to C, D. (C–J) Depletion of Vangl2 inhibits MCC intercalation into the superficial cell layer. Four-cell embryos were unilaterally injected with Vangl2 MO (10 ng per blastomere) and GFP RNA (0.1 ng) for lineage tracing with or without HA-Vangl2 RNA (0.1 ng). The embryos were fixed at indicated stages and immunostained for GFP and acetylated tubulin (Ac-tub) (C, D) or centrin (E–J) to mark MCCs. (E–J) Vangl2 RNA injection partly rescues MCC intercalation defect caused by Vangl2 MO. (E–G) En face view. (H–J) Embryo sections. Arrows point to MCCs in the superficial cell layer. Asterisks indicate a deficiency in MCC migration. The apical surface position (at the top) and MCC boundaries are shown by dotted lines. Scale bars in E, H are 10 µm. (K–M) Quantitation of data for the experiments shown in (C–J) and Fig. S1. The percentage of MCCs that fail to migrate and remain in the deep cell layer was calculated separately for Ac-tub (K), centrin (K, L) or Rab11 (M) relative to the total number of MCCs. Number of scored MCCs (n) is shown on top of each bar. All graphs present means±s. e. Statistical significance was determined by the Student's t-test (**, p=0.014). Data are representative of two to four independent experiments, 4–7 embryos were examined for each treatment.
	Fig. 2. Expression of prickle3 transcripts in Xenopus early embryos. Embryos were subjected to whole-mount in situ hybridization with Pk3 anti-sense RNA probe or sense probe as a control. Pk3 transcripts are present in the animal pole ectoderm of stage 10 gastrulae (A), and non-neural ectoderm of stage 15 neurulae (B). (C) Stage 18 embryo. Epidermal ectoderm is strongly positive (arrowhead), as compared to neuroectoderm. (D) Stage 21 embryo, anterior region, with staining in the profundal and posterior region placodes (arrowhead). (E) Stage 24 embryo, the otic vesicle and the posterior placodal region are positive (arrowheads). (F) Stage 26 embryos, tailbud. An embryo hybridized with the sense probe is shown on bottom. Anterior is to the left. Lateral view is shown in all panels, except dorsal view in C. (G, H) Cross-sections of stage 18 embryos. Arrowheads point to the staining in neural folds (G) and in both inner and outer layers of the epidermis (H). Positions of nuclei in the epidermis are outlined by dotted lines. Abbreviations: n, notochord, s, somites, np, neural plate.
	Fig. 3. Interference with Pk3 and Disheveled leads to MCC intercalation defects. (A–C) Role of Pk3 in MCC intercalation. Four-cell embryos were injected with control morpholino (COMO) or Pk3 MO1 (25 ng per blastomere), along with GFP RNA (0.1 ng) as a lineage tracer. Stage 22 embryos were cryosectioned and immunostained for Centrin and GFP. Arrows indicate MCCs integrated into the superficial cell layer. Asterisks point to MCCs that failed to migrate. Dashed lines mark the boundaries of the epidermis, apical is up. (D–G) Effects of Disheveled mutant constructs on MCC intercalation. Xdd1 and Xdd2 RNAs (0.5–1 ng each) were coinjected with GFP RNA into four-cell embryos, embryos were either lysed at stage 11 for protein analysis (D) or fixed at stage 22 and immunostained for GFP and Centrin (E, F). (D) Immunoblot analysis with anti-Myc antibodies showing Xdd1 and Xdd2 expression levels. Scale bar in B is 20 μm, also applies to (A, E, F). (C, G) Quantification of data. Scoring has been done as described in Fig. 1. All graphs show means±s. e. At least 6–10 embryos were examined per each treatment. n, number of examined cells.
	Fig. 4. KIF13B is a Disheveled-interacting protein that synergizes with PCP signaling to regulate MCC intercalation. (A) KIF13B co-immunoprecipitates with Disheveled. HEK293T cells were transfected with Myc-Dvl2 and FLAG-KIF13B. KIF-13B complexes were pulled down from cell lysates using anti-FLAG antibodies coupled to agarose, and both KIF13B and Myc-Dvl2 were visualized by western blotting. (B) Depletion of KIF13B affects MCC integration into the superficial cell layer. Control MO or KIF13B MO (40 ng each) were injected into 8-cell stage embryos. MCC migration was analyzed in cryosections of stage 22 embryos by immunostaining for centrin. Depletion of KIF13B induced MCC migration defects (arrow). Scale bar is 15 μm. (C). Quantification of MCC migration defects. Rescue experiments were performed by co-injecting KIF13B MO with 150 pg of human MYC-tagged KIF13B RNA. Data are presented as means±s. e. Statistical significance was determined by the Student's t-test (*, p<0.05). (D) Synergistic effects of KIF13B, Vangl2 and Disheveled on MCC intercalation. Eight-cell stage embryos were injected with Vangl2 MO (5 ng), KIF13B MO (10 ng), or Xdd2 mRNA (0.25 ng), separately or in combination as indicated. MCC migration was quantified as in (C).
	Fig. 5. Polarized distribution of PCP proteins in embryonic neuroectoderm. (A) Scheme of neurula stage embryo and a transverse section of the Xenopus neural plate at the hindbrain level. Dashed line indicates section plane. The boxed area indicates an approximate location of images. (B) Neural plate section with apically directed Vangl2/Pk complexes shown at low magnification. Eight-cell embryos were injected into animal-dorsal blastomeres with RNAs encoding HA-Vangl2, Pk and GFP-CAAX (0.1 ng each). The embryos were cultured until stage 15, cross-sectioned and immunostained with indicated antibodies. The apical surface is at the top as indicated by dashed lines. Midline (M) is to the right. Arrows point to the polarized Vangl2/Pk complex in deep neuroctodermal cells. (C–C′) The localization of the Vangl2/Pk complex in embyos coinjected with HA-Vangl2 and Pk RNAs as compared to the lineage tracer GFP-CAAX. np, neural plate; s, somite; n, notochord. Scale bar in C” is 10 μm, also applies to (D–D′). (D–D′) HA-Vangl2 is not significantly polarized in the neural plate in the absence of overexpressed Pk. (E) A protrusion directed towards the outer surface (arrow) is detectable in a single GFP-CAAX-marked cell. (F, F′) A single deep layer cell containing an apical protrusion with enriched Vangl2/Pk complex (arrow). Scale bar is 10 μm, also applies to E, G. (G) Staining of neuroectoderm with anti-Vangl2 antibodies reveals apically enriched endogenous Vangl2 in an inner layer cell.
	Fig. 6. Radial intercalation of deep layer cells is inhibited in embryos deficient in PCP signaling. Four-to-eight-cell embryos were unilaterally injected into prospective dorsal or ventral ectoderm with control MO (A, CO MO, 15–20 ng) or Vangl2 MO (B, 15–20 ng). Coinjected GFP RNA (0.1 ng) is a lineage tracer. Embryos were cross-sectioned at stage 15 and immunostained for β-catenin to mark cell boundaries and GFP to identify the injected side of the embryo. Dashed lines mark notochord and somite boundaries. Vertical white bars indicate the thickness of the neural plate, which is increased in Vangl2-depleted tissues. Scale bar is 20 μm. M, midline (dashed line); Np, neural plate; s, somites; n, notochord. (C–E) Thickness of non-neural ectoderm is controlled by PCP signaling. (C) Injected embryo shown at low magnification. White boxed areas approximately correspond to those shown at higher magnification in C′ (injected side) and C′ (control uninjected side). Scale bar is 100 μm in C, 20 μm in C′, also applies to C′, D, E. Abbreviations: e, ectoderm; n, notochord; s, somites. (D) Control MO does not change ectoderm morphology. (E) The effect of Vangl2 MO on ectoderm thickness in stage 10.5 embryo. Up to 5–6 cell layers are visible after Vangl2 depletion (white bar) as compared with the uninjected side, containing 2 to 4 cell layers. Bar, 40 µm. (F–H) Thick ectoderm in embryos expressing the dominant interfering Disheveled constructs Xdd1 and Xdd2. Shown are representative sections of a control embryo (F) or embryos injected with 1 ng of Xdd1 (G) or Xdd2 (H) RNA into a ventral animal blastomere and immunostained for GFP and β-catenin to mark cell boundaries. White bars indicate ectoderm thickness (C–H).
	prickle3 (prickle 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage15, lateral view, anterior left, dorsal up.
	prickle3 (prickle 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage18, dorsal view, anterior left.
	prickle3 (prickle 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21,, head region, lateral view, anterior left, dorsal up.
	prickle3 (prickle 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, head region, lateral view, anterior left, dorsal up.
	prickle3 (prickle 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26/27, lateral view, anterior left, dorsal up.
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	Fig. S2
	Fig S3
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