Ossipova O , Kim K , Sokol SY .

R01 NS040972 NINDS NIH HHS , R21 HD078874 NICHD NIH HHS

	Figure 1. Polarized F-actin cables in the Xenopus neural plate. F-actin was visualized by phalloidin staining in the Xenopus neural plate at the beginning of neurulation. Representative explants are shown at stage 13 (st. 13, A), st. 14 (B) and st.15 (C). Boxed regions are magnified in (A’-C’). Apical constriction of neural plate cells is first evident along the dorsolateral hinge regions (arrowheads), and later Biology Open Accepted manuscript throughout the neural plate. F-actin cables become more pronounced in the mediolateral rather than anteroposterior orientation, forming stripes along anteroposterior cell faces (arrows). The anteroposterior (A-P) axis of the neural plate is indicated. Dashed line, the neural plate midline. Bar, 20 μm.
	Figure 2. Non-homogeneous subcellular distribution of Vangl2 along the anteroposterior axis. Early embryos were injected with 100 pg of GFP-CAAX RNA to label cell boundaries. At early neural plate stage, embryos were fixed and immunostaned with anti-Vangl2 and anti-GFP antibodies. En face view of the neural plate is shown, anterior is to the top. (A) Neural plate explant with the approximate position of the imaged area (B-B’’) (boxed). Dashed line indicates the neural midline, also applies to (D, E). The anteroposterior (A-P) and the mediolateral (M-L) axes are shown. (B) anti-Vangl2 and (B’) anti-GFP staining (B’’) Merged image. Arrows point to anterior Vangl2. (C) Double staining with anti-Vangl2 and anti-ZO1 reveals the anterior localization of Vangl2. (D) Cross-section of a neurula stage embryo injected with Vangl2 MO. Vangl2 immunostaining is reduced by the unilateral injection of Vangl2 MO (20 ng) (asterisk). Arrow points to apical Vangl2 at the uninjected side. (E) Apotome imaging of a normal control embryo costained with anti-Vangl2 and anti-ZO1. Z-stacks reveal apical/subapical staining of anterior Vangl2 patches at the midline (yellow arrow) as compared to the lateral/basolateral staining in non-neural ectoderm (green arrow). The midline is indicated. Scale bar, 20 μm.
	Figure 3. Vangl2 polarity is directed by Prickle. Early embryos were injected with HA-Vangl2 (150 pg) and YFP-Pk RNAs (100 pg) either separately or together as indicated. At early neural plate stage, embryos were fixed and immunostaned with anti-HA or anti-GFP antibodies. (A) HA-Vangl2 is homogeneously distributed at cell boundaries. (B-B’) The complex of Vangl2/Pk is polarized at the anterior end of each cell. HA-Vangl2 (B) and YFP-Pk (B’). (C, C’) The anterior localization of the Vangl2/YFP-Prickle complex in an expressing cell is visualized by YFP and membrane RFP (mRFP) epifluorescence. (D, E) Early embryos were coinjected with zPk1ΔPL RNA (1.5 to 2 ng) and GFP as lineage tracer (100 pg). (D, D’) Immunofluorescence reveals Vangl2 anterior accumulation (arrows) in the neural plate, with the exception of the cells containing zPk1ΔPL (marked by GFP, asterisks). Dorsal view of the neural plate midline is shown, anterior is to the top. (E) Quantitation of the data shown in D. Scale bars are 20 μm in B, D, and 5 μm in C.
	Figure 4. A role of Wnt signaling in establishing the Vangl2 polarity. (A, B) Early embryos were injected with DN-Wnt5a RNA (0.3 ng) and GFP RNA (0.1 ng) as a lineage tracer. Neural plate cells mosaically expressing this construct reveal lack of Biology Open Accepted manuscript Vangl2 enrichment at the anterior of each cell (asterisks) as compared to non-expressing cells (arrows). Control LacZ RNA (1.5 ng) coinjected with GFP RNA (0.1 ng) had no effect on anterior distribution of Vangl2. Dorsal view of the neural plate is shown, anterior is to the top. Bar, 20 μm. (C) Quantification of data from the experiments with DN-Wnt constructs showing mean frequencies of cells with anterior Vangl2 +/- s. d. At least 5-10 embryos were examined per each treatment. Numbers of scored cells are shown on top of each bar. (D) Vangl2 is phosphorylated in response to Wnt5a in Xenopus ectoderm. Early embryos were injected with HA-Vangl2 mRNA (0.1 ng) together with Wnt5a RNA (0.5 ng). Cell lysates were prepared at the midgastrula stage (st. 11) for western analysis with anti-HA antibodies. Wnt5a causes HA-Vangl2 to migrate slower in whole embryo lysates (st. 11). Asterisk indicates a non-specific protein band reflecting protein loading.
	Figure 5. Wnt-induced Vangl2 phosphorylation in the establishment of the Vangl2 polarity. (A-C) Different activities of overexpressed Vangl2 and Vangl2SA RNAs on neural plate closure. (A) Normal neurulation in embryos overexpressing wild-type Vangl2 RNA (100 pg). (B) Neural tube defects in embryos overexpressing Vangl2SA RNA (100 pg). (C) Quantification. (D) Immunoblot analysis with anti-HA antibodies shows comparable levels of Vangl2 constructs. Anti-β-catenin antibodies reveal protein loading. Note faster migration of non-phosphorylatable Vangl2. (E, F) Phosphorylation of Vangl2 is critical for the establishment of AP-PCP. YFP-Pk mRNA (50-100 pg) was unilaterally coinjected with HA-Vangl2 RNA or HA-Vangl2SA RNAs (100 pg each) at the 8-cell stage. mRFP RNA (100 pg) is a lineage tracer. AP-PCP is assessed by the recruitment of Pk to anterior (arrows) or lateral (arrowheads) cell edges near the neural midline (st 14/15) in the presence of HA-Vangl2 or HA-Vangl2SA. Bar, 20 μm.
	Figure 6. Feedback regulation of AP-PCP by the ROCK/Myosin II pathway. (A-B) Eight cell embryos were unilaterally coinjected with the lineage tracer (GFP RNA, 100 pg) and DN-ROCK RNA (100 pg, A) or Mypt1T696A RNA (100 pg, B) and Vangl2 polarity was assessed by immunostaining at the neural plate stage. Both the interference with ROCK signaling (A, A’) and the dephosphorylation of Myosin II light chain by the myosin phosphatase Mypt1 (B, B’) inhibit Vangl2 polarity. (C) Quantification of the data shown in A, B. (D) Model of AP-PCP. Wnt activity gradient leads to the polarization of the Vangl2/Pk complex to the anterior cell domain. Myosin II activity, a putative target of PCP signaling, mediates feedback regulation of core PCP protein localization. Bar, 20 μm.
	Fig. 1. Polarized F-actin cables in the Xenopus neural plate.F-actin was visualized by phalloidin staining in the Xenopus neural plate at the beginning of neurulation. Representative explants are shown at stage 13 (st. 13, A), st. 14 (B) and st.15 (C). Boxed regions are magnified in A′–C′. Apical constriction of neural plate cells is first evident along the dorsolateral hinge regions (arrowheads), and later throughout the neural plate. F-actin cables become more pronounced in the mediolateral rather than anteroposterior orientation, forming stripes along anteroposterior cell faces (arrows). The anteroposterior (‘A–P’) axis of the neural plate is indicated. Dashed line, the neural plate midline. Scale bar, 20 µm.
	Fig. 2. Non-homogeneous subcellular distribution of Vangl2 along the anteroposterior axis.Early embryos were injected with 100 pg of GFP-CAAX RNA to label cell boundaries. At early neural plate stage, embryos were fixed and immunostained with anti-Vangl2 and anti-GFP antibodies. En face view of the neural plate is shown, anterior is to the top. (A) Neural plate explant with the approximate position of the imaged area (B–B″) (boxed). Dashed line indicates the neural midline, also applies to (D,E). The anteroposterior (‘A–P’) and the mediolateral (‘M–L’) axes are shown. (B) anti-Vangl2 and (B′) anti-GFP staining (B″) Merged image. Arrows point to anterior Vangl2. (C) Double staining with anti-Vangl2 and anti-ZO1 reveals the anterior localization of Vangl2. (D) Cross-section of a neurula stage embryo injected with Vangl2 MO. Vangl2 immunostaining is reduced by the unilateral injection of Vangl2 MO (20 ng) (asterisk). Arrow points to apical Vangl2 at the uninjected side. (E) Apotome imaging of a control embryo costained with anti-Vangl2 and anti-ZO1. Z-stacks reveal apical/subapical staining of anterior Vangl2 patches at the midline (yellow arrow) as compared to the lateral/basolateral staining in non-neural ectoderm (green arrow). The midline is indicated. Scale bars, 20 µm.
	Fig. 3. Vangl2 polarity is directed by Prickle.Early embryos were injected with HA-Vangl2 (150 pg) and YFP-Pk RNAs (100 pg) either separately or together as indicated. At early neural plate stage, embryos were fixed and immunostaned with anti-HA or anti-GFP antibodies. (A) HA-Vangl2 is homogeneously distributed at cell boundaries. (B,B′) The complex of Vangl2/Pk is polarized at the anterior end of each cell. HA-Vangl2 (B) and YFP-Pk (B′). (C,C′) The anterior localization of the Vangl2/YFP-Prickle complex in an expressing cell is visualized by YFP and membrane RFP (mRFP) epifluorescence. (D,E) Early embryos were coinjected with zPk1ΔPL RNA (1.5 to 2 ng) and GFP as lineage tracer (100 pg). (D,D′) Immunofluorescence reveals Vangl2 anterior accumulation (arrows) in the neural plate, with the exception of the cells containing zPk1ΔPL (marked by GFP, asterisks). Dorsal view of the neural plate midline is shown, anterior is to the top. (E) Quantitation of the data shown in D. See also Fig. 4C for control RNA effect. Error bars represent s.d. Co, control cells on the uninjected side. Scale bars are 20 µm in B,D, and 5 µm in C.
	Fig. 4. A role of Wnt signaling in establishing the Vangl2 polarity.(A,B) Early embryos were injected with DN-Wnt5a RNA (0.3 ng) and GFP RNA (0.1 ng) as a lineage tracer. Neural plate cells mosaically expressing this construct reveal lack of Vangl2 enrichment at the anterior of each cell (asterisks) as compared to non-expressing cells (arrows). A', B' are single-channel images corresponding to A and B. Control LacZ RNA (1.5 ng) coinjected with GFP RNA (0.1 ng) had no effect on anterior distribution of Vangl2. Dorsal view of the neural plate is shown, anterior is to the top. Scale bar, 20 µm. (C) Quantification of data from the experiments with DN-Wnt constructs showing mean frequencies of cells with anterior Vangl2±s.d. At least 5–10 embryos were examined per each treatment. Numbers of scored cells are shown on top of each bar. (D) Vangl2 is phosphorylated in response to Wnt5a in Xenopus ectoderm. Early embryos were injected with HA-Vangl2 mRNA (0.1 ng) together with Wnt5a RNA (0.5 ng). Cell lysates were prepared at the midgastrula stage (st. 11) for western analysis with anti-HA antibodies. Wnt5a causes HA-Vangl2 to migrate slower in whole embryo lysates (st. 11). Asterisk indicates a non-specific protein band reflecting protein loading.
	Fig. 5. Wnt-induced Vangl2 phosphorylation in the establishment of the Vangl2 polarity.(A–C) Different activities of overexpressed Vangl2 and Vangl2SA RNAs on neural plate closure. (A) Normal neurulation in embryos overexpressing wild-type Vangl2 RNA (100 pg). (B) Neural tube defects in embryos overexpressing Vangl2SA RNA (100 pg). (C) Quantification. (D) Immunoblot analysis with anti-HA antibodies shows comparable levels of Vangl2 constructs. Anti-β-catenin antibodies reveal protein loading. Note faster migration of non-phosphorylatable Vangl2. The image of lane containing uninfected embryo lysate (Co) is obtained from the same gel. (E,F) Phosphorylation of Vangl2 is critical for the establishment of AP-PCP. YFP-Pk mRNA (50–100 pg) was unilaterally coinjected with HA-Vangl2 RNA or HA-Vangl2SA RNAs (100 pg each) at the 8-cell stage. mRFP RNA (100 pg) is a lineage tracer. AP-PCP is assessed by the recruitment of Pk to anterior (arrows) or lateral (arrowheads) cell edges near the neural midline (st 14/15) in the presence of HA-Vangl2 or HA-Vangl2SA. Scale bar, 20 µm.
	Fig. 6. Feedback regulation of AP-PCP by the ROCK/Myosin II pathway.(A,B) Eight cell embryos were unilaterally coinjected with the lineage tracer (GFP RNA, 100 pg) and DN-ROCK RNA (100 pg, A) or Mypt1T696A RNA (100 pg, B) and Vangl2 polarity was assessed by immunostaining at the neural plate stage. Both the interference with ROCK signaling (A,A′) and the dephosphorylation of Myosin II light chain by the myosin phosphatase Mypt1 (B, B′) inhibit Vangl2 polarity. Arrows point to polarized Vangl2 staining. Asterisks indicate treated cells marked by GFP with non-polarized Vangl2. (C) Quantification of the data shown in A,B. Error bars represent s.d. (D) Model of AP-PCP. Wnt activity gradient leads to the polarization of the Vangl2/Pk complex to the anterior cell domain. Myosin II activity, a putative target of PCP signaling, mediates feedback regulation of core PCP protein localization. Scale bar, 20 µm.

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