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.

Adler, The frizzled/stan pathway and planar cell polarity in the Drosophila wing. 2012, Pubmed

Adler, The frizzled/stan pathway and planar cell polarity in the Drosophila wing. 2012, Pubmed
Aigouy, Cell flow reorients the axis of planar polarity in the wing epithelium of Drosophila. 2010, Pubmed
Antic, Planar cell polarity enables posterior localization of nodal cilia and left-right axis determination during mouse and Xenopus embryogenesis. 2010, Pubmed , Xenbase
Bastock, Strabismus is asymmetrically localised and binds to Prickle and Dishevelled during Drosophila planar polarity patterning. 2003, Pubmed
Bertet, Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation. 2004, Pubmed
Blair, Twinstar, the Drosophila homolog of cofilin/ADF, is required for planar cell polarity patterning. 2006, Pubmed
Borovina, Vangl2 directs the posterior tilting and asymmetric localization of motile primary cilia. 2010, Pubmed
Burnside, Analysis of morphogenetic movements in the neural plate of the newt Taricha torosa. 1968, Pubmed
Casal, Two separate molecular systems, Dachsous/Fat and Starry night/Frizzled, act independently to confer planar cell polarity. 2006, Pubmed
Choi, The involvement of lethal giant larvae and Wnt signaling in bottle cell formation in Xenopus embryos. 2009, Pubmed , Xenbase
Ciruna, Planar cell polarity signalling couples cell division and morphogenesis during neurulation. 2006, Pubmed
Classen, Hexagonal packing of Drosophila wing epithelial cells by the planar cell polarity pathway. 2005, Pubmed
Colas, Towards a cellular and molecular understanding of neurulation. 2001, Pubmed
Curtin, Mutation of Celsr1 disrupts planar polarity of inner ear hair cells and causes severe neural tube defects in the mouse. 2003, Pubmed
Darken, The planar polarity gene strabismus regulates convergent extension movements in Xenopus. 2002, Pubmed , Xenbase
Davidson, Neural tube closure in Xenopus laevis involves medial migration, directed protrusive activity, cell intercalation and convergent extension. 1999, Pubmed , Xenbase
Devenport, Planar polarization in embryonic epidermis orchestrates global asymmetric morphogenesis of hair follicles. 2008, Pubmed
Devenport, The cell biology of planar cell polarity. 2014, Pubmed
Dollar, Regulation of Lethal giant larvae by Dishevelled. 2005, Pubmed , Xenbase
Etheridge, Murine dishevelled 3 functions in redundant pathways with dishevelled 1 and 2 in normal cardiac outflow tract, cochlea, and neural tube development. 2008, Pubmed
Fernandez-Gonzalez, Myosin II dynamics are regulated by tension in intercalating cells. 2009, Pubmed
Gao, Wnt signaling gradients establish planar cell polarity by inducing Vangl2 phosphorylation through Ror2. 2011, Pubmed
Goto, The planar cell polarity gene strabismus regulates convergence and extension and neural fold closure in Xenopus. 2002, Pubmed , Xenbase
Gray, Planar cell polarity: coordinating morphogenetic cell behaviors with embryonic polarity. 2011, Pubmed
Gray, The planar cell polarity effector Fuz is essential for targeted membrane trafficking, ciliogenesis and mouse embryonic development. 2009, Pubmed , Xenbase
Harris, Mouse mutants with neural tube closure defects and their role in understanding human neural tube defects. 2007, Pubmed
Hashimoto, Planar polarization of node cells determines the rotational axis of node cilia. 2010, Pubmed
Heisenberg, Forces in tissue morphogenesis and patterning. 2013, Pubmed
Hikasa, Wnt signaling in vertebrate axis specification. 2013, Pubmed , Xenbase
Itoh, GEF-H1 functions in apical constriction and cell intercalations and is essential for vertebrate neural tube closure. 2014, Pubmed , Xenbase
Itoh, Graded amounts of Xenopus dishevelled specify discrete anteroposterior cell fates in prospective ectoderm. 1997, Pubmed , Xenbase
Itoh, Centrosomal localization of Diversin and its relevance to Wnt signaling. 2009, Pubmed , Xenbase
Itoh, Specific modulation of ectodermal cell fates in Xenopus embryos by glycogen synthase kinase. 1995, Pubmed , Xenbase
Jenny, Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling. 2003, Pubmed , Xenbase
Jiang, Ascidian prickle regulates both mediolateral and anterior-posterior cell polarity of notochord cells. 2005, Pubmed
Keller, The cellular basis of the convergence and extension of the Xenopus neural plate. 1992, Pubmed , Xenbase
Keller, Shaping the vertebrate body plan by polarized embryonic cell movements. 2002, Pubmed
Kiecker, A morphogen gradient of Wnt/beta-catenin signalling regulates anteroposterior neural patterning in Xenopus. 2001, Pubmed , Xenbase
Kimura, Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). 1996, Pubmed
Kirschner, Evolvability. 1998, Pubmed
Lake, Strabismus regulates asymmetric cell divisions and cell fate determination in the mouse brain. 2009, Pubmed
Lecuit, Force generation, transmission, and integration during cell and tissue morphogenesis. 2011, Pubmed
Li, Sfrp5 coordinates foregut specification and morphogenesis by antagonizing both canonical and noncanonical Wnt11 signaling. 2008, Pubmed , Xenbase
Lu, PTK7/CCK-4 is a novel regulator of planar cell polarity in vertebrates. 2004, Pubmed , Xenbase
Mahaffey, Cofilin and Vangl2 cooperate in the initiation of planar cell polarity in the mouse embryo. 2013, Pubmed
Marlow, Zebrafish Rho kinase 2 acts downstream of Wnt11 to mediate cell polarity and effective convergence and extension movements. 2002, Pubmed , Xenbase
Montcouquiol, Identification of Vangl2 and Scrb1 as planar polarity genes in mammals. 2003, Pubmed
Nagaoka, The Wnt/planar cell polarity pathway component Vangl2 induces synapse formation through direct control of N-cadherin. 2014, Pubmed
Nagaoka, Vangl2 regulates E-cadherin in epithelial cells. 2014, Pubmed
Nandadasa, N- and E-cadherins in Xenopus are specifically required in the neural and non-neural ectoderm, respectively, for F-actin assembly and morphogenetic movements. 2009, Pubmed , Xenbase
Nishimura, Planar cell polarity links axes of spatial dynamics in neural-tube closure. 2012, Pubmed
Olofsson, Prickle/spiny-legs isoforms control the polarity of the apical microtubule network in planar cell polarity. 2014, Pubmed
Ossipova, Role of Rab11 in planar cell polarity and apical constriction during vertebrate neural tube closure. 2014, Pubmed , Xenbase
Ossipova, Vangl2 cooperates with Rab11 and Myosin V to regulate apical constriction during vertebrate gastrulation. 2015, Pubmed , Xenbase
Ossipova, PAR-1 phosphorylates Mind bomb to promote vertebrate neurogenesis. 2009, Pubmed , Xenbase
Peng, Asymmetric protein localization in planar cell polarity: mechanisms, puzzles, and challenges. 2012, Pubmed
Qian, Wnt5a functions in planar cell polarity regulation in mice. 2007, Pubmed , Xenbase
Rolo, Morphogenetic movements driving neural tube closure in Xenopus require myosin IIB. 2009, Pubmed , Xenbase
Sawyer, Apical constriction: a cell shape change that can drive morphogenesis. 2010, Pubmed , Xenbase
Schroeder, Neurulation in Xenopus laevis. An analysis and model based upon light and electron microscopy. 1970, Pubmed , Xenbase
Schwarz-Romond, The ankyrin repeat protein Diversin recruits Casein kinase Iepsilon to the beta-catenin degradation complex and acts in both canonical Wnt and Wnt/JNK signaling. 2002, Pubmed , Xenbase
Ségalen, The Fz-Dsh planar cell polarity pathway induces oriented cell division via Mud/NuMA in Drosophila and zebrafish. 2010, Pubmed
Sepich, Wnt/PCP signaling controls intracellular position of MTOCs during gastrulation convergence and extension movements. 2011, Pubmed
Shindo, PCP and septins compartmentalize cortical actomyosin to direct collective cell movement. 2014, Pubmed , Xenbase
Shook, Morphogenic machines evolve more rapidly than the signals that pattern them: lessons from amphibians. 2008, Pubmed
Simons, Planar cell polarity signaling: from fly development to human disease. 2008, Pubmed
Skoglund, Convergence and extension at gastrulation require a myosin IIB-dependent cortical actin network. 2008, Pubmed , Xenbase
Sokol, Analysis of Dishevelled signalling pathways during Xenopus development. 1996, Pubmed , Xenbase
Song, Planar cell polarity breaks bilateral symmetry by controlling ciliary positioning. 2010, Pubmed
Suzuki, Molecular mechanisms of cell shape changes that contribute to vertebrate neural tube closure. 2012, Pubmed
Tada, Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway. 2000, Pubmed , Xenbase
Tada, Convergent extension: using collective cell migration and cell intercalation to shape embryos. 2012, Pubmed , Xenbase
Takeuchi, The prickle-related gene in vertebrates is essential for gastrulation cell movements. 2003, Pubmed , Xenbase
Tatin, Planar cell polarity protein Celsr1 regulates endothelial adherens junctions and directed cell rearrangements during valve morphogenesis. 2013, Pubmed
Torban, Genetic interaction between members of the Vangl family causes neural tube defects in mice. 2008, Pubmed
Vicente-Manzanares, Non-muscle myosin II takes centre stage in cell adhesion and migration. 2009, Pubmed
Vladar, Planar cell polarity signaling: the developing cell's compass. 2009, Pubmed
Vladar, Microtubules enable the planar cell polarity of airway cilia. 2012, Pubmed
Walentek, Wnt11b is involved in cilia-mediated symmetry breakage during Xenopus left-right development. 2013, Pubmed , Xenbase
Wallingford, The continuing challenge of understanding, preventing, and treating neural tube defects. 2013, Pubmed
Wallingford, Convergent extension: the molecular control of polarized cell movement during embryonic development. 2002, Pubmed , Xenbase
Wallingford, Cloning and expression of Xenopus Prickle, an orthologue of a Drosophila planar cell polarity gene. 2002, Pubmed , Xenbase
Wang, Tissue/planar cell polarity in vertebrates: new insights and new questions. 2007, Pubmed
Weiser, Rho-regulated myosin phosphatase establishes the level of protrusive activity required for cell movements during zebrafish gastrulation. 2009, Pubmed
Winter, Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton. 2001, Pubmed
Wolda, Overlapping expression of Xwnt-3A and Xwnt-1 in neural tissue of Xenopus laevis embryos. 1993, Pubmed , Xenbase
Wu, Wg and Wnt4 provide long-range directional input to planar cell polarity orientation in Drosophila. 2013, Pubmed
Yan, Rho1 has multiple functions in Drosophila wing planar polarity. 2009, Pubmed
Yin, Cooperation of polarized cell intercalations drives convergence and extension of presomitic mesoderm during zebrafish gastrulation. 2008, Pubmed