Chuykin I , Itoh K , Kim K , Sokol SY .

R35GM122492 NIH HHS , NS100759 NIH HHS , GM122492 NIH HHS , R01 NS100759 NINDS NIH HHS , R35 GM122492 NIGMS NIH HHS

Xla Wt + fzd3 MO (Fig.1.C,C')
Xla Wt + fzd3 MO (Fig.S2.A,B)
Xla Wt + vangl2 (Fig.S4.A,B)

	Fig. 1. Frizzled3 is required for neural plate planar polarity. (A) Experimental scheme. One dorsal animal blastomere of a 16-cell Xenopus embryo was co-injected with Fz3 MO1 or control (Co) MO (5 ng each) and GFP RNA (200 pg). Stage 16 embryos were fixed and the neural plates were dissected and stained with anti-Vangl2 and anti-GFP antibodies. (B–C′) Representative en face images of neural plates from the injected embryos. Vangl2 (red) is enriched at the anterior cell edges (arrows). GFP is a lineage tracer (green). A-P, anterior-posterior axis. Scale bar: 30 µm. (B,B′) Co MO, (C,C′) Fz3 MO1. Many GFP-positive cells lack Vangl2 polarization (asterisk). (D) Quantification of the mean±s.d. frequencies for cells with anteriorly enriched Vangl2. Numbers of scored cells (n) are shown on top of each bar. Three embryos were scored for each group (20–30 cells per embryo). Data represent three different experiments. *P<0.05 (two-tailed unpaired Student's t-test).
	Fig. 2. Fz3 reduces the interaction between Vangl2 and Pk3 proteins in Xenopus embryos. (A) Vangl2 biotinylation by BLN–Pk3 is decreased in the presence of Fz3. Biotin and mRNAs encoding FLAG–BLN–Pk3 (400 pg), HA–Vangl2 or ΔPBD RNAs (100 pg each) and Fz3–FLAG (100 or 500 pg) as indicated, were injected into the animal region of 4- to 8-cell embryos. Protein lysates were immunoprecipitated (IP) with anti-HA antibody from stage 13 Xenopus embryos. Proteins were detected by immunoblotting (IB) with anti-biotin, anti-HA and anti-FLAG antibodies as indicated. Black arrowheads point to Vangl2 and Pk3, white arrowhead points to the slower migrating Vangl2 in pulldowns and lysates. Asterisks indicate endogenous proteins labeled by the anti-biotin antibody. (B) Lysates of embryos overexpressing Pk3 and Fz3 show that Pk3 increases Vangl2 mobility, whereas Fz3 reduces it. (C) Physical interaction of HA–Vangl2 and FLAG–Pk3 in transfected HEK293T cells. HA–Vangl2, but not the construct lacking the presumed Pk3-binding domain (ΔPBD, residues 298–382), is detected in pulldowns of FLAG–Pk3 from lysates of the transfected HEK293T cells. (D) HA-tagged Vangl2 but not ΔPBD is biotinylated by FLAG–BLN–Pk3. Embryo microinjection details and abbreviations are as in A. (E) Model. The formation of a complex between Vangl2 and Pk3 results in the biotinylation of Vangl2 by BLN–Pk3, green circles depict biotin. Fz3 causes an upshift of Vangl2 due to phosphorylation (circled red P) and inhibits the Vangl2–Pk3 interaction. Data is representative of three experiments.
	Fig. 3. Fz3 decreases the amount of the Vangl2–Pk3 complex in vivo. (A) Experimental scheme; 8-cell embryos were injected into each animal blastomere with GFP–Pk3 (200 pg), HA–Vangl2 (50 pg) or Fz3–FLAG (400 pg) mRNA as indicated. Ectoderm explants were dissected at stage 8.5–9, cultured until stage 13, and cross-linked with 2 mM DSP for 30 min. After immunoprecipitation (IP) with GFP-trap beads, protein levels were assessed by immunoblotting (IB) with anti-HA and anti-GFP antibodies. (B) Vangl2 and GFP–Pk3 co-precipitation in control and Fz3-expressing ectoderm explants (right). No binding is detected without cross-linking (left). Black arrowheads point to Vangl2 and Pk3, white arrowhead points to the slower migrating Vangl2 band in the lysates. A nonspecific band reflects protein loading (asterisk). The numbers are band intensity ratios for HA–Vangl2 and GFP–Pk3 in precipitated complexes. Data are representative of three experiments.
	Fig. 4. The interaction between Vangl2 and Prickle3 is enhanced in Fz3-depleted embryos. (A) Experimental scheme. 4- to 8-cell embryos were sequentially injected into dorsal animal blastomeres with BLN–Pk3 and HA–Vangl2 DNAs (50 pg each), and Fz3 MO1 or Fz3 MO2 with biotin. Embryo lysates were collected at stage 18 for immunoprecipitation (IP) and immunoblotting (IB). (B) Vangl2 and Pk3 biotinylation in control and Fz3 morphant embryos in pulldowns were assessed with anti-biotin antibodies. Total exogenous Vangl2 levels were analyzed by anti-HA antibodies. Owing to low abundance and insufficient sensitivity of the detection, the levels and the activity of FLAG–BLN–Pk3 were assessed in the second sequential FLAG pulldown. Biological triplicates were performed for each experimental condition with 20 embryos per sample. (C) Band intensity ratios of biotinylated to total Vangl2 levels in pulldowns, evaluated with anti-biotin and anti-HA antibodies, respectively. Results are mean±s.d. (n=3). *P<0.05; **P<0.001 (two-tailed unpaired Student's t-test). (D) Anti-Fz3 antibody detects Fz3 (arrowhead) in lysates of stage 16 control embryos or embryos injected with Fz3-FLAG mRNA (25 pg); asterisks indicate non-specific bands. (E) Levels of the endogenous Fz3 protein (arrowhead) in control, Fz3 MO1- and Fz3 MO2-injected stage 16 embryos; ERK1 is a loading control. Data in D,E are representative of three experiments.
	Fig. 5. Phosphorylation of Vangl2 at threonine residues is increased in Fz3-expressing embryos. (A) Scheme of Vangl2 constructs with the modified cytoplasmic N-terminal region (amino acids 1–84, blue) used to analyze Vangl2 phosphorylation. Phosphorylated (p-)serine and threonine (S/T) residues in clusters I and II are indicated. (B,C) Immunoprecipitations (IP) with anti-HA antibodies from lysates of embryos expressing Vangl2 constructs with or without Fz3 were immunoblotted (IB) with phospho-specific antibodies as indicated. Anti-HA antibodies show total Vangl2 levels. Asterisk in B marks non-specific band recognized by anti-HA antibody in lysates. (C) Phosphothreonine phosphorylation in Vangl2 requires T76 and T78. A Vangl2 upshift is visible for the T76T78>AA mutant (white arrowheads). Data are representative of three experiments.
	Fig. 6. Fz3-induced Vangl2 phosphorylation is necessary for the inhibition of the Vangl2–Pk3 interaction. (A) Scheme of Vangl2 constructs with alanine substitutions. Conserved serine and threonine (S/T) clusters I and II are indicated. (B,C) Effect of Fz3 on the biotinylation of various Vangl2 constructs by BLN–Pk3. Embryos were injected dorsoanimally with mRNAs encoding Vangl2 constructs (50 pg each), BLN–Pk3 (200 pg), or Fz3–FLAG mRNA (200 pg or 400 pg) (B) and 400 pg (C); and collected at stage 13. (B) The Vangl2-2A mutant is partly resistant to the Fz3 inhibitory effect. (C) Vangl2-7A is completely resistant to Fz3 and is not upshifted (black and white arrowheads). Immunoblotting (IB) with anti-biotin, anti-HA and anti-FLAG antibodies was done as indicated. Asterisks represent a non-specific band. Data are representative of three experiments.
	Fig. 7. Fz3-induced Vangl2 phosphorylation is required for PCP. (A–C) Two dorsal blastomeres of 16-cell embryos were co-injected with HA–Vangl2 or HA–Vangl2-7A RNA (50 pg) and GFP–Pk3 RNA (200 pg). (A–B′) Representative fixed stage 14 neural plates have been imaged for GFP–Pk3 (direct GFP fluorescence) and immunostained for HA–Vangl2 (A,A′) or HA–Vangl2-7A (B,B′). (A,A′) Polarized anterior PCP complexes are indicated by arrows. (B,B′) Cells lacking anterior GFP–Pk3 are marked by asterisks. Scale bar: 30 µm. Anteroposterior (AP) axis is indicated. (C) Quantification of GFP–Pk3 fluorescence for PCP complexes containing wild-type Vangl2 (blue) and those containing Vangl2-7A (orange) in mosaically expressing cells. Mean±s.d. is shown along the cell circumference for multiple cells as a function of the circular angle from 0 to 360 degrees relative to the AP axis (as shown on the scheme). Numbers of scored cells per each group are indicated. Data are representative of three independent experiments. (D) Model. Pk3 binding to Vangl2 promotes the formation of the anterior PCP complex in neuroectoderm cells. The anterior accumulation of the complex is reinforced by Fz3 that triggers T76T78 phosphorylation of Vangl2 and inhibits the Vangl2–Pk3 interaction. Presumptive posterior Fz3 localization is shown by the dashed line.
	Fig. S1. Fz3 depletion does not affect Vangl2 membrane localization at the mediolateral cell borders. (A) Vangl2- specific fluorescence at the mediolateral cell borders (dashed boxes) was compared between control and Fz3 MO1-injected neural plate cells at stage 16 (from the experiment shown in Fig. 1). GFP (green) is a lineage tracer. Anteroposterior (A-P) axis is indicated. (B) Quantification of mean fluorescence intensity at the mediolateral cell borders in control and Fz3 MO1-injected neuroectoderm cells. Standard deviations are indicated, cell numbers are shown above each bar.
	Fig. S2. Depletion of Fz3 causes neural tube closure defects. (A) Two dorsal animal blastomeres were injected with control morpholino (Co MO) (A, left and middle) or Fz3 MO (A, right), 10 ng each. Dorsal view of stage 17 embryos is shown, anterior is at the top. Arrows point to the neural folds. (B) Quantification of the experiments shown in A, showing frequencies of mild (light grey) or severe (dark grey) neural fold defects. The number of scored embryos per group is shown above each bar. Data are representative of three experiments.
	Fig. S3. Vangl2 T76/T78 phosphorylation is necessary for the inhibition of Pk3 cortical localization by Fz3. (A-D’) Two dorsal blastomeres of 16 cell embryos were coinjected with mRNAs encoding HA-Vangl2 and T76T78>AA, 100 pg each and GFP-Pk3, 500 pg, with or without Fz3-FLAG mRNA, 400 pg. Vangl2 and Pk3 distribution was analyzed at stage 10.5-11 by anti-HA immunostaining and GFP fluorescence. (A-D’) The localization of Pk3 and Vangl2 without (A-A’, C-C’) or with (B-B’, D-D’) Fz3 in superficial ectoderm cells. Scale bar, 30 μm. (E) Quantification of data in (B-B’) and (D-D’) is shown as mean frequencies ± s. d. of the cells containing cortical GFP-Pk3 patches. Numbers of scored cells are shown above each bar; 20 to 50 cells were scored per embryo with five embryos taken for each experimental condition. Data are representative of three experiments.
	Fig. S4. Expression of Vangl2 T76T78>AA mutant causes neural tube defects. Four-to-eight cell embryos were unilaterally injected with mRNAs encoding Vangl2 or T76T78>AA (2A) constructs, 100, 200 or 300 pg each. (A) Embryo injected with 100 pg of Vangl2 mRNA is indistinguishable from an uninjected embryo (left). Embryo injected with 200 pg of Vangl2 mRNA exhibits mild neural tube defect (middle). Embryo injected with 200 pg of 2A mRNA shows a severe neural tube defect (right). Anterior is at the top. Neural tube defects (arrows) are shown at the injected side (asterisk). (B) Frequencies of mild and severe neural fold defects are shown. Numbers of scored embryos per group are above each bar. Data are representative of two experiments.

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
Bastock, Strabismus is asymmetrically localised and binds to Prickle and Dishevelled during Drosophila planar polarity patterning. 2003, Pubmed
Belotti, Molecular characterisation of endogenous Vangl2/Vangl1 heteromeric protein complexes. 2012, Pubmed
Butler, Planar cell polarity in development and disease. 2017, Pubmed
Chen, Asymmetric homotypic interactions of the atypical cadherin flamingo mediate intercellular polarity signaling. 2008, Pubmed
Chien, Mechanical strain determines the axis of planar polarity in ciliated epithelia. 2015, Pubmed , Xenbase
Choi-Rhee, Promiscuous protein biotinylation by Escherichia coli biotin protein ligase. 2004, Pubmed
Chu, Prickle3 synergizes with Wtip to regulate basal body organization and cilia growth. 2016, Pubmed , Xenbase
Chuykin, Par3 interacts with Prickle3 to generate apical PCP complexes in the vertebrate neural plate. 2018, Pubmed , Xenbase
Darken, The planar polarity gene strabismus regulates convergent extension movements in Xenopus. 2002, Pubmed , Xenbase
Davey, Planar cell polarity in moving cells: think globally, act locally. 2017, Pubmed
Davey, PCP Signaling between Migrating Neurons and their Planar-Polarized Neuroepithelial Environment Controls Filopodial Dynamics and Directional Migration. 2016, Pubmed
Deardorff, A role for frizzled 3 in neural crest development. 2001, Pubmed , Xenbase
Devenport, The cell biology of planar cell polarity. 2014, Pubmed
Gao, Wnt signaling gradients establish planar cell polarity by inducing Vangl2 phosphorylation through Ror2. 2011, Pubmed
Goodrich, Principles of planar polarity in animal development. 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
Greene, Abnormalities of floor plate, notochord and somite differentiation in the loop-tail (Lp) mouse: a model of severe neural tube defects. 1998, Pubmed
Hildebrand, Shroom, a PDZ domain-containing actin-binding protein, is required for neural tube morphogenesis in mice. 1999, Pubmed
Jenny, Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling. 2003, Pubmed , Xenbase
Jessen, Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements. 2002, Pubmed
Kelly, Frizzled-Induced Van Gogh Phosphorylation by CK1ε Promotes Asymmetric Localization of Core PCP Factors in Drosophila. 2016, Pubmed
Kibar, Ltap, a mammalian homolog of Drosophila Strabismus/Van Gogh, is altered in the mouse neural tube mutant Loop-tail. 2001, Pubmed
Kim, An improved smaller biotin ligase for BioID proximity labeling. 2016, Pubmed
Kunimoto, Disruption of Core Planar Cell Polarity Signaling Regulates Renal Tubule Morphogenesis but Is Not Cystogenic. 2017, Pubmed
Mahaffey, Cofilin and Vangl2 cooperate in the initiation of planar cell polarity in the mouse embryo. 2013, Pubmed
Mancini, The dorsal blastopore lip is a source of signals inducing planar cell polarity in the Xenopus neural plate. 2021, Pubmed , Xenbase
Mattson, A practical approach to crosslinking. 1993, Pubmed
McGreevy, Shroom3 functions downstream of planar cell polarity to regulate myosin II distribution and cellular organization during neural tube closure. 2015, Pubmed
Montcouquiol, Asymmetric localization of Vangl2 and Fz3 indicate novel mechanisms for planar cell polarity in mammals. 2006, Pubmed
Murdoch, Severe neural tube defects in the loop-tail mouse result from mutation of Lpp1, a novel gene involved in floor plate specification. 2001, Pubmed
Newman-Smith, Reciprocal and dynamic polarization of planar cell polarity core components and myosin. 2015, Pubmed
Newport, A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage. 1982, Pubmed , Xenbase
Nikolopoulou, Neural tube closure: cellular, molecular and biomechanical mechanisms. 2017, Pubmed
Nishimura, Planar cell polarity links axes of spatial dynamics in neural-tube closure. 2012, Pubmed
Ossipova, Role of Rab11 in planar cell polarity and apical constriction during vertebrate neural tube closure. 2014, Pubmed , Xenbase
Ossipova, Planar polarization of Vangl2 in the vertebrate neural plate is controlled by Wnt and Myosin II signaling. 2015, Pubmed , Xenbase
Park, The planar cell-polarity gene stbm regulates cell behaviour and cell fate in vertebrate embryos. 2002, Pubmed , Xenbase
Peng, Asymmetric protein localization in planar cell polarity: mechanisms, puzzles, and challenges. 2012, Pubmed
Reis, Identification of the centrosomal maturation factor SSX2IP as a Wtip-binding partner by targeted proximity biotinylation. 2021, Pubmed , Xenbase
Roux, A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. 2012, Pubmed
Shafer, Vangl2 promotes Wnt/planar cell polarity-like signaling by antagonizing Dvl1-mediated feedback inhibition in growth cone guidance. 2011, Pubmed
Shi, Expression of Xfz3, a Xenopus frizzled family member, is restricted to the early nervous system. 1998, Pubmed , Xenbase
Sokol, Spatial and temporal aspects of Wnt signaling and planar cell polarity during vertebrate embryonic development. 2015, Pubmed , Xenbase
Strutt, The role of RhoA in tissue polarity and Frizzled signalling. 1997, Pubmed
Strutt, Reciprocal action of Casein Kinase Iε on core planar polarity proteins regulates clustering and asymmetric localisation. 2019, Pubmed
Strutt, Dynamics of core planar polarity protein turnover and stable assembly into discrete membrane subdomains. 2011, Pubmed
Tree, Prickle mediates feedback amplification to generate asymmetric planar cell polarity signaling. 2002, Pubmed
Turner, Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. 1994, Pubmed , Xenbase
Vladar, Microtubules enable the planar cell polarity of airway cilia. 2012, Pubmed
Wang, The role of Frizzled3 and Frizzled6 in neural tube closure and in the planar polarity of inner-ear sensory hair cells. 2006, Pubmed
Winter, Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton. 2001, Pubmed
Wu, The frizzled extracellular domain is a ligand for Van Gogh/Stbm during nonautonomous planar cell polarity signaling. 2008, Pubmed
Yang, Wnt-induced Vangl2 phosphorylation is dose-dependently required for planar cell polarity in mammalian development. 2017, Pubmed
Yin, Comparison of phenotypes between different vangl2 mutants demonstrates dominant effects of the Looptail mutation during hair cell development. 2012, Pubmed