Escobedo N , Contreras O , Muñoz R , Farías M , Carrasco H , Hill C , Tran U , Pryor SE , Wessely O , Copp AJ , Larraín J .

087259 Wellcome Trust , 087525 Wellcome Trust , 7R01DK080745-04 NIDDK NIH HHS , G0801124 Medical Research Council , R01 DK080745 NIDDK NIH HHS

	Fig. 1. Sdc4 and Vangl2 expression during mouse development. (A-E) Whole-mount in situ hybridization with an antisense RNA probe for Sdc4. Embryos at E8.5, dorsal view (A), and E9.5, lateral view (B) and rostral view (C). Transverse sections (D,E) are at the levels indicated on embryos in A and B, respectively. (F-H) β-Galactosidase activity in Sdc4lacZ/+ embryos at E9.0. Lateral view (F) shows expression in otic vesicle (arrowhead) and hindgut (arrow). Dorsal view of caudal region (G) shows expression in non-neural ectoderm on the outside of the neural fold of the posterior neuropore (arrowhead) and overlying recently closed neural tube (arrow). Section (H) is from the level indicated by the line in G. Arrowhead indicates the non-neural ectoderm. (I,J) Immunostaining of transverse sections through wild-type E9.0 embryos at the hindbrain level using anti-Sdc4 (green, arrows) and anti-pan-cadherin (red). Expression is detected in the neural folds (I, arrow), foregut (J, arrowhead) and hindgut (J, arrow). (K) X-Gal staining of the whole cochlea from Sdc4lacZ/+ embryo at E18.5. Inset: section showing lacZ expression in the outer hair cells (bracket) and in the inner hair cell (arrow). (L) Immunostaining of Sdc4 (green) on transverse section through the otic vesicle in wild-type E9.0 embryo. (M,N) Sections through the closed neural tube (M) and open PNP (N) of an E9.5 embryo subjected to whole-mount in situ hybridization using an antisense RNA probe for Vangl2. cm, cephalic mesenchyme; hf, head folds; hg, hindgut; nt, neural tube; ov, otic vesicle; PNP, posterior neuropore. Scale bars: 500 μm in A,B; 300 μm in G; 10 μm in I,L; 250 μm in K.
	Fig. 2. Genetic interaction between Sdc4 and Vangl2 in neural tube closure. (A) HSPGs are upregulated in Sdc4-null fibroblasts. Extracts from wild-type or Sdc4-null mouse embryonic fibroblasts (MEFs) were analyzed by western blot using anti-Stub, anti-Sdc4 or anti-tubulin antibodies. (B,C) Reduction of Sdc4 levels on a Vangl2Lp/+ background results in neural tube defects. (B) A newborn mouse, genotyped as Sdc4lacZ/lacZ;Vangl2Lp/+, exhibiting open sacral spina bifida. The edges of the incomplete neural arches are exposed (arrowheads in magnified view). (C) The incidence of spina bifida in the Sdc4;Vangl2 compound mice. The number of mice observed with each genotype is given at the top of each bar. The frequency of spina bifida in Sdc4lacZ/lacZ;Vangl2Lp/+ mice is significantly greater than in Sdc4lacZ/+;Vangl2Lp/+ littermates (Fisher exact test; P=0.006). (D) Interaction of Sdc4 and xVangl2 in Xenopus embryos. Eight-cell stage Xenopus embryos were co-injected in the two dorsal-animal blastomeres with the indicated amounts of Sdc4 and xVangl2 morpholinos. Phenotypes were classified at stage 20 as type I (severe gastrulation and neural tube closure defects; red) and type II (impairment of neural tube closure; green). The graph summarizes three independent experiments, with numbers of embryos given at the top of each bar. The percentage of embryos with defective neural tube closure is significantly greater with both morpholinos than each morpholino alone (Chi-square test, P<0.001).
	Fig. 3. Sdc4 and Vangl2 interact in cochlear stereociliary bundle orientation. (A-C) The interaction between Sdc4 and Vangl2 disrupts planar cell polarity in the cochlea. Stereociliary bundles were stained with phalloidin (red) and kinocilia with anti-acetylated-tubulin (green) in cochleae isolated from E18.5 embryos with the indicated genotypes. Arrow indicates supernumerary cells outside the inner hair cell (IHC). Middle panels depict schematic representations of the hair bundle orientation based on the top panel. Bottom panel summarizes the distribution of hair bundle orientation in the IHC, outer hair cell (OHC) 1, OHC2 and OHC3 for each of the three genotypes. Scale bar: 10 μm in A.
	Fig. 4. Sdc4 and Vangl2 interact in wound healing. Wound healing is delayed in Sdc4;Vangl2 compound mutant mice. (A,B) Representative macroscopic views of wound healing in the back skin of mice with the indicated genotypes. (C) Summary of the data from a total of at least 9-12 wounds (three or four mice) for each genotype. Data are represented as mean±s.e.m. *P<0.001. Scale bar: 3 mm in A.
	Fig. 5. Vangl2 regulates Sdc4 steady-state levels. (A) Sdc4 and Vangl2 colocalize at the subcellular level. Confocal analysis of HeLa cells by double immunofluorescence using antibodies against endogenous Sdc4 (green) and Vangl2 (red). (B) siVangl2 increases Sdc4 steady-state levels. HEK293 cells were co-transfected as indicated and xSdc4-Flag steady-state levels were evaluated by western blot. (C) Vangl2 reduces Sdc4 steady-state levels. HEK293 cells were co-transfected with the indicated DNAs and xSdc4-Flag steady-state levels were evaluated by western blot. Vangl2Lp is more active than wild-type Vangl2 in this assay. (D,E) Cells transfected with xSdc4-Flag (D) without or (E) with mVangl2 were incubated with cycloheximide (40 μg/ml) for different times. The half-life of xSdc4 protein in the different conditions was estimated from experiments in triplicate. (F) Sdc4 is absent from otic vesicles of Vangl2Lp/Lp mice. Double immunofluorescence using antibodies against Sdc4 (green) and pan-cadherin (red) was performed on transverse sections from wild-type and Vangl2Lp/Lp mutant mice at E9.5. (G) Interaction of Sdc4 and xVangl2 in Xenopus embryos. Eight-cell stage Xenopus embryos were co-injected in the two dorsal-animal blastomeres with the indicated amounts of Sdc4 morpholino and xVangl2 synthetic mRNA. Phenotypes were classified at stage 20 as type I (severe gastrulation and neural tube closure defects; red) and type II (impairment of neural tube closure; green). The graph summarizes three independent experiments, with numbers of embryos given at the top of each bar. Co-injection of xSdc4-Mo + xVangl2 mRNA resulted in a significantly greater proportion of embryos with defective neural tube closure than the individual suboptimal amounts of xSdc4-Mo and xVangl2 mRNA (Chi-square test, P<0.001). (H) Reduced levels of HSPG in Vangl2Lp mice. Homogenates from E14.5 wild-type, Vangl2Lp/+ and Vangl2Lp/Lp mice were analyzed by western blot using anti-Stub and anti-tubulin antibodies. Scale bars: 10 μm in A; 20 μm in F.
	Fig. 6. Inhibition of HSPG sulfation induces severe NTDs in Vangl2Lp/+ embryos. Embryos cultured from E8.5 (prior to closure 1) for 24 hours in the presence or absence of sodium chlorate. (A) Embryos at E8.5 prior to culture. A range of stages is used: between three somites (left) and no somites (right). (B,C) Side views of control (con) embryos after 24 hours culture without chlorate. Both wild-type (B) and Vangl2Lp/+ (C) embryos have undergone normal closure 1. Neurulation is nearing completion in the hindbrain, while the posterior neuropore remains open, as expected at this stage. (D,E) Side views (with dorsal views in insets) of embryos cultured for 24 hours in chlorate (chlor). Axial rotation is incomplete and cranial neural tube closure is delayed. Despite these abnormalities, the wild-type embryo (D) has undergone closure 1 (arrow in inset), whereas the Vangl2Lp/+ embryo (E) has failed in closure 1 (asterisk in inset) and exhibits craniorachischisis. (F-I) Transverse sections stained with Haematoxylin and Eosin at the level of the dashed lines in B-E, respectively. A closed neural tube is seen in all groups except the Vangl2Lp/+ embryo cultured in chlorate, in which the neural tube is wide open (asterisk in I). The gut has failed to close in this embryo, whereas it is closed in all other embryos. g, gut; hb, hindbrain; hf, open head folds; nt, neural tube; pnp, posterior neuropore. Scale bars: 0.5 mm in A; 0.5 mm in B-E; 0.15 mm in F-I.

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