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Fig. S1. Immunodetection of endogenous Vangl2 protein.
(A, B) Basolateral localization (arrowhead) of Vangl2 (A) and b1-integrin (B) in embryonic ectoderm at stage 11. (C) Immunoblot of ectodermal cell lysates with anti- Vangl2 antibody reveals a 60 kDa band (arrowhead) corresponding to endogenous Vangl2, misexpressed Xenopus CFP-Vangl2 and mouse HA-VL2, as indicated.(D-G) Vangl2 immunostaining on cross-sections of st. 10- and st. 10.5 embryos. Dashed boxes in D and E correspond to the magnified images shown in D’ and E’. Vangl2 is detected at the basolateral membrane and in cytoplasmic vesicles near the apical cortex, and appears more abundant in the superficial cell layer. Scale bar in A is 20 μm. Both apical (arrow) and basolateral staining is detected in the constricting blastopore cells of st. 10.5 embryo. D-G, Scale bar is 20 μm. |
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Fig. 1. Subcellular distribution of Vangl2 at the blastopore lip. (A-D) Parasagittal sections of stage (st) 11 embryos showing the blastopore area (arrows). (A) Immunohistochemical staining demonstrating apical and basolateral localization of endogenous Vangl2. (B) Bright-field view of a similar embryo section shows extensive pigmentation in the constricting blastopore cells. (C) Staining with antibodies against pMLC. (D) Basolateral localization of β1-integrin in bottle cells. (E,E′) Subcellular distribution of Vangl2 in the superficial and the inner ectoderm cells. (E) Mosaic distribution of Vangl2 MO with GFP RNA as a lineage tracer (red) confirms Vangl2 antibody specificity. (E′) Green channel only. Arrowheads point to Vangl2 staining in E,E′; asterisks indicate lack of staining. Two embryo sections are shown side-by-side to illustrate Vangl2 depletion. Scale bars: 20 µm. |
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Fig. S5. Vangl2 is dissociated from the cell membrane in Rab11-depleted embryos.
Embryos were coinjected with CO MO or Vangl2 MO (30 ng each) and 100 pg of GFP RNA as lineage tracer (not shown). A, B, Morphology of control (A) and Rab11-depleted (B) embryos at st. 11, vegetal views are shown. C, D, Rab11 depletion interferes with the membrane localization of Vangl2, including the apical surface of the constricting blastopore cells (arrow). E, F, Basolateral membrane localization of Vangl2 is inhibited in ectoderm cells depleted of Rab11. Scale bar in E, also refers to C, D and F, is 20 μm. |
