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Fig. S3. Rab11 subcellular localization before gastrulation.
(A-C) Cross-sections of late blastula embryos (n=10) stained for endogenous Rab11. A, Representative embryo at stage 9.5 is shown, B, Dorsal marginal zone area reveals enriched apical (asterisk) and apical junctional (arrowhead) staining. C, Rab11 is mostly at the junctions (arrowheads) in the ventral marginal zone. Scale bar is 20 μm in all panels. |
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Fig. 5. Rab11 is apically localized at the blastopore lip in a Vangl2-dependent manner. (A-C) Cross-sections of early gastrula embryos [stage (st) 10+] immunostained for Rab11. An, animal; Veg, vegetal; D, dorsal; V, ventral axes are indicated. The dashed box in A corresponds to the magnified image shown in B. The inset in A shows a schematic of a sectioned gastrula-stage embryo. (D-D′) Co-immunostaining of Rab11 and pMLC. In D′, bottle cell morphology is indicated by the dashed line. BL, blastopore lip. (E,F) Rab11 localization at the blastopore requires Vangl2. Rab11 staining at the blastopore area of embryos co-injected with 30 ng of Vangl2 MO and 100 pg of GFP RNA. A cross-section of a stage 10.5 embryo is shown. The arrow in E points to bottle cells; the asterisk in F indicates inhibited apical constriction. CO, control. (G,H) Sections of ectoderm immunostained for Rab11, apical is to the top. (G) Rab11 is at the apical cell junctions (arrow) in control ectoderm. (H) Rab11 is more cytoplasmic in cells depleted of Vangl2. Scale bars: 20 µm in B,G, 10 µm in D. |
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Fig. 7. Myosin V regulates endogenous and Shroom-mediated apical constriction. (A-C) Early embryos were injected with 2 ng of RNAs encoding GFP (A) or the carboxy-terminal tail of Myosin V fused to GFP (GFP-MyoVT, B). Blastopore defects of stage (st) 11 MyoVT embryos (B), as compared with the control (A) are shown. (C) Quantification of the results at the early and late gastrula stages. Co, control. (D,E) GFP-MyoVT alters the normal pattern of Rab11 distribution. BL, blastopore lip. (D) Transverse section of a stage 11 blastopore, stained for endogenous Rab11. The animal-vegetal (An-Vg) axis is indicated. Arrow points to Rab11 staining in bottle cells. Scale bar: 20 µm. (E,E′) Transverse section of a stage 11 defective blastopore from a MyoVT-expressing embryo. MyoVT forms cytoplasmic aggregates that colocalize with endogenous Rab11 protein. (F,G) GFP-MyoVT interferes with Shroom-mediated constriction. Scoring was carried out for embryos with strong (++) and mild (+) ectopic constriction. |
