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Fig. 2. Vangl2 and Daam1 are required for blastopore formation. (A-D) Blastopore defects in embryos depleted of Vangl2 and Daam1. Embryos were injected with control (CO) MO, Vangl2 MO or Daam1 MO, as indicated, and lacZ RNA (150 pg) as a lineage tracer. The vegetal view is shown. Dorsal is to the top. A schematic of a gastrula embryo is shown in the inset in A. (A-C) Blastopore formation (arrowhead) is inhibited in stage (st) 10+ embryos by Vangl2 MO or Daam1 MO. (D) Quantification of the effect. (E,E′) Myosin II light chain (MLC) phosphorylation at the blastopore lip (arrow) in control embryos. (F,F′) Vangl2 MO inhibits Myosin II activation (asterisk) in apically constricting cells. Scale bar: 20 µm. |
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Fig. 6. The involvement of Rab11 in blastopore formation and myosin II activation. (A-F) Embryos were co-injected with RNAs encoding wild-type Rab11 or Rab11S25N and GFP RNA as a lineage tracer. Alternatively, Rab11 was depleted with a specific Rab11 MO. Co, control. (A-C) En face view of the injected embryo morphology at stage (st) 10.5. The arrow points to the inhibited blastopore lip. (D-F) Inhibition of pMLC staining in Rab11S25N-expressing embryos. A cross-section is shown with animal pole to the right. The arrow shows lack of the pMLC signal. BL, blastopore lip; Veg, vegetal; An, animal. Scale bar: 20 µm. (G) Quantification of the results. The total number of embryos per group is shown at the top of each column. (H) Mesendodermal marker expression in embryos (stage 10.5) with manipulated Rab11 function. (I) Rab11 rescues blastopore formation in Vangl2-depleted embryos. Both dorsal blastomeres of four-cell embryos were injected with 30 ng of Vangl2 MO and 1 ng of RNA encoding wild-type Rab11, Rab11Q70L or GFP. Data are shown as the frequency of embryos with dorsal lip defects scored at stage 10+. |
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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. 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. |
