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Fig. 2. Rab11 involvement in neural tube closure. a-g, Four-cell embryos were injected unilaterally with Rab11 RNAs (1 ng), or MOs (20 ng) as indicated. Neural fold and cell morphology was assessed in whole embryos (a-d, dorsal view, anterior is at the top) and in cryosections (e-g). Rab11S25N and Rab11 MO, but not wild-type Rab11 or control MO, inhibit neural tube closure. Arrows point to neural folding defects. b, d, Quantification of data presented in (a) and (c). e, Sections confirm the effect of Rab11S25N on neural fold morphology. β-catenin staining reveals cell boundaries. f, Ratios of apical width to apicobasal length (AW/ABL) in the neural groove cells that are adjacent to midline and express Rab11 constructs (stages 16/17; n, number of examined cells). Means +/- s. d. are shown on the graph. Rab11-expressing cells are indistinguishable from uninjected control cells (data not shown). g, Rab11 constructs do not affect the neural progenitor fate marker Sox3. h, Medial polarization of GFP-Sec15 in the neural plate of stage 15 embryos. Top view, red rectangle in the inset indicates approximate image position. F-actin marks cell boundaries. i-k, Rab11 polarization depends on PCP. Immunostaining for endogenous Rab11 in cross-sections of embryos unilaterally injected with Diversin MO (20 ng, i), Vangl2 MO (20 ng, j) or Xdd1 RNA (1 ng, k). Arrows point to polarized Rab11 on the uninjected side, asterisks indicate lack of Rab11 polarity. Scale bar in j (also refers to i, k), is 20 μm. M, midline position. GFP-CAAX or GFP RNA (100 pg) served as lineage tracers as indicated. Each result was verified in 3 to 6 independent experiments.
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Fig. 3. Rab11 is required for myosin II activation, but does not affect apicobasal polarity. Four-cell embryos were unilaterally injected with Rab11 (2 ng, a) or Rab11S25N RNA (1 ng, b) or Rab11 MO (c-f), and cultured until neurula stages. Transverse cryosections of the neural groove (stage 17-18) are shown. * indicates the injected right side, marked by the lineage tracer GFP-CAAX RNA (50 pg, green, a, b) or GFP RNA (50 pg, green, c-f). a-c, Myosin II activation was assessed by pMLC-specific immunostaining. Rab11S25N (b, bâ) and Rab11 MO (c, câ), but not wild-type Rab11 (a, aâ), inhibit apical pMLC staining (red), as compared to the uninjected side. No significant effect is observed on ZO1 (d), aPKC (e) and Sox3 (f). Scale bar is 20 μm (a-f). Asterisk indicates lack of apical pMLC. M, midline position. These results are representative from 4-7 independent experiments.
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Fig. 4. Rab11 protein levels in ectodermal cells with manipulated PCP signaling. Embryos were animally injected with Vangl2 MO (20 ng) or Diversin MO (30 ng) (a) or Rab11 (100 pg), Shroom (0.3 ng) and Vangl2 (1 ng) RNAs (b) and the levels of Rab11 in isolated neural (dorsal, D) and non-neural (ventral, V) explants (stage 14, a) or ectoderm explants made at stage 12 (b) were determined by immunoblot analysis. WE, whole embryo. α-Tubulin is a loading control. The quality of dissection for neural and non-neural explants was assessed by the pan-neural marker Sox3. a, Although Rab11 levels are higher in neuroectoderm, there was no change after Diversin and Vangl2 depletion. b, Rab11 levels were not affected in ectoderm explants after Shroom and/or Vangl2 RNA injection.
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Fig. 5. Role and localization of Rab11 during ectopic Shroom-induced apical constriction. a, Scheme of experiments. b, c, Apical recruitment of Rab11 in response to microinjected Shroom RNA (300 pg). b, Brightfield image showing pigment accumulation in a constricted cell (arrow). (bâ) anti-Rab11 antibody apical staining of embryo cryosection (arrow). c, En face view of apical Rab11 in Shroom-positive cells. GFP RNA is a lineage tracer (red). Arrow points to the constricted apical surface, asterisk indicates cell body. d, e, Localization of CFP-Vangl2 in stage 11 uninjected ectoderm (d) and at the site of Shroom RNA injection (e, eâ). Colocalization of CFP-Vangl2 and myc-Shroom (arrowhead). f, g, Rab11 polarity in the cells adjacent to Shroom-containing cells. f, junctional Rab11 staining in control ectoderm. g, Rab11 localization (arrow) in cells adjacent to a Shroom-expressing cell (asterisk). Green arrow indicates Rab11 planar polarity, cell boundary is demarcated by dashed line. h, i, GFP-Rab11 (100 pg ) and Shroom (300 pg ) RNAs were injected into adjacent blastomeres. Membrane-targeted Cherry RNA (400 pg) was co-injected with GFP-Rab11 RNA to mark cell boundaries. h, GFP-Rab11 is uniformly present at cell borders in control ectoderm (asterisks). i, Arrows point to Rab11 polarized towards Shroom-expressing ectoderm (strongly pigmented area at the bottom). Top view is shown, scale bar, 20 μm. j, Requirement of Rab11 for Shroom-induced constriction. Shroom RNA (300 pg) was coinjected with wild-type Rab11 or Rab11S25N RNA (1 ng each) animally, into four-cell embryos as indicated. Representative embryos are shown at stage 10, numbers indicate percentage of embryos with depicted phenotype. Apical constriction area is heavily pigmented (arrowheads).
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Fig. 6. Planar polarity of Rab11 endosomes during epithelial wound healing. a, Representative blastula embryo (stage 9), 30 min after wounding. Arrowheads point to polarized cells. b, Rab11 staining in control ectoderm, c, Representative ectodermal explant with Rab11 polarization relative to the wound position. Costaining with α-catenin antibodies indicates cell boundaries. Dashed green arrow indicates the direction toward the wound. d, e, Live images of GFP-Rab11 polarization (white arrows) in ectoderm during wound healing. d, control ectoderm; e, explant with wound healing for 1 hr. Wound (W) position is indicated by broken line. eâ, Inset shows two individual cells with polarized GFP-Rab11. f-h, Rab11 is essential for wound healing in ectodermal explants. Ectodermal explants were prepared at late blastula stages from the uninjected embryos (f), or embryos injected with wild-type Rab11 (h) or Rab11S25N (g) RNAs (1 ng each), and allowed to heal for 1 hr at room temperature. Scale bars are 20 μm in (b) and (d), also refer to (c, e), and 300 μm in (f), also refers to (g, h). Results are representative of 3-5 independent experiments.
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Figure 1: Rab11 distribution reveals planar polarity along the mediolateral axis of the Xenopus neural plate.
(a,b) Scheme (a) and a representative transverse cryosection (b) of the neural plate stained with anti-Rab11 monoclonal antibodies at stage 14/15. (b′) Higher magnification is shown for image within the white square in b. Costaining for phosphorylated myosin II light chain (pMLC, b′) reveals cell boundaries. (c–e) Embryo sections were co-stained with Rab11 and β-catenin (c,c′), ZO-1 (d) and aPKC (e) antibodies at higher magnification. (f) Polarized localization of exogenous Rab11. Four-cell embryos were injected with 100 pg of green fluorescent protein (GFP)-Rab11 RNA and stained with anti-GFP antibodies at midneurula stages (st 16). (b–f) White arrows point to polarized Rab11 distribution. Dashed green arrows and blue arrows in a, directed towards the midline (M) indicate cell and tissue polarity. Neural plate and individual cell boundaries are shown by broken lines. (g–i) En face view of the neural plate with polarized Rab11 and Diversin (white arrows). (g) Dorsal view of a neural plate explant (stage 16) with white rectangle indicating approximate location of images in h and i. A, anterior; L, lateral; M, medial; P, posterior. (h) Endogenous Rab11 is medially enriched along the mediolateral (ML) axis. (i,i′) Both exogenous GFP-Rab11 (green vesicles) and Diversin-RFP (i′, cytoplasmic red staining) show medial polarization along the ML axis (white arrows) and partly co-localize. Antibody specificity is indicated at the upper right corner of each panel. The anteroposterior (AP) axis is indicated. Scale bar in b and c (also refers to (d–f) is 10 μm.
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