XB-ART-49942Development. January 1, 2015; 142 (1): 99-107.
Vangl2 cooperates with Rab11 and Myosin V to regulate apical constriction during vertebrate gastrulation.
Core planar cell polarity (PCP) proteins are well known to regulate polarity in Drosophila and vertebrate epithelia; however, their functions in vertebrate morphogenesis remain poorly understood. In this study, we describe a role for PCP signaling in the process of apical constriction during Xenopus gastrulation. The core PCP protein Vangl2 is detected at the apical surfaces of cells at the blastopore lip, and it functions during blastopore formation and closure. Further experiments show that Vangl2, as well as Daam1 and Rho-associated kinase (Rock), regulate apical constriction of bottle cells at the blastopore and ectopic constriction of ectoderm cells triggered by the actin-binding protein Shroom3. At the blastopore lip, Vangl2 is required for the apical accumulation of the recycling endosome marker Rab11. We also show that Rab11 and the associated motor protein Myosin V play essential roles in both endogenous and ectopic apical constriction, and might be involved in Vangl2 trafficking to the cell surface. Overexpression of Rab11 RNA was sufficient to partly restore normal blastopore formation in Vangl2-deficient embryos. These observations suggest that Vangl2 affects Rab11 to regulate apical constriction during blastopore formation.
PubMed ID: 25480917
PMC ID: PMC4299144
Article link: Development.
Grant support: HD078874 NICHD NIH HHS , NS40972 NINDS NIH HHS , R01 NS040972 NINDS NIH HHS , R21 HD078874 NICHD NIH HHS , R56 NS040972 NINDS NIH HHS
Genes referenced: actl6a daam1 itgb1 myl2 myo5b rab11a rhoa rock1 shroom3 vangl2
Antibodies referenced: Itgb1 Ab1 Phosphorylated myosin light chain Ab1 Rab11a Ab2 Rab11a Ab3 Rab11a Ab4 Vangl2 Ab2 Vangl2 Ab3
Morpholinos referenced: daam1 MO2 rab11a MO1 vangl2 MO2
Article Images: [+] show captions
|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.|
|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.|
|Fig. 3. Roles of downstream PCP components in bottle cell formation. (A-D) Early blastopore defects in embryos expressing dominant interfering constructs for PCP and Rock pathway components. Embryos were injected with GFP, N-Daam1 or RhoA-N19 RNAs (2 ng each) or DN-Rock RNA (0.1 ng), together with lacZ RNA (0.15 ng) as lineage tracer. Vegetal view of stage (st) 10+ embryos is shown. Dorsal is to the top. (E-G) Embryos expressing N-Daam1 and RhoA-N19 display delayed blastopore closure at stage 11. Arrows in A and E indicate blastopore lip in control GFP-expressing embryos. (H) Quantification of the effect. N-Dm1, N-Daam1; N19, RhoA-N19.|
|Fig. 4. Interference with the PCP pathway prevents Shroom-induced apical constriction. Early embryos were animally injected with FLAG-Shroom RNA (50 pg) and other RNAs or MOs as indicated. (A-D) Vangl2 depletion inhibits Shroom-dependent ectopic apical constriction (arrowhead). CO, control. (A-C) Representative embryo images at stage (st) 10.5. (D) Quantification of the data, numbers of embryos with strong, mild and no ectopic constriction are presented. Strong (++) apical constriction involves hyperpigmentation and the accompanying tissue invagination, mild (+) constriction is frequently visible as diffuse hyperpigmentation in the absence of tissue indentation. (E-H) Inhibitory effects of N-Daam1 and zPk1ΔPL on Shroom-dependent apical constriction in stage 10 embryos. (H) Quantification of the data. +indicates ectopic apical constriction; ‘normal’ indicates no constriction.|
|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.|
|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+.|
|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.|
|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.|
|Fig. S2. Inhibition of blastopore lip formation in Vangl2-depleted embryos. (A-E) Early embryos were injected with Vangl2 MO or control MO either dorsally or ventrally as indicated. LacZ RNA (150 pg) has been coinjected a lineage tracer. Vegetal views of stage 11 embryos are shown. E, Quantitation of the experiments shown in A-D. (F, G) En face view of the dorsal lip in stage 10+ embryos. Vangl2 was depleted by a unilateral injection of control or Vangl2 MO (30 ng each). F-actin staining in control (F) and Vangl2-depleted (G) blastopore cells. Arrow points to reduced apical F-actin in Vangl2-depleted cells (marked by GFP, green). Animal pole is at the top. Scale bar in F (also applies to G) is 10 μm. Dashed line indicates the midline. Arrowheads point to F-actin-enriched areas, which are reduced by Vangl2 depletion.|
|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.|
|Fig. S4. Rescue of dorsal blastopore in Vangl2-depleted embryos by Rab11 RNA. (A, B) Embryos were injected with 30 ng of Vangl2 MO and 1 ng of Rab11Q70L RNA as indicated. (C) Uninjected control embryo. Vegetal views reveal the comparative morphology of the dorsal lip (arrow) for representative embryos from data in Fig. 6I.|
|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.|