Role of Rab11 in planar cell polarity and apical constriction during vertebrate neural tube closure

Epithelial folding is a critical process underlying many morphogenetic events including vertebrate neural tube closure, however, its spatial regulation is largely unknown. Here we show that during neural tube formation Rab11-positive recycling endosomes acquire bilaterally symmetric distribution in the Xenopus neural plate, being enriched at medial apical cell junctions. This mediolateral polarization was under the control of planar cell polarity (PCP) signaling, was necessary for neural plate folding, and was accompanied by the polarization of the exocyst component Sec15. Our further experiments demonstrate that similar PCP-dependent polarization of Rab11 is essential for ectopic apical constriction driven by the actin-binding protein Shroom and during embryonic wound repair. We propose that anisotropic membrane trafficking plays key roles in diverse morphogenetic behaviors of individual cells and propagates in a tissue by a common mechanism that involves PCP.


Introduction
Epithelial tissue folding is a universal mechanism for germ layer rearrangements at gastrulation, neural tube closure and organogenesis. During vertebrate neurulation, dorsal ectoderm forms the neural plate that bends to generate the neural groove [1][2][3] . At a later stage, the bilateral neural folds fuse at the midline into the neural tube. Actomyosin contractility and cell junction remodeling are believed to be major driving forces for epithelial folding in Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms

Planar polarization of Rab11 in the neural plate
Immunostaining of sectioned early neurula embryos at stages 14-15 revealed striking polarization of Rab11 in the plane of the neuroepithelium (Fig. 1a-c, Supplementary Fig. 1ad). Even before neural plate folding became morphologically apparent, Rab11 became localized to one apical corner of neuroepithelial cells that is closer to the dorsal midline, thereby establishing mirror-image planar polarity in the early neural plate. The bilateral staining progressively changed towards apical midline staining at the neural fold stages (Fig.  1a, b, Supplementary Fig. 1a, b). Other apical or basolateral markers, such as atypical PKC, ZO1 and β-catenin, did not reveal similar polarization (Fig. 1c-e). The observed pattern was often detected as comet-shape cytoplasmic staining directed towards a cell junction (marked by ZO1, Fig. 1d). To independently confirm that Rab11 is indeed unilaterally polarized in individual cells, we examined early embryos injected with RNA encoding Rab11-GFP (Fig.  1f). Mosaically expressed exogenous Rab11-GFP was similarly distributed in a gradient with the highest concentration at the medial apical corner of the cell (Fig. 1f). Lack of staining in the neighboring cells, which did not receive Rab11-GFP RNA, allowed us to unequivocally define the position of Rab11-GFP relative to the cell border. En face views of the neural plate immunostained for both endogenous and exogenous Rab11 confirmed the polarization of Rab11 in the plane of the tissue, in a manner reminiscent of the distribution of PCP proteins in Drosophila epithelia 11 (Fig. 1g-i). Interestingly, Diversin, a vertebrate homologue of the fly PCP protein Diego, revealed a similar polarization (Fig. 1i, i'), providing insight into the role of PCP proteins in neural tube closure. These findings suggest that anisotropic Rab11-dependent recycling is an early event in neural tube closure.

Rab11 is required for Myosin II activation and neural tube closure
To test whether Rab11-mediated membrane trafficking is required for neural plate folding, we studied the phenotype of embryos microinjected with RNA encoding Rab11S25N, a dominant negative construct 23 , which is distributed throughout the cytoplasm ( Supplementary Fig. 1e, f). In the majority of injected embryos (85 %, n= 53), the neural plate remained open at the injected side, yet this defect was rescued by wild-type Rab11 RNA (Fig. 2a, b). Similar results have been obtained using a specific morpholino antisense oligonucleotide (MO) (Fig. 2c, d), which effectively depletes Rab11 in Xenopus embryos 23 . The accumulation of phosphorylated Myosin II light chain (pMLC), a key RhoA-dependent regulatory process in neural tube closure 7-10 , failed to occur (Fig. 3a-c), complementing changes in neural fold and individual cell morphology (Fig. 2e, f; Supplementary Fig. 2a, b). This observation indicates that Rab11 is required for Myosin II activation in the neural plate. Despite these changes in the contractile machinery, the apical-basal polarity markers aPKC and ZO1 were not affected (Fig. 3d, e). Together, our observations show that Rab11 is not just a marker for neuroepithelial progenitor polarity, but an essential modulator of neural tube closure. Lack of changes in the neural tissue markers Sox3 (Fig. 2g, Fig. 3f), Nrp1 and Rx1 ( Supplementary Fig. 2c, d) indicates that Rab11 is not involved in cell fate determination during neural induction. To further understand how Rab11 regulates neural plate folding, we examined the localization of exocyst, a protein complex that targets post-Golgi secretory vesicles for subsequent membrane fusion during exocytosis 26 . We find that the Rab11-interacting exocyst component Sec15 27 polarizes in the neural plate similarly to Rab11 (Fig. 2h). This result suggests that Sec15 promotes neural tube closure by directing exocytosis near the medial surface of each cell.

PCP proteins regulate Rab11 localization in the neural plate
Given the medial polarization of Diversin in the neural plate, we hypothesized that PCP signaling could function upstream of Rab11 in neural plate closure. Indeed, neural tube closure defects were apparent in Diversin-depleted embryos ( Supplementary Fig. 3). Moreover, interference with the function of Diversin and two other core PCP proteins, Vangl2 and Dishevelled, inhibited Rab11 polarization in the neural plate ( Fig. 2i-k, Supplementary Fig. 4a). In these experiments the total amount of Rab11 protein remained unaffected (Fig. 4a). Despite the major changes in neural plate morphology and the decrease of myosin II phosphorylation in Vangl2-depleted embryos ( Supplementary Fig. 4b), Vangl2 MO did not alter the fate of neural progenitors, which remained positive for Sox3 ( Supplementary Fig. 4a). Finally, coinjection of RNAs encoding Rab11S25N and Vangl2 strongly enhanced the neural tube closure defects observed in separate RNA injections, indicating that these molecules functionally interact ( Supplementary Fig. 4c, d). These experiments indicate that the polarization of Rab11 endosomes along the mediolateral axis of the neural plate is controlled by the PCP pathway.

Rab11 functions during ectopic apical constriction
Since neural plate folding is largely driven by apical constriction of neuroepithelial cells, we suspected that Rab11 plays a role in this process. We wanted to examine an alternative model, in which morphogenesis can be separated from neural induction and patterning that accompany neural tube closure. Ectopic apical constriction is induced in blastula ectoderm by overexpression of Shroom, a protein linked to actomyosin contractility and essential for neural tube closure 10,28 . We first assessed Rab11 localization during Shroom-induced apical constriction, which took place in the majority of injected embryos (Fig. 5). We observed strong apical recruitment of both Rab11 and Vangl2 in the constricting Shroomexpressing cells, which are easily identified by abundant pigment granules near their apical surfaces and elongated morphology ( Fig. 5a-e, Supplementary Fig. 5a-c). Moreover, both endogenous and exogenous Rab11 polarized towards the site of Shroom RNA injection in the cells adjacent to the Shroom-expressing tissue ( Fig. 5f-i, Supplementary Figs. 5a, b, e). Therefore, our lineage tracing analysis suggests that a Shroom-induced signal triggers noncell-autonomous Rab11 polarization in the surrounding cells. Although it is currently unclear whether Rab11 has the same function in Shroom-expressing and nonexpressing cells, Shroom-dependent apical constriction was inhibited by Rab11S25N in more than 80 % of injected embryos (n = 92) (Fig. 5j). Consistent with this morphological effect, Rab11S25N inhibited apical pMLC accumulation induced by Shroom, but had no significant effect on aPKC or ZO1 ( Supplementary Fig. 5h, i, and data not shown). Of interest, overexpressed Vangl2 interfered with Shroom-induced constriction and inhibited Rab11 apical accumulation and planar polarization in Shroom-expressing ectoderm and during neural tube closure (Supplementary Fig. 5c-g, j, k), although total Rab11 protein levels in Vangl2expressing cells did not change (Fig. 4b). This Rab11 polarization in cells that respond to Shroom was inhibited by Vangl2 both in the cell-autonomous and non cell-autonomous manner ( Supplementary Fig. 5c-g). Together, these results are consistent with the possibility that the PCP pathway interacts with vesicular trafficking machinery to drive ectopic apical constriction in response to Shroom.

Rab11 and PCP proteins in embryonic wound healing
Coordinated changes in cell shape that are similar to apical constriction take place during embryonic wound repair 10 . We further addressed Rab11 function in morphogenesis, by removing a small piece of tissue from blastula ectoderm, a tissue lacking detectable planar polarity (Fig. 6a). In this model, wound healing is initiated within minutes after the surgery with pigmented superficial ectoderm spreading eventually over the inner cell layer. Planar polarization of Rab11 became apparent in the superficial layer of the healing tissue in both embryos and ectoderm explants in 30 to 60 min after the wound was inflicted (Fig. 6a-c and Supplementary Fig. 6a, b). We found that both endogenous and exogenous Rab11containing recycling endosomes are polarized in individual cells towards the direction of the wound (Fig. 6a-e, Supplementary Fig. 6a, b). Additionally, both Rab11 and Vangl2 protein complexes became apically enriched in inner ectoderm cells, which are known to participate in apical constriction during wound closure 10 (Supplementary Fig. 6b, asterisk, Supplementary Fig. 7). Moreover, wound healing was defective in embryos and explants that expressed Rab11S25N, Rab11 MO or Vangl2 MO (Fig. 6f-h, Supplementary Fig. 6c-i), indicating that both Rab11 and Vangl2 are required for this process. Similar to our observations of neural plate folding, Vangl2 depletion inhibited Rab11 polarization and pMLC accumulation (data not shown). Cumulatively, our findings suggest that polarized Rab11-mediated recycling is involved in diverse models of epithelial morphogenesis and point to the conservation of mechanisms regulating neural tube closure and embryonic wound healing.

Discussion
Several models of epithelial folding have been proposed including coordinated regulation of actomyosin contractility 2, 3, 6, 9 , repositioning of adherens junctions by PAR proteins 5 and enhanced endocytosis 20 . Despite the recent progress, it remained unclear whether tissue folding has any directionality. Our experiments visualizing the graded distribution of Rab11 recycling endosomes are the first to reveal the cell polarity corresponding to the axis of bending or constriction. We also find that this polarity requires core PCP proteins, reinforcing the known connection between vesicular trafficking and PCP 19,[29][30][31][32] . The unique localization of Rab11 and the exocyst component Sec15 suggests a model with an essential role of anisotropic membrane trafficking in neural tube closure. In this model, PCP signals are conveyed to individual cells to polarize Rab11-mediated membrane trafficking and allow apical constriction to proceed directionally, towards the midline, by expanding the lateral membrane domain at the expense of the shrinking apical domain. Although our experiments demonstrate a requirement of Rab11 for myosin II activation in the neural plate, we note that the proposed mechanism does not support the classical 'purse string' model with isotropic actomyosin contractility along the mediolateral axis. The same general mechanism may act to polarize membrane trafficking in individual cells in response to wounding, leading to cell reorientation and tissue repair.
The observed regulation of Rab11-dependent vesicular trafficking by core PCP molecules reiterates novel roles of these proteins in modulating cell shape and behavior during tissue folding and embryonic wound healing. Nevertheless, Rab11 roles in cell elongation, radial intercalation or convergent extension during neural tube closure 1 , and relevant cargo proteins for Rab11 vesicles remain to be elucidated. Consistent with the possibility that Vangl2 trafficking depends on Rab11, CFP-Vangl2 membrane localization became more diffuse and shifted to the cytoplasm in cells depleted of Rab11 or expressing Rab11S25N ( Supplementary Fig. 8). This finding supports a positive feedback loop, in which PCP proteins that regulate Rab11 are themselves targeted to their specific locations by Rab11dependent trafficking 31,33,34 .

Xenopus embryo injections, apical constriction and wound healing assays
In vitro fertilization, culture and staging of Xenopus laevis embryos were carried out following standard techniques 43

Immunohistochemistry and immunoblot analysis
To examine protein subcellular localization, neural plate explants expressing GFP-or RFPtagged constructs were dissected at stages 15-17 from embryos fixed for 1 hr with MEMFA (0.1 M MOPS, pH 7.4, 2mM EGTA, 1mM MgSO4 and 3.7% formaldehyde), washed with 1× PBS and mounted for observation with the Vectashield mounting medium (Vector). Ectoderm explants expressing GFP-tagged constructs were dissected at stage 11 and processed as described above. For F-actin staining, Alexa 568-conjugated phalloidin (5 units/ml, Molecular Probes) was used. For detection of Rab11 and pMLC on cryosections, embryos were devitellinized, fixed with 2% trichloracetic acid (TCA) solution for 30 min at room temperature and washed with 0.3% Triton X100 in 1× PBS for 30 min 44 . For en face view of Rab11 and ZO1 in the neural plate, explants were fixed with TCA (38), and stained with primary antibodies overnight, secondary antibody for 2 hrs, washed and mounted for imaging.
Immunoblot analysis was done with dissected tissues (neural plate explants, stage 14, or animal caps, stage 12) with anti-Rab11, anti-Sox3 and anti-αTubulin antibodies using standard techniques 45 . Briefly, embryos were resuspended in lysis buffer (0.5% Triton X100, 10 mM Tris-HCI at pH 7.5, 50 mM NaCl, 1 mM EDTA), containing protease inhibitor cocktail (Roche). Proteins were resolved by SDS-PAGE. An equivalent of 0.2-0.5 embryo was loaded per lane. After protein transfer to the Immobilon-P PVDF membrane (Millipore), the membrane was incubated sequentially with primary and secondary antibodies, conjugated to horseradish peroxidase. Peroxidase activity was detected by enhanced chemiluminescence.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.   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.