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First published online 17 July 2007
doi: 10.1242/jcs.004556

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Regulation of otic vesicle and hair cell stereocilia morphogenesis by Ena/VASP-like (Evl) in Xenopus

Department of Genetics, Cell Biology and Development and Developmental Biology Center, University of Minnesota, Minneapolis, MN 55455, USA

View larger version (47K): [in this window] [in a new window]   Fig. 1. Distribution of Xevl transcripts in the Xenopus inner ear. (A) Diagram of the otic region. (B-E) In situ hybridizations on transverse sections through the otic region. The neural tube is at the top of the panels, the otic vesicle is located ventrolateral to the neural tube, and the vestibulocochlear ganglion resides between the otic vesicle and the neural tube. Nieuwkoop and Faber stages are indicated in the upper right corners. (B) Xevl is enriched in the ventromedial region of the otic vesicle at stage 25. (C) At stage 30, Xevl continues to be enriched in the ventromedial region of the otic vesicle as well as in cells delaminating from this region (arrow). (D) At stage 35, Xevl expression is found in the ventromedial region of the otic vesicle that will give rise to the sensory epithelium of the saccular maculae (arrowhead) as well as in the vestibulocochlear ganglion (arrow). (E) At stage 45, Xevl is enriched at the presumptive sensory epithelium (arrowhead) and is weakly expressed in the vestibulocochlear ganglion (arrow). ov, otic vesicle; vg, vestibulocochlear ganglion. Bar, 100 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 2. Xevl knockdown disrupts otic vesicle development. (A) Western blot analysis using a Xevl polyclonal antibody shows a marked reduction in Xevl protein production in embryos injected with XevlMO compared with embryos injected with control MO (coMO). Levels of -tubulin are unaffected by injection of the XevlMO. Numbers on right indicate molecular mass markers (kDa). (B) Quantitative analysis of Xevl knockdown and rescue experiments indicating the percentage of embryos displaying perturbed otic vesicle development. (C-G) Head region of Xenopus embryos at stage 35 (C) Diagram showing the olfactory placode (ol), lens placode (lens), otic vesicle (ov), epibranchial placodes (epi), and lateral line placodes (unlabeled). (D) XEya1 expression on the uninjected side of the embryo. (E) XEya1 expression on the XevlMO-injected side of the embryo. Otic vesicle size is reduced in 86% of Xevl-depleted embryos (n=121) with 72% exhibiting a strong reduction in size and 14% displaying a mild reduction. Otic vesicle size was unaffected in 14% of injected embryos. (F) XEya1 expression on the uninjected side of a rescued embryo. (G) XEya1 expression on the side of the embryo injected with Xevl-GFP mRNA and XevlMO. Expression of Xevl-GFP results in rescue of XEya1 expression with 43% displaying normal otic vesicles, 21% exhibiting a mild phenotype and 36% exhibiting a strong phenotype (n=77). Arrow marks the otic vesicle in D-G.

View larger version (153K): [in this window] [in a new window]   Fig. 3. Xevl regulates epithelial morphology in the otic vesicle. Confocal images of whole-mount preparations showing (A,A') -catenin, (B,B') vinculin, and (C,C') occludin immunostaining in the otic vesicle. Uninjected otic vesicles at stage 35 comprise a hollow sphere of columnar epithelial cells. (A-C) The otic epithelium exhibits apical-basal polarity with enhanced -catenin, vinculin and occludin localization at apical junctions (arrows). (D-F) Xevl depletion results in a smaller otic vesicle, disruption of columnar morphology, and loss of apical localization of (D,D') -catenin, (E,E') vinculin and (F,F') occludin. Arrows in A-F mark regions shown at higher magnification in A'-F'. Bars, 50 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 4. Xevl is required for cellular adhesion. (A) Confocal image of a whole-mount embryo at stage 35, showing Xevl immunostaining in the otic vesicle; a higher magnification image shown in A'. Xevl is enriched apically throughout the otic vesicle epithelium (arrow) and at cellular adhesions. Xevl is also highly expressed in the ventromedial sensory epithelium where mechanosensory hair cells form (arrowhead). (B) Dissociated otic vesicles from uninjected stage 35 embryos form large aggregates. (C) By contrast, otic vesicle cells from XevlMO-injected embryos form smaller and more loosely adherent aggregates. Insets in B and C show higher magnification of the aggregates indicated by dashed boxes. Bars, 50 µm.

View larger version (76K): [in this window] [in a new window]   Fig. 5. Xevl localizes to sensory structures of the inner ear. Confocal imaging of frozen sections of stage 35 embryos. Dorsal is up and the otic vesicle is to the right in all images. (A) Xevl is present in the vestibulocochlear ganglion (arrow) as well as in cells within the otic vesicle sensory epithelium (arrowhead). (B) Islet-1 localization in the vestibulocochlear ganglion (arrow) and within the otic vesicle sensory epithelium (bracket). (C) The merged image reveals Xevl (red) is more strongly expressed in the cells that also have strong islet-1 staining (green). DAPI staining is in blue. (D-F) Xevl (D, red in F) colocalizes with neural neurofilament (nf; E, green in F) in neurons of the vestibulocochlear ganglion and the neurites that extend to innervate the otic vesicle epithelium (arrowhead in F) and the hindbrain (arrow in F). Bar, 20 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 6. Vestibulocochlear ganglion size and organization is perturbed by Xevl depletion. Dorsal is up and the otic vesicle is to the right in all images. (A) Expression of XNeuroD in the vestibulocochlear ganglion (arrow) and ventromedial sensory epithelium (arrowhead) of control stage 35 embryos. (B) Xevl depletion diminishes the size of the vestibulocochlear ganglion (arrow) and reduces the amount of XNeuroD-positive cells in the sensory epithelium and the vestibulocochlear ganglion. The Xevl-depleted otic vesicle also exhibits a loss of columnar morphology in the thickened sensory epithelium compared with the control otic vesicle (arrowhead, 100% affected; n=9). (C-H) Confocal images of vibrotome-sectioned embryo at stage 42 showing islet-1 (C,F; red in E,H) and laminin (D,G; green in E,H) immunostaining of the vestibulocochlear ganglion. (F,H) Xevl depletion causes a reduction in the number of islet-1-labeled cells and an overall reduction in the size of the vestibulocochlear ganglion (arrow). (G,H) In addition, the laminin-rich matrix surrounding the vestibulocochlear ganglion (arrow) is disrupted in Xevl-depleted embryos. Bar, 20 µm. A longer exposure time was necessary for the Xevl-depleted otic vesicle to show islet-1 in the vestibulocochlear ganglion, making islet-1 levels appear higher in the neural tube and otic vesicle of Xevl-depleted embryos compared with controls.

View larger version (98K): [in this window] [in a new window]   Fig. 7. Neurite outgrowth is reduced in Xevl-depleted otic vesicles. Vibrotome sections of the otic vesicle region of stage-35 embryos with dorsal oriented up and the otic vesicle to the right in each image. (A,B) Uninjected embryos exhibit extensive neurofilament-positive projections into the vestibulocochlear ganglion (arrowhead) and the ventromedial sensory epithelium (arrow) of the otic vesicle. (C,D) Xevl-depleted otic vesicles exhibit shorter and fewer sensory projections in both the vestibulocochlear ganglion (arrowhead) and the sensory epithelium (arrow). Neurofilament is shown in green in B,D. Preparations were co-stained with spectrin (red in B,D) to outline cell boundaries. Bar, 50 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 8. Xevl depletion in hair cells disrupts spectrin localization and stereocilia formation. (A-D) Images of frozen sectioned embryos with dorsal oriented up and the otic vesicle on the right of each image. (A) Xevl is present in hair cells of the ventromedial sensory epithelium and is enriched at the apical aspect of these cells (arrow). (B) Xevl (red) and actin (phalloidin; green) colocalize at the apical region of hair cells (arrow) at the base of stereocilia. (C,D) Higher magnification view of Xevl localization in hair cells (arrows) showing the colocalization of Xevl (red) and actin (green) at the apical margin of the hair cells. (E,F) Image of a dissected otic vesicle showing that Xevl (E, red in F) localizes to the cuticular plate at the base of each actin-rich stereocilium (green in F). (G) Images of frozen sectioned embryos showing that spectrin is localized to the cuticular plate at the apical region of hair cells (arrow). (H) Spectrin (red) is at the base of each stereocilium and colocalizes with actin (green) at the apical membrane of hair cells in uninjected embryos. (I) In XevlMO-injected embryos, spectrin levels at the cuticular plate are markedly reduced and hair cells lose their columnar shape (arrowhead). (J) Xevl depletion also results in a reduction in actin staining (green) at the apical portion of hair cells and few stereocilia are present. Spectrin is shown in red in the merged image. Bars, 20 µm, except for E and F where bar, 5 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 9. Xevl is required for stereocilia formation. Confocal immunofluorescence images of dissected Xenopus otic vesicles at stage 45 stained with actin (phalloidin; A,B, red in C-H), spectrin (green in C-E) and espin (green in F-H). (A) Uninjected otic vesicles possess stereocilia with a staircase of actin filaments (arrows). (B) XevlMO-injected otic vesicles have shorter stereocilia that are more uniform in length and exhibit a splayed appearance (arrows). (C) Uninjected otic vesicles possess strong spectrin localization at the cuticular plate (arrow). (D,E) Xevl-depleted embryos display a decrease in spectrin staining (arrows) that correlate with defects in stereocilia morphology. (F) In uninjected embryos, espin staining is strongly associated with stereocilia (arrow). (G,H) Xevl depletion causes a reduction in espin levels in stereocilia (arrow). Bars, 5 µm.

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