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The inner ear is derived from a thickening in the embryonic ectoderm, called the otic placode. This structure undergoes extensive morphogenetic movements throughout its development and gives rise to all components of the inner ear. Ena/VASP-like (Evl) is an actin binding protein involved in the regulation of cytoskeletal dynamics and organization. We have examined the role of Evl during the morphogenesis of the Xenopus inner ear. Evl (hereafter referred to as Xevl) is expressed throughout otic vesicle formation and is enriched in the neuroblasts that delaminate to form the vestibulocochlear ganglion and in hair cells that possess mechanosensory stereocilia. Knockdown of Xevl perturbs epithelial morphology and intercellular adhesion in the otic vesicle and disrupts formation of the vestibulocochlear ganglion, evidenced by reduction of ganglion size, disorganization of the ganglion, and defects in neurite outgrowth. Later in embryogenesis, Xevl is required for development of mechanosensory hair cells. In Xevl knockdown embryos, hair cells of the ventromedial sensory epithelium display multiple abnormalities including disruption of the cuticular plate at the base of stereocilia and disorganization of the normal staircase appearance of stereocilia. Based on these data, we propose that Xevl plays an integral role in regulating morphogenesis of the inner earepithelium and the subsequent development of the vestibulocochlear ganglion and mechanosensory hair cells.
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.
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.
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.
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.
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.
