Molecular Basis For The Morphogenesis Of The Inner Ear

National Institute On Deafness And Other Communication Disorders

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Abstract

This years major accomplishments are in the following areas: 1) Live imaging of hair bundle polarity acquisition demonstrates a critical timeline for transcription factor Emx2. The mechano-transduction channels that are responsible for detecting sound and head positions in the mammalian inner ear reside in the stereociliary bundle (also known as the hair bundle), which is erected on the apical surface of the sensory HC (HC). Understanding how this unique apparatus forms during development is important as many mutations that cause hearing loss are associated with failing of assembly of the mechano-transduction channel and/or hair bundle. Notably, the position where the hair bundle is located on the apical HC surface is also important as the orientation of the hair bundle dictates the directional sensitivity of the HC. Previously, we demonstrated that the presence of a transcription factor, Emx2, within a sensory HC alters its hair bundle orientation by 180 degrees. While all HCs of the cochlea express Emx2, this transcription factor is only regionally expressed in two of the vestibular organs, maculae of the utricle and saccule. As a result, in both maculae, a line of polarity reversal (LPR) demarcates the border of the Emx2 positive and negative domains, across which hair bundles are oriented in opposite directions. Based on scanning electron micrographs and immunostaining results, it is thought that the normal process of hair bundle establishment involves the mother centriole docking at the center of the apical HC surface and forming the base of the kinocilium. The protruding kinocilium on the apical surface then migrates to its destinated peripheral position before the microvilli-based stereocilia staircase is built next to it. The presence of Emx2 causes the hair bundle to be built at the opposite side of the apical surface away from its normal default location. While the downstream effector(s) of Emx2 that mediate this effect are unclear, we used live imaging to investigate how hair bundle is normally established in HCs across the LPR in the utricle by following the movements of the centrioles during HC differentiation. We aimed to determine whether the kinocilium in the Emx2-positive HCs first moves to the default position similar to the Emx2-negative HCs before reversing its course to the opposite end of the cell or the kinocilium moves directly from the center towards its destination. Our results showed that the kinocilium in the Emx2-positive HCs moves directly towards its destinated position opposite from the Emx2-negative HCs, suggesting that Emx2-HCs are already pre-staged by Emx2 prior to hair bundle establishment. Furthermore, ectopic Emx2 expression in HCs using transgenic mice or adeno-associated virus indicate that the timing of Emx2 requirement is short occurring within 10-12 hours, suggesting that Emx2 does not mediate hair bundle reversal via a cascade of transcriptional events. This study was published in the journal eLife. https://elifesciences.org/articles/60432 2) Emx2 regulates HC rearrangement but not positional identity within neuromasts The ability of Emx2 to reverse hair bundle orientation in mammalian HCs appears conserved in neuromasts of the zebrafish lateral line system. In each neuromast, HCs are born as a pair and in half of the time, the two sibling HCs switch positions with each other. The significance of this rearrangement is not clear, but the resulting pattern in a neuromast oriented along the anterior-posterior (A-P) direction, for example, is that the anterior-positioned HC always expresses Emx2 with its hair bundle orientation pointing towards the posterior, whereas the hair bundle of the posterior-positioned, Emx2-negative sibling HC, points towards the anterior. While results of gain- and loss-of-function Emx2 mutants indicate that Emx2 is necessary and sufficient to alter hair bundle orientation in HCs of the neuromast, it was not clear whether Emx2 was mediating this effect by changing HCs positions or by changing hair bundle orientation cell autonomously, similar to their mammalian counterparts. To address this question, we generated an Emx2 reporter line (Emx2:Gfp) to track the Emx2-positive HCs by knocking in Gfp to the Emx2 locus using crispr. Live imaging of nascent hair-cell pair formation in Emx2:Gfp larvae indicates that Emx2 is activated at the time when the two sibling HCs are in the midst of switching positions with each other. The activation of Emx2 in HCs appeared random. When Emx2 was activated in the posterior HC, the two siblings invariably switched positions, whereas when Emx2 was activated in the anterior HC, the two siblings remained in positions. In addition, in Emx2 knockouts, the duration of the cell rearrangement process was prolonged, whereas in Emx2 gain-of-function neuromasts, the duration is shortened. Taken together results of Emx2 onset and the cell rearrangement phenotypes in Emx2 mutants suggest that Emx2 could be involved in the positioning of the HCs within the neuromast. This notion, however, was refuted by the normal location of Emx2:Gfp HCs in Emx2 knockouts. Further analyses indicate that Emx2 facilitates HC rearrangement by delaying the apical protrusion of its nascent HC, but it does not affect the final positions of HCs. Therefore, we concluded that Emx2 mediates HC orientation in zebrafish in a similar conserved manner as mammalian HCs by changing hair bundle orientation in a cell autonomous fashion. This work was published in the journal, eLife https://elifesciences.org/articles/60432 3) Emx2 mediates neuronal selectivity in the maculae of the vestibular system The vestibular ganglion of the inner ear is comprised of bipolar neurons that relay sensory inputs from the five main vestibular sensory organs to the brain. Unlike the highly ordered tonotopically-organized sensory neurons of the auditory ganglion in the cochlea or the spatially organized retinal ganglion neurons in the eye, the organization of the vestibular neurons is not apparent, and the neurons are not segregated based on individual sensory organs that they innervate or their central targets in the brain. Identifying the underlying molecular and functional organization of the vestibular ganglion is an essential piece of the puzzle for understanding the vestibular system and addressing the etiology of balance disorders. The line of polarity reversal (LPR), which is established by Emx2, divides HCs with opposite hair bundle orientations into two regions within the maculae. Notably, the vestibular ganglionic neurons that innervate HCs across the LPR are also segregated, with neurons that innervate the Emx2-positive region project to the cerebellum and neurons that innervate the Emx2-negative region project to the brainstem. Since we have shown previously that Emx2 regulates both the hair bundle orientation as well as neuronal selectivity in zebrafish neuromasts, we asked whether Emx2 also regulates the neuronal selectivity in the two maculae. Using lipophilic dye tracing technique in Emx2 gain- and loss-of-function mouse mutants, we found that Emx2 expressed in the sensory epithelium of maculae is indeed required for neuronal segregation. In Emx2 knockout mutants, the neurons that normally innervate the Emx2-positive sensory epithelium fail to reach the sensory organ, whereas neurons that normally innervate the Emx2-negative sensory region are expanded to the entire organ. In the Emx2 gain-of-function mouse models, where Emx2 is expressed in the entire sensory epithelium or all the sensory HCs, neuronal innervation pattern is disrupted. Taken together these results with our previous results in zebrafish indicate that Emx2 has a conserved role in regulating both hair bundle orientation and neuronal selectivity. (manuscript in preparation)