Molecular Basis For The Morphogenesis Of The Inner Ear

National Institute On Deafness And Other Communication Disorders

Investigators

Linked publications, trials & patents

Abstract

This years major accomplishments are in the following areas: 1)Function of bidirectional sensitivity in the otolith organs established by transcription factor Emx2 The inner ear is a major contributor for maintaining our sense of balance. A better understanding of how vestibular information is being encoded by the inner ear and processed centrally in the brain will facilitate the design of strategies to alleviate vestibular deficits, which are common in the elderly population. We utilized a genetic approach to generate mutant mice with specific structural defects in the vestibular organs to address the functional significance of these structural features. Each of the two otolith organs of the inner ear, responsible for detecting linear acceleration of the head, are innervated by two parallel afferent projections to the brainstem and cerebellum. A line of polarity reversal (LPR) can be drawn within the otolith organ, across which hair bundles of sensory hair cells are arranged in mirror images. Previously, we have demonstrated that the restricted expression of transcription factor Emx2 in one region of the otolith organ reverses the hair bundle orientation within the region and thus establishes the LPR. The relationship between the LPR and the neuronal segregation pattern is not clear. We demonstrated that afferents of the otolith organ with different central projections are segregated across the LPR; ones that innervate the Emx2-positive domain largely project to the cerebellum and others that innervate the Emx2-negative domain project to the brainstem. Notably, in the Emx2 knockout mouse embryos, the vestibular neurons with central projection to the cerebellum that normally innervate hair cells in the Emx2 domain fail to reach their destinated sensory epithelium. Together, these results indicate that Emx2 regulates bidirectional sensitivity of the otolith organs on two levels: hair bundle orientation as well as neuronal selectivity. To address the functional significance of this bidirectional sensitivity, we generated conditional knockout (cKO) of Emx2 in hair cells. These cKO mice are viable but they lack the LPR because hair bundles in the region that normally expresses Emx2 are reverted to the default position similar to hair bundle in the Emx2-negative domain. However, unlike the KO, neuronal projection to the cerebellum in the Emx2 cKO is largely normal, thus allowing us to address the functional consequence of loss of the LPR. Additionally, we generated another mouse mutant which lacks bidirectional sensitivity in the otolith organs, Tmie cKO, in which the LPR is present but hair cells in the Emx2-positive domain are rendered inactive due to the lack of mechanotransduction channels in hair cells. By subjecting Emx2 cKO and Tmie cKO mutants to a number of behavioral tests such as swimming, rotarod, and balance beam, we determined that the bidirectional sensitivity in the otolith organs is important for mice to swim comfortably and to traverse a narrow balance beam efficiently. Manuscript in review, https://assets.researchsquare.com/files/rs-1471702/v1/c29013e9-4e9b-417a-bbb5-28cb8cb3449b.pdf?c=1648475319 2)Requirements of Sonic Hedgehog in spiral ganglion formation of the mouse inner ear The mammalian cochlea of the inner ear is a structurally sophisticated organ responsible for detecting sound. Sensory hair cells in the organ of Corti convert sound in waveforms into chemical signals which are released and relayed to the brain as neural signals via neurons of the spiral ganglion. Insults to either cochlear hair cells or spiral ganglion neurons (SGN) could result in hearing loss. Notably, formation of the cochlea and SG are coupled during development. A better understanding of the molecular pathways that coordinate the formation of these two structures will provide insights into the etiology of neurosensory hearing loss in humans. Previously, we have shown that the ligand, Sonic hedgehog (Shh), secreted by the developing spiral ganglion regulates the growth of the cochlear duct and timing of cell cycle exit of hair cells. Given the importance of Shh in cochlear formation, we investigated whether Shh has any role in SG development. Using conditional knockout and lineage tracing of Shh in the developing SG of mice, we found that SGN are generated from the base of the cochlear duct towards the apex over time, supporting previous birthdating results using H3-thymidine. Additionally, we found Shh is expressed transiently in nascent SGN and it is not expressed in neuronal precursors or mature neurons. However, Shh secreted by nascent SGN is required to maintain the proliferation of adjacent neural progenitors. The reduction of Shh signaling has an accumulative effect on subsequent SGN production. Together, these results indicate that as Shh synchronizes the production of SGN from base to apex, it concomitantly coordinates cochlear duct outgrowth and timing of HC differentiation. (Manuscript in preparation) 3)The function of Emx2 in the mammalian cochlea Many genetic pathways are involved in the formation of the structurally complex organ of Corti within the mammalian cochlea. Deciphering some of the molecular pathways involved in its formation provides a fundamental understanding of its developmental process and can pave the way for future therapeutic approaches for alleviating neurosensory hearing loss. The transcription factor Emx2 is important for establishing the bidirectional sensitivity of the otolith organs by regulating hair bundle orientation and neuronal selectivity. The requirement of Emx2 in cochlear hair cells appears to be earlier in development than hair cells in the otolith organs. In Emx2 knockout cochlea, outer hair cells are absent and inner hair cells, though present, are disorganized and often appear as doublets. The lack of outer hair cell formation and the disorganized inner hair cell patterning in Emx2 KO negate the analysis of Emx2s role in hair bundle orientation in the cochlea. To address whether Emx2 has a similar role in establishing hair bundle orientation in cochlear hair cells similar to the vestibular hair cells, we generated conditional knockout of Emx2 in nascent hair cells using Atoh1cre. Our results indicate that in the Emx2 cKO, hair bundle orientation is reversed in the outer but not inner hair cells. In outer hair cells, there is a longitudinal and a radial gradient in the severity of hair bundle reversal phenotype; bundle reversal is more severe at the base of the cochlea than the apex and more severe in the first than third row of outer hair cells. Together, these results suggest that Emx2 has a conserved role of establishing hair bundle orientation among outer hair cells in the cochlea, similar to vestibular hair cells.