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)Function of bidirectional sensitivity in the otolith organs established by transcription factor Emx2 The vestibular system of the inner ear is responsible for detecting position and movements of our head. This system, together with our vision and receptors at our muscle and joints, maintain our sense of balance. As we age, our ability to maintain balance declines but therapeutics and interventions to combat this decline are limited. A better understanding of the vestibular system at the fundamental level will help in the design of strategies to alleviate this problem in the future. Five sensory organs comprise the vestibular system of the mouse inner ear; three cristae that are responsible for detecting angular acceleration and two otolith organs for detecting linear acceleration. Each sensory organ consists of hair cells (HC) and supporting cells. Erected on the surface of each hair cell is the mechanotransducer apparatus known as the stereociliary bundle or hair bundle. This hair bundle, in the shape of a staircase, is directionally sensitive. During head motions, only bending the staircase-shaped bundle towards the tall side of the staircase, results in activation of the HC. Bending the bundle in the opposite direction results in inhibition. Notably, a line of polarity reversal (LPR) can be drawn, which divides each otolith organ into two regions with opposite hair bundle orientation. Previously, we have demonstrated that the restricted expression of the transcription factor Emx2 to one of the two regions reverses the hair bundle orientation in all the HCs within the region from its default orientation and thus establishes the LPR. Interestingly, HCs located within the Emx2-positive and -negative regions appear to be innervated by afferent neurons that project to different parts of the brain. Whether the neuronal segregation occurs across the LPR is not clear. In this study, we demonstrated that: 1) afferents of the otolith organ with different central projections are segregated across the LPR, 2) this neuronal segregation is regulated by Emx2, and 3) neuronal selection most likely requires Emx2 in both HCs and supporting cells. Thus, our results indicate that Emx2 regulates bidirectional sensitivity of the otolith organs on two levels: hair bundle orientation as well as neuronal selectivity. While the importance of bidirectional sensitivity maybe intuitive, its role in mediating specific behavior in mice was not known. To address this question, we generated two bidirectional sensitivity mutants. One is a tissue-specific knockout of Emx2 in HCs (Emx2 cKO), in which all hair bundles in the otolith organs are unidirectional. The other is Tmie cKO, in which HCs in the Emx2-positive domain are rendered inactive by the lack of mechanotransduction channels, even though their hair bundle orientation and LPR are normal. Although both mutants only have unidirectional sensitivity, there should be a gain of sensitivity to the responsive direction of stimulus in the Emx2 cKO compared to controls, due to more HCs responding, but not in the case of Tmie cKO. We conducted several vestibular tests on these two mutants such as vestibular evoked potential in response to jerk stimuli, swimming, rotarod, and balance beam. We concluded that the bidirectional sensitivity in the otolith organs is important for mice to swim comfortably and for mice to traverse a narrow balance beam efficiently. While both mutant strains appear to have similar behavioral deficits, the differences in vestibular evoked potential response to directional jerk stimuli between the two mutants support our hypothesis that there is indeed a gain in specific-directional response in Emx2 cKO that is absent the Tmie cKO. Future experiments will focus on identifying behavioral differences caused by this sensitivity difference between the two mutant strains. Manuscript published, https://pubmed.ncbi.nlm.nih.gov/36280667/ 2)Requirements of Sonic Hedgehog in spiral ganglion formation of the mouse inner ear Neurons of the spiral ganglion (SGN) have an indispensable role in auditory function by relaying sound information received by HCs in the cochlea to nuclei in the brainstem. SGN have been implicated in age-related hearing loss as well as hidden hearing loss. Thus, a better understanding regarding the molecular mechanism underlying spiral ganglion formation will provide insights into treatments in the future. A salient feature of the mammalian cochlea is its tonotopic organization such that high frequency sounds are detected by the base of the cochlea and low frequency sounds detected at the apex. While many features of cochlear HCs support this function, the tonotopic map is maintained all the way up to the auditory cortex, starting with the SGN. The establishment of the tonotopic axis for the SGN however, is not clear. It is not known whether the tonotopic differences between basal and apical SGN are inherently different from each other, or they are acquired secondary to the tonotopic axis established by the cochlear HCs. In the brain, the timing of neuronal birthdates dictates a neurons fate and positional identity. Interestingly, in the spiral ganglion, there is a sequential production of SGN during development such that early-born neurons are located by the cochlear base where high-frequency sounds are being detected, but late-born neurons are located by the apex. This sequential neuronal production supports the notion that basal and apical SGN are intrinsically different from each other. However, our results from analyses of Sonic hedgehog (Shh) conditional mutants, suggest otherwise. We found that Shh, a secreted molecule, is expressed transiently in nascent SGN, which signals adjacent neuroblasts to undergo transit amplification before differentiating into SGN. In the absence of Shh, the sequential order of neuronal production is maintained but the total number of SGN was reduced. However, instead of SGN sparsely located along the cochlear duct in the mutants, we observed an accumulative loss of SGN by the cochlear apex, presumably resulting from later-born neurons filling the void generated by the reduced number of earlier-born neurons. These results suggest that SGN do not have a positional identity based on its birthdate. We are currently investigating whether the fate of the SGN is dependent on the neuronal birthdate. (Manuscript in preparation) 3)Deciphering the individual components of the striolar/central zones in the vestibular organs The vestibular system of the inner ear is important for maintaining balance. Yet, the processing of vestibular information at the sensory organ level is not well understood. A morphologically distinct region that is present in each of the five vestibular organs known as the central zone in the cristae and striola in the otolith organs. Previously, we have shown that the striolae are important for generating the vestibular evoked potential in response to jerk stimuli in mice. Considering the striola is distinct from its surrounding sensory tissue on multiple levels such as their innervating afferents, properties of Type I HCs and supporting cells, we are generating conditional mouse mutants to address which of these components is more important to mediate striolar functions. These experiments are currently underway.