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) Retinoic acid synthesis is required for patterning the common crus of the inner ear The vestibular system of the inner ear is responsible for detecting head positions and movements to maintain balance. Detection of angular head movements is dependent on the three semicircular canals – anterior, posterior and lateral – which are arranged orthogonally to each other. The anterior and posterior semicircular canals, aligned with the respective sagittal and coronal plane, are joined by a stalk-like structure known as the common crus at one end. In Goldenhar syndrome, characterized by malformations of the eye, ear, face and spine, the absence of the common crus has been reported despite the presence of intact canals. The cause of this anomaly is not known, and gaining insight into the molecular mechanisms underlying vestibular development could have clinically relevance. During development, the anterior and posterior canals, along with the common crus, arise from a vertical outpouching of the otocyst – the embryonic inner ear. As this pouch grows, opposing epithelia in two central regions of the pouch merged, fused and resorbed, leaving behind the peripheral to form the anterior and posterior canals. These canals are joined at one end by the common crus. Whether the common crus and the canals are molecularly similar structures, formed by regulated epithelial resorption, or the common crus is molecularly defined and distinct from the adjoining canals has remained unclear. Our in ovo study support the hypothesis that the common crus is molecularly distinct from the canals. First, we found that in the developing chicken inner ear, the presumptive common crus – but not the region that destined to become canals – expresses two genes, Aldh1a2 and Aldh1a3, each of which encodes a retinoic synthesizing enzyme. Second, inhibition of retinoic acid synthesizing enzyme using citral blocked common crus formation, confirming the importance of this signaling pathway. Additionally, we found that Fibroblast growth factor, which we have previously shown to promote canal formation, antagonizes retinoic acid activity required for patterning the common crus. Conversely, ectopic expression of retinoic acid inhibits canal formation. These findings indicate that the formation of canals and the common crus is mediated by distinct molecular mechanisms that regulate each other. Our results also provide a developmental explanation for the clinical observation of the absence of common crus in Goldenhar syndrome (manuscript published 2025). 2) Selective deletion of type I hair cells in striolar/central zones of the vestibular organs Each of the five vestibular sensory organs of the mammalian inner ear is zonally divided into the central striola and the surround extrastriola in the two otolith organs, and the central and peripheral zones in the three cristae. While these regions are molecular and cellular distinct from each other, their functions in decoding various vestibular sensory inputs, however, remains unclear. A better understanding of how head movement and orientation information is being processed at the level of the inner ear will help to design better strategies and therapeutics for patients with vestibular dysfunction in the long run. Previously, we have generated a developmental mouse mutant in which the striolar/central zones took on extrastriolar/peripheral zone identity. These mutant mice, named as “centerless” mutant, are viable and allowed for the first time, the investigation of striolar/central zone functions in mammals. Striolar/central zones were postulated to specialize in generating rapid responses such as vestibular ocular reflexes (VOR) and vestibular evoked potential (VsEP), both of which are short latency responses, occurring within milliseconds of the stimulus. In the centerless mutant, VsEP is absent, but VOR is normal suggesting sensory processing at the end-organ level is regionally segregated. Additionally, these mice exhibit head tremor and have difficulty in traversing a narrow balance beam. While VOR and VsEP are responses initiated at the inner ear, it was not clear whether the behavioral phenotypes were attributed to the inner ear defects alone or they could be confounded by retinoic acid signaling defects in the brain as well. To resolve this issue, we generated a second mouse mutant, in which type I hair cells in the striolar/central zones were selectively ablated postnatally. This model alleviates two key limitations of the centerless mutants. First, the cre driver used is specific for the inner ear avoiding disruption of retinoic acid signaling in the brain. Second, postnatal ablation minimizes potential secondary effects on central projections. Remarkably, this model recapitulates many of the phenotypes observed in the centerless mutant, even though in a milder form. VsEP is reduced, while VOR remains normal. These mice also exhibit no delay in crossing a narrow balance beam compared to controls. However, they show diminished head motion at higher frequencies during rest but increased head motion during balance beam task, both consistent with vestibular deficits. More importantly, both mutant strains display pronounced head tremor at birth, strongly suggesting that this phenotype arises from the loss of striolar/central zone functions. Interestingly, transient head oscillations have also been reported in human patients with chronic bilateral vestibular loss when challenged with weighted head mass. These findings position both mouse mutants as valuable tools for investigating mechanistic basis of vestibular dysfunction and for understanding clinically relevant phenotypes. (manuscript in preparation) 3) Investigating vestibular circuitry in the brainstem Patients with unilateral vestibular dysfunction often recover over time from central compensation. In contrast, individuals with bilateral vestibular dysfunction typically experience chronic balance impairments, as compensation is less effective. Understanding the cellular basis of central compensation could provide therapeutic strategies for patients with vestibular deficits. The vestibular ganglion afferents, which innervate the five vestibular sensory organs in the inner ear, project to four vestibular nuclei in the brainstem as well as to the cerebellum. However, the precise circuitry among the brainstem vestibular nuclei remain poorly understood. As a first step towards elucidating the mechanisms underlying central compensation, we aimed to identify brainstem vestibular neurons involved in generating vestibular-evoked potentials (VsEP), which are initiated by striola activation of the two otolith organs in response to jerk stimuli. To map neuronal activation, we examined expression of c-fos, an immediate early gene, used as a proxy for neuronal activity in brainstem vestibular nuclei following jerk stimuli. Mice were subjected to jerk stimulation and sacrificed thirty minutes later for analysis of c-fos expression in the brainstem. We observed that cfos-positive neurons were predominantly concentrated in the descending vestibular nuclei (DVN) of the brainstem but sparse labeling in the other vestibular nuclei. To confirm this activity depended on jerk stimulations, we used TMIE knockout mice, which lacks mechanotransduction function in hair cells. In these mutants, all hair cells are nonfunctional. Following jerk stimulation, VsEPs were absent and no cfos expression was detected in the DVN, confirming that the observed cfos activation in wildtype mice were driven by activities of the inner ear. These findings represent an important step towards mapping the central pathways involved in vestibular processing.