Auditory and vestibular function are dependent of the formation of a functional inner ear. While there are multiple components for both of these systems, this laboratory focuses on the development of the sensory epithelia, which contain mechanosensory hair cells and associated cells called supporting cells and on the innervation of those hair cells by neurons from the VIIIth (acousticovestibular) cranial nerve. All three of these cell types are derived from the otocyst, a placodal structure that forms adjacent to the hindbrain early in development. Identifying the factors the specify each of these cell types and then direct their assembly into functional units is a key goal of the Section on Developmental Neuroscience. During the previous year, different members of the laboratory have examined several different aspects of these developmental processes. The ability of mammals, including humans, to discriminate a broad spectrum of frequencies is dependent on an elongated auditory sensory structure, called the organ of Corti, which extends along the entire length of the spiral of the cochlea. One of the most striking aspects of this organ is a precise alignment of mechanosensory hair cells into 4 rows that extend along the spiral. Previous studies from our laboratory and others have suggested that the precursor cells that give rise to hair cells may become organized into these rows through a conserved developmental process referred to as convergent extension (CE). However, the role of CE was only inferred through analysis of fixed tissue. To examine the role of CE in cochlear extension directly, we combined mouse genetics, in vitro explants and confocal live-imaging to study the outgrowth of the cochlear duct over time. Analysis of those movies demonstrated, as expected, that cochlear cells actively migrate outwards through the generation of cellular protrusions directed in the direction of migration. However, contrary to expectations, limited CE was observed. Instead analysis of the data indicated that radial intercalation (RI), the directed movement of cells towards the basement membrane, provides a significant driving force for cochlear extension. These results provide the first visualization of the cellular movements that drive cochlear extension. Moreover, they demonstrate that existing dogma regarding the processes that mediate cochlear outgrowth is incorrect. Moving forward, these findings should have significant implications in terms of understanding the genetic and mechanistic bases for developmental defects that lead to shortened cochleae and losses in frequency discrimination. Another remarkable aspect of the auditory sensory epithelium of the inner ear is the uniform orientation of mechanosensory stereociliary bundles towards the outer edge of the cochlear spiral. Bundles are only mechanosensitive within a single plane of deflection and the biophysical structure of the organ of Corti is such that incoming sounds create oscillations along the same deflection plane. Therefore, bundles that are not oriented towards the outer edge will show significantly decreased activity in response to sound, which leads to a loss of hearing acuity in model systems. Work over the last 15 years has established a link between the conserved planar cell polarity pathway and the positioning of primary cilia in the process of uniform cellular orientation. Further, hearing loss is associated with a number of mutations in cilia-related genes, called ciliopathies. As part of a previous study to identify novel factors that intereact with cilia genes, MACF was identified as a possible interacting partner. A study of Macf mutant mice indicated defects in cilia formation and outgrowth in both the retina and cochlea. Surprisingly, the defects in cilia formation in the inner ear had no effect on orientation of stereociliary bundles. This result, along with others, suggests that the role of cilia in hearing may be more complex than originally thought. Two particular challenges in studying the inner ear have been its relatively small size and high cellular diversity. As a result, there are many different types of unique cells, but only limited numbers of each cell type. However, over the last 2-3 years, new technologies have been developed to be able to isolate single cells or even single nuclei, and then to obtain useful mRNA expression data from those individual cells. Because of the obvious potential benefit to the inner ear field, we have spent time adapting these technologies for use in the inner ear. At this point we have successfully developed protocols for single cell RNA-seq using both microfluidics and fluorescent activated cell sorting to isolate cells of interest. In addition, in collaboration with other laboratories at NIH we have developed a microfluidic based approach to capture single nuclei prior to RNA-seq.
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