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 that 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. One hypothesis that arose from this study was the idea that outer hair cells become aligned, in part, as a result of restrictions in their ability to move. In particular, our results suggested that constraining developing hair cells along the medial-lateral axis to a relatively narrow region of epithelium would force their migration along the orthogonal basal-apical axis which could play a role in alignment. To test this, we are using cell-type specific mouse mutants to selectively eliminate constraint along the medial-lateral axis. Our prediction is that this change will lead to defects in outgrowth and alignment. In addition, another remarkable aspect of the cochlea is the precise coiling that occurs during development. We have begun to examine how selective growth of cells in different regions of the cochlea could play a role in inducing coiling. The results of this study should have implications in understanding human disorders that lead to shortened or misshapen cochleae such as Mondini Disorders or cochlear hypoplasias. A significant goal of the Section on Developmental Neuroscience is the characterization of cell types within the mammalian cochlea. In the past, a particular problem has been that there are a fairly large number of different cells types in the cochlea, at least 9 that we can identify based on morphology, but comparatively small numbers of any specific cell type. For instance there are only approximately 1000 inner hair cells in one mouse cochlea. These two conditions have made it difficult to develop transcriptional profiles for known or unknown cell types. The recent development of methods for isolation and capture of mRNAs from single cells offers an exciting opportunity to more fully characterize tissues like the cochlea. Therefore, we collected approximately 10,000 cells per time point at 6 different time points spanning cochlear development. The resulting data set allowed us to determine the total number of unique cells within the cochlea, about 18. Future analyses will allow us to determine genes and gene pathways that play a role in specifying many of those cell types. As part of our initial analysis of the single cell data, we were able to identify two genes, Lrrn1 and Sall1, that were expressed in subsets of cochlear cells. We then obtained or generated mutants for each of these genes and are presently analyzing the inner ear phenotypes in these animals. Finally, we have happily shared our expertise in single cell isolation and analysis with other laboratories at the NIDCD and NIH. This has resulted in the publication of several manuscripts over the past year with members of the Section on Developmental Neuroscience as authors.
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