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. A key step in development of the inner ear is the formation of mechanosensory hair cells. Previous work from our laboratory, as well as several others, has demonstrated that the transcription factor Atoh1 plays a key role in the induction of a hair cell fate. However, while Atoh1 clearly plays a key role in initiating the developmental program that leads to hair cell formation, the specific downstream targets that are activated by Atoh1 have not been determined. To identify potential Atoh1 targets, we dissected inner ear tissue from Atoh1 wildtype and mutant tissue and performed transcriptional analysis to identify genes that were differentially regulated between the two conditions. In addition to multiple known hair cell specific genes, a riboflavin transporter gene was also identified. Mutations in this gene, Rft2, have also been linked to human deafness. Hair cell specific transcription was confirmed by in situ hybridization. Next, we generated a mouse mutant for Rft2 using a KOMP construct. While hair cell number and phenotype appeared unaffected in these mice, in vitro studies in which cochlear explants were treated with a hair cell toxin, gentamycin, demonstrated an increased sensitivity to this toxin in the absence of Rft2 and/or riboflavin. The inner ear contains a comparatively large number of unique cell types, however the number of cells of each cell type present in a given ear is fairly limited. As a first step towards characterizing different inner ear cell types, we isolated and characterized approximately 300 cells from the P1 cochlea and utricle. Cells were isolated from triple-transgenic mice (LfngGfp;Gfi1cre;R26RtdTomato) in which virtually all supporting cells express Gfp and all hair cells express tdTomato. Following dissection of sensory regions, epithelial cells were dissociated and single cells were captured using a Fluidigm microfluidics platform. Each cell was then imaged prior to lysis, reverse transcription and initial amplification using SMARTer/Nextera kits. A unique, cell-specific, bar code was then applied to the cDNA from each sample and samples from 48 cells were then combined for multiplex RNA-Seq on an Illumina Hi-Seq. Individual reads were de-multiplexed and aligned to the Ensembl mouse genome. Average expression data from combined single cells showed a strong correlation with RNA-Seq results from tube controls containing 200-300 cells. Based on these results we were able to identify new lineage relationships within the inner ear as well as to model the changes in gene expression that occur during hair cell development. To build on these studies we have expanded both the number and type of cells analyzed as well as developing new methods to isolate and analyze single cells. In particular, we have collected and analyzed cochlear cells at embryonic day 16 (E16) and at post-natal day 7. While still ongoing, our initial analysis has revealed intriguing and unknown lineage relationships within the developing cochlea. In particular, our transcriptional analysis suggests that inner and outer hair cells, which were thought to arise from a common progenitor pool, may actually arise from two unique and separate progenitors. We are in the process of testing this hypothesis (see below) but if confirmed, the result could alter the development of strategies for targeting of regenerative therapies to different regions of the inner ear. In addition, we have collected and characterized cells from the developing spiral ganglion at both E16 and P1. These experiments were done in an effort to characterize different cell types within the spiral ganglion. The neurons within the spiral ganglion relay all auditory information to the brain. Between 10 and 15 neurons typically innervate each individual hair cell, however our understanding of what different types of information are conveyed by each of these neurons is extremely limited, in part because no good characterizations of these different neurons have been developed. By using single-cell RNA-Seq we hope to be able to identify unique neuronal cell types within the spiral ganglion. Preliminary analysis indicates the presence of at least three or four transcriptionally distinct neural cell types at P1. Future experiments will validate specific candidates within those populations and characterize morphological or physiological differences between those cells. One of the more striking aspects of the sensory epithelium of the cochlea, referred to as the organ of Corti, is a remarkable level of cellular organization. Two distinct types of hair cells, inner and outer hair cells, are asymmetrically distributed along the medial-to-lateral axis of the organ. Similarly, associated supporting cells also differ along the same medial-to-lateral axis. The results of our single cell sequencing studies suggested that these cells may arise from unique medial and lateral progenitors. To understand factors that might regulate the formation of these progenitor types, we examined the role of Glycogen Synthase Kinase-3 (GSK3), an enzyme that modulates multiple morphogenic signals during development. Using an in vitro system we blocked GSK3 during the formation of the organ of Corti. Subsequent analysis of cell types indicated a change in the ratio of inner hair cells to outer hair cells without any change in the total number of hair cells. To determine whether this effect represented a change in the fates of different progenitors, we used mouse genetics to label the lateral progenitor pool prior to inhibition of GSK3. Subsequent analysis indicated that blocking GSK3 does lead to a change in the developmental fates of lateral progenitors suggesting first, that the fates of these cells are not restricted and second, that GSK3 may play a role in the determination of medial and lateral progenitor cells. A final project in the laboratory has combined mouse genetics, in vitro explants and confocal live-imaging to study the outgrowth of the cochlear duct over time. A key step in the evolution of hearing and in particular in the ability of mammals to perceive different sound frequencies is the formation of an elongated auditory organ. However the factors the regulate this elongation are unknown. To directly visualize this developmental process, we labeled subsets of developing cochlear cells with a fluorophore and then generated time lapse movies of these cells as the duct extends. Analysis of those movies demonstrates that cochlear cells actively migrate outwards through the generation of cellular protrusions directed in the direction of migration. Moreover, inhibition of MyosinII disrupts both cochlear elongation and cellular migration, suggesting the this molecule plays a key role in regulating cellular outgrowth. The results of these studies have important implications for understanding developmental defects that may result in shortened cochlea and correlated losses in frequency discrimination.
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