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. Based on these results, Atoh1 has been proposed as a possible candidate gene for the development of gene therapy approaches to potentially repair defects in auditory or vestibular function. However, an important un-answered question was whether forced expression of Atoh1 alone is sufficient to generate functional hair cells. To address this question, adenoviral vectors expressing Atoh1 we introduced into explant cultures of adult utricles. Results indicated that while most supporting cells will respond to forced expression of Atoh1 with changes in morphology and gene expression that are consistent with the formation of immature hair cells, these cells fail to express genes and proteins consistent with mature hair cells. While these experiments were performed in vitro rather than in vivo, they suggest that Atoh1 may not be sufficient to induce functional hair cells. Many sensory systems, including the auditory system, are organized based on a separation of complex sensory input into more fundamental components. In the auditory system, this separation occurs based on frequency. As a result, sounds are separated based on individual frequencies which then stimulate different regions of the organ of Corti. This organization, referred to as tonotopy, is preserved through the auditory brainstem and into the auditory cortex where component frequencies are reassembled to allow perception of the original sound. Separation of frequencies in the organ of Corti is based on graded changes in multiple characteristics of the cells and physical structures of the cochlea, leading to different optimal resonances along its length. Similar structural changes are observed in the avian functional equivalent of the mammalian organ of Corti, referred to as the basilar papilla. Because of greater ease in manipulation at embryonic stages, we opted to initially examine this phenomenon in an avian system, the chicken basilar papilla. Following a series of experiments to determine when cells along the basilar papilla begin to develop frequency-specific characteristics, gene expression profiling was performed to identify potential signaling molecules that could influence tonotopic identity. Results indicated graded expression of the soluble molecule, Bmp7, along the tonotopic axis of the basilar papilla. Subsequent experiments both in vitro and in vivo, using windowed eggs, indicated that changes in the gradient of Bmp7 lead to changes in the tonotopic gradient such that cells along the length of the basilar papilla are all tuned to the same frequency. Subsequent experiments demonstrated that the effects of Bmp7 are mediated through activation of Tak1, a down-stream signaling pathway. These results provide the first information regarding the factors that act to specify tonotopic organization in the auditory periphery and should lead to valuable discoveries related to tonotopic organization throughout the auditory system. Both the auditory and vestibular sensory epithelia contain hair cells and supporting cells. Electrophysiological data suggest that both of these populations are not homogenous and, instead are made of up different subtypes that serve different functions. However, our understanding of how these different types of hair cells and supporting cells develop is limited by the fact that we do not have markers that allow us to discriminate the different types. Therefore, to address this problem, we utilized newly available protocols for the isolation and profiling of individual cells to generate transcriptional profiles for individual hair cells and supporting cells. To accomplish this, transgenic mice were generated in which hair cells and supporting cells express unique fluorescent tags. Then cells from the utriclar (vestibular) or cochlear (auditory) sensory epithelia were dissociated and separated using Fluidigm microfluidics chips. Individual cells were visualized and then RNA was isolated form those cells, reverse-transcribed and then used to generate libraries for profiling of their transcriptomes. Based on the results of these experiments we were able to identify several different subtypes of both hair cells and supporting cells. We are now working to correlate the molecular profiles of these cells with unique electrophysiological characteristics.
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