Hair Cell Development in the Mammalian Cochlea

National Institute On Deafness And Other Communication Disorders

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Abstract

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. With the lifting of all remaining COVID restrictions, productivity in the laboratory has largely returned to pre-pandemic levels. A project using single cell RNA sequencing to profile the development of spiral ganglion neurons was published in P.N.A.S. in November of 2023. This study used single cell RNAsequencing to explore the transcriptional changes that occur as neurons develop within the spiral ganglion. By collecting developing neurons at different time points and then using single cell RNA-Seq to generate transcriptional profiles, we were able to assemble a developmental trajectory for each of four neuronal subtypes that are present in the functioning spiral ganglion. In addition, we were able to identify candidate transcription factors that may influence the formation of different subtypes. As a next step, we are now developing/generating conditional mouse mutants for several of these factors. Preliminary data suggest that deletion of Tle4, a gene that was identified in our single cell study, leads to a decrease the percentage of neurons that develop with several of the known phenotypes, suggesting that this factor plays a role in specific of those specific cellular phenotypes. In July, 2023, we published the results of a study examining the role of Leucine Rich Repeat Neuronal 1 (Lrrn1) in patterning of the cochlear sensory epithelium. Analysis of cochlear development using single cell RNAseq had identified Lrrn1 as being expressed at one edge of the inner ear sensory epithelium. To examine the role of Lrrn1, we generated Lrrn1 mutant mice. Analysis of their ears indicated a disruption in cellular patterning within the ear. In subsequent studies we demonstrated that Lrrn1 acts to enhance activation of Notch1 in cells located adjacent to the sensory epithelium, preventing them from becoming hair cells. POU4F3 is a human deafness gene that has been reported to be required for hair cell survival. However, one of the Research Fellows in the laboratory discovered that some hair cells persist in the vestibular system of Pou4f3 mutant mice. To determine how these cells differ from wild type cells, we are using single cell RNAseq to compare wild type and Pou4f3 negative hair cells. Results indicate that Pou4f3 negative cells are arrested in an immature state. This provides a potential opportunity to use gene therapy to restore normal function to vestibular hair cells. We are in the process of testing this hypothesis using viral vectors to re-express Pou4f3 in Pou4f3 mutant mice. Variants in Sall1, a transcriptional repressor, cause Townes-Brocks syndrome in humans which includes hearing loss. Sall1 is a member of a family of genes, Sall1-4 of which Sall1,2 and 3 are expressed in the cochlea. To better understand the role of the Sall genes in inner ear function, we are generating single and compound mutants for the different Sall genes. The transcription factor Prox1 is expressed only in the region of the cochlea that will give rise to outer hair cells. To determine the role of Prox1 we generated a conditional deletion in this gene within the cochlea. Assessment of auditory function indicates significant hearing loss in these mice while phenotypic analysis indicates a loss of hair cells in adult mice. We will use single cell RNAseq to explore transcriptional changes underlying these defects. In collaborations with external researchers, we have performed transcriptional profiling of the developing cochlear nucleus and examined the transcriptional similarities between neurons generated in inner ear organoids and endogenous spiral ganglion neurons. Finally, an existing collaboration with Dr. Michael Burger to examine effects of peripheral inputs on the structure of the auditory CNS resulted in the awarding of an R01 to Dr. Burger in 2022. Because of employee turnover in Dr. Burgers lab, we are just now beginning to move forward with these collaborative experiments.