Hair Cell Development in the Mammalian Cochlea

National Institute On Deafness And Other Communication Disorders

Investigators

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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. Overall activity in the laboratory continued to be significantly affected by COVID-mandated reductions in occupancy, and by general emotional fatigue among members of the laboratory. With COVID restrictions slowly improving, laboratory productivity is returning to normal levels. A project using single cell RNA sequencing to profile the development of spiral ganglion neurons was submitted for consideration for publication at P.N.A.S. While the manuscript has not been accepted yet, the remaining concerns are minor and the editor has informed us that it will be accepted. This study sought to understand how developing spiral ganglion neurons become specified into one of four distinct physiological phenotypes. 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 neuronal subtype and to identify candidate transcription factors that may influence the formation of different subtypes. For several of these candidate genes, we have obtained conditional mutant mice and are in the process of generating spiral ganglion-specific deletions. We will then use a combination of morphology and single cell RNAseq on mutant neurons to determine changes in identity/function. Previous results from an analysis of cochlear development using single cell RNAseq had identified a gene called Lrrn1 as 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. Subsequent studies demonstrated that Lrrn1 interacts with Notch1, a gene that has been shown to be important for inner ear patterning. This project was headed by a post-bac in the laboratory who left during COVID. We have now finished up this work and it will be submitted within the next few weeks. 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. In addition, there is data to suggest that Sall1 variants in Townes-Brocks patients may generate functional dominant negative proteins that suppress the function of all three Sall transcription factors. To test this, we will develop a gene therapy approach to target Sall1 for inactivation to determine whether Sall2 and Sall3 can functionally compensate. If this is the case, treatments targeting Sall1 could be developed. 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 this year.