Transcriptomic Analysis of Cochlear Outer Hair Cell Heterogeneity in Juvenile and Adult Mice

Abstract

Outer Hair Cells (OHCs) are sensory receptor cells in the mammalian cochlear organ of Corti (OC). OHCs in different regions of the OC vary in their biological properties, ultimately contributing to the sensitivity and frequency selectivity along the cochlea. Previous findings have shown that various genes are differentially expressed in OHCs along the OC, but the molecular profiles have not been further characterized. Single cell RNA sequencing (scRNA-seq) has allowed the study of individual cells' transcriptome profile and has proven to be the best way to study the heterogeneity of cell populations. Juvenile and adult cochlear OHC transcriptome profiles have proved challenging to obtain because OHCs are difficult to maintain once leaving their unique environment in the cochlea, limiting the study of heterogeneous molecular and biological processes at the adult stage. As a result, there are currently no scRNA-seq studies identifying heterogeneity among OHCs of the mammalian cochlea. Our group has successfully performed scRNA-seq and bioinformatic analysis on juvenile and adult mammalian cochlear OC cells. Bioinformatic analyses of OC cells at three ages led to the hypothesis that mammalian cochlear OHCs are composed of a molecularly heterogeneous cell population. We identified three distinct OHC clusters and differentially expressed genes present and persisting across time. Further, we hypothesized that the OHCs exhibit a longitudinal gradient along the tonotopic axis of the cochlea, corresponding with the identified clusters. Unbiased trajectory and regression analysis revealed gradient expressing genes (GEGs) that change as a function of Pseudospace. Published literature revealed previously identified gradient-expressing genes. In situ RNAscope confirmed bioinformatic findings on OHC heterogeneity and clustering analysis corresponding to spatial localization along the cochlea's tonotopic axis. Genes expressed in a base-to-apex and apex-to-base gradient were revealed. We further hypothesized that the detected GEGs result in a change in molecular pathway activity taking place along the tonotopic axis of the cochlea. Gene ontology (GO) analysis uncovered a difference in GO term activity along the tonotopic axis. This work has, for the first time at the single-cell level, identified and characterized molecular heterogeneity present among the OHCs of the mammalian cochlea and suggested the clinical therapeutics of the spatial gradient for OHC susceptibility to damage.