Mature cochlear hair cell transcriptional diversity revealed with single-cell RNA sequencing

In adults, the most common causes of hearing loss are noise exposure and aging, with one out of every three people having some degree of hearing loss by age 65. Permanent hearing loss, called permanent threshold shift (PTS), results mainly from loss of auditory hair cells within the cochlea due to loud noise, aging, ototoxic chemicals and/or genetic predisposition. In mammals, lost hair cells do not regenerate naturally. Exposure to loud but not deafening levels of sound causes a temporary hearing loss, called temporary threshold shift (TTS), which recovers over time without hair cell degeneration. Despite several advances in the field, the molecular and cell biological mechanisms of TTS recovery are not fully understood. This gap in knowledge is fundamentally due to our inability to efficiently isolate healthy mature auditory hair cells. The molecular biological characterization of mature auditory hair cells would provide a proper baseline for comparing how gene expression changes in hair cells during TTS noise trauma. The pursuit of this baseline and setting the stage for evaluating TTS at the single-cell level is the focus of this dissertation. Molecular and biochemical studies of the mammalian cochlea that would help identify possible biological therapeutic interventions for hearing loss are currently limited by the paucity of hair cells, as well as the difficulty associated with accessing viable mature cochlear cells that are embedded in dense bone. Single-cell RNA sequencing (scRNA-seq) and computational data analysis have helped to alleviate some of these limitations. These technological advances are accompanied by the availability of transgenic reporter mice and cell sorting methods to isolate viable individual cells from the mature cochlea. To date, no studies have evaluated the mature cochlear hair cell transcriptomic landscape with single-cell resolution, with the oldest age evaluated using single-cell RNA sequencing technology being post-natal day (P)15, an age where mice have just acquired the ability to hear. Although a manual "hand-picking" method has been established to isolate hair cells, this method is technically challenging, with few labs able to effectively isolate hair cells for bulk RNA sequencing. A more in-depth overview of the transcriptomic studies that have been performed on auditory hair cells is outlined in chapter 1 and 2. In Chapter 3, I present a method to reproducibly isolate mature auditory hair cells from postnatal day (P) 28 mice using fluorescent-activated cell sorting (FACS) for downstream single-cell RNA sequencing transcriptomic gene expression characterization of hair cells during homeostasis and in response to noise trauma. The bioinformatic analysis of our single-cell RNA sequencing of hair cells revealed distinct groups of 418 inner hair cells (IHC) and 22 outer hair cells (OHC). A surprising result of this study was the identification of three distinct subpopulations of IHCs. One population is suggestive of a physiologically active state, and another is indicative of a recovery hair cell state, while the third was determined to be either compromised or dying IHCs. Differential gene expression analysis allowed us to identify distinct marker genes that are associated with each subgroup of cells. To confirm that two types of IHCs are present within the cochlea, I performed single mRNA molecule in situ hybridization to validate the differentially expressed genes between the subpopulations of IHCs and between IHCs and OHCs in whole-mount P28 cochlear tissue preparations. In Chapter 4, I establish a method to visualize the mRNA within the adult cochlear wholemount tissue, published in Cell STAR Protocols (Diaz et al. 2021). The method utilizes hybridization chain reaction, a recently described new mRNA visualization method (Choi et al. 2018). I used this technological advance to validate our single-cell RNAseq findings. The published protocol provides a tool for the scientific community to further explore mRNA expression within the mature mouse cochlea, which has been a challenging endeavor to date. In Chapter 5, I describe the engineering of a system to induce temporary and permanent threshold shift noise damage in live mice. The system also quantitatively measures hearing thresholds in live mice to validate transient noise damage and to monitor recovery. With this system and the above-mentioned hair cell isolation and single-cell RNA sequencing technology, I evaluate the changes in gene expression following TTS-noise exposure and identify measurable changes in gene expressions during hair cell damage. Cochlear hair cells play a fundamental role in our ability to hear, making them an ideal target for therapeutic intervention. By characterizing the transcriptional landscape of mature hair cells with single-cell resolution during homeostasis and in response to noise trauma, I have uncovered novel sub-populations of hair cells and identified early responding genes that are triggered in response to noise damage. These findings also pose additional unanswered questions, that are discussed throughout this dissertation. Together, this dissertation provides new candidate genes for hearing disorders and has the potential to identify new molecular targets to be used for diagnostic, preventive, and therapeutic strategies

Keywords: Developmental Biology.
Advisor: Heller Stefan
Grillet Nicolas
Nusse Roel
Wysocka Joanna