Regulatory landscape of the organ of Corti: Identification of Notch-dependent targets that regulate supporting cell fate

Hearing loss, or deafness, affects hundreds of millions of people worldwide. Most hearing loss cases are associated with death or damage of sensory hair cells (HCs) in the organ of Corti. In humans, HCs are generated during embryonic development but are not regenerated if lost. Recent studies focus on identifying treatments that promote HC regeneration that can potentially restore hearing. It has been shown that blocking Notch signaling, a key regulator of HC differentiation, can induce HC regeneration in prenatal mice, but this capacity to regenerate is lost a few weeks after birth. The mechanisms underlying the loss of regenerative potential and what factors promote HC regeneration are currently unclear. Here we aim to define the potential Notch targets that promote trans-differentiation of non-sensory supporting cells (SCs) to sensory HCs in the organ of Corti and to characterize the effect of these target genes on the regeneration of HCs. We have obtained RNA-seq data from SCs treated with Notch inhibitors, which will be complemented with CUT&RUN, a chromatin profiling strategy, on Rbpj, a transcriptional regulator that mediates downstream transcriptional activities of Notch receptor. Integration of these high-throughput assays will enable us to build a gene regulatory network that determines SCs identity during development and differentiation. Finally, we will employ a CRISPR-ON via adeno-associated virus (AAV) to induce direct trans-differentiation in cochlear cultures that we have developed to characterize the effect of identified target genes on HC regeneration. Overall, our data will provide detailed knowledge of the new targets that have the potential to contribute to the regenerative treatments for hearing loss.