Investigation of Molecular Cues Driving IHC vs. OHC Differentiation Using an Insm1 Knockout Mouse Model

The mammalian cochlea contains inner hair cells (IHCs), which transmit auditory signals to the brain, and outer hair cells (OHCs), which amplify and refine these signals. Loss of either cell type results in irreversible hearing impairment, as neither can regenerate. Current therapeutic approaches are limited by an incomplete understanding of the signaling pathways governing IHC versus OHC development. The Insm1 mutant mouse model, in which OHCs transdifferentiate into IHCs, provides a unique system for studying IHC formation. In the absence of INSM1, approximately half of all OHCs lose their identity, acquiring the characteristic morphology and molecular markers of an IHC fate. This transdifferentiation occurs in a gradient, with medial OHCs more likely to transition, suggesting the involvement of a graded IHC-inducing morphogen that INSM1 normally represses. In our organotypic cochlear explants from Insm1 mutants, we observed OHC-to-IHC transdifferentiation mirroring in vivo findings, evidenced by the loss of the OHC marker, BCL11b, and gain of the IHC marker, VGLUT3. Pharmacological modulation of candidate signaling pathways demonstrated that transdifferentiation is dynamic, supporting the hypothesis of morphogen involvement. Enhancing or inhibiting specific candidate signaling pathways either promoted near-complete transdifferentiation or suppressed it. Here, we propose to leverage the Insm1 mutant model to identify the morphogen(s) driving IHC and OHC development, using spatial transcriptomics and genetic models to validate their roles and determine their cellular sources. Additionally, we will investigate how downstream transcriptional effectors are recruited to chromatin in response to morphogens, to activate IHC-specific gene expression programs. These insights will advance our understanding of cochlear development and help to guide strategies for targeted hair cell regeneration to ultimately restore hearing.