Outer and Inner Hair Cell Development

The developing mammalian cochlea generates two types of mechanosensors: Inner hair cells (IHCs), endowed with a prominent presynaptic apparatus, transmit sound information to neurons and the brain; outer hair cells (OHCs), equipped for electromotility, are used for amplification and sharp frequency discrimination of sounds. Loss of OHCs is the leading cause of hearing loss, and hence their regeneration could be the most valuable means to restore hearing. In the first cycle of this grant, we found that the zinc-finger protein INSM1, expressed in nascent OHCs, acts during a brief critical period of their early development to consolidate their fate, preventing OHCs from transdifferentiating into IHCs. In the absence of INSM1, OHCs are born properly specified but, within a couple of days, nearly half of them cease expressing OHC markers and proceed to transdifferentiate into IHCs. We hypothesize that INSM1 consolidates the fate of OHCs by modifying their epigenome, closing regulatory elements required for IHC differentiation. To test this and alternative hypotheses, we will perform ATAC-seq on developing OHCs with and without INSM1 (which are susceptible to transdifferentiate into IHCs) as well as in IHCs ectopically expressing INSM1. In the previous funding cycle, we also found that the distribution of transdifferentiated hair cells varies in a graded fashion along the neural to abneural axis, suggesting that INSM1 normally consolidates the OHC fate by preventing their responding to an IHC-inducing morphogen. In order to identify such a morphogen, which presumably determines the IHC vs OHC distribution in the cochlea, we will culture mutant organs of Corti and alter their extent of transdifferentiation with agonists and antagonists of candidate morphogens and their signaling pathways. These studies use the transdifferentiation of OHCs into IHCs as a novel means for elucidating the molecular mechanisms (chromatin modifications, morphogens, and resulting gene expression) by which the complementary IHCs and OHCs are generated in the developing cochlea. Finally, in the previous funding cycle we identified an OHC-specific transcriptions factor, BCL11b which, together with its paralog BCL11a, is necessary for the precise alignment of OHCs in parallel rows and with stereocilia pointing perpendicular to the reticular lamina, essential features for their function as cochlear amplifiers. We will explore how BCL11b+a achieve this task by genetic, anatomical, physiological, transcriptomic and epigenomic examinations, the same approaches we are or already have successfully employed with INSM1. We predict that the studies here proposed will provide an unprecedented advance in understanding how OHCs form, and align themselves, so as to serve their role in hearing.