Disabling age-related hearing loss (ARHL) afflicts greater than 50% of the population over the age of 70. The progressive loss of sensory hair cells (HCs) of the cochlea, primarily the outer HCs (OHCs), is either the cause or the final common pathology of most forms of ARHL. However, the molecular basis of ARHL is unknown, impeding development of targeted interventions. Furthermore, attempts to reprogram progenitor cells towards cochlear HC fates result primarily in immature HC-like cells, similar to vestibular HCs. For these reasons, the study of the molecular pathways underlying OHC differentiation and survival is critical. Transcription factors (TFs) are key regulators of gene expression. When associated with disease, identifying the upstream regulators and downstream targets of a TF is an effective route to determine additional key players in the same process. In this competitive renewal we build upon our previous success, identifying critical roles for the RFX and IKZF2 TFs in OHC differentiation, function and survival, and follow up with two specific aims designed to elucidate their molecular mechanism within the HC/OHC genetic programs: Group 1 RFX TFs (RFX1/2/3), are master regulators of ciliogenesis. However, while OHCs lacking Rfx1/3 die within 12 hours from the onset of hearing, they have normal bundle development. RFX2 is transiently expressed in the developing HCs, possibly preventing a ciliogenesis defect with loss of Rfx1/3. We therefore hypothesize that group 1 RFX TFs have a dual role in HC development, with an early role in hair bundle formation, and later role in terminal differentiation and survival.
Specific Aim 1 : To identify the mechanisms by which RFX TFs support embryonic as well as early post-natal cochlear hair cell differentiation. IKZF2 is a key regulator of OHC gene expression, precisely regulated to express only in OHCs starting from P4. We hypothesize that: (1) deletion of IKZF2 target genes will reveal genes essential for OHC function and survival; (2) IKZF2 is required for maintenance of mature OHCs; and (3) Ikzf2 expression is regulated by a master-regulator of OHC maturation.
Specific Aim 2 : To identify the mechanism by which IKZF2 leads to OHC differentiation, to determine its role in mature OHCs, and to identify its upstream regulatory network. To accomplish these aims we combine novel protocols for OHC-specific multi-omic analyses with state- of-the-art bioinformatics, and characterization of novel mouse models to validate the roles of candidate genes in hearing. Successful completion of these aims will: (1) impact our understanding of OHC differentiation and survival; (2) functionally characterize the roles of key RFX and IKZF2 target genes in hearing; (3) result in transcriptomic and epigenetic maps of differentiating and mature OHCs which will be applied to inform studies for HC regeneration/identifying candidate genes for ARHL; and (4) generate numerous mouse models for RFX and IKZF2 target genes, which will be readily available to the scientific community. Additionally, all of the data generated as part of this proposal will be shared via the gEAR (umgear.org).
The loss or damage of sensory cells in the inner ear, specifically the outer hair cells (OHCs), is a critical cause of age-related and noise-induced hearing loss, which are now worldwide epidemics. This competitive renewal builds on our success in identifying transcription factors critical for the functional maturation and survival of OHCs to now define their role within the OHC genetic program. Successful completion of this research will: impact our understanding of inner ear sensory cell development and survival; advance research on the molecular basis of age-related hearing loss; and, inform the design of future therapeutic interventions such as hair cell regeneration therapy.
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