PROJECT SUMMARY In order to successfully develop regenerative therapies to treat a large percentage of individuals affected by hearing and vestibular disorders, we must first understand the transcriptional regulatory networks necessary for inner ear hair cell development and maintenance. Thus, the National Institute on Deafness and Other Communication Disorders (NIDCD) has set its first priority (Priority Area 1 of the 2017-2021 Strategic Plan) to identify the molecules and genetic and epigenetic changes that take part in the development of the peripheral and central auditory and vestibular pathways. Members of the group-1 Regulatory Factor X (RFX) transcription factor (TF) family (RFX1, RFX,2 and RFX3), which are known as master regulators of ciliogenesis, have been recently found by our laboratory to play a key role in hair cell development. Specifically, the expression of Rfx1 and Rfx3 (Rfx1/3) is necessary for the terminal differentiation of outer hair cells and their survival after the onset of hearing. Mice without Rfx1/3 expression have a late, mild vestibular phenotype. We have found that Rfx2, the final member of the group-1 RFX TFs, is also expressed in the auditory and vestibular hair cells. Therefore, we hypothesize that Rfx2 compensates for the loss of Rfx1/3 in the Rfx1/3 conditional knockout (cKO) mice and that to uncover the full role of the group-1 RFX TFs in hair cell development, all three group-1 RFX TFs must be deleted. We predict that loss of expression of Rfx1, Rfx2, and Rfx3 (Rfx1/2/3) from hair cells will result in a severe planar cell polarity (PCP) defect as well as robust auditory and vestibular dysfunction. Specific Aim 1 will elucidate the role of the group-1 RFX TFs in early hair cell development and PCP through detailed histological analyses of the Rfx2Gt (a gene trap knockout model), Rfx1/2/3 cKO, and control littermate mice. Additionally, we will complete the spatiotemporal characterization of the expression of Rfx2 by X-gal staining of inner ear sections. Specific Aim 2 will explore the role the group-1 RFX TFs in vestibular function by conducting behavioral and electrophysiological testing on the Rfx2Gt, Rfx1/2/3 cKO, and control littermate mice. Specific Aim 3 will identify downstream targets of the group-1 RFX TFs in vestibular hair cell development with the use of single cell RNA-sequencing and validation of direct targets with RNAScope. The proposed work is impactful as it will determine whether RFX TFs will also regulate ciliogenesis and PCP in the inner ear HCs, identify new genes important for inner ear function, and contribute to the body of work leading to the development of TF cocktails for HC regeneration.

PROJECT NARRATIVE Loss of hair cells (the sensory cells of the inner ear) accounts for much of hearing loss and vestibular disorders, which in the United States afflict at least 50 and 15 percent of people over 70, respectively. Recent data suggest a critical role for the group-1 RFX transcription factors in hair cell development and maturation. Understanding the roles of group-1 RFX transcription factors and their target genes in hair cell development will shed necessary light on pathways important for hair cell regeneration and development of therapeutics for hearing and balance dysfunction.