A Triple Line of Defense: The Role of the Group 1 RFX Transcription Factors in the Inner Ear

项目总结 为了成功地开发再生疗法来治疗大部分受 听力和前庭障碍，我们必须首先了解转录调控网络 内耳毛细胞的发育和维护。因此，国家聋人研究所和其他机构 传播障碍(NIDCD)已将其第一优先事项(2017-2021年战略计划的优先领域1)设定为 确定参与外周发育的分子以及遗传和表观遗传变化 以及中枢听觉和前庭通路。1组调控因子X(RFX)转录的成员 因子(TF)家族(RFX1、RFX、2和RFX3)被认为是纤毛发生的主要调节因子。 最近被我们实验室发现在毛细胞发育中起关键作用。具体来说，Rfx1的表达 和Rfx3(Rfx1/3)是外毛细胞终末分化和发病后存活所必需的 倾听的声音。没有Rfx1/3表达的小鼠有较晚的、温和的前庭表型。我们发现Rfx2，即 在听觉和前庭毛细胞中也表达-1RFX转录因子的最后一个成员。因此，我们 假设Rfx2补偿了Rfx1/3条件性基因敲除(CKO)小鼠中Rfx1/3的丢失 要揭示-1组RFX-F在毛细胞发育中的全部作用，所有三个-1组-1 RFX-F必须是 已删除。我们预测毛细胞中Rfx1、Rfx2和Rfx3(Rfx1/2/3)的表达缺失将导致严重的 平面细胞极性(PCP)缺陷以及强烈的听觉和前庭功能障碍。具体目标1将 通过详细的组织学研究阐明第1组RFX转录因子在毛细胞早期发育和PCP中的作用 Rfx2Gt(一种基因陷阱敲除模型)、Rfx1/2/3 CKO和对照小鼠的分析。另外， 我们将通过内耳X-Gal染色来完成Rfx2表达的时空表征 横断面。特指目标2将通过传导来探讨第1组RFX转录因子在前庭功能中的作用 对Rfx2Gt、Rfx1/2/3CKO和对照仔鼠进行行为和电生理测试。特定的 AIM 3将确定前庭毛细胞发育中第1组RFX转录因子的下游靶点 使用RNAScope对单细胞RNA测序和直接靶标进行验证。拟议的工作是有影响力的 由于它将确定RFX TF是否也将调节内耳HCS中的纤毛发生和PCP，因此确定新的 对内耳功能很重要的基因，并对导致转铁蛋白发育的工作机构做出贡献 用于HC再生的鸡尾酒。

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.