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

三重防线：1 组 RFX 转录因子在内耳中的作用

• 批准号: 10312450
• 负责人: Kathleen Michelle Gwilliam
• 金额: $ 4.26万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-10-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10312450
• 关键词: Adult; Affect; Age; Area; Auditory; Auditory Brainstem Responses; Behavioral; Binding; Binding Sites; Cell Count; Cell Differentiation process; Cells; Cochlea; Confocal Microscopy; DNA Binding Domain; DNA cassette; Data; Defect; Development; Disease; Ear; Early Promoters; Electrophysiology (science); Embryo; Epigenetic Process; Equilibrium; Evoked Potentials; Exhibits; Family; Financial compensation; Flow Cytometry; Functional disorder; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Hair Cells; Hearing; Hearing problem; Histologic; Individual; Knock-out; Knockout Mice; Knowledge; Laboratories; Labyrinth; LacZ Genes; Life; Light; Maintenance; Modeling; Molecular; Morphology; Mus; Mutation; Names; National Institute on Deafness and Other Communication Disorders; Outer Hair Cells; Pathway interactions; Peripheral; Phenotype; Play; Prevalence; Process; RFX regulatory factor; RFX1; RFX3; Reporting; Research; Role; Sampling; Scanning Electron Microscopy; Secondary to; Sensorineural Hearing Loss; Sensory; Stains; Strategic Planning; Suggestion; System; TNFSF5 gene; Testing; Tissue-Specific Gene Expression; Tissues; Transcript; United States; Utricle structure; Validation; Vestibular Hair Cells; Work; auditory pathway; base; behavior test; cilium biogenesis; conditional knockout; equilibration disorder; hair cell regeneration; hearing impairment; member; mutant; planar cell polarity; postnatal; promoter; regenerative therapy; single-cell RNA sequencing; spatiotemporal; therapeutic development; transcription factor; vestibular pathway

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.

项目概要 为了成功开发再生疗法来治疗大部分受此影响的个体 听力和前庭疾病，我们必须首先了解必要的转录调控网络 内耳毛细胞的发育和维护。因此，国家耳聋和其他研究所 沟通障碍 (NIDCD) 已将其首要任务（2017-2021 年战略计划的优先领域 1）设定为 识别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 组调节因子 X (RFX) 转录的成员 因子 (TF) 家族（RFX1、RFX,2 和 RFX3）被称为纤毛发生的主要调节因子，已被 我们的实验室最近发现它在毛细胞发育中发挥着关键作用。具体来说，Rfx1的表达 Rfx3 (Rfx1/3) 对于外毛细胞的终末分化及其发病后的存活是必需的 的听力。没有 Rfx1/3 表达的小鼠具有晚期、轻度的前庭表型。我们发现 Rfx2， group-1 RFX TF 的最后一个成员也在听觉和前庭毛细胞中表达。因此，我们 假设 Rfx2 补偿了 Rfx1/3 条件敲除 (cKO) 小鼠中 Rfx1/3 的损失，并且 为了揭示 group-1 RFX TF 在毛细胞发育中的全部作用，所有三个 group-1 RFX TF 必须 已删除。我们预测毛细胞中 Rfx1、Rfx2 和 Rfx3 (Rfx1/2/3) 表达的丧失将导致严重的 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 通过详细的组织学阐明 group-1 RFX TF 在早期毛细胞发育和 PCP 中的作用 对 Rfx2Gt（基因陷阱敲除模型）、Rfx1/2/3 cKO 和对照同窝小鼠进行分析。此外， 我们将通过内耳X-gal染色完成Rfx2表达的时空表征 部分。具体目标 2 将通过进行以下实验来探索第 1 组 RFX TF 在前庭功能中的作用 对 Rfx2Gt、Rfx1/2/3 cKO 和对照同窝小鼠进行行为和电生理测试。具体的 目标 3 将通过使用确定前庭毛细胞发育中 1 组 RFX TF 的下游靶标 使用 RNAScope 进行单细胞 RNA 测序和直接靶点验证。拟议的工作具有影响力 因为它将确定 RFX TF 是否也会调节内耳 HC 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，并有助于导致 TF 发展的工作主体 用于 HC 再生的鸡尾酒。