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

三重防线：第 1 组 RFX 转录因子在内耳中的作用

基本信息

批准号：
10505836
负责人：
Kathleen Michelle Gwilliam
金额：
$ 4.43万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
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/10505836
关键词：
Adult Affect Age Area Auditory Auditory Brainstem Responses Behavioral Binding Binding Sites Cell Count Cell Differentiation process Cells Cochlea Compensation Confocal Microscopy DNA Binding Domain DNA cassette Data Defect Development Disease Ear Early Promoters Electrophysiology (science)Embryo Epigenetic Process Equilibrium Evoked Potentials Exhibits Family Flow Cytometry Functional disorder Gene Expression Gene Expression Regulation Genes Hair Cells Hearing Hearing problem Histologic Individual Knock-out Knockout Mice Knowledge Laboratories Labyrinth LacZ Genes Life Maintenance Modeling Molecular Morphology Mus Mutation Names National Institute on Deafness and Other Communication Disorders Outer Hair Cells Pathway interactions Peripheral Persons 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 System TNFSF5 gene Testing Tissue-Specific Gene Expression Tissues Transcript United States Utricle structure Validation Vestibular Hair Cells Work auditory pathway 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 transcription regulatory network vestibular pathway

项目摘要

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.

项目摘要为了成功开发再生疗法，以治疗大部分受影响的人听力和前庭疾病，我们必须首先了解所需的转录监管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）已将其第一个优先级（2017-2021战略计划的优先区域1）设置为确定参与周围发展的分子以及遗传和表观遗传变化以及中央听觉和前庭途径。 1组调节因子X（RFX）转录的成员因子（TF）家族（RFX1，RFX，2和RFX3）被称为纤毛生成的主要调节剂最近，我们的实验室发现了在毛细胞发育中发挥关键作用。具体而言，RFX1的表达 RFX3（RFX1/3）对于外毛细胞的末端分化及其在发作后的存活中是必需的听力。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， 1组RFX TFS的最终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，并且要揭示组1 RFX TF在毛细胞发育中的全部作用，所有三个组1 RFX TF必须为删除。我们预测RFX1，RFX2和RFX3（RFX1/2/3）的表达丧失将导致严重平面细胞极性（PCP）缺陷以及强大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了第1组RFX TF在早期毛细胞开发和PCP中的作用 RFX2GT（基因陷阱基因敲除模型），RFX1/2/3 CKO和控制同窝仔的小鼠的分析。此外，我们将通过内耳的x-gal染色来完成RFX2表达的时空表征部分。特定的目标2将通过进行前庭功能探讨组1 RFX TF的作用 RFX2GT，RFX1/2/3 CKO和控制同窝仔的行为和电生理测试。具体的 AIM 3将在前庭毛细胞开发中识别第1组RFX TF的下游目标用RNASCOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPOPE的直接靶标的验证和验证。拟议的工作有影响因为它将确定RFX TF是否还会调节内耳HC中的纤毛生成和PCP，请识别新的对于内耳功能重要的基因，并为导致TF发展的工作体系做出贡献 HC再生的鸡尾酒。