Transcriptional regulation of hair cell development in the hearing organ

摘要 每500名新生儿中就有1人受到听力损失的影响，65岁以上的人中有1人受到影响。这种障碍通常是 由机械感觉毛细胞(HC)的丧失引起的，这是一种在成熟阶段永久存在的缺陷 哺乳动物的耳蜗线。因此，已经开发了人工方法来通过“转换”来再生HCS。 在小鼠身上，他们已经被证明成功地产生了类HC细胞。然而，这些细胞 未完全成熟且寿命短，表明并不是所有阻碍HC再生的主要障碍都 克服困难。HC再生途径建立在生理性HC发育研究的基础上。 这些结果表明转录因子‘声调同源1’(Atoh1)是HC的基础。 但它对基因表达的影响取决于上下文。这些影响在HCS和 其他表达Atoh1的细胞。因此，Atoh1的功能受到其他转录因子的微调。驾驭 Atoh1再生HCS的全部潜力将需要鉴定新的修饰剂 Hcs中的Atoh1活性。我们的初步数据表明，Atoh1活性和HC成熟受 转录因子‘胸腺细胞选择相关HMG盒蛋白’(TOX)。中国人Tox基因的敲除 小鼠(Tox∆/∆)造成HC丢失和耳聋，并对这些小鼠的Corti器官进行rna-seq分析。 揭示了多种基因在异常水平上的表达。‘异常低表达’组 包括Atoh1靶基因、RE1沉默转录因子(REST)靶基因和转录 抑制子编码基因蓖麻锌指1(CASZ1)。CASZ1在Corti器官中的靶向性突变 培养表明CASZ1是抑制几个异常表达的基因所必需的 在科尔蒂的毒素∆/∆器官中含量很高。条件性CASZ1基因敲除小鼠的初步鉴定 研究发现，CASZ1的HC特异性缺失会导致外部HC(OHC)变性和听力损失。此外, 我们先前对休息功能的分析表明，围产期休息活动需要下调 HC成熟。拟议研究的目的是确定TOX在人卵巢癌成熟过程中的作用。 HCS。我们的中心假设是TOX通过调节Atoh1、Rest和 CASZ1在发展初级保健服务方面的活动。我们建议通过两个具体目标来检验这一假设：目标1) 确定TOX在耳蜗发育的不同阶段对HC成熟的影响及其程度 它支持Atoh1活性、静息调节和人工诱导的HC样细胞的产生；目的 2)检测CASZ1对HC形态和耳蜗基因表达的影响，并鉴定基因 抑制复合体，介导CASZ1依赖的对某些间接靶基因的抑制 HCS中的毒物。这些目标将使用从rna-seq到体细胞的各种方法来实现。 基因组编辑。拟议的研究具有重要意义，因为发现了Atoh1的新修饰物 活动对于改进碳氢化合物再生方法的合理设计是必要的。

SUMMARY Hearing loss affects 1 in 500 newborns and ~1 in 3 individuals over the age of 65. This disorder is often caused by the loss of mechanosensory hair cells (HCs), a deficiency that is permanent in the mature mammalian cochlea. Therefore, artificial approaches have been developed to regenerate HCs by `converting' non-HCs into HCs; and in mice they have proven successful in generating HC-like cells. However, these cells fail to mature fully and their lifespan is short, indicating that not all major barriers to HC regeneration have been overcome. The HC-regenerating approaches have been based on studies of physiological HC development. These have shown that the transcription factor `Atonal homolog 1' (ATOH1) is fundamental for HC development but that its effects on gene expression are context dependent. These effects differ in HCs versus other ATOH1-expressing cells. Thus, ATOH1 function is fine-tuned by other transcription factors. Harnessing the full potential of ATOH1 for the regeneration of HCs will require the identification of novel modifiers of ATOH1 activity in HCs. Our preliminary data suggest that ATOH1 activity and HC maturation are regulated by the transcription factor `thymocyte selection‐associated HMG box protein' (TOX). Knockout of the Tox gene in mice (Tox∆/∆) caused HC loss and deafness, and RNA-seq analysis of the organ of Corti in these mice revealed that a variety of genes are expressed at abnormal levels. The `abnormally low expression' group includes ATOH1 target genes, `RE1-silencing transcription factor' (REST) target genes, and the transcriptional repressor-encoding gene castor zinc finger 1 (Casz1). Targeted mutagenesis of Casz1 in organ of Corti cultures revealed that CASZ1 is needed for the repression of several genes that are expressed at abnormally high levels in the Tox∆/∆ organ of Corti. Our preliminary characterization of conditional Casz1 knock-out mice revealed that HC-specific deletion of Casz1 causes outer HC (OHC) degeneration and hearing loss. In addition, our previous analyses of REST function showed that perinatal downregulation of REST activity is needed for HC maturation. The objective of the proposed research is to define the role of TOX in the maturation of HCs. Our central hypothesis is that TOX supports cochlear HC maturation by modulating ATOH1, REST, and CASZ1 activities in developing HCs. We propose to test this hypothesis through 2 specific aims: Aim 1) determine the effects of TOX on HC maturation during various phases of cochlear development, and the extent to which it supports ATOH1 activity, REST regulation, and artificially induced production of HC-like cells; Aim 2) determine the effects of CASZ1 on HC morphology and cochlear gene expression, and identify gene repressor complexes that mediate the CASZ1-dependent repression of some of the indirect target genes of TOX in HCs. These aims will be achieved using a variety of methods ranging from RNA-seq to somatic cell genome-editing. The proposed studies are significant because identification of novel modifiers of ATOH1 activity will be necessary for improving the rational design of HC-regenerating approaches.