The role of the let-7 microRNA gradient in the development of tonotopic specialization in the cochlea

项目摘要 耳蜗是听觉系统中负责探测声音的器官。它容纳了一个螺旋形的- 含有将声波转化为神经元的机械感觉毛细胞的形状感觉上皮 信号。这种感觉上皮是有规律地组织起来的，这样它就能在 螺旋的底部和顶端的低频声音。声调专业化认证包括分级的 毛细胞从底部到顶端的增大，以及电生理的渐进性变化 毛细胞和神经元的特性。然而，控制耳蜗强直的外在因素和内在因素 专业化在很大程度上是未知的。这项提案的主要目的是描述 LIN28b/let-7轴在听觉毛细胞强直性特化中的作用促进生长的RNA结合蛋白 Lin28b和功能相反的let-7家族的miRNAs调节数百个基因的表达， 包括参与新陈代谢和细胞生长的基因。我们之前已经证明了LIN28B和LET-7 MiRNAs以相反的梯度表达，通知感觉前体细胞周期退出和毛细胞 差异化。我们的初步数据显示，在耳蜗成熟过程中，let-7在基底层高表达。 而促生长基因let-7在顶端外毛细胞中表达。基于这些 发现我们假设let-7miRNA的基底部-顶端梯度指导着 耳蜗强直性特化，特别是毛细胞大小和毛细胞的强直性差异。 特定的基因表达。小鼠耳蜗单听特化的发展尚未得到证实。 有很好的特点，但人们认为在听力开始时，立位的变化开始明显 (第11页)。在目标1中，我们建议确定毛细胞/立体纤毛大小的色调差异何时出现在 通过分析小鼠耳蜗术前(P7)、听力期间(P14)和听力开始后(P21，P30)的这些特征 和P60)。此外，我们还将确定小鼠耳蜗声强直特化的标志物。在目标2中， 我们将利用现有的转基因小鼠株系来消除成熟耳蜗中let-7miRNA的梯度(P1-miRNA)。 P12)。在P30，我们将评估这些操作对特定频率听力的影响，以及 毛细胞的特异化。我们预测，Lin28b的过度表达将产生更典型的 高频听力中毛细胞的特性和功能缺陷。相反，let-7G过度表达将 导致“心尖”毛细胞特性的丧失和低频听力的缺陷。我们的发现将为我们提供见解 研究调节毛细胞强直特性的分子机制。这将有利于毛细胞的努力 再生疗法，因为沿着耳蜗生出适当的特化毛细胞将是关键 对有意义的声音的感知。

Project Summary The cochlea is the organ in the auditory system responsible for the detection of sound. It houses a spiral- shaped sensory epithelium containing mechanosensory hair cells that transduce sound waves into neuronal signals. This sensory epithelium is tonotopically organized such that it detects high frequency sounds at the base of the spiral and low frequency sounds at the apex. The tonotopic specializations include a graded increase in hair cell size from the base to the apex, as well as graded changes in the electrophysiological properties of hair cells and neurons. However, the extrinsic and intrinsic factors controlling cochlear tonotopic specialization are largely unknown. The main aim of this proposal is to characterize the role of the LIN28B/let-7 axis in tonotopic specialization of auditory hair cells. The pro-growth RNA-binding protein LIN28B and the functionally opposing let-7 family of miRNAs regulate the expression of hundreds of genes, including genes involved in metabolism and cell growth. We have previously shown that LIN28B and let-7 miRNAs are expressed in opposing gradients that inform sensory progenitor cell cycle exit and hair cell differentiation. Our preliminary data show that during cochlear maturation, let-7 is highly expressed in basal outer hair cells, whereas pro-growth let-7 target genes are expressed in apical outer hair cells. Based on these findings we hypothesize that the basal-apical gradient of let-7 miRNA instructs the development of cochlear tonotopic specialization, specifically tonotopic differences in hair cell size and hair cell- specific gene expression. The development of tonotopic specialization in the mouse cochlea has not been well characterized, but it is thought that tonotopic changes start to become evident around the onset of hearing (P11). In aim 1, we propose to establish when tonotopic differences in hair cell/stereocilia size arise in the murine cochlea, by analyzing these features prior (P7), during (P14) and after the onset of hearing (P21, P30 and P60). In addition, we will characterize markers for tonotopic specialization in the murine cochlea. In aim 2, we will use existing transgenic mouse lines to abolish the gradient of let-7 miRNA in the maturing cochlea (P1- P12). At P30, we will assess the effects of these manipulations on frequency-specific hearing, as well as the tonotopic specializations of hair cells. We predict that LIN28B overexpression will produce a more `apical' identity of hair cells and functional deficits in high frequency hearing. Conversely, let-7g overexpression will lead to loss of `apical' hair cell identity and deficits in low frequency hearing. Our findings will provide insight into the molecular mechanisms regulating tonotopic identity of hair cells. This will benefit efforts in hair cell regeneration therapies, as generating appropriately specialized hair cells along the cochlea will be critical to the perception of meaningful sound.