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

项目概要 耳蜗是听觉系统中负责检测声音的器官。它有一个螺旋- 含有机械感觉毛细胞的形状感觉上皮，可将声波转变成神经元 信号。这种感觉上皮是按音调组织的，因此它可以检测高频声音 螺旋的底部和顶部的低频声音。音调专业包括分级 毛细胞大小从基部到顶端增大，以及电生理学的分级变化 毛细胞和神经元的特性。然而，控制耳蜗音位的外在和内在因素 专业化很大程度上是未知的。该提案的主要目的是描述 听觉毛细胞音位特化中的 LIN28B/let-7 轴。促生长 RNA 结合蛋白 LIN28B 和功能相反的 let-7 miRNA 家族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。我们之前已经展示过 LIN28B 和 let-7 miRNA 以相反的梯度表达，通知感觉祖细胞周期退出和毛细胞 差异化。我们的初步数据表明，在耳蜗成熟过程中，let-7 在基底细胞中高度表达。 外毛细胞中表达，而促生长 let-7 靶基因在顶端外毛细胞中表达。基于这些 根据我们的发现，我们假设 let-7 miRNA 的基底-顶端梯度指导着 耳蜗音位特化，特别是毛细胞大小和毛细胞的音位差异 特定的基因表达。小鼠耳蜗音位特化的发展尚未被证实 特征良好，但人们认为音调变化在听力开始时开始变得明显 （P11）。在目标 1 中，我们建议确定毛细胞/立纤毛大小何时出现音调差异。 小鼠耳蜗，通过分析听力出现前 (P7)、听力出现期间 (P14) 和听力出现后 (P21、P30) 的这些特征 和 P60）。此外，我们还将描述小鼠耳蜗中音调专业化的标记。在目标 2 中， 我们将使用现有的转基因小鼠品系来消除成熟耳蜗中let-7 miRNA的梯度（P1- 第 12 页）。在 P30，我们将评估这些操作对特定频率听力的影响，以及 毛细胞的音调特化。我们预测 LIN28B 过度表达将产生更多的“顶端” 毛细胞的特性和高频听力的功能缺陷。相反，let-7g 过度表达会 导致“顶端”毛细胞特性丧失和低频听力缺陷。我们的研究结果将提供见解 进入调节毛细胞音位特性的分子机制。这将有利于毛细胞的努力 再生疗法，因为沿着耳蜗生成适当特化的毛细胞对于 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.