Outer and Inner Hair Cell Development

项目摘要 发育中的哺乳动物耳蜗产生两种类型的机械传感器：内毛细胞（IHC），赋予 具有突出的突触前器官，将声音信息传递给神经元和大脑;外毛细胞 （OHC），配备了电动性，用于放大和尖锐的声音频率歧视。 OHC的丧失是听力损失的主要原因，因此它们的再生可能是最有价值的。 就是恢复听力。在该基金的第一个周期中，我们发现锌指蛋白INSM 1，表达于 在新生的人道主义协调员中，在其早期发展的短暂关键时期采取行动，以巩固其命运， 防止OHC转分化为IHC。在缺乏INSM 1的情况下，OHC正常生成 但是，在几天内，他们中的近一半停止表达OHC标志物，并继续进行 转分化为IHC。我们假设INSM 1通过改变OHC的表达， 表观基因组，关闭IHC分化所需的调控元件。为了测试这个和另一个 假设，我们将对具有和不具有INSM 1的OHC进行ATAC-seq（其易受 转分化成IHC）以及异位表达INSM 1的IHC中。在上一个融资周期，我们 还发现转分化毛细胞的分布沿着神经纤维以梯度方式变化， 这表明INSM 1通常通过阻止OHC对神经外轴的反应来巩固OHC的命运。 诱导IHC的形态发生素。为了鉴定这样的形态原，它可能决定了免疫组化与 OHC在耳蜗中的分布，我们将培养突变的Corti器官并改变它们的程度。 用候选形态发生素的激动剂和拮抗剂及其信号传导途径进行转分化。 这些研究使用OHC向IHC的转分化作为阐明分子生物学特性的新手段。 机制（染色质修饰，形态发生素和由此产生的基因表达）， 在发育中的耳蜗中产生互补的IHC和OHC。最后，在上一个供资周期中， 我们鉴定了一种OHC特异性的转录因子BCL 11b，它与它的辅因子BCL 11 a一起， 这对于OHC在平行行中的精确对齐以及静纤毛垂直于 网状板，其作为耳蜗放大器的功能的基本特征。我们将探讨BCL 11b +a 通过遗传学、解剖学、生理学、转录组学和表观基因组学检查来实现这一任务， 我们正在或已经成功地使用了INSM 1。我们预测这里的研究 建议将提供前所未有的进步，了解OHC如何形成，并调整自己， 以发挥他们在听力方面的作用。

Project Summary The developing mammalian cochlea generates two types of mechanosensors: Inner hair cells (IHCs), endowed with a prominent presynaptic apparatus, transmit sound information to neurons and the brain; outer hair cells (OHCs), equipped for electromotility, are used for amplification and sharp frequency discrimination of sounds. Loss of OHCs is the leading cause of hearing loss, and hence their regeneration could be the most valuable means to restore hearing. In the first cycle of this grant, we found that the zinc-finger protein INSM1, expressed in nascent OHCs, acts during a brief critical period of their early development to consolidate their fate, preventing OHCs from transdifferentiating into IHCs. In the absence of INSM1, OHCs are born properly specified but, within a couple of days, nearly half of them cease expressing OHC markers and proceed to transdifferentiate into IHCs. We hypothesize that INSM1 consolidates the fate of OHCs by modifying their epigenome, closing regulatory elements required for IHC differentiation. To test this and alternative hypotheses, we will perform ATAC-seq on developing OHCs with and without INSM1 (which are susceptible to transdifferentiate into IHCs) as well as in IHCs ectopically expressing INSM1. In the previous funding cycle, we also found that the distribution of transdifferentiated hair cells varies in a graded fashion along the neural to abneural axis, suggesting that INSM1 normally consolidates the OHC fate by preventing their responding to an IHC-inducing morphogen. In order to identify such a morphogen, which presumably determines the IHC vs OHC distribution in the cochlea, we will culture mutant organs of Corti and alter their extent of transdifferentiation with agonists and antagonists of candidate morphogens and their signaling pathways. These studies use the transdifferentiation of OHCs into IHCs as a novel means for elucidating the molecular mechanisms (chromatin modifications, morphogens, and resulting gene expression) by which the complementary IHCs and OHCs are generated in the developing cochlea. Finally, in the previous funding cycle we identified an OHC-specific transcriptions factor, BCL11b which, together with its paralog BCL11a, is necessary for the precise alignment of OHCs in parallel rows and with stereocilia pointing perpendicular to the reticular lamina, essential features for their function as cochlear amplifiers. We will explore how BCL11b+a achieve this task by genetic, anatomical, physiological, transcriptomic and epigenomic examinations, the same approaches we are or already have successfully employed with INSM1. We predict that the studies here proposed will provide an unprecedented advance in understanding how OHCs form, and align themselves, so as to serve their role in hearing.