Hair Cell Development

描述(申请人提供)：数以百万计的人因衰老和过早暴露在噪音等危险环境中而导致毛细胞退化，导致不可逆转的听力损失。与鸟类等其他物种不同，哺乳动物的内耳不会自发地替换受损的毛细胞。任何旨在替换受损毛细胞的干预措施都需要了解毛细胞的发育控制，以便通过操纵决定因素来诱导毛细胞的再生。 毛细胞的发育受祖细胞的增殖、细胞命运决定和终末分化的控制。然而，对于控制前体细胞的细胞周期退出以及毛细胞和支持细胞的有丝分裂后静止状态的维持的基因和相关通路的识别，人们知之甚少。 在本申请中，我们提出了实现四个目标。首先，为了验证我们的假设，即视网膜母细胞瘤基因(Rb)在内耳感觉上皮细胞周期退出中起关键作用。这将使用条件Rb基因敲除小鼠模型来完成，在该模型中，Rb在感觉上皮细胞中被取消。此外，还将研究两种毛细胞特异性RB条件小鼠模型，以了解RB是否是维持有丝分裂后毛细胞所必需的。这些小鼠模型也将为我们提供一些问题的答案，包括Rb是否参与毛细胞的分化，或者在缺乏pRb功能的情况下是否可以产生功能性毛细胞。 其次，为了验证我们的假设，即成熟毛细胞的静止状态需要Rb。在成熟的LOX-P Rb椭圆体器官培养系统中，急性缺失Rb后，将研究成熟毛细胞的增殖潜力。新获得的毛细胞将通过摄取FM1-43染料和膜片钳技术进行研究，以确定它们是否具有功能。成熟的毛细胞重新进入细胞周期以产生功能毛细胞将为未来的治疗提供巨大的毛细胞再生潜力。 第三，利用功能基因组学方法，我们将确定内耳中受RB控制的通路。识别Rb途径中的关键分子将为我们提供微调控制毛细胞有丝分裂后状态的过程的可能性。 最后，利用分化后的Rb缺失毛细胞的增殖特性，我们将建立富含毛细胞特性的细胞系。这些细胞系将被鉴定为毛细胞特有的基因表达。这样的细胞系总体上将对听力研究具有重要价值。 该项目的完成将有助于更好地了解毛细胞的细胞周期控制机制，并有助于开辟毛细胞再生的新途径。

DESCRIPTION (provided by applicant): Millions of people suffer from irreversible hearing loss due to hair cell degeneration as a result of aging and early exposure to hazardous environment such as noise. Unlike other species such as birds, the mammalian inner ear does not replace the damaged hair cells spontaneously. Any intervention aiming at replacing damaged hair cells requires understanding the developmental controls of the hair cells, so that by manipulating the determining factors it will be possible to induce the regeneration of hair cells. The development of the hair cell is controlled by proliferation of progenitor cells, initiation of cell fate determination and the terminal differentiation. Little is known, however, about the identity of genes and associated pathways that control the cell cycle exit of progenitor cells and the maintenance of the quiescent post mitotic status of the hair cells and the supporting cells. In this application we propose to accomplish fours aims. First, to test our hypothesis that the retinoblastoma gene (Rb) is critically involved in the cell cycle exit of the sensory epithelium in the inner ear. This will be accomplished using the conditional Rb knockout mouse model in which Rb is abolished in the sensory epithelial cells. Furthermore two hair cell- specific Rb conditional mouse models will be studied, to understand whether Rb is required for the maintenance of post mitotic hair cells. These mouse models will also provide us with the answers to questions including whether Rb is involved in the differentiation of hair cells, or whether functional hair cells can be produced in the absence of pRb function. Second, to test our hypothesis that Rb is required for the quiescent status of mature hair cells. The proliferation potential of mature hair cells will be studied, after acute deletion of Rb in the mature lox-P Rb utricle organ culture system. The newly derived hair cells will be studied to determine if they are functional, by dye FM1-43 uptake and patch clamping. The re-entry into the cell cycle by the mature hair cells to produce the functional hair cells will have enormous potential for hair cell regeneration for future therapy. Third, using the functional genomic approach, we will identify the pathways controlled by Rb in the inner ear. Identification of critical molecules involved in the Rb pathway will offer us the possibility to fine-tune the process controlling the hair cell post mitotic status. Lastly, by taking advantage of proliferating properties of differentiated Rb-null hair cells, we will establish cell lines enriched with hair cell properties. The cell lines will be characterized for hair-cell-specific gene expression. Such cell lines will be of great value to hearing research in general. Completion of the project will provide better understanding of the mechanism involved in cell cycle control of hair cells, and help to open new avenues in hair cell regeneration.