Hair Cell Development

描述（由申请人提供）：数百万人由于老化和早期暴露于危险环境（如噪音）而导致毛细胞退化，从而遭受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增殖、细胞命运决定的启动和终末分化的控制。然而，很少有人知道的身份的基因和相关的途径，控制祖细胞的细胞周期退出和毛细胞和支持细胞的有丝分裂后的静止状态的维持。 在本申请中，我们提出实现四个目标。首先，为了验证我们的假设，视网膜母细胞瘤基因（Rb）是关键参与细胞周期退出的感觉上皮在内耳。这将使用条件性Rb敲除小鼠模型来实现，其中Rb在感觉上皮细胞中被消除。此外，将研究两种毛细胞特异性Rb条件性小鼠模型，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pRb功能缺失的情况下是否可以产生功能性毛细胞。 第二，为了验证我们的假设，Rb是所需的静止状态的成熟毛细胞。将研究在成熟lox-P Rb椭圆囊器官培养系统中急性缺失Rb后，成熟毛细胞的增殖潜力。将通过染料FM 1 -43摄取和膜片钳研究新衍生的毛细胞以确定它们是否具有功能。成熟毛细胞重新进入细胞周期产生功能性毛细胞将具有巨大的毛细胞再生潜力，用于未来的治疗。 第三，使用功能基因组学方法，我们将确定Rb在内耳中控制的途径。Rb通路中关键分子的鉴定将为我们提供微调毛细胞有丝分裂后状态的过程控制的可能性。 最后，通过利用分化的Rb-空毛细胞的增殖特性，我们将建立富含毛细胞特性的细胞系。将对细胞系的毛细胞特异性基因表达进行表征。这样的细胞系将对听力研究有很大的价值。 该项目的完成将使我们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.