Cell cycle in development and regeneration of the inner ear

描述(申请人提供)：哺乳动物感觉毛细胞的丧失会导致永久性耳聋，因为再生不会发生。再生能力的丧失与Corti器官内的特殊支持细胞无法开始分裂以应对毛细胞死亡有关。我们对这个问题采取了发展的方式。我们的希望是，通过彻底了解Corti器官细胞在胚胎发生期间停止分裂的过程，我们将深入了解为什么没有再生。通过这样做，我们希望为耳聋问题的治疗干预提供工具和目标。在Corti器官的发育过程中，细胞增殖的控制与细胞分化和构图的过程密切相关(Ruben，1968)。我们已经证明，周期蛋白依赖的激酶抑制物p27Kip1是这种协调的定时所必需的。在p27Kip1突变小鼠中，细胞周期退出延迟，导致细胞过剩，毛细胞组织的有序模式被打乱，并导致耳聋(Chen和Segil，1999)。尽管人们普遍认为p27Kip1的丰度是通过控制蛋白质周转在转录后水平上进行调节的，但我们的结果表明，p27Kip1的转录调控在很大程度上(尽管不是全部)决定了成熟器官中的细胞数量。更多的初步数据表明，在Corti器官形成过程中，Notch通路信号可能是调节p27转录的关键因素。在特定的目标1中，我们分析了Notch信号在Corti器官胚胎发生过程中对p27Kip1转录的时空调控中的作用。尽管p27Klp1的转录调控很重要，但我们观察到，在Skp2突变的小鼠中，细胞周期退出和Corti结构的器官也存在缺陷。Skp2是SCF-泛素连接酶复合体的一部分，参与调节p27Kip1蛋白的周转。在特定的目标2中，我们讨论了转录后机制在p27Kip1调控中的作用。最后，在特定的目标3和4中，我们通过研究出生后支持细胞中p27Kip1的调控，直接解决了再生问题。我们最近开发了一种技术，使我们能够提纯出生后的支持细胞，并在体外培养它们。在这样做的过程中，我们发现围产期的支持细胞保持着重新进入细胞周期并进行分裂的能力，而来自P14小鼠的支持细胞则无法做到这一点。P14支持细胞下调p27Kip1的能力变化是导致细胞分裂受阻导致再生不足的部分原因。这一特定的目的是研究p27调节随年龄变化的分子基础，我们假设这是哺乳动物内耳缺乏再生的基础。

DESCRIPTION (provided by applicant): Loss of sensory hair cells in mammals results in permanent deafness because regeneration does not occur. The loss of regenerative ability is tied to the inability of the specialized supporting cells within the organ of Corti to begin dividing in response to hair cell death. We have taken a developmental approach to this problem. Our hope is that by thoroughly understanding the process by which the cells of the organ of Corti stop dividing during embryogenesis, we will gain insight into why regeneration does not occur. In doing so, we hope to provide tools and targets for therapeutic intervention into the problem of deafness. During development of the organ of Corti, control of cell proliferation is tightly coordinated with the process of cell differentiation and patterning (Ruben, 1968). We have shown that the cyclin-dependent kinase inhibitor p27Kip1 is required for timing this coordination. In p27Kip1 mutant mice, cell cycle exit is delayed, leading to supernumerary cells, a disruption of the orderly pattern of hair cell organization, and deafness (Chen and Segil, 1999). Although p27Kip1 abundance is widely believed to be regulated at the post-transcriptional level through control of protein turnover, our results indicate that transcriptional regulation of p27Kip1 is largely, though not entirely, responsible for the determining the number of cells in the mature organ. Additional preliminary data indicates that Notch pathway signaling may be a key player in regulating p27 transcription during organ of Corti formation. In Specific Aim 1, we analyze the role of Notch signaling in the spatial and temporal regulation of p27Kip1 transcription during embryogenesis of the organ of Corti. In spite of the importance of p27Klp1 transcriptional regulation, we have observed that in Skp2 mutant mice, there is also a defect in cell cycle exit and organ of Corti structure. Skp2 is part of the SCF-ubiquitin ligase complex that is involved in regulating p27Kip1 protein turnover. In Specific Aim 2, we address the role of post-transcriptional mechanisms in the regulation of p27Kip1 Finally, in Specific Aims 3 and 4, we address the problem of regeneration directly, by studying p27Kip1 regulation in postnatal supporting cells. We have recently developed techniques that allow us to purify postnatal supporting cells and grow them in vitro. In doing so, we have discovered that perinatal supporting cells retain the capacity to reenter the cell cycle and divide, while supporting cells from P14 mice are unable to do so. Changes in the ability of P14 supporting cells to down-regulate p27Kip1 are partly responsible for the block to cell division that results in the lack of regeneration. This specific aim investigates the molecular basis for the age-dependent change in p27 regulation that we hypothesize underlies the lack of regeneration in the mammalian inner ear.