THE ROLE OF E2F2 MODULATION OF RB1 IN COCHLEAR HAIR CELLS AND SUPPORTING CELLS

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 视网膜母细胞瘤1蛋白（pRb或RB 1）的靶向操作表明，Rb 1是正常毛细胞（HC）细胞周期控制所必需的，包括完全分化和有丝分裂静止（Mantela et al. 2005; Sage et al. 2005）。然而，全球Rb 1缺陷小鼠出生前死亡，并表现出异常模式的细胞增殖在内耳。因此，需要靶向切除内耳毛细胞中的Rb 1，以避免有害的整体效应。最近显示小鼠HC中的特异性Rb1缺失导致早期异常增殖，随后导致成年HC大量丢失（Weber et al. 2005; Sage et al. 2006）。这表明，正如Rb1基因或其相关转录因子E2Fs基因缺失所预期的那样，成人HC中Rb1失衡导致细胞凋亡（Ikeda et al. 1996; Kastner et al. 1998; Giangrande et al. 2000; Hallstrom and Nevins 2003; Mantela et al. 2005）。然而，CNS和心脏细胞中的数据显示，一些细胞群能够重新进入细胞周期并分化而不发生细胞凋亡，这表明pRb消融的影响可能是细胞类型依赖性的（Ferguson et al. 2002; MacPherson et al. 2003; Marino et al. 2003; Maclellan et al. 2005）。Rb1基因操作的目前结果表明，需要一种替代方法来研究pRb操纵HC再生的潜力。为了实现这一目标，我们利用了几种新的分子生物学技术，这些技术在我们的原始提案中有更详细的描述，并在我们的具体目标描述中简要强调（见下文）。此外，根据COBRE内部理事会的建议，本研究增加了两个新目标。首先，将评估Rb1在内耳毛细胞中诱导HC再生的潜力（S.A. 2）。我们的第三个目标将包括使用转录组学的方法，以确定可能的基因调控网络的分化的毛细胞和支持细胞在耳蜗。我们的最终目标是操纵有丝分裂后的SC重新进入细胞周期，并评估其自发分化为HC的潜力。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Targeted manipulation of retinoblastoma 1 protein, (pRb or RB1), suggested that Rb1 is required for normal hair cell (HC) cell cycle control, including full differentiation and mitotic quiescence (Mantela et al. 2005; Sage et al. 2005). However, global Rb1 deficient mice die before birth and exhibit an aberrant pattern of cell proliferation in the inner ear. Thus, a targeted excision of Rb1 in inner ear HCs is needed to avoid the deleterious global effects. Specific Rb1 deletion in the mouse HCs was recently shown to cause an aberrant proliferation at an early age, followed by a massive loss of HCs in the adults (Weber et al. 2005; Sage et al. 2006). This suggests that Rb1 imbalance in adult HCs leads to apoptosis, as expected from the deletion of either the Rb1 gene or its associated transcription factors: the E2Fs genes (Ikeda et al. 1996; Kastner et al. 1998; Giangrande et al. 2000; Hallstrom and Nevins 2003; Mantela et al. 2005). Nevertheless, data in the CNS and cardiac cells have shown the ability of some cell populations to re-enter the cell cycle and differentiate without apoptosis, suggesting that the impact of pRb ablation is likely cell-type dependent (Ferguson et al. 2002; MacPherson et al. 2003; Marino et al. 2003; Maclellan et al. 2005). The current results in Rb1 gene manipulation suggest that an alternative approach to study the potential of pRb manipulation for HC regeneration is needed. In order to accomplish this goal we have taken advantage of several of the novel molecular biology technologies which are described more in detail in our original proposal and briefly highlighted in the description of our specific aims (see bellow). Additionally, as per the COBRE internal council's suggestion, two new aims were added to this study. Initially, the potential of Rb1 manipulation in the inner ear hair cells to induce HCs regeneration will be assessed (S.A. 2). Our third aim will consist of using a transcriptomic approach to identify possible gene regulatory network for the differentiation of hair cells and supporting cells in the cochlea. Our ultimate goal is to manipulate postmitotic SC to re-enter the cell cycle and evaluate their potential to spontaneously differentiate into HCs.