Hair Cell Development

描述（由申请人提供）：耳聋的主要原因之一是内耳毛细胞的损失，内耳毛细胞是检测声音的感觉细胞。在哺乳动物中，毛细胞在胚胎发育期间产生，并在一生中保持静止。哺乳动物的内耳，与鸡或鱼等低等脊椎动物的内耳不同，在受损后不能再生毛细胞。缺失负生长基因（Rb1和p27kip1）导致胚胎和新生儿内耳细胞周期重新进入。然而，我们已经证明，在成人内耳中，Rb1缺失不足以诱导增殖。此外，增殖的毛细胞和支持细胞最终会死亡。因此，成熟内耳不能重新进入细胞周期和增殖细胞的凋亡是毛细胞再生的两个主要挑战。这一建议旨在具体解决这两个问题。我们发现FGF信号是斑马鱼神经鞘毛细胞再生所必需的。为了达到特定的目的1，我们将通过诱导小鼠模型验证这一假设，即通过Rb1缺失激活FGF可能导致体内成人内耳细胞周期重新进入。我们已经在体外观察到这样的事件。通过免疫染色检测增殖细胞中FGF活性的相关性将支持FGF在细胞周期再进入中的关键作用。利用诱导小鼠模型对支持细胞进行遗传标记，我们将鉴定来自支持细胞转分化的毛细胞。在目的1b中，我们将通过免疫染色、FM1-43摄取和转导电流记录来表征再生毛细胞的分化、突触形成和功能。在第二个目标中，我们将评估IGF1和p53两种通路在增殖毛细胞存活和凋亡中的作用。我们有证据表明IGF1激活或p53阻断可保护Rb1-/-耳蜗毛细胞免于凋亡。在目的2a中，我们将通过阻断IGF1功能诱导细胞凋亡来确定IGF1在Rb1-/-耳蜗毛细胞存活中的必要性。此外，我们将特异性阻断两个IGF1信号通路：PI3K/Pdk1/Akt和Raf/Mek/Erk，以评估它们各自在Rb1-/-耳蜗毛细胞存活中的作用。在目的2b中，我们将通过一种特异性抑制剂阻断p53的功能，并将p53失活与Rb1-/-耳蜗毛细胞存活联系起来。我们将进一步研究IGF1激活或抑制后对p53通路的影响。IGF1激活后p53通路失活，反之亦然，表明IGF1拮抗p53功能促进存活。最后，我们将在p53缺失的背景下诱导成人内耳细胞周期再进入，并研究增殖毛细胞的长期存活。成熟内耳细胞周期的重新进入和增殖毛细胞的存活将使功能性毛细胞再生成为可能。

DESCRIPTION (provided by applicant): One of the major causes for deafness is the loss of inner ear hair cells, the sensory cells that detect sounds. In mammals, hair cells are born during embryonic development and are maintained in quiescence throughout life. Mammalian inner ear, unlike the counterpart in lower vertebrates such as chick or fish, does not regenerate hair cells after damage. Deletion of negative growth genes (Rb1 and p27kip1) led to cell cycle re-entry in embryonic and neonatal inner ear. However, we have shown in adult inner ear, Rb1 deletion is not sufficient to induce proliferation. Further, the proliferating hair cells and supporting cells will ultimately die. Thus the inability to re-enter cell cycle by mature inner ear and apoptosis of proliferating cells present two main challenges to hair cell regeneration. This proposal is designed to specifically address the two issues. We showed that FGF signaling is necessary for hair cell regeneration in zebrafish neuromasts. For the specific aim 1, we will test the hypothesis that, with an inducible mouse model, FGF activation with Rb1 deletion could lead to cell cycle re-entry in adult inner ear in vivo. We have observed such events in vitro. Correlation of FGF activity in the proliferating cells by immunostaining will support the crucial role of FGF in cell cycle re-entry. Using an inducible mouse model to mark supporting cells genetically, we will identify hair cells derived from supporting cell transdifferentiation. In the aim 1b, we will characterize regenerated hair cells in differentiation, synapse formation and function by immunostaining, FM1-43 uptake and transduction current recording. In the second aim, we will evaluate two pathways, IGF1 and p53, for their roles in survival and apoptosis of proliferating hair cells. We have the evidence that IGF1 activation or p53 blockade protect Rb1-/- cochlear hair cells from apoptosis. In the aim 2a, we will determine the necessity of IGF1 in Rb1-/- cochlear hair cell survival by blocking IGF1 function to induce apoptosis. Further, we will specifically block two IGF1 signaling pathways: PI3K/Pdk1/Akt and Raf/Mek/Erk, to assess their respective role in the survival of Rb1-/- cochlear hair cells. In the aim 2b, we will block p53 function by a specific inhibitor and correlate p53 inactivation with Rb1-/- cochlear hair cell survival. We will further study the effects on the p53 pathway after IGF1 activation or inhibition. Inactivation of the p53 pathway after IGF1 activation, or vice versa, is an indication that IGF1 antagonizes p53 function to promote survival. Finally we will induce cell cycle re-entry in the adult inner ear on the p53-null background, and study long-term survival of proliferating hair cells. Cell cycle re-entry in mature inner ear and the survival of proliferating hair cells will make it possible to regenerate functional hair cells. PUBLIC HEALTH RELEVANCE: The proposal is designed to demonstrate that FGF activation and deletion of retinoblastoma gene can lead to cell cycle re-entry and regeneration of hair cells, the sensory cells that detect sounds, in adult mouse inner ear in vivo. It will demonstrate that manipulation of IGF1 (insulin-like growth factor) and p53 could promote survival of regenerated hair cells. Success of the proposal will lay the foundation for the study of functional recovery of hearing by regenerated hair cells.