Hair Cell Development

描述(申请人提供)：耳聋的主要原因之一是内耳毛细胞的丧失，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出生，并在整个生命中保持静止。哺乳动物的内耳与鸡或鱼等低等脊椎动物的内耳不同，内耳受损后不会再生毛细胞。负生长基因(Rb1和p27kip1)的缺失导致胚胎和新生儿内耳的细胞周期重新进入。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。这项建议旨在具体解决这两个问题。我们的研究表明，在斑马鱼神经肥大的毛细胞再生过程中，成纤维细胞生长因子信号是必需的。对于特定的目标1，我们将通过一个可诱导的小鼠模型来检验这样的假设，即在活体内，Rb1缺失的成纤维细胞生长因子激活可以导致细胞周期重新进入成年内耳。我们已经在体外观察到了这样的事件。免疫组织化学染色检测增殖细胞中成纤维细胞生长因子活性的相关性将支持成纤维细胞生长因子在细胞周期重入中的重要作用。使用可诱导的小鼠模型来标记支持细胞的遗传学，我们将鉴定来自支持细胞转分化的毛细胞。在目标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，我们将评估两个通路，IGF1和P53，它们在增殖的毛细胞存活和凋亡中所起的作用。我们有证据表明IGF1的激活或P53的阻断可以保护Rb1/-耳蜗毛细胞免于凋亡。在目标2a中，我们将通过阻断IGF1的功能诱导凋亡来确定IGF1在Rb1/-耳蜗毛细胞生存中的必要性。此外，我们将专门阻断两个IGF1信号通路：PI3K/PDK1/Akt和Raf/MEK/Erk，以评估它们在Rb1/耳蜗毛细胞生存中的各自作用。在目标2b中，我们将用一种特定的抑制物来阻断P53的功能，并将P53的失活与Rb1-/-耳蜗毛细胞的存活联系起来。我们将进一步研究IGF1激活或抑制对P53通路的影响。IGF1激活后P53通路失活，或反之亦然，表明IGF1拮抗P53功能以促进生存。最后，我们将在p53缺失的背景下诱导成年内耳细胞周期重新进入，并研究增殖毛细胞的长期存活。细胞周期在成熟内耳的重新进入和增殖的毛细胞的存活将使再生有功能的毛细胞成为可能。

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.