Regeneration and reinnervation of mechanosensory hair cells

描述（由申请人提供）： 内耳是所有脊椎动物中调节听觉和平衡功能的主要感觉器官。听觉和前庭感觉上皮共同的是感觉毛细胞，其对声损伤非常敏感。哺乳动物内耳毛细胞的损失是不可逆的，并导致听力障碍、耳聋和平衡功能障碍。与哺乳动物相比，鱼类不仅在一生中产生新的毛细胞，而且具有再生功能性毛细胞的先天能力。以斑马鱼为再生动物模型，本研究的重点是长寿毛细胞前体的形成和调控以及新生毛细胞神经再生的遗传学。该提案的长期目标是提供对斑马鱼毛细胞更新的分子和遗传理解，并利用这些信息开发新的策略，迫使哺乳动物毛细胞再生和神经再生。具体目标1是集中在有丝分裂后的毛细胞前体介导的非有丝分裂的毛细胞再生在斑马鱼的形成和调节。使用一种新鉴定的斑马鱼品系，我们发现Notch mRNA在胚后感觉上皮中表达，并且3-分泌酶活性的抑制剂在体内诱导早熟毛细胞形成。本研究的目的是（a）确定Notch信号在调节前体分化中的作用，（B）确定有丝分裂后前体在感觉上皮生长和再生过程中的功能，（c）鉴定长寿毛细胞前体的有丝分裂来源，以及（d）确定斑马鱼非有丝分裂毛细胞再生的遗传基础。具体目标2将确定新生感觉毛细胞的神经元再支配所需的基因。听觉功能的恢复需要细胞更新和重新连接到中枢神经系统的不可分割的联系过程。然而，毛细胞再生和神经再支配可以在体内分离，在斑马鱼中使用分子遗传学和突变体鉴定。通过利用简单的视觉和行为分析，我们将重点放在（i）斑马鱼突变体，不再生毛细胞和（ii）突变体再生，但不重新神经支配新生的毛细胞。克隆这些突变体中受影响的基因座将鉴定新的基因，或具有新功能的已知基因，这些基因是新毛细胞形成和突触连接重建所需的。 公共卫生相关性： 7.听力损失是退伍军人群体中最常见的一种个体残疾。近60%的暴露于爆炸物的士兵遭受永久性听力损失，28%的从战区返回的士兵听力下降。目前，超过80万名退伍军人因与服务有关的听力障碍而获得残疾补偿，预计到2011年，每年将增长18%，支付总额为11亿美元。这些研究结果将为感觉毛细胞再生和神经再支配的内在机制提供新的见解。由于再生不会发生在哺乳动物中，因此这些信息对于指导当前的研究工作和设计旨在改善或逆转人类听力损失影响的新策略是必要的。

DESCRIPTION (provided by applicant): Project Summary/Abstract The inner ear is the primary sensory organ mediating hearing (auditory) and balance (vestibular) function in all vertebrates. Common to both the auditory and vestibular sensory epithelia are sensory hair cells, which are acutely sensitive to acoustic trauma. Loss of hair cells in the mammalian inner ear is irreversible and causes hearing impairment, deafness and balance dysfunction. In contrast to mammals, fish not only produce new hair cells throughout life but also have the innate capacity to regenerate functional hair cells. Using zebrafish as a regenerative animal model, this proposal is focused on the formation and regulation of long-lived hair cell precursors and the genetics of nascent hair cell reinnervation. The long-term goal of this proposal is to provide a molecular and genetic understanding of hair cell renewal in zebrafish and use this information to develop novel strategies that force hair cell regeneration and reinnervation in mammals. Specific Aim 1 is focused on the formation and regulation of postmitotic hair cell precursors that mediate non-mitotic hair cell regeneration in zebrafish. Using a newly identified strain of zebrafish, we show that Notch mRNA is expressed in the postembryonic sensory epithelium and that inhibitors of 3-secretase activity induce precocious hair cell formation in vivo. The goals of this Aim are to (a) determine the role of Notch signaling in regulating precursor differentiation, (b) determine the function of postmitotic precursors during growth and regeneration in the sensory epithelium, (c) identify the mitotic source of long-lived hair cell precursors, and (d) determine the genetic basis of non-mitotic hair cell regeneration in zebrafish. Specific Aim 2 will identify genes required for neuronal reinnervation of nascent sensory hair cells. Restoration of auditory function requires the inextricably linked processes of cell renewal and reconnection to the central nervous system. However, hair cell regeneration and reinnervation can be dissociated in vivo using molecular genetics and mutant identification in zebrafish. By exploiting simple visual and behavioral assays we will focus on (i) zebrafish mutants that do not regenerate hair cells and (ii) mutants that regenerate, but do not reinnervate nascent hair cells. Cloning of the affected loci in these mutants will identify novel genes, or known genes with novel functions, required for new hair cell formation and reestablishment of synaptic connectivity. PUBLIC HEALTH RELEVANCE: 7. Project Narrative Hearing loss is the single most common individual disability in the veteran population. Nearly 60% of soldiers exposed to explosive blasts suffer permanent hearing loss and 28% of troops returning from a war zone have diminished hearing. Currently, more than 800,000 veterans are receiving disability compensation for service-connected hearing disorders, which is expected to grow by 18% a year with payments totaling $1.1 billion annually by 2011. Results of these proposed studies will provide new insight to the innate mechanisms of sensory hair cell regeneration and reinnervation. Since regeneration does not occur in mammals, this information is necessary to direct current research efforts and design novel strategies aimed toward ameliorating or reversing the effects of hearing loss in humans.