Origin of hair cells in development and regeneration

描述(申请人提供)：听觉和前庭障碍是由于调节听力和平衡的感觉毛细胞受到不可逆转的损害而引起的。该项目的目标是确定斑马鱼内耳中新的感觉毛细胞(干细胞)的来源。感觉性毛细胞的产生贯穿鱼类的整个生命，受损的毛细胞很容易再生。我们将测试胚胎感觉前体细胞在胚胎后生长过程中保持不变的假设，并在成年斑马鱼感觉上皮中提供持久的毛细胞来源。我们将：A)确定感觉毛细胞的胚胎来源，B)确定感觉上皮中毛细胞的胚胎后来源，以及C)确定胚胎感觉细胞前体是否能在成熟的感觉上皮中产生具有再生能力的细胞。斑马鱼胚胎和幼虫的内耳是光学透明的，可以在实验中接触到。这项提案中概述的实验结合了实验胚胎学和最先进的显微镜来鉴定体内的毛细胞前体细胞。我们将利用笼中荧光素葡聚糖的双光子依赖去化来标记单个耳部胎盘细胞，追踪它们在发育过程中的谱系，并确定它们的最终命运，从而开发出一张高分辨率的斑马鱼耳部胎盘的命运图。这张命运图将确定感觉毛细胞的胚胎来源。利用斑马鱼幼虫，我们将确定正常翻转时和激光消融后后巩膜再生毛细胞的来源。我们将通过双光子成像对后巩膜的细胞进行体内感觉细胞群体动力学的研究。我们的努力最终将检验一种假设，即感觉(干细胞)谱系在耳道胎盘中出生，在整个胚胎发育过程中保持，并成为幼虫耳朵中的祖细胞。斑马鱼中感觉干细胞的鉴定和未来的特征将有助于在其他脊椎动物(最重要的是哺乳动物)中鉴定它们，最终目标是开发干细胞疗法来改善耳聋和平衡障碍的影响。

DESCRIPTION (provided by applicant): Auditory and vestibular disorders result from irreversible damage to sensory hair cells that mediate hearing and balance. The goal of this project is to identify sources of new sensory hair cells (stem cells) in the zebrafish inner ear. Sensory hair cell production continues throughout the life of fish and damaged hair cells are readily regenerated. We will test the hypothesis that embryonic sensory progenitor cells are maintained during postembryonic growth and provide a persistent source of hair cells in the adult zebrafish sensory epithelia. We will: A) Determine the embryonic source of sensory hair cells, B) Determine the postembryonic source of hair cells in a sensory epithelium, and C) Determine if embryonic sensory cell progenitors give rise to regeneration-competent cells in a mature sensory epithelium. The inner ears of zebrafish embryos and larvae are optically transparent and experimentally accessible. Experiments outlined in this proposal combine experimental embryology and state-of-the-art microscopy to identify hair cell progenitors in vivo. We will develop a high-resolution fate map of the zebrafish otic placode using two-photon dependant uncaging of caged fluorescein dextran to label single otic placode cells, trace their lineages during development and determine their ultimate fates. This fate map will identify the embryonic source of sensory hair cells. Using zebrafish larvae, we will determine the source of regenerated hair cells in the posterior crista during normal turnover and after laser ablation. We will investigate sensory cell population dynamics in vivo by two-photon imaging of cells in the posterior crista. Our efforts will culminate in a test of the hypothesis that sensory (stem) cell lineages are born in the otic placode, maintained throughout embryogenesis, and become progenitor cells in the larval ear. Identification and future characterization of sensory stem cells in the zebrafish will aid their identification in other vertebrates (most importantly mammals) with the ultimate goal of developing stem cell therapies to ameliorate the effects of deafness and balance disorders.