Origin of hair cells in development and regeneration

描述（由申请人提供）：听觉和前庭疾病是由介导听力和平衡的感觉毛细胞的不可逆损伤引起的。该项目的目的是确定斑马鱼内耳中新的感觉毛细胞（干细胞）的来源。感觉毛细胞的产生贯穿鱼的一生，受损的毛细胞很容易再生。我们将测试胚胎感觉祖细胞在胚胎后生长过程中维持的假设，并为成年斑马鱼的感觉上皮提供持续的毛细胞来源。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，C)确定胚胎感觉细胞祖细胞是否在成熟的感觉上皮中产生具有再生能力的细胞。斑马鱼胚胎和幼体的内耳是透明的，实验上可以接近。实验概述在这个建议结合实验胚胎学和最先进的显微镜鉴定毛细胞祖在体内。我们将开发一个高分辨率的斑马鱼耳基板命运图，使用双光子依赖的笼状荧光素葡聚糖释放来标记单个耳基板细胞，在发育过程中追踪它们的谱系并确定它们的最终命运。这张命运图将确定感觉毛细胞的胚胎来源。使用斑马鱼幼虫，我们将确定正常翻转和激光消融后后嵴再生毛细胞的来源。我们将通过后嵴细胞的双光子成像来研究体内感觉细胞的种群动态。我们的努力最终将在一个假设的测试中达到高潮，即感觉（干细胞）细胞系出生在耳位，在胚胎发育过程中保持，并在幼虫耳中成为祖细胞。斑马鱼感觉干细胞的鉴定和未来表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治疗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.