Origin of hair cells in development and regeneration

描述（由申请人提供）：听觉和前庭障碍是由介导听觉和平衡的感觉毛细胞的不可逆损伤引起的。该项目的目标是确定斑马鱼内耳中新感觉毛细胞（干细胞）的来源。感觉毛细胞的生产在鱼的整个生命过程中持续进行，受损的毛细胞很容易再生。我们将测试的假设，胚胎感觉祖细胞维持在胚后生长，并提供一个持久的来源，成年斑马鱼的感觉上皮毛细胞。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后来源，和C）确定胚胎感觉细胞祖细胞是否在成熟感觉上皮中产生再生能力细胞。斑马鱼胚胎和幼体的内耳是光学透明的，可以通过实验获得。本提案中概述的实验结合了联合收割机实验胚胎学和最先进的显微镜技术，以在体内鉴定毛细胞祖细胞。我们将开发一个高分辨率的斑马鱼耳基板的命运地图使用双光子依赖uncaging的笼状荧光素葡聚糖标记单个耳基板细胞，在发展过程中跟踪他们的谱系，并确定他们的最终命运。这个命运图将确定感觉毛细胞的胚胎来源。使用斑马鱼幼虫，我们将确定在正常营业额和激光消融后的后嵴再生毛细胞的来源。我们将通过对后嵴细胞的双光子成像来研究感觉细胞群体在体内的动态。我们的努力将最终在测试的假设，即感觉（干）细胞系出生在耳基板，保持整个胚胎发育，并成为祖细胞在幼虫的耳朵。斑马鱼感觉干细胞的鉴定和未来特征将有助于它们在其他脊椎动物（最重要的是哺乳动物）中的鉴定，最终目标是开发干细胞疗法，以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.