Characterizing hair cell regeneration in the larval zebrafish inner ear

项目总结 内耳感觉毛细胞死亡是听力和前庭功能障碍的常见原因 人类。这种损伤是永久性的，因为哺乳动物不能有效地再生内耳感觉。 超过新生儿期的补丁。相比之下，非哺乳类脊椎动物，如鸟类和鱼类，可以 在一生中再生毛细胞。哺乳动物和非哺乳动物之间在再生能力上的差异 哺乳动物脊椎动物还没有得到很好的了解。在能够再生毛细胞的物种中，无感觉的 毛细胞周围的支持细胞可以作为毛细胞的祖细胞。内耳毛细胞的活体研究 再生和支持细胞受到内耳在哺乳动物骨头骨中的位置的限制 鸟。因此，斑马鱼的侧线，一种检测水流变化的外部感官系统， 已被开发为研究活着的支持细胞和毛细胞再生的替代模型 动物。然而，侧线再生机制的程度与 耳朵的位置尚不清楚。斑马鱼内耳是一个尚未被研究的系统，有可能弥合这一差距。 哺乳动物内耳和侧线之间的研究。这个项目研究了人的毛细胞再生。 斑马鱼内耳在幼体阶段，此时感官器官变得完全功能和 保持可供活体成像使用。我们的目标是鉴定斑马鱼内耳和毛细胞的前体 确定它们在再生过程中何时以及如何产生新的毛细胞。在鸟类的内耳中 哺乳动物，支持细胞通过增殖和分化或直接转分化再生毛细胞。 在斑马鱼的侧线上，只使用了前一种机制。在本提案的目标1中，我们将使用一部小说 毛细胞消融的方法确定再生的时间进程和细胞是否增殖， 转分化，或两者都用于斑马鱼的内耳。侧线中的毛细胞再生 主要由空间上和功能上不同的一组支撑单元组成。是否有相似的支持细胞亚型 斑马鱼内耳中是否存在尚不清楚。在目标2中，我们将验证假定的内耳支持细胞亚型 通过单细胞RNA-seq分析鉴定。然后我们将产生标记支持细胞的转基因系 亚型和使用命运映射来识别毛细胞前体。总之，这些实验将表现出 斑马鱼毛细胞再生的时机、机制及支持细胞亚型的多样性 内耳。这项工作将有助于建立斑马鱼内耳作为一个优秀的体内模型系统。 未来对支持细胞及其在保守的再生机制中的作用的深入研究。正在扩张 我们对斑马鱼等动物内耳毛细胞再生机制的了解 最终导致人类听力和前庭衰退的治疗方法的进步。

PROJECT SUMMARY Death of sensory hair cells in the inner ear is a common cause of auditory and vestibular dysfunction in humans. This damage is permanent due to the inability of mammals to effectively regenerate inner ear sensory patches beyond the neonatal stage. In contrast, non-mammalian vertebrate species such as birds and fish can regenerate hair cells throughout life. This discrepancy in regeneration capacity between mammals and non- mammalian vertebrates is not well understood. In species that can regenerate hair cells, the nonsensory support cells that surround hair cells can act as hair cell progenitors. In vivo studies of inner ear hair cell regeneration and support cells are limited by the inner ear’s location within the bony skull of mammals and birds. As a result, the zebrafish lateral line, an external sensory system for detecting changes in water flow, has been developed as an alternative model for studying support cells and hair cell regeneration in living animals. However, the degree to which the mechanisms of regeneration in the lateral line are similar to those of the ear is unclear. The zebrafish inner ear is an understudied system that has the potential to bridge the gap between mammalian inner ear and lateral line studies. This project examines hair cell regeneration in the zebrafish inner ear during the larval stage, at which point the sensory organs become fully functional and remain accessible for in vivo imaging. We aim to identify hair cell precursors in the zebrafish inner ear and determine when and how they give rise to new hair cells during regeneration. In the inner ear of birds and mammals, support cells regenerate hair cells by proliferating and differentiating or by direct transdifferentiation. In the zebrafish lateral line, only the former mechanism is used. In Aim 1 of this proposal, we will use a novel method of hair cell ablation to determine the timecourse of regeneration and whether cell proliferation, transdifferentiation, or both are used in the zebrafish inner ear. Hair cells in the lateral line are regenerated primarily by a spatially and functionally distinct group of support cells. Whether a similar support cell subtype exists in the zebrafish inner ear is unknown. In Aim 2, we will validate putative inner ear support cell subtypes identified through single-cell RNA-seq analysis. We will then generate transgenic lines labeling support cell subtypes and use fate mapping to identify hair cell precursors. Together these experiments will characterize the timing and mechanism of hair cell regeneration and the diversity of support cell subtypes in the zebrafish inner ear. This work will help to establish the zebrafish inner ear as an outstanding in vivo model system for future in-depth studies of support cells and their roles in conserved mechanisms of regeneration. Expanding our knowledge of the mechanisms of inner ear hair cell regeneration in animals such as the zebrafish will ultimately lead to the advancement of treatments for auditory and vestibular decline in humans.