Single cell analysis of mitotic regeneration in the mouse vestibular system

项目概要（摘要） 感音神经性听力损失和前庭功能障碍是最常见的感觉障碍， 全球百万人1-3听觉和前庭功能需要机械敏感的毛细胞，伴有毛细胞损失 导致永久性听力损失和使人丧失能力的前庭功能障碍/机能减退。最近，新生儿 小鼠椭圆囊是依赖毛细胞检测线性加速度的五个前庭器官之一， 具有大量的祖细胞6，7。然而，虽然有丝分裂和非有丝分裂的存在， 哺乳动物中的机制现在很清楚，我们目前缺乏对时间，位置和机制的了解 细胞命运的决定。在其他系统中，如皮肤，众所周知，命运决定是在皮肤的下游做出的。 干细胞和他们的过境扩增人口，但我们还不知道这些假定的人口的命运 在内耳39.一个在许多器官中组织稳态和干细胞维持的中央调节器是 Wnt途径8，并且该信号级联在内耳12中被上调。我假设受伤后， 有丝分裂再生导致新生儿椭圆囊感觉上皮细胞的不同谱系， 激活引导更多的支持细胞采用有丝分裂细胞谱系。深入了解 损伤后驱动有丝分裂再生的事件顺序将揭示再生的潜在途径 毛细胞和支持细胞，最终目的是恢复听力和平衡功能。 作为一名外科医生兼科学家，我热衷于治疗听力和平衡障碍患者， 有能力解决上面提到的科学问题我对基础科学的兴趣源于我的 大学期间，他致力于体感皮层的遗传学和发育， 嗅觉系统在医学院期间，我看到永久性听力损失患者缺乏治疗， 一个机会，在著名科学家的指导下研究毛细胞再生， Heller和Roel Nusse博士（Jan等人，2013年，发展）。作为耳鼻喉科的住院医生，我专注于获得 临床和外科专业知识来治疗病人，并有机会继续基础科学研究 在康斯坦丁娜·斯坦科维奇医生的指导下专注于听力损失。为了获得先进的外科技术和学习 研究内耳再生的最先进技术，我完成了T32资助的临床医生科学家培训 耳科与神经耳科课程。这个程序进一步使我能够收集初步的数据和图表我的 作为加州大学旧金山分校的一名新教员，我为这项提案制定了目标。我在内耳生物学方面受过广泛的训练， 我的知识是缺乏先进的小鼠遗传学，新的单细胞RNAseq技术，和先进的 生物信息学在著名的干细胞医生-科学家Ophir Klein博士的指导下， 加州大学旧金山分校，和专家内耳外科医生，科学家，博士艾伦程作为我的共同导师在斯坦福大学，我有信心， 该奖项将通过R 01资助机制为我的科学独立做好准备。

PROJECT SUMMARY (ABSTRACT) Sensorineural hearing loss and vestibular dysfunction are most common sensory disorders affecting millions worldwide 1–3. Auditory and vestibular functions require mechanosensitive hair cells, with hair cell loss leading to permanent hearing loss and disabling vestibular dysfunction/hypofunction. Recently, the neonatal mouse utricle, one of five vestibular organs that relies on hair cells to detect linear acceleration, was shown to harbor robust numbers of progenitor cells 6,7. However, while the existence of both mitotic and non-mitotic mechanisms in mammals is now clear, we currently lack understanding of the timing, location, and mechanisms of cell fate decisions. In other systems, like the skin, it is known that fate decisions are made downstream of stem cells and their transit amplifying populations, but we do not yet know the fates of these putative populations in the inner ear 39. A central regulator of tissue homeostasis and stem cell maintenance across many organs is the Wnt pathway 8, and this signaling cascade is upregulated in the inner ear 12. I hypothesize that following injury, mitotic regeneration leads to different cell lineages in the neonatal utricular sensory epithelium, and that Wnt activation directs more supporting cells to adopt the mitotic cell lineage. Gaining an in-depth understanding of the sequence of events that drive mitotic regeneration post injury will reveal potential approaches to regenerate hair cells and supporting cells, with the ultimate goal of restoring hearing and balance functions. As a surgeon-scientist with a passion for treating patients with hearing and balance disorders, I am well equipped to tackle the scientific questions outlined. My interests in the basic sciences stem from my undergraduate years working on the genetics and development of the somatosensory cortex and studying the olfactory system. During medical school, I saw the lack of therapies of patients with permanent hearing loss as an opportunity, working on hair cell regeneration under the tutelage of renowned scientists, including Dr. Stefan Heller and Dr. Roel Nusse (Jan et al., 2013, Development). As a resident in otolaryngology, I focused on gaining the clinical and surgical expertise to treat patients and had the opportunity to continue basic science research with a focus on hearing loss under Dr. Konstantina Stankovic. In order to gain advanced surgical skills and learn state of the art techniques to study inner ear regeneration, I completed the T32 funded Clinician Scientist Training Program in Otology & Neurotology. This program further allowed me to collect preliminary data and chart my goals for this proposal as a new faculty member at UCSF. While I have extensive training in inner ear biology, my knowledge is lacking in advanced mouse genetics, new single cell RNAseq technologies, and advanced bioinformatics. Under the guidance of renowned stem cell physician-scientist Dr. Ophir Klein as my mentor at UCSF, and expert inner ear surgeon-scientist, Dr. Alan Cheng as my co-mentor at Stanford, I am confident this award will prepare me for scientific independence through the R01 grant mechanism.