Single cell analysis of mitotic regeneration in the mouse vestibular system

项目摘要(摘要) 感觉神经性听力损失和前庭功能障碍是最常见的影响 全球数以百万计的人1-3.听觉和前庭功能需要机械敏感的毛细胞，毛细胞丢失 导致永久性听力损失和前庭功能障碍/功能减退。最近，这位新生儿 小鼠椭圆体是五个前庭器官之一，依靠毛细胞来检测线性加速度，研究表明 6，7.然而，尽管有丝分裂和非有丝分裂细胞的存在 哺乳动物的机制现在已经清楚，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，我们知道命运的决定是在 干细胞及其转运放大种群，但我们还不知道这些假定种群的命运 在内耳39。在许多器官中，组织动态平衡和干细胞维持的一个中心调节器是 Wnt通路8，这个信号级联在内耳12中上调。我假设在损伤之后， 有丝分裂再生导致新生儿椭圆体感觉上皮不同的细胞谱系，WNT 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一系列事件将揭示潜在的再生途径 毛细胞和支持细胞，最终目标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，我很好 有能力解决概述的科学问题。我对基础科学的兴趣源于我的 本科期间致力于体感皮质的遗传和发育，并研究 嗅觉系统。在医学院期间，我认为永久性听力损失患者缺乏治疗是 在包括斯特凡博士在内的著名科学家的指导下，研究毛细胞再生的机会 Heller和Roel Nusse博士(Jan等人，2013年，发展)。作为耳鼻喉科的一名住院医生，我专注于 临床和外科专业知识治疗患者，并有机会继续基础科学研究 在康斯坦蒂娜·斯坦科维奇博士的领导下，重点关注听力损失。为了获得先进的外科技能和学习 为了研究最先进的内耳再生技术，我完成了T32资助的临床医生科学家培训 耳科学与神经病学课程。这一程序进一步允许我收集初步数据并绘制我的 作为加州大学旧金山分校的一名新教员，这项提案的目标。虽然我在内耳生物学方面有广泛的训练， 我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。在著名干细胞医生兼科学家Ophir Klein博士的指导下，我在 加州大学旧金山分校，内耳外科专家兼科学家郑家纯博士作为我在斯坦福的共同导师，我相信这一点 该奖项将通过R01奖励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.