Mouse vestibular regeneration and function

摘要： 感觉毛细胞是平衡功能所必需的。前庭毛细胞变性导致平衡 功能障碍/功能减退表现为头晕和眩晕。而哺乳动物的耳蜗却没有能力 再生失去的毛细胞，有限程度的自发再生发生在前庭器官--椭圆囊 检测到线加速。最近使用命运映射技术的研究已经准确地将支持细胞定位为 再生毛细胞的前体。然而，目前尚不清楚再生的毛细胞是否具有完整的功能 如果器官功能恢复的话。在初步实验中，我们已经确定了毛细胞退化和 小鼠成熟椭圆囊的再生和前庭诱发电位恢复后的丢失 (VsEP)在体内。这项建议的第一个目标是确定增加毛细胞再生是否会改善 前庭功能恢复。具体地说，通过命运映射标记的再生毛细胞通过 组织学和电生理学评估束形态、机械敏感性、基底侧向电流和 突触特性包括囊泡释放。同时，测量VsEP响应并将其与 组织学和电生理检查。下一步，通过转基因方法过量表达Atoh1，我们将 Atoh1过表达毛细胞的组织学和电生理学研究及VsEP 回应。在第二个目标中，我们将确定Atoh1基因缺失是否会阻止毛细胞再生和恢复 VsEP反应的比例。同时，命运映射的存活毛细胞将通过组织学进行可能的修复检查 和电生理学。为了不偏不倚地了解毛细胞祖细胞和幸存者的基因特征 毛细胞，第三个目的是用单细胞RNA检测受损的成熟小鼠椭圆囊 测序技术。这里的第一个目标是发现毛细胞祖细胞和 在受损的椭圆囊中存活的毛细胞。其次，我们将检测未受损的基因表达 Atoh1过表达后损伤椭圆囊。最后，我们将使用生物信息学方法来描绘 自发的和Atoh1增强的支持细胞-毛细胞转变的轨迹并验证这一点 组织学上。总而言之，我们将应用最先进的技术(前庭生理学，毛细胞生理学， 单细胞RNA-SEQ，生物信息学策略)研究转基因小鼠的前庭毛细胞再生 模特们。我们已经组建了一个专家团队，他们共同努力收集有希望的初步数据。 在这个为期5年的计划结束时，我们将1)确定毛细胞再生之间的关系 和功能恢复以及2)在哺乳动物毛细胞中发现并在时间上排序的新基因 再生。

Abstract: Sensory hair cells are required for balance function. Vestibular hair cell degeneration causes balance dysfunction/hypofunction manifested as dizziness and vertigo. While the mammalian cochlea lacks the ability to regenerate lost hair cells, a limited degree of spontaneous regeneration occurs in the utricle, a vestibular organ detecting linear acceleration. Recent studies using fate-mapping techniques have pinpointed supporting cells as precursors of regenerated hair cells. However, it is not clear whether regenerated hair cells are fully functional and if organ function recovers. In preliminary experiments we have characterized hair cell degeneration and regeneration in the mature mouse utricle and also a loss followed by recovery of vestibular evoked potentials (VsEP) in vivo. The first aim of this proposal is to determine if increasing hair cell regeneration improves the recovery of vestibular function. Specifically, regenerated hair cells labeled via fate-mapping are probed via histology and electrophysiology to assess bundle morphology, mechanosensitvity, basolateral currents, and synaptic properties including vesicle release. In parallel, VsEP responses are measured and compared to histologic and electrophysiological measures. Next, by overexpressing Atoh1 via a transgenic approach, we will study the histology and electrophysiology of Atoh1-overexpressing hair cells and also the overall VsEP responses. In the second aim, we will determine if Atoh1 deletion prevents hair cell regeneration and the recovery of VsEP responses. In parallel, fate-mapped, surviving hair cells will be examined for possible repair via histology and electrophysiology. To gain an unbiased insight into the genetic signature of hair cell progenitors and surviving hair cells, the third aim is designed to examine the damaged mature mouse utricle using single cell RNA sequencing technologies. Here the first goal is to discover the genetic landscape of hair cell progenitors and surviving hair cells in the damaged utricle. Secondly, we will examine the gene expression of the undamaged and damaged utricle after Atoh1 overexpression. Lastly, we will use bioinformatic approaches to delineate the trajectory of the spontaneous and Atoh1-enhaced supporting cell-hair cell transition and validate this histologically. In summary, we will apply state-of-the art technologies (vestibular physiology, hair cell physiology, single cell RNA-seq, bioinformatic strategies) to study vestibular hair cell regeneration in transgenic mouse models. We have assembled a team of experts who have worked together to collect promising preliminary data. At the end of this 5-year proposal, we will have 1) determined the relationship between hair cell regeneration and functional recovery and 2) revealed and temporally ordered novel genes during mammalian hair cell regeneration.