Mouse vestibular regeneration and function

摘要： 感觉毛细胞需要平衡功能。前庭毛细胞退化导致平衡 功能障碍/功能减退，表现为头晕和眩晕。虽然哺乳动物的耳蜗缺乏 再生失去的毛细胞，有限程度的自发再生发生在椭圆囊，前庭器官 检测线性加速度。最近的研究使用命运映射技术已经确定了支持细胞， 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全的功能 以及器官功能是否恢复在初步实验中，我们已经描述了毛细胞变性的特征， 在成熟的小鼠椭圆囊的再生，也是前庭诱发电位的损失后恢复 （VsEP）。这项建议的第一个目的是确定增加毛细胞再生是否能改善毛细胞的生长。 恢复前庭功能。具体地，通过命运映射标记的再生毛细胞通过 组织学和电生理学，以评估束形态、机械敏感性、基底外侧电流和 突触特性，包括囊泡释放。同时，测量VsEP响应并与 组织学和电生理学测量。接下来，通过转基因方法过表达Atoh 1，我们将 研究Atoh 1过表达毛细胞的组织学和电生理学以及整体VsEP 应答在第二个目标中，我们将确定Atoh 1缺失是否会阻止毛细胞再生和恢复。 的VsEP响应。与此同时，将通过组织学检查命运映射的存活毛细胞的可能修复 和电生理学。为了公正地了解毛细胞祖细胞的遗传特征， 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。在这里，第一个目标是发现毛细胞祖细胞的遗传景观， 受损胞果中残存的毛细胞其次，我们将检查未受损的基因表达， Atoh 1过表达后胞囊受损。最后，我们将使用生物信息学方法来描述 自发和Atoh 1增强的支持细胞-毛细胞转变的轨迹，并验证了这一点 组织学上总之，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）研究转基因小鼠中前庭毛细胞再生 模型我们已经组建了一个专家小组，他们共同努力收集有希望的初步数据。 在这5年的提案结束时，我们将1）确定毛细胞再生之间的关系 和功能恢复和2）揭示和时间排序的新基因在哺乳动物毛细胞 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.