Genomic approaches to inner ear hair cell regeneration

描述（由申请人提供）：内耳毛细胞损伤是人类耳聋和平衡障碍的主要原因，影响着世界上10%的人口。哺乳动物的毛细胞不能再生。然而，鸟类（和其他低等脊椎动物）可以再生这些细胞。这是三位研究人员的合作建议，他们是研究内耳毛细胞并将基因组方法应用于分析的先驱。我们研究的长期目标是开发再生和维持内耳毛细胞的方法。为了实现这一目标，我们首先需要确定导致感觉再生的分子遗传途径。到目前为止，我们对这些过程的了解受到基因组工具的限制，这些工具要么不具有成本效益，要么不全面。随着高通量下一代DNA测序方法的发展，我们现在可以获得毛细胞再生过程中转录组的全面数字图谱。我们拟利用这些方法对鸟类和小鼠内耳感觉上皮基因表达进行综合分析。我们将研究三个模型系统：(1)禽小囊毛细胞再生的完整转录组图谱（mRNA和微rna）。除此之外，我们将使用ChIP-Seq方法获得大多数禽类细胞特异性增强子元件的图谱和增强子相关辅助因子p300的抗体。通过将这三组数据相关联，我们希望能够深入了解哪些基因、哪些microrna和哪些远程控制元件在禽类毛细胞再生过程中是活跃和重要的。(2)小鼠胞囊在出生时具有有限的增殖能力，但在出生后大约两周就失去了这种能力。我们将通过比较出生时（P0）和P16时以及抗生素杀死毛细胞后的小鼠胞室来探索这一过程（再次通过mRNA和微rna Next Gen测序）。我们期望观察到的差异将使我们深入了解这两个时间点在其扩散能力方面的差异。(3)经γ分泌酶抑制剂DAPT处理的P0小鼠胞囊支持细胞向毛细胞的超转化，但这种能力在P12和P16之间丢失。在我们的第三个目标中，我们将在DAPT治疗过程中获得全面的mRNA和微rna谱，特别关注P12和P16之间增殖能力下降的时期。从这些研究中获得的所有数据集都将通过独立的方法进行验证，所有数据都将在万维网上可访问。此外，我们期望在数据中添加相当程度的注释、路径和网络分析，以帮助其他人进行解释和使用。该项目将提供关于鸡和小鼠毛细胞发育/再生的三组非常详细和重叠的信息。在本项目结束时，我们将了解在不同处理下鸟类毛细胞再生和小鼠出生后早期阶段表达的所有蛋白质编码基因和微rna。我们的团队对所有提出的方法都很有经验（并且已经储存了许多样本），我们的目标在这个资助机制的两年时间框架内很容易实现。

DESCRIPTION (provided by applicant): Damage to inner ear hair cells is a leading cause of human deafness and balance disorders and affects >10% of the world's population. Mammals cannot regenerate their hair cells. However, birds (and other lower vertebrates) can regenerate these cells. This is a collaborative proposal from three investigators who have been pioneers in studying inner ear hair cells and applying genomic methods to their analysis. The long term goal of our research is to develop methods to regenerate and maintain inner ear hair cells. In order to achieve this goal, we first need to identify the molecular genetic pathways that will lead to sensory regeneration. Until now our insights into these processes were limited by genomic tools that were either not cost effective or were not comprehensive. With the development of high throughput Next Generation DNA sequencing methods we can now derive comprehensive digital maps of the transcriptome during the process of hair cell regeneration. We propose to use these methods to conduct a comprehensive analysis of inner ear sensory epithelia gene expression in birds and mice. We shall investigate three model systems: (1) A complete transcriptome map (mRNA and micro-RNA) of avian utricle hair cell regeneration. In addition to this we will derive a map of the majority of avian utricle-specific enhancer elements using ChIP-Seq methods and an antibody to the enhancer-associated co-factor p300. By correlating these three sets of data we expect to gain insights into which genes, which microRNAs and which long range control elements are active and important during avian hair cell regeneration. (2) The mouse utricle has a limited proliferative capacity at birth, but then loses this capacity by about two weeks after birth. We shall explore this process (again by mRNA and micro-RNA Next Gen sequencing) by comparing mouse utricles at birth (P0) and at P16 and after antibiotic killing of the hair cells at both these time points. We expect to observe differences that should provide us with insights into how these two time points differ in their proliferative capacity. (3) P0 mouse utricles treated with the gamma secretase inhibitor DAPT undergo a hyper-conversion of supporting cells to hair cells, but this capacity is lost between P12 and P16. In our third aim we will derive comprehensive mRNA and micro-RNA profiles across a DAPT treatment time course paying particular attention to the period between P12 and P16 when proliferative capacity decreases. All of the data sets derived in these studies will be validated by independent methods and all of the data will be accessible on the World Wide Web. In addition we anticipate adding a considerable degree of annotation, pathway and network analysis to the data to aid others in interpretation and utility. This project will provide three exceptionally detailed and overlapping sets of information on the development/regeneration of chicken and mouse hair cells. At the end of this project we will know all of the protein coding genes and micro-RNAs that are expressed during avian hair cell regeneration and early postnatal mouse stages under various treatments. Our groups are experienced in all of the proposed methods (and have already stockpiled many of the samples) and our goals are readily achievable within the two year time frame of this funding mechanism. PUBLIC HEALTH RELEVANCE: Damage to inner ear hair cells is a leading cause of irreversible human hearing loss and balance disorders, which affect >10% of the world's population. Mammals cannot regenerate their hair cells, however, birds (and other lower vertebrates) can regenerate these cells and recover hearing and balance functions. The long-term goal of our research is to develop methods to regenerate and maintain inner ear hair cells by studying the regenerative process in birds and comparing it to mammalian hair cell growth.