Genomic approaches to inner ear hair cell regeneration

内耳毛细胞再生的基因组方法

• 批准号: 7933799
• 负责人: Michael Lovett
• 金额: $ 49.86万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-17 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7933799
• 关键词: Ablation; Affect; Aminoglycosides; Antibiotic Therapy; Antibiotics; Antibodies; Attention; Biological Models; Birds; Birth; Cells; Chickens; Code; Complementary DNA; DNA Sequence; Data; Data Set; Development; EP300 gene; Elements; Enhancers; Epithelium; Equilibrium; Funding Mechanisms; Gene Expression; Gene Expression Profile; Genes; Genetic Enhancer Element; Genome; Genomics; Goals; Hair Cells; Hearing; Hour; Human; Internet; Laboratories; Labyrinth; Lead; Mammals; Maps; Measurement; Measures; Messenger RNA; Methods; MicroRNAs; Molecular Genetics; Mus; Natural regeneration; Pathway Analysis; Pathway interactions; Population; Process; Proteins; Regulatory Element; Research; Research Personnel; Sampling; Sensory; Staging; Supporting Cell; Time; Tissue Banks; Tissues; Transcription factor genes; Utricle structure; Vertebrates; cell growth; comparative; cost effective; deafness; digital; equilibration disorder; experience; gamma secretase; hair cell regeneration; hearing impairment; inhibitor/antagonist; insight; killings; next generation; postnatal; programs; public health relevance; regenerative; tool

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.

描述（由申请人提供）：对内耳毛细胞的损害是人类耳聋和平衡疾病的主要原因，并影响了世界人口的10％。哺乳动物无法再生其毛细胞。但是，鸟类（和其他下脊椎动物）可以再生这些细胞。这是三位研究人员的协作提议，他们一直在研究内耳毛细胞并将基因组方法应用于其分析中。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，我们首先需要确定将导致感觉再生的分子遗传途径。到目前为止，我们对这些过程的见解受到基因组工具的限制，这些工具要么不具有成本效益或不全面。随着高吞吐量下一代DNA测序方法的发展，我们现在可以在毛细胞再生过程中得出转录组的全面数字图。我们建议使用这些方法对鸟类和小鼠中内耳感觉上皮基因表达进行全面分析。我们将研究三个模型系统：（1）鸟类毛细胞再生的完整转录组图（mRNA和微RNA）。除此之外，我们还将使用ChIP-Seq方法和与增强子相关的辅助因子p300的抗体获取大多数鸟类特异性增强子元件的图。通过将这三组数据相关联，我们期望获得有关哪些基因，microRNA和哪些远距离控制元件在鸟类毛细胞再生过程中具有活性和重要的基因。 （2）小鼠乌特莱斯出生时的增殖能力有限，但随后在出生后约两周失去了这种能力。我们将通过比较出生时（P0）和P16时的小鼠尿素（在P16）以及在这两个时间点对毛细胞的抗生素杀死后，通过比较小鼠的尿素（P0）来探索这个过程（再次通过mRNA和Micro-RNA下一代测序）。我们希望观察到差异，这些差异应该为我们提供有关这两个时间点的增殖能力如何不同的见解。 （3）用伽马泌尿酶抑制剂DAPT处理的P0小鼠尿素经历了支持细胞对毛细胞的超转换，但这种能力在p12和p16之间丧失。在我们的第三个目标中，我们将在DAPT治疗时间过程中得出全面的mRNA和微RNA谱，并特别关注P12和P16之间的周期，而增殖能力降低。这些研究中得出的所有数据集将通过独立方法验证，所有数据都将在万维网上访问。此外，我们预计将在数据中添加相当多的注释，途径和网络分析，以帮助他人进行解释和效用。该项目将提供有关鸡肉和小鼠毛细胞开发/再生的三个非常详细且重叠的信息集。在该项目结束时，我们将知道在各种治疗下在鸟类毛细胞再生和早期产后小鼠阶段表达的所有蛋白质编码基因和微RNA。我们的小组在所有提议的方法中都有经验（并且已经储存了许多样本），并且我们的目标很容易在这种资金机制的两年时间内实现。 公共卫生相关性：内耳毛细胞的损害是不可逆转的人类听力损失和平衡疾病的主要原因，这影响了世界人口> 10％的人口。哺乳动物无法再生其毛细胞，但是，鸟类（和其他下脊椎动物）可以再生这些细胞并恢复听力和平衡功能。我们研究的长期目标是通过研究鸟类的再生过程并将其与哺乳动物的毛细胞生长进行比较，开发用于再生和维持内耳毛细胞的方法。