A genetic program for organ regeneration in Zebrafish

斑马鱼器官再生的遗传程序

• 批准号: 10473893
• 负责人: Jeremy Sandler
• 金额: $ 7.42万
• 依托单位: STOWERS INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-28 至 2023-09-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10473893
• 关键词: ATAC-seq; Address; Air; Automobile Driving; Back; Binding; Binding Sites; Bioinformatics; Biological Assay; Biological Models; Cells; Cessation of life; ChIP-seq; Chromatin; Chromatin Structure; Cochlea; Complex; Computer Analysis; Core Facility; Data; Detection; Development; Doctor of Philosophy; Ectopic Expression; Enhancers; Environment; Epigenetic Process; Equilibrium; Event; Evolution; Fishes; Force of Gravity; Gene Expression; Gene Transfer Techniques; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Goals; Hair Cells; Hearing; Heart; Histones; Homeostasis; Hour; Human; Individual; Kidney; Knowledge; Labyrinth; Learning; Limb structure; Link; Mammals; Maps; Modeling; Movement; Mus; Mutagenesis; Mutate; Mutation; Natural regeneration; Nerve; Nervous system structure; Organ; Organism; Orthologous Gene; Phenotype; Problem Solving; Process; Quality of life; Regenerative capacity; Regenerative research; Regulatory Element; Research; Research Project Grants; Resolution; Role; Sensory Hair; Signal Pathway; Signal Transduction; Space Perception; Surface; System; Testing; Therapeutic Intervention; Time; Tissues; Training; Vertebrates; Vestibular Hair Cells; Water; Zebrafish; cell injury; cell type; cellular transduction; chromatin remodeling; differential expression; experience; experimental study; functional restoration; gene network; gene regulatory network; genetic approach; hair cell regeneration; hearing restoration; improved; lateral line; member; neuromast; organ regeneration; permanent hearing loss; programs; response; sensory system; single-cell RNA sequencing; sound; spatiotemporal; temporal measurement; time use; tool; transcription factor; vibration

Project Summary Sensory hair cells in the mammalian inner ear and vestibular system are respectively responsible for hearing through the transduction of air vibrations to sound, and the detection of gravity and providing balance. Once they are damaged or killed, sensory hair cells do not regenerate, leading to permanent hearing loss and vestibular disfunction. Despite research efforts, regeneration of mammalian hair cells is limited, and hearing and vestibular function have never been restored. Thus, there is a pressing need to further investigate hair cell regeneration. We have identified zebrafish as an excellent model to address this need. Sensory hair cells of mammals and zebrafish are functionally and genetically homologous, and hair cells in zebrafish rapidly regenerate and exist in a state of constant turnover. Zebrafish are an established research organism, they are genetically tractable with mutation and transgenesis, and rapid regeneration of hair cells is ideal for performing assays. A number of genes are known to influence hair cell regeneration in zebrafish, but how they interact in a gene regulatory network (GRN) is still unknown. I believe that understanding the global epigenetic landscape and complex genetic regulatory interactions between genes is essential to making progress on hair cell regeneration. My objective is to assemble a GRN describing the genetic and regulatory interactions that drive hair cell regeneration, and to test the GRN by manipulating individual components. I hypothesize that the loss of regeneration in mammals is caused by evolutionary changes to the hair cell GRN. This project will define the regulatory interactions driving hair cell regeneration in zebrafish and identify specific genes and components of the GRN that are essential for this process as targets for mammalian studies. I will characterize the global regulatory landscape using ChIP-seq and ATAC-seq in a fine time scale during hair cell regeneration and in homeostasis. I will then perform a bioinformatic analysis to build a GRN integrating ATAC-seq and ChIP-seq with scRNA-seq data. I will search for enriched transcription factor binding motifs associated with genes changing during hair cell regeneration, and use these enriched motifs to draw genetic connections between transcription factors and target genes. Lastly, I will test GRN connections by mutating genes and enhancers involved in regeneration, and assay phenotypes to determine the key genes driving the process. I will clone enhancers of orthologous genes from mice into zebrafish to determine where the GRN has changed between the two species, and attempt to restore connections by adding transcription factor binding back to mouse enhancers. My rigorous PhD training has prepared me to carry out this research project and expand upon my previous experience testing GRNs and carrying out mutagenesis and phenotypic assays. With the support of core facilities, I have already generated high quality preliminary ATAC-seq and ChIP-seq data, used it to find new GRN connections, and demonstrated regeneration phenotypes by identifying and mutating key regeneration genes. Through this research, I expect to make significant contributions towards understanding hair cell regeneration, providing new gene and regulatory targets to be used in mammalian studies and eventually therapeutic interventions.

项目摘要 哺乳动物内耳和前庭系统中的感觉毛细胞分别负责通过 将空气振动转换为声音，检测重力并提供平衡。一旦它们被损坏或被杀死， 感觉毛细胞不能再生，导致永久性听力损失和前庭功能障碍。尽管做出了研究努力， 哺乳动物毛细胞的再生有限，听力和前庭功能从未恢复。因此，在那里 是进一步研究毛细胞再生的迫切需要。我们已经确定斑马鱼是一个很好的模型来解决 这种需要。哺乳动物和斑马鱼的感觉毛细胞在功能和基因上是同源的，而在 斑马鱼迅速再生，并以不断更替的状态存在。斑马鱼是一种成熟的研究有机体，它们 毛细胞在基因上易于突变和转基因，毛细胞的快速再生是进行分析的理想选择。 已知有许多基因影响斑马鱼毛细胞的再生，但它们如何在基因调控中相互作用 网络(GRN)仍未知。我认为，理解全球表观遗传格局和复杂的基因 基因之间的调节相互作用对于毛细胞再生方面的进展至关重要。我的目标是组装 GRN描述了驱动毛细胞再生的遗传和调控相互作用，并通过以下方法测试GRN 操纵单个组件。我假设哺乳动物再生能力的丧失是由进化引起的 毛细胞GRN的更改。该项目将定义推动毛细胞再生的调控相互作用 斑马鱼，并确定在这一过程中必不可少的GRN的特定基因和组件作为目标 哺乳动物研究。我将在一个很好的时间尺度上，使用CHIP-SEQ和ATAC-SEQ来描述全球监管格局 在毛细胞再生和动态平衡期间。然后我将执行生物信息学分析，以建立一个GRN集成 具有scRNA-seq数据的ATAC-SEQ和CHIP-SEQ。我将寻找与以下相关的丰富的转录因子结合基序 基因在毛细胞再生过程中的变化，并使用这些丰富的基序来绘制 转录因子和靶基因。最后，我将通过突变相关基因和增强子来测试GRN连接 再生，并分析表型以确定驱动这一过程的关键基因。我将克隆同源基因的增强子 从老鼠到斑马鱼的基因，以确定GRN在这两个物种之间的变化，并试图恢复 通过将转录因子结合回小鼠增强剂来连接。我严格的博士训练使我做好了准备 开展这一研究项目，并扩展我以前测试GRN和进行突变和 表型分析。在核心设施的支持下，我已经生成了高质量的初步ATAC-SEQ和 ChIP-SEQ数据，用它来寻找新的GRN连接，并通过识别和突变来展示再生表型 关键的再生基因。通过这项研究，我希望对了解毛细胞做出重大贡献 再生，提供新的基因和调控靶点，用于哺乳动物研究并最终用于治疗 干预措施。