A genetic program for organ regeneration in Zebrafish

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听觉系统进行听觉。 将空气振动转换为声音，以及检测重力和提供平衡。一旦它们被损坏或杀死， 感觉毛细胞不能再生，导致永久性听力丧失和前庭功能障碍。尽管研究工作， 哺乳动物毛细胞的再生是有限的，听力和前庭功能从未恢复。因此 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， 这种需要。哺乳动物和斑马鱼的感觉毛细胞在功能和遗传上是同源的， 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生物，它们 在突变和转基因的情况下，毛细胞在遗传上易于处理，并且毛细胞的快速再生对于进行测定是理想的。 已知许多基因影响斑马鱼毛细胞再生，但它们如何在基因调控中相互作用， 网络（GRN）仍然未知。我相信，了解全球表观遗传景观和复杂的遗传 基因间的相互调节对毛细胞再生的进展至关重要。我的目标是 描述驱动毛细胞再生的遗传和调节相互作用的GRN，并通过以下方式测试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造成的， 毛细胞GRN的变化。该项目将定义驱动毛细胞再生的调控相互作用， 斑马鱼，并确定特定的基因和GRN的组成部分，这是必不可少的这一过程作为目标， 哺乳动物研究我将在一个很好的时间尺度上描述使用ChIP-seq和ATAC-seq的全球监管格局 在毛细胞再生和体内平衡的过程中。然后，我将进行生物信息学分析，以建立一个GRN整合 ATAC-seq和ChIP-seq与scRNA-seq数据。我将寻找与转录因子结合基序相关的富集的转录因子结合基序， 基因在毛细胞再生过程中发生变化，并利用这些丰富的基序来绘制毛细胞之间的遗传联系。 转录因子和靶基因。最后，我将通过突变基因和增强子来测试GRN连接， 再生，并测定表型以确定驱动该过程的关键基因。我会克隆一个增强器 将小鼠的基因植入斑马鱼，以确定两个物种之间GRN的变化，并试图恢复 通过将转录因子结合回小鼠增强子来连接。我严格的博士训练让我准备好 开展这项研究项目，并扩大我以前的经验，测试GRNs和进行诱变， 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， ChIP-seq数据，用它来寻找新的GRN连接，并通过识别和突变来证明再生表型。 关键再生基因通过本研究，希望能对毛细胞的研究做出重要贡献 再生，提供新的基因和调控目标，用于哺乳动物研究，并最终治疗 干预措施。

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.