Atoh7 cis regulation and gene regulatory network analysis during retinal ganglion cell development

视网膜神经节细胞发育过程中Atoh7顺式调控及基因调控网络分析

• 批准号: 10480882
• 负责人: Joel B Miesfeld
• 金额: $ 24.9万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10480882
• 关键词: ATAC-seq; ATOH7 gene; Adult; Alleles; Architecture; BHLH Protein; Bacterial Artificial Chromosomes; Binding; Binding Sites; Bioinformatics; Biological Assay; Biological Models; Blindness; Brain; CRISPR screen; California; Cell Differentiation process; Cell Transplantation; Cell division; Cells; Chromatin; Chromatin Structure; Community Developments; Competence; Complex; DNA; DNA Sequence; Data; Data Analyses; Development; Disease; Elements; Enhancers; Environment; Eye; Flow Cytometry; Generations; Genetic Enhancer Element; Genetic Models; Genetic Transcription; Glaucoma; Goals; Human; Image; In Vitro; Individual; KDM1A gene; Laboratory mice; Learning; Lysine; Mentors; Messenger RNA; Methods; Mitotic; Modeling; Muller' s cell; Multipotent Stem Cells; Mus; Neuroglia; Neurons; Nucleic Acid Regulatory Sequences; Optic Nerve; Organoids; Pathogenicity; Pathway Analysis; Patients; Pattern; Population; Regulation; Regulatory Element; Reporter; Repression; Research; Research Personnel; Retina; Retinal Ganglion Cells; Role; Scientist; Solid; Stereotyping; Structure; System; Techniques; Testing; Therapeutic; Time; Tissues; Tracer; Transgenes; Transgenic Mice; Transgenic Organisms; Universities; Untranslated RNA; Vision; Work; Xenopus; Zebrafish; career development; cell fate specification; cell type; chromatin immunoprecipitation; cofactor; competence factor; gene regulatory network; gene repression; genetic corepressor; histogenesis; improved; induced pluripotent stem cell; malformation; mutant; neurogenesis; notch protein; prevent; progenitor; promoter; relating to nervous system; research and development; retinal progenitor cell; stem cells; success; tool; transcription factor; transcriptome; transcriptome sequencing

Project Summary/Abstract Retinal ganglion cells (RGCs) connect the eyes to the brain. They are essential for vertebrate vision and pathogenic targets in glaucoma. One therapeutic goal of vision scientists is to fully understand the factors required for RGC development, so these cells can be generated in vitro. The proneural basic helix-loop-helix (bHLH) protein ATOH7 is expressed transiently in a subpopulation of early retinal progenitor cells, which give rise to the 7 major cell types of the retina but is only essential as a competence factor for RGC genesis. Loss of ATOH7 causes optic nerve aplasia and severe secondary retinovascular malformations. Cre-lox lineage data show only 55% of RGCs descend from Atoh7+ progenitors. What factors control genesis of the other 45% of RGCs? Why do only some Atoh7+ cells become RGCs? In humans with nonsyndromic congenital retinal nonattachment (NCRNA), a remote 5’ conserved enhancer for ATOH7 is deleted, preventing development of RGCs and leading to total blindness. This DNA segment is obviously vital, but its exact role is unknown. In transgene reporter mice, this ‘shadow’ enhancer (SE) appears to be wholly redundant with the ‘primary’ (promoter-adjacent) enhancer (PE), despite is requirement in human NCRNA. In preliminary studies, we observed that Atoh7 SE deletion mice retain optic nerves. How do these dual enhancer elements coordinately regulate the rapid onset and offset of Atoh7 expression? Here, we propose to investigate functional differences between the human NCRNA and mouse SE deletion, to determine how specific DNA sequences control the level, timing and pattern of ATOH7 expression, to analyze ATOH7 transcriptional repression, and to identify cofactors influencing ATOH7+ cell fate decisions during RGC genesis. First, we will apply a multi-species approach to test the necessity and sufficiency of each ATOH7 regulatory element and determine precisely how each component contributes to the dynamic tissue and cellular expression pattern. Second, we will investigate mechanisms of ATOH7 transcriptional repression via Notch effector RPBJ and Kdm1a, using a high-throughput zebrafish screen, transgenic reporters and RNAseq. Third, we will use single-cell and pooled ATACseq and RNAseq methods to profile retinal progenitors in detail as they progress through stages of Atoh7 expression. These data will illuminate mechanisms controlling ATOH7 transcription, the onset of retinal neurogenesis and RGC fate specification; the action of binary enhancers generally; and the potential generation of RGCs in vitro for cell transplantation. My work toward these goals will be aided by the strong research and career development community at the University of California, Davis and my established team of mentors. Together, the proposed research and environment will provide a solid platform for my continued career development as a vision scientist – learning new techniques and model systems, and interacting with a wide variety of scientists (short term goals), which will pave the way for me to become an independent academic researcher probing gene regulatory networks that control ATOH7, RGC fate and retinal histogenesis (long term goals).

项目总结/摘要 视网膜神经节细胞（RGC）连接眼睛和大脑。它们对脊椎动物的视觉和致病靶点至关重要 在青光眼中。视觉科学家的一个治疗目标是充分了解RGC发展所需的因素， 这些细胞可以在体外产生。前神经碱性螺旋-环-螺旋（bHLH）蛋白ATOH 7瞬时表达 在早期视网膜祖细胞的亚群中，其产生视网膜的7种主要细胞类型，但仅 是研资局成立的重要能力因素。ATOH 7的缺失导致视神经发育不全和严重的继发性 视网膜血管畸形Cre-lox谱系数据显示仅55%的RGC来自Atoh 7+祖细胞。什么 其他45%的RGCs的发生是由哪些因素控制的？为什么只有一些Atoh 7+细胞成为RGC？在人类 非综合征性先天性视网膜不附着（NCRNA），ATOH 7的远程5'保守增强子缺失， 阻止RGC的发展并导致完全失明。这个DNA片段显然至关重要，但它的确切作用是 未知在转基因报告小鼠中，这种“影子”增强子（SE）似乎与“初级”增强子完全多余。 （启动子-邻近）增强子（PE），尽管在人NCRNA中需要。在初步研究中，我们观察到， Atoh 7 SE缺失小鼠保留视神经。这些双重增强子元件是如何协调调节快速起效的 和Atoh 7表达的偏移量？在这里，我们建议研究人类NCRNA和 小鼠SE缺失，以确定特定DNA序列如何控制ATOH 7表达的水平、时间和模式， 分析ATOH 7转录抑制，并鉴定影响ATOH 7+细胞命运决定的辅因子， RGC起源。首先，我们将应用多物种的方法来测试每个ATOH 7的必要性和充分性 调节元件，并精确地确定每个组件如何有助于动态组织和细胞表达 格局其次，我们将研究ATOH 7通过Notch效应子RPBJ转录抑制的机制， Kdm 1a，使用高通量斑马鱼筛选，转基因报告基因和RNAseq。第三，我们将使用单细胞和 合并ATACseq和RNAseq方法，以详细描述视网膜祖细胞在Atoh 7阶段的进展 表情这些数据将阐明控制ATOH 7转录、视网膜神经发生的发生和 RGC命运特化;二元增强子的作用一般;以及RGC在体外对细胞的潜在产生。 移植我对这些目标的工作将得到强大的研究和职业发展社区的帮助， 加州大学戴维斯分校和我的导师团队。总之，拟议的研究和环境 作为一名视觉科学家，我将为我的职业发展提供一个坚实的平台-学习新技术， 模型系统，并与各种科学家（短期目标）互动，这将为我铺平道路， 成为一名独立的学术研究人员，探索控制ATOH 7，RGC命运和视网膜的基因调控网络 组织发生（长期目标）。