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).

项目概要/摘要 视网膜神经节细胞（RGC）将眼睛与大脑连接起来，它们对于脊椎动物的视力和致病目标至关重要。 视觉科学家的治疗目标之一是充分了解 RGC 发育所需的因素，因此 这些细胞可以在体外产生原神经碱性螺旋-环-螺旋 (bHLH) 蛋白 ATOH7。 在早期视网膜祖细胞的亚群中，它产生视网膜的 7 种主要细胞类型，但仅 ATOH7 的缺失会导致视神经发育不全和严重的继发性病变。 Cre-lox 谱系数据显示，仅 55% 的 RGC 源自 Atoh7+ 祖细胞。 控制其他 45% RGC 发生的因素 为什么只有一些 Atoh7+ 细胞会成为患有 RGC 的人类？ 非综合征性先天性视网膜不附着 (NCRNA)，ATOH7 的远程 5' 保守增强子被删除， 该 DNA 片段显然至关重要，但它的确切作用是阻止 RGC 的发育并导致完全失明。 未知。在转基因报告小鼠中，这种“影子”增强子（SE）似乎与“主要”增强子完全冗余。 （启动子相邻）增强子 (PE)，尽管是人类 NCRNA 中所必需的，但在初步研究中，我们观察到这一点。 Atoh7 SE 缺失小鼠保留视神经，这些双增强子元件如何协调调节快速起效。 在这里，我们建议研究人类 NCRNA 和 Atoh7 表达之间的功能差异。 小鼠 SE 缺失，以确定特定 DNA 序列如何控制 ATOH7 表达的水平、时间和模式， 分析 ATOH7 转录抑制，并确定影响 ATOH7+ 细胞命运决定的辅助因子 首先，我们将应用多物种方法来测试每个 ATOH7 的必要性和充分性。 调节元件并精确确定每个成分如何促进动态组织和细胞表达 其次，我们将研究通过 Notch 效应器 RPBJ 和 ATOH7 转录抑制的机制。 Kdm1a，使用高通量斑马鱼筛选、转基因生产和RNAseq 第三，我们将使用单细胞和RNAseq。 汇集 ATACseq 和 RNAseq 方法来详细分析视网膜祖细胞在 Atoh7 阶段的进展 这些数据将阐明控制 ATOH7 转录、视网膜神经发生和发生的机制。 RGC 命运规范；二元增强子的一般作用以及细胞体外 RGC 的潜在生成 我实现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建立的导师团队共同提出了拟议的研究和环境。 将为我作为视觉科学家的持续职业发展提供一个坚实的平台——学习新技术和 模型系统，并与各种各样的科学家互动（短期目标），这将为我铺平道路 成为一名独立学术研究员，探索控制 ATOH7、RGC 命运和视网膜的基因调控网络 组织发生（长期目标）。