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).

项目摘要/摘要 视网膜神经节细胞（RGC）将眼睛连接到大脑。它们对于脊椎动物视力和致病靶标至关重要 视觉科学家的一个治疗目标是充分了解RGC开发所需的因素，因此 这些细胞可以在体外产生。主底基螺旋 - 环螺旋（BHLH）蛋白ATOH7瞬时表达 在早期视网膜祖细胞的亚群中，这引起了视网膜的7种主要细胞类型，但仅是 作为RGC起源的能力因素必不可少的。 ATOH7的损失会导致视神经性和严重的次级 视网膜畸形。 Cre-Lox谱系数据显示，RGC的55％来自ATOH7+祖细胞。什么 因素控制其他45％的RGC的起源？为什么只有一些ATOH7+细胞成为RGC？在人类中 非型环境先天性视网膜非辅助（NCRNA），删除了ATOH7的远程5'配置增强子， 防止RGC的发展并导致完全失明。该DNA段显然至关重要，但其确切的作用是 未知。在传输记者小鼠中，这种“阴影”增强器（SE）似乎完全多余 （启动子 - 染色）增强子（PE），在人类NCRNA中需要任务。在初步研究中，我们观察到 ATOH7 SE缺失小鼠保留视神经。这些双重增强子元素如何协调调节快速发作 和AtoH7表达的抵消？在这里，我们建议研究人NCRNA和 小鼠SE删除，以确定特定的DNA序列如何控制ATOH7表达的水平，时机和模式， 分析ATOH7的转录表示，并确定辅助因子会影响ATOH7+细胞脂肪的决策 RGC起源。首先，我们将采用多物种方法来测试每个ATOH7的必要和充分性 调节元件并精确确定每个成分如何有助于动态组织和细胞表达 图案。其次，我们将通过Notch效应子RPBJ和 KDM1A，使用高通量斑马鱼屏幕，转基因记者和RNASEQ。第三，我们将使用单细胞和 汇总ATACSEQ和RNASEQ方法在ATOH7的阶段进行详细介绍视网膜祖细胞 表达。这些数据将阐明控制ATOH7转录，永久神经发生和 RGC命运规范；通常，二元增强剂的作用；以及在体外RGC的潜在产生的细胞 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 加利福尼亚大学，戴维斯大学和我成熟的导师团队。拟议的研究和环境在一起 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，并与各种科学家（短期目标）进行互动，这将为我铺平道路 成为控制ATOH7，RGC命运并保持的独立学术研究人员探测基因调节网络 组织发生（长期目标）。