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: 10662505
• 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 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10662505
• 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; 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; Reporter Genes; 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; autosome; 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; neural; neurogenesis; notch protein; postmitotic; prevent; progenitor; promoter; 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)连接眼睛和大脑。它们对脊椎动物的视力和致病靶点是必不可少的。 青光眼。视觉科学家的一个治疗目标是充分了解RGC发育所需的因素，因此 这些细胞可以在体外产生。神经碱性螺旋-环-螺旋(BHLH)蛋白ATOH7瞬时表达 在早期视网膜祖细胞的一个亚群中，它们产生了视网膜的7种主要细胞类型，但只有 对于RGC的形成来说，是一个必不可少的能力因素。ATOH7基因缺失导致视神经再生障碍和严重继发性 视网膜血管畸形。Cre-lox谱系数据显示，只有55%的RGC来自Atoh7+祖细胞。什么 其他45%的视网膜节细胞的发生受因素控制？为什么只有部分Atoh7+细胞成为RGC？在人类中， 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， 阻止视网膜节细胞的发展并导致完全失明。这个DNA片段显然是至关重要的，但它的确切作用是 未知。在转基因报告鼠中，这种“影子”增强子(SE)似乎与“初级”完全多余。 (启动子相邻的)增强子(PE)，尽管在人类ncRNA中是必需的。在初步研究中，我们观察到 Atoh7 SE缺失的小鼠保留了视神经。这些双重增强子元件是如何协调调节快速发病的 和Atoh7表达的偏移量？在这里，我们建议研究人类ncRNA和ncRNA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素 RGC的起源。首先，我们将采用多物种方法来测试每种ATOH7的必要性和充分性。 并精确地确定每种成分对动态组织和细胞表达的贡献 图案。第二，我们将研究ATOH7通过Notch效应器RPBJ和 KDM1A，使用高通量斑马鱼筛选，转基因记者和RNAseq。第三，我们将使用单细胞和 联合ATACseq和RNAseq方法在视网膜前体细胞经历Atoh7阶段时详细描述他们的情况 表情。这些数据将阐明控制ATOH7转录的机制，视网膜神经发生的开始和 RGC命运规范；二元增强剂的一般作用；以及RGC体外对细胞的潜在生成 移植。我为实现这些目标所做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老牌导师团队。总之，拟议的研究和环境 将为我作为视觉科学家的继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 成为独立的学术研究人员，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)。