Elucidating the role of pioneer factors in RPC developmental competence

阐明先驱因素在 RPC 发展能力中的作用

• 批准号: 10313677
• 负责人: Patrick Leavey
• 金额: $ 4.6万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-29 至 2024-08-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10313677
• 关键词: ATAC-seq; Address; Age related macular degeneration; Amacrine Cells; Amphibia; Automobile Driving; Binding; Biological Assay; Birth; Blindness; Cell Death; Cell Line; Cell Therapy; Cells; Chimeric Proteins; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Competence; Complex; Computer software; Development; Disease; Electroporation; Epigenetic Process; Exhibits; Factor Analysis; Family; Fibrinogen; Fishes; Gene Expression; Generations; Genes; Genomic Segment; Genomics; Glaucoma; Goals; Histology; In Vitro; Individual; Location; Modeling; Molecular; Molecular Mechanisms of Action; Muller' s cell; Mus; Mutant Strains Mice; Neurons; Nuclear Translocation; Organizational Change; Pattern; Phenotype; Plasmids; Play; Population; Predictive Factor; Process; Production; Proliferating; Regenerative capacity; Regulation; Regulator Genes; Resolution; Retina; Retinal Cone; Retinal Diseases; Retinal Ganglion Cells; Role; Route; Site; System; Testing; Therapeutic Intervention; Tissues; Validation; Work; cell fate specification; cell type; cold blooded vertebrate; epigenomics; experimental study; gain of function; horizontal cell; in vivo; induced pluripotent stem cell; loss of function; neurogenesis; overexpression; pluripotency; response to injury; retinal progenitor cell; single-cell RNA sequencing; small molecule; transcription factor

PROJECT SUMMARY Temporal patterning drives retinal cellular diversity through complex gene regulatory networks (GRNs). These GRNs are headed by transcription factors (TFs) that promote stage-specific cell birth while repressing GRNs associated with other developmental timepoints. This cross play is especially noticeable when comparing the GRNs controlling generation of early and late-born cell types in retinal progenitor cells (RPCs). Notably, over the course of differentiation, mammalian retinal cells lose the regenerative capacity seen in fish and amphibian models. This loss of regenerative capacity permits for the cell death associated with leading causes of blindness such as age-related macular degeneration and glaucoma. Key to therapeutic interventions is an understanding of how to stimulate the birth of specific cell types to allow for successful regrowth of the cell populations damaged in the course of these diseases. This understanding hinges on identifying which TFs within the GRNs drive cell fate specification and the loss of pluripotency for the early and late-stage RPCs. Because the genomic organizational states between the early and late-stage RPCs is so distinct, I hypothesize that the TFs driving the GRNs act in a pioneering capacity to drive the birth of temporally restricted cell types. If this holds true, the TFs associated with early-born cell types will be able to drive the birth of cones, amacrines, and horizontal cells in late-stage RPCs through the opening of genomic regions inaccessible at that temporal window. Likewise, late-stage TFs will induce the production of bipolar cells and Müller glia in the early RPC population through genomic organizational changes. To address these hypotheses, I propose two Aims. Aim 1: Functional analysis of top candidate transcription factors for temporal patterning regulation. This work will help me narrow down the list of candidate regulators of temporal patterning through validation of their impact on cell fate in gain-of and loss-of-function experiments in the developing mouse retina. Aim 2: Determine whether TFs that regulate transition from early to late-stage states have pioneering activity. I will take established temporal patterning regulators and assay how they control the dynamics of epigenetic modulation. Once systems for in vitro and in vivo characterization have been established, I will take candidates identified in Aim 1 and use this pipeline to phenotype their pioneering activity. Through the establishment of a set of PFs that drive early and late-stage cell fate specification in the retina, I will be able to better address current barriers to successful iPSC-derived cell-based therapeutic approaches to glaucoma and age-related macular degeneration.

项目总结 时间模式通过复杂的基因调控网络(GRN)驱动视网膜细胞的多样性。这些 GRN由转录因子(TF)领导，在抑制GRN的同时促进特定阶段细胞的诞生 与其他发育时间点相关。这种交叉使用在比较 GRN控制视网膜祖细胞(RPC)中早生和晚生细胞类型的产生。值得注意的是，结束了 在分化过程中，哺乳动物的视网膜细胞失去了鱼类和两栖动物的再生能力 模特们。这种再生能力的丧失允许与主要原因相关的细胞死亡 失明，如老年性黄斑变性和青光眼。治疗干预的关键是 了解如何刺激特定细胞类型的诞生，以实现细胞的成功再生 在这些疾病的过程中受损的人口。这一理解取决于确定哪些TF 在GRN中，驱动细胞命运规范和丧失早期和晚期RPC的多能性。 由于早期和晚期RPC之间的基因组组织状态是如此截然不同，即 假设推动GRN的TF以开创性的能力推动临时性 受限的单元格类型。如果这是真的，与早期出生的细胞类型相关的TF将能够驱动 晚期RPC中通过基因组区域开放的锥体、无核细胞和水平细胞的诞生 在该时间窗口不可访问。同样，晚期转铁蛋白将诱导双极细胞的产生和 通过基因组结构的变化，早期RPC群体中的Müler胶质细胞。要解决这些问题 假设，我提出了两个目标。目标1：转录因子的功能分析 图案化管理。这项工作将帮助我缩小时间模式的候选调节器列表 通过在功能获得和功能丧失实验中验证它们对细胞命运的影响 小鼠视网膜。目标2：确定规范从早期国家向晚期国家过渡的技术框架是否有 开拓性的活动。我将采用已建立的时间模式调节器，并分析它们是如何控制 表观遗传调控的动力学。一旦用于体外和体内表征的系统已经 一旦确定，我将选择目标1中确定的候选人，并利用这条渠道来表现他们的开拓性 活动。通过建立一套PFS来驱动早期和晚期细胞命运规范 视网膜，我将能够更好地解决目前成功的基于iPSC的细胞治疗的障碍 青光眼和老年性黄斑变性的治疗方法。