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.

项目概要 时间模式通过复杂的基因调控网络（GRN）驱动视网膜细胞多样性。 GRN 以转录因子 (TF) 为首，转录因子促进特定阶段细胞的诞生，同时抑制 GRN 与其他发育时间点相关的这种交叉作用在比较时尤其明显。 值得注意的是，GRN 控制视网膜祖细胞 (RPC) 中早期和晚期细胞类型的生成。 在分化过程中，哺乳动物视网膜细胞失去了鱼类和两栖动物的再生能力 模型中再生能力的丧失导致了与以下主要原因相关的细胞死亡。 治疗干预的关键是年龄相关性黄斑变性和青光眼等失明。 了解如何刺激特定细胞类型的诞生以使细胞成功再生 这种理解取决于确定哪些转录因子。 GRN 内的驱动因素驱动细胞命运规范以及早期和晚期 RPC 的多能性丧失。 由于早期和晚期 RPC 之间的基因组组织状态非常不同，我 促进推动 GRN 的 TF 发挥先锋作用，推动暂时性的诞生 如果这是真的，那么与早期出生的细胞类型相关的转录因子将能够驱动 通过基因组区域的开放，晚期 RPC 中视锥细胞、无长突细胞和水平细胞的诞生 同样，晚期 TF 将诱导双极细胞的产生和 早期 RPC 群体中的穆勒神经胶质细胞通过基因组组织变化。 假设，我提出两个目标 目标 1：时间候选转录因子的功能分析。 这项工作将帮助我缩小时间模式候选调节剂的范围。 通过在发育中的功能获得和丧失实验中验证它们对细胞命运的影响 目标 2：确定调节从早期状态到晚期状态转变的 TF 是否具有 我将采用已建立的时间模式调节器并分析它们如何控制 一旦体外和体内表征系统已经建立。 确定后，我将采用目标 1 中确定的候选者，并使用此管道来对他们的开创性进行表型分析 通过建立一组驱动早期和晚期细胞命运规范的 PF。 视网膜，我将能够更好地解决当前 iPSC 衍生细胞疗法成功的障碍 青光眼和年龄相关性黄斑变性的治疗方法。