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: 10477965
• 负责人: Patrick Leavey
• 金额: $ 4.68万
• 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/10477965
• 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; 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; gene regulatory network; 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.

项目摘要 时间模式通过复杂的基因调节网络（GRN）驱动视网膜细胞多样性。这些 GRN由转录因子（TF）领导，这些因子促进了特定阶段的细胞出生，同时反映了GRNS 与其他发育时间点相关联。在比较 控制残留祖细胞（RPC）中的早期和晚期细胞类型的GRN。值得注意的是，结束了 分化的过程，哺乳动物视网膜细胞失去了鱼类和两栖动物的再生能力 型号。与主要原因有关的细胞死亡的再生能力允许的损失 失明，例如与年龄相关的黄斑变性和青光眼。治疗干预的关键是 了解如何刺激特定细胞类型的诞生以成功回归细胞 在这些疾病过程中，种群损害了。这种理解取决于识别哪些TFS 在GRNS中，驱动细胞命运规格以及早期和晚期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是如此独特，所以我 假设TFS驾驶GRNS行为以开创性的能力推动临时出生 受限的细胞类型。如果这是正确的，那么与早期出生的单元类型相关的TFS将能够驱动 通过基因组区域的开放，晚期RPC中锥，悬浆和水平细胞的诞生 在那个临时窗口中无法访问。同样，后期TFS将诱导双极细胞的产生和 MüllerGlia通过基因组组织变化在RPC早期人群中。解决这些 假设，我提出了两个目标。目标1：临时候选候选转录因子的功能分析 图案法规。这项工作将有助于我缩小临时图案的候选监管机构的清单 通过验证其对开发中的功能丧失实验和功能丧失实验的影响 小鼠视网膜。目标2：确定调节从早期到后期过渡的TF是否有 开创性活动。我将采用既定的临时模式调节器，并分析他们如何控制 表观遗传调制的动力学。一旦体外和体内表征的系统已经 成立，我将带AIM 1中确定的候选人，并使用此管道表型表型 活动。通过建立一组PFS，该PFS在 视网膜，我将能够更好地解决成功基于IPSC的细胞疗法的当前障碍 青光眼和与年龄相关的黄斑变性的方法。