Elucidating the role of pioneer factors in RPC developmental competence

项目摘要 时间模式通过复杂的基因调控网络（GRNs）驱动视网膜细胞多样性。这些 GRNs由转录因子（TF）领导，这些转录因子促进阶段特异性细胞出生，同时抑制GRNs 与其他发育时间点相关。这种交叉发挥是特别明显的比较时， GRNs控制视网膜祖细胞（RPC）中早期和晚期出生细胞类型的生成。值得注意的是， 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。 模型这种再生能力的丧失导致了细胞死亡， 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，以使细胞成功再生 在这些疾病的过程中受到损害的人口。这种理解取决于确定哪些TF 在GRNs内的基因驱动细胞命运特化以及早期和晚期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不同， 我假设，驱动GRN的TF以开拓性的能力驱动时间的诞生， 限制细胞类型。如果这是真的，与早期出生的细胞类型相关的TF将能够驱动细胞生长。 通过基因组区域的开放，在晚期RPC中产生视锥细胞、无长突细胞和水平细胞 在那个时间窗口无法访问。同样，晚期TF将诱导双极细胞的产生， Müller胶质细胞在早期RPC人口通过基因组组织的变化。解决这些 假设，我提出两个目标。目的1：对颞叶神经节细胞凋亡的最佳候选转录因子进行功能分析。 模式调节这项工作将帮助我缩小时间模式的候选调节者名单 通过在开发中的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：确定调节从早期状态向晚期状态过渡的TF是否具有 开拓性活动。我将采用已建立的时间模式调节器，并分析它们如何控制 表观遗传调节的动力学。一旦用于体外和体内表征的系统已经被 建立，我将采取目标1中确定的候选人，并使用此管道表型他们的开拓性 活动通过建立一组驱动早期和晚期细胞命运特化的PF， 视网膜，我将能够更好地解决目前的障碍，成功的iPSC衍生的细胞为基础的治疗 青光眼和老年性黄斑变性的治疗方法。

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.