A Roadmap to Uncover RPE Plasticity

摘要 退行性视网膜疾病是一个巨大的公共卫生负担，需要创新的策略， 替换视网膜神经元。理想的解决方案将克服与终端差异化相关的先天障碍， 内源性再生视网膜神经元。视网膜色素上皮（RPE）细胞对此有希望 这些细胞可以重新编程，在胚胎视网膜中产生神经视网膜。其原因 不太清楚的是，RPE细胞在早期羊膜发育过程中丧失神经能力。本研究 建议全面绘制发育可塑性阶段RPE的基因调控图谱， 以探测这些细胞分化时出现的重编程的分子障碍。视网膜色素上皮细胞 鸡在胚胎第4天（E4）是可塑的，并且可以在视网膜切除术后重编程为神经视网膜， 用FGF 2治疗然而，到胚胎第5天（E5），RPE细胞的命运变得受限， 能力被废除。先前的研究表明，E4 RPE细胞通过激活 神经视网膜转录因子，如VSX 2，SIX 6和PAX 6，以及RPE的同时抑制 差异化方案。同时，表观遗传重编程事件重置DNA甲基化和平衡， 染色质转化为更活跃的构型，以促进细胞身份的随后变化。但不 了解转录因子网络如何与动态染色质景观相互作用以确定RPE 神经能力我们的初步证据表明，前神经基因仍然是可诱导的， E5 RPE，但早期的RPE分化程序作为一个内在的障碍，神经身份， 现阶段这种分化程序由RPE转录因子MITF和OTX 2引导，并且与其他细胞分化程序不同。 来自这些因子的可接近性足迹在染色质景观中是可观察到的。目前的提案将 通过绘制与RPE能力相关的全基因组表观遗传模式， 限制，包括DNA和组蛋白修饰，这些修饰先前已被证明有助于RPE 重编程（Aim 1）。此外，将在单个细胞中分析RPE分化和重编程。 分辨率，能够精确识别描述可塑性和命运限制的转录组学特征 RPE（目标2）。同时，关键效应转录因子如MITF和OTX 2的DNA结合活性， 将在分化或重编程的不同阶段跨RPE进行分析。这些因素，以及 关键的转录靶点，将使用CRISPR-Cas9进行干扰，目的是恢复RPE神经元 在更高级的分化阶段的能力（目标3）。总之，这些发现将提供一个 与RPE能力限制相关的染色质状态的扩展视图，同时探索 染色质-转录因子的相互作用驱动观察到的表型。这些结果将提供 了解RPE分化和这种细胞类型用于神经元的潜力的必要见解 替代战略。

Abstract Degenerative retinal diseases represent an enormous public health burden and demand innovative strategies to replace retinal neurons. Ideal solutions will overcome innate barriers associated with terminal differentiation to endogenously regenerate retinal neurons. Retinal pigment epithelium (RPE) cells hold promise for this application, as these cells can reprogram to produce neural retina in embryonic amniotes. For reasons that are not well understood, RPE cells lose neural competence during early amniotic development. The present study proposes to comprehensively map the gene regulatory landscape of RPE at plastic stages of development and to probe the molecular barriers to reprogramming that emerge as these cells differentiate. RPE cells of the chicken are plastic at embryonic day 4 (E4) and can be reprogrammed to neural retina following retinectomy and treatment with FGF2. However, by embryonic day 5 (E5), RPE cell fate becomes restricted and reprogramming capacity is abrogated. Previous studies have demonstrated that E4 RPE cells reprogram through the activation of neural retina transcription factors, such as VSX2, SIX6, and PAX6, and the simultaneous repression of RPE differentiation programs. Concomitantly, an epigenetic reprogramming event resets DNA methylation and poises chromatin into a more active configuration to facilitate the ensuing change in cell identity. However, it is not understood how transcription factor networks interact with a dynamic chromatin landscape to determine RPE neural competence. Our preliminary evidence suggests that pro-neural genes remain comparably inducible in the E5 RPE, but that an incipient RPE differentiation program serves as an inherent barrier to neural identity at this stage. This differentiation program is led by the RPE transcription factors MITF and OTX2, and distinct accessibility footprints from these factors are observable in the chromatin landscape. The current proposal will build on these findings by mapping genome-wide epigenetic patterns associated with RPE competence restriction, including DNA and histone modifications that have been previously demonstrated to facilitate RPE reprogramming (Aim 1). Additionally, RPE differentiation and reprogramming will be profiled at a single cell resolution, enabling the precise identification of transcriptomic features that delineate plastic and fate restricted RPE (Aim 2). In parallel, the DNA binding activity of key effector transcription factors, such as MITF and OTX2, will be profiled across the RPE at different stages of differentiation or reprogramming. These factors, as well as key transcriptional targets, will be perturbed using CRISPR-Cas9 with the intention of recovering RPE neural competence at more advanced stages of differentiation (Aim 3). Together, these findings will provide an expansive view of chromatin states associated with RPE competence restriction, while simultaneously probing the chromatin – transcription factor interactions that drive the observed phenotypes. These results will provide imperative insights toward understanding RPE differentiation and the potentiality of this cell type for use in neuron replacement strategies.