Regulatory mechanisms for retinal ganglion cell genesis

项目总结/摘要 单个视网膜细胞类型如何从视网膜祖细胞（RPC）中产生是一个活跃的研究领域，因为它是 对于理解视网膜细胞多样性的产生和开发治疗策略至关重要 来治疗视网膜退化疾病许多转录因子参与个体视网膜色素瘤的产生， 细胞类型已经确定。单细胞技术已经导致了前所未有的进步， 不同谱系轨迹的细胞关系和表观遗传景观的潜在变化。 单个转录因子在塑造表观遗传景观以驱动多能RPC中的作用 具体的命运也开始显露。单细胞RNA-seq研究的一个主要发现是， 在命运决定之前，视网膜谱系经历一种共享的状态，即过渡性RPC（tRPC）。tRPC 是多能的，并且共表达涉及不同视网膜细胞类型的基因，例如RGC的Atoh7和Otx2 和Neurod1用于光感受器。我们通过CUT & Tag绘制的Atoh7和Otx2结合位点的图谱表明， 共表达的转录因子竞争谱系特异性基因的增强子的一般范例 驱动tRPC走向不同的细胞命运。我们实验室的重点是控制RGC发生的机制。 在这个应用程序中，我们建议解决几个关键的知识差距， 从tRPC中分离出RGC谱系。第一个是上游输入的缺失分支，如我们的scRNA所示- Atoh7无效视网膜的seq分析。我们假设SoxC因子通过平行的功能来履行这一角色 基于先前关于SoxC因子在RGC发生中的作用的发现，用Atoh7促进RGC发生。 我们旨在解决的第二个缺口是Atoh7对RGC谱系特异性的分子基础。这是 基于几种前神经bHLH转录因子，包括Atoh7和Neurod1， 与E盒基序结合，在tRPC中共表达，但只有Atoh7促进RGC形成。使用异位 我们已经获得了令人信服的证据，表明RGC特异性的视网膜移植， Atoh7位于bHLH结构域中。我们将进一步探索Atoh7的RGC特异性的分子基础， 在体内使用敲入小鼠系。最后，我们将研究导致固定的调节机制 RGC的命运。我们将利用从scATAC-seq和CUT & Tag中鉴定的候选增强子 实验的关键RGC特异性转录因子基因Pou4f2，并检查他们的贡献， Pou4f2的最终表达。我们的实验旨在使用组合方法来解决这些差距 小鼠遗传学、免疫组织化学、基因组学、转录组学和单细胞技术。这些 提出的实验旨在了解控制RGC出现的关键基因调控事件 来自tRPC的谱系。这一发现将进一步深入了解支撑细胞免疫的分子基础。 视网膜的多样性，并为制定治疗退行性视网膜疾病的策略提供指导。

Project Summary/Abstract How individual retinal cell types arise from retinal progenitor cells (RPCs) is an active field of research, as it is critical to understanding the generation of cellular diversity in the retina and developing therapeutic strategies to treat degenerative retinal diseases. Many transcription factors involved in the generation of individual retinal cell types have been identified. Single-cell technologies have led to unprecedented progress in discerning the cellular relationships of the different lineage trajectories and the underlying changes in the epigenetic landscape. The roles of individual transcription factors in shaping the epigenetic landscape to drive multipotent RPCs to specific fates are also beginning to be revealed. A major finding from the single-cell RNA-seq studies is that all the retinal lineages undergo a shared state, namely transitional RPCs (tRPCs), before fate determination. tRPCs are multipotent and co-express genes involved in the different retinal cell types such as Atoh7 for RGCs and Otx2 and Neurod1 for photoreceptors. Our mapping of binding sites for Atoh7 and Otx2 by CUT&Tag suggests a general paradigm via which the co-expressed transcription factors compete at enhancers of lineage-specific genes to drive tRPCs to different cell fates. The focus of our lab has been on the mechanisms controlling RGC genesis. In this application, we propose to address several key knowledge gaps regarding the emergence of the RGC lineage from tRPCs. The first is the missing branch of upstream input as indicated by our scRNA- seq analysis of the Atoh7-null retina. We hypothesize that the SoxC factors fulfill this role by function in parallel with Atoh7 to promote RGC genesis based on previous findings on the roles of the SoxC factors in RGC genesis. The second gap we aim to address is the molecular basis for the specificity of Atoh7 for the RGC lineage. This is based on the fact that several proneural bHLH transcription factors, including Atoh7 and Neurod1, which all bind to the E box motif, are co-expressed in tRPCs, but only Atoh7 promotes RGC formation. Using ectopic expression in retinal explants, we have obtained compelling evidence which suggests that the RGC-specificity of Atoh7 resides in the bHLH domain. We will further explore the molecular basis for the RGC specificity of Atoh7 in vivo using knockin mouse lines. Lastly, we will investigate the regulatory mechanisms leading to the fixation of the RGC fate. We will leverage the candidate enhancers identified from our scATAC-seq and CUT&Tag experiments for the key RGC specific transcription factor gene Pou4f2 and examine their contributions to the eventual expression of Pou4f2. Our experiments are designed to address these gaps using a combined approach of mouse genetics, immunohistochemistry, genomics, transcriptomics, and single cell techniques. These proposed experiments aim to understand key gene regulatory events controlling the emergence of the RGC lineage from tRPCs. The findings will lead to further insights into the molecular basis underpinning the cellular diversity in the retina and offer guidance for developing strategies to treat degenerative retinal diseases.