Regulatory mechanisms for retinal ganglion cell genesis

项目总结/文摘

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.