Atoh7 interacting proteins involved in retinal ganglion cell genesis

About This Grant

Project Summary Retinal ganglion cells (RGCs) send visual information from the retina to the brain via the optic nerve. Their degeneration underlies several major eye diseases affecting vision, including glaucoma, hereditary optic neuropathies, and ischemic optic neuropathies. Normally, RGCs do not regenerate; thus, RGC loss in these diseases is not reversible. One potential strategy for replenishing lost RGCs in patients is to reprogram stem cells and/or glial cells to functional RGCs. Knowledge about how RGCs are generated during embryonic development will greatly facilitate such efforts. During development, RGCs originate from naïve proliferating retinal progenitor cells (RPCs). Key transcription factors functioning at the different stages of RGC differentiation have been identified, and the mechanisms by which these transcription factors interact with enhancers to regulate target genes and promote RGC formation are being unraveled. These transcription factors likely interact with other factors to carry out their functions, but this is a much understudied area. The current proposal focuses on the factors that interact with Atoh7 to promote RGC formation. Atoh7 is a bHLH proneural transcription factor that is specifically required for RGC genesis. Atoh7 activates downstream genes by binding to E-box sequences in the target gene enhancers. However, multiple proneural bHLH transcription factors are expressed in the developing retina, and they are all capable of binding to similar if not identical E-boxes, yet only Atoh7 is capable of efficiently promoting RGC genesis. Using both ex vivo and in vivo assays, we have now shown that the differences between Atoh7 and other related bHLH transcription factors such as Neurod1 in promoting RGC formation lie within the bHLH domain. We have further narrowed down the responsible differences between Atoh7 and Neurod1 to six amino acid (a.a) residues. The locations of these six a.a. residues suggest that they likely provide interfaces for interaction with additional protein patterners, indicating a likely mechanism for Atoh7 to promote RGC differentiation. Our current proposal aims to identify these interacting proteins and characterize their functions. We will achieve our aims using proximity biotin labeling with our newly generated knock-in Atoh7 allele that expresses a fusion protein of Atoh7 and the biotin ligase BioID2. Our preliminary results demonstrate that this is a very feasible approach. Our specific Aims are: 1) To identify proteins interacting with Atoh7 in the developing retina and to characterize the specificity of the interactions. 2). To investigate the function of Atoh7 interacting proteins in RGC genesis. The proteins we will identify that are associated with Atoh7 will provide new research directions regarding how RGC specific gene regulation is achieved and how the RGC lineage is established. The findings will be significant not only for understanding the fundamental process of retinal cell differentiation but also for guiding efforts to regenerate RGCs to treat related retinal diseases.