Project Summary Retinal ganglion cells (RGCs) are the interneurons that transmit visual information from the eye to the brain. Degeneration or death of RGCs results in a number of blinding conditions, including glaucoma. RGCs can be classified into many subtypes, each with distinct morphologies, functions, and gene expression profiles. While RGC subtypes have been identified in many vertebrate model organisms, RGC subtypes in the human retina have not been well-characterized molecularly. Moreover, the mechanisms controlling the specification of RGC subtypes remain poorly understood, especially in the human retina. In this project, I will study human retinas and organoids to identify human RGC subtypes and determine mechanisms controlling their generation. My work will yield mechanistic insights into human RGC subtype specification and facilitate the use of stem cell-derived organoids in regenerative therapies for retinopathies like glaucoma. One of the major goals of this project is to molecularly classify the RGC subtypes of the adult human retina. Based on published single cell RNA sequencing (scRNA-seq) data in human and macaque retinas, I generated a testable gene expression code to uniquely identify each RGC subtype. I will use this gene expression code to guide combinatorial expression analysis (i.e. immunohistochemistry and RNA FISH) and identify human RGC subtypes. I have already distinguished three human RGC subtypes using my gene expression code. I will also characterize the morphologies of these RGC subtypes. I will use combinatorial IHC and the lipophilic dye DiD to molecularly and morphologically classify human RGC subtypes (Aim 1). A major challenge to studying human RGC biology is the limited access to genetically and pharmacologically manipulatable human tissue. Human retinal organoids provide a powerful model to study developing human tissue in controlled conditions. The Johnston lab advanced the use of human retinal organoids to study the mechanisms controlling photoreceptor fate specification. I showed that organoids are a tractable model system to study RGC biology by developing an RGC axon outgrowth assay, which demonstrated that RGCs rapidly extend axons, recapitulating their in vivo capabilities. Furthermore, I identified two subtypes of RGCs in organoids. To identify the repertoire of RGC subtypes in human organoids, I will assess expression based on the testable gene expression code and complement this approach by conducting scRNA-seq over a time course of organoid development (Aim 2). The transcription factors EOMES/TBR2, TBR1, and TBX5 have been implicated in RGC subtype specification. To investigate mechanisms that specify human RGC subtypes, I will utilize CRISPR mutagenesis to knock out and viral transfection to ectopically express these three transcription factors and examine changes in RGC subtype populations in human retinal organoids (Aim 3).