Project Summary: Photoreceptor (PR) subtype patterning in the retina generates primate-specific trichromatic color and high-acuity spatial vision. Networks of factors drive cells along poorly understood developmental trajectories to reach terminal PR fates. Misspecification of PRs results in diseases causing blindness. My central goal is to determine the mechanisms that specify PRs and how these change to generate PR diversity among primates. PRs express light-sensitive proteins called opsins, which confer distinct functions. Short-wavelength sensitive cones (S cones) express S-opsin, long/medium-wavelength sensitive cones (L/M cones) express L/M-opsin, and dim light sensitive rods express Rhodopsin (Rho). In humans, opsin expression follows a temporal order: S opsin, then L/M opsin, and lastly Rho. The Johnston Lab found that retinoic acid (RA) and thyroid hormone (TH) signaling specify cone subtypes in human retinal organoids. Early, low TH and high RA signaling specify S cones. Later, high TH and low RA yields L/M cones. The mechanism by which TH and RA signaling regulate PR development is unclear. Comparative developmental biology is a powerful approach for revealing general mechanisms of development and how tweaking these mechanisms generates diversity between species. In contrast to humans, the initiation of opsin expression in the marmoset is inverted: Rho, then L/M opsin, followed by S opsin, suggesting that marmoset PR subtypes are born in a different order and/or mature at different rates compared to human PRs. I developed primate retinal organoid technology and determined that opsin expression in organoids recapitulates species-specific developmental patterns. I will genetically and pharmacologically manipulate retinal organoids to compare mechanisms of PR specification between primates and identify changes that diversify retinal patterning between species. During the K99 phase, I will determine how TH and RA regulate PR subtype specification and maturation in human and marmoset organoids. During the R00 phase, I will elucidate how TH and RA signaling regulates the cell fate trajectories of human and marmoset PRs during development. I will identify and validate candidate cis-regulatory changes that drive differences in PR specification, by knocking out associated genes, reciprocally swapping putative divergent regulatory elements in stem cells, and assessing effects in human and marmoset organoids. These comparative approaches will untangle the regulatory networks underlying the divergence of PR development between primate species. My goal is to become a leader in the field of neuronal fate specification, with a focus on understanding mechanisms of cell fate determination in primate retinal development. My mentoring team and the Johns Hopkins University community will provide the ideal environment for achieving in this goal.