Table_1_Single cell transcriptomics identifies distinct choroid cell populations involved in visually guided eye growth.docx

IntroductionPostnatal ocular growth is regulated by a vision-dependent mechanism, termed emmetropization, which acts to minimize refractive error through coordinated growth of the ocular tissues. The choroid participates in the emmetropization process via the production of scleral growth regulators that control ocular elongation and refraction.MethodsTo elucidate the role of the choroid in emmetropization, we used single-cell RNA sequencing (scRNA-seq) to characterize the cell populations in the chick choroid and compare gene expression changes in these cell populations when the eye is undergoing emmetropization. Emmetropization was modeled by inducing form deprivation myopia in 3 -4 day old chicks via application of translucent occluders to one eye for a period of 10 days followed by 24 hrs of unrestricted vision (by removing the occluder). Numerous studies have previously shown that visual form deprivation stimulates axial elongation and myopia, and upon removal of the occluder, the myopic chick eye rapidly slows its rate of axial elongation to recover from the induced myopia and regain emmetropia. The contralateral, untreated eyes served as controls.ResultsUMAP clustering analysis identified 24 distinct cell clusters in all chick choroids. 7 clusters were identified as fibroblast subpopulations; 5 clusters represented different populations of endothelial cells; 4 clusters were CD45+ macrophages, T cells and B cells; 3 clusters were Schwann cell subpopulations; and 2 clusters were identified as melanocytes. Additionally, single populations of RBCs, plasma cells and neuronal cells were identified. Significant changes in gene expression between control and treated choroids were identified in 17 cell clusters, representing 95% of total choroidal cells. The majority of gene expression changes were relatively small (DiscussionOur results, for the first time, provide a comprehensive profile of the major choroidal cell types and their gene expression changes during the process of emmetropization and provide insights into the canonical pathways and upstream regulators that coordinate postnatal ocular growth.