Single Cell Transcriptomics Identifies Distinct Choroid Cell Populations Involved in Visually Guided Eye Growth

Postnatal ocular growth is regulated by a vision-dependent mechanism, termed emmetropization, which acts to minimize refractive error through coordinated growth of the ocular tissues. Many studies suggest that the ocular choroid participates in the emmetropization process via the production of scleral growth regulators that control ocular elongation and refractive development. To 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 during conditions in which the eye is undergoing emmetropization. UMAP 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 significant gene expression changes were relatively small (< 2 fold). The highest changes in gene expression were identified in a rare cell population (0.11% - 0.49% of total choroidal cells). This cell population expressed high levels of neuron-specific genes as well as several opsin genes suggestive of a rare neuronal cell population that is potentially light sensitive. Our 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 as well as insights into the canonical pathways and upstream regulators that coordinate postnatal ocular growth. Overall design: To investigate the gene expression changes in choroidal cell populations during the process of emmetropization, we isolated cells from choroid tissue digests of choroids isolated from chick eyes recovering from form deprivation myopia and contralateral control eyes. Following FACS sorting of live cells, live cells were processed for sc-RNA sequencing. Comparative gene expression profiling analyses were performed for three samples of choroidal cell populations from control eyes and three sets of choroidal cell populations from recovering eyes.