Fig. 2: A population of endothelial cells that express progenitor-associated genes is present in the optic nerve.

a Schematic diagram of the strategy for 3′ and 5′ scRNA-seq for sorted ECs (CD45^negCD31⁺) from retina or retina plus optic nerve. Experiments using 10 mice were pooled three times and analyzed. b A uniform manifold approximation (UMAP) of ECs identified by scRNA-seq of sorted 44,358 total (retina and retina plus optic nerve) cells (CD45^negCD31⁺) at 8-weeks of age. Batch corrections were performed by Harmony. c UMAPs of the levels of gene expression (Pecam1 [CD31], Bst1 [CD157], Procr [CD201] and Lrg1. d Velocity estimate projected onto UMAP demonstrating a directionality of RNA dynamics. e UMAP projection of endothelial cluster colored by RNA velocity–derived pseudotime (0 = early, 1 = late) as computed with scVelo, demonstrating a continuous progression along the inferred cellular trajectory. f mRNA levels of Bst1 [CD157] in vascular endothelial cluster in cells from retina only (left) and retina plus optic nerve (right). A distinct Bst1 positive population was present through integration of the optic nerve into the retina. g Distribution of the percentage of Bst1 positive cells in each cluster. h Bar plots of the fractions of cells form retina or retina plus optic nerve in all cells (top), cluster 10 (middle) and Bst1 positive cells in cluster 10 (bottom), respectively. *p = 7.308e-09, **p < 2.2e-16 by two-sided Fisher’s exact test. i Scatter plot of average expression (x‑axis) versus log₂ fold change (y‑axis) for genes upregulated in cluster 10. Predicted transcription factors are colored, with Atf3, Irf1 and Stat3 (logFC > 1) highlighted in red. Highly expressed marker genes—including Lrg1, Mctp1 and Ptgs2—are labeled, and the Atf3‑binding motif sequence logo is overlaid at the top left. Statistical significance was determined using a two-sided Wilcoxon Rank Sum test. P-values were adjusted with the Bonferroni correction.