Fig. 6: Single-cell RNA sequencing identified Schlemm’s canal endothelial cells.

A Cdh5 + endothelial cells were identified from the total cell population and reclustered using a new set of principal components, resulting in four endothelial cell clusters. B Expression analysis of marker genes present in the four unique endothelial populations. Examination of the endothelial populations in (A) revealed that Ccl21a-positive cells in cluster 1 were comprised of two distinct populations. One of these populations was nearly absent in Angpt1ΔNC eyes, suggesting that they might represent SC endothelial cells. C We, therefore, isolated these cells and reclustered them, allowing detailed analysis. D Expression analysis revealed that the presumptive lymphatic endothelial cells (LECs) expressed classic lymphatic markers such as Prox1, Pdpn, and Lyve1. In contrast, Schlemm’s canal endothelial cells (SCs) expressed Plvap, Tek and Ptprb in addition to Flt1 while retaining low-level expression of Prox1 and Flt4. E Consistent with our histological results, very few SC endothelial cells were identified in Angpt1ΔNC mice. To exclude the possibility that was due to sampling variability, we performed bootstrapping analysis, picking a sample of endothelial cells of equal size to our knockout sample from the WT dataset. 10,000 iterations were performed, revealing that Angpt1 knockout mice have far fewer SC endothelial cells then controls. Distribution of simulated samples is shown as a violin plot compared to the true proportion of SC endothelial cells identified in Angpt1 knockout eyes. F Dendrogram illustrating the transcriptomic relationship between endothelial cell clusters identified. G Identification of unique markers of Schlemm’s canal (SC), ocular lymphatic (LEC) and blood vascular (BEC) endothelium.