Interplay between Netrin-1 and Norrin controls arteriovenous zonation of blood–retina barrier integrity

The integrity of the blood–retina barrier (BRB) is crucial for phototransduction and vision, by tightly restricting transport of molecules between the blood and surrounding neuronal cells. Breakdown of the BRB leads to the development of retinal diseases. Here, we show that Netrin-1/Unc5b and Norrin/Lrp5 signaling establish a zonated endothelial cell gene expression program that controls BRB integrity. Using single-cell RNA sequencing (scRNA-seq) of postnatal BRB-competent mouse retina endothelial cells (ECs), we investigate >100 BRB genes encoding Wnt signaling components, tight junction proteins, and ion and nutrient transporters. We find that BRB gene expression is zonated across arteries, capillaries, and veins and regulated by opposing gradients of the Netrin-1 receptor Unc5b and Lrp5-β-catenin signaling between retinal arterioles and venules. Mice deficient for Ntn1 or Unc5b display more BRB leakage at the arterial end of the vasculature, while Lrp5 loss of function causes predominantly venular BRB leakage. ScRNA-seq of Ntn1 and Unc5b mutant ECs reveals down-regulated β-catenin signaling and BRB gene expression that is rescued by Ctnnb1 overactivation, along with BRB integrity. Mechanistically, we demonstrate that Netrin-1 and Norrin additively enhance β-catenin transcriptional activity and Lrp5 phosphorylation via the Discs large homologue 1 (Dlg1) scaffolding protein, and endothelial Lrp5-Unc5b function converges in protection of capillary BRB integrity. These findings explain how arteriovenous zonation is established and maintained in the BRB and reveal that BRB gene expression is regulated at the level of endothelial subtypes.