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-ß-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 ß-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 ß-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. Overall design: Mutant and corresponding control mice were injected with TAM P6-P8 and sacrified at P12. Retinas were dissected, pooled according to genotype and cells were isolated with endothelial cell enrichment.

血-视网膜屏障（blood–retina barrier, BRB）的完整性对于光转导与视觉功能至关重要，其通过严格限制血液与周围神经元细胞间的分子转运发挥屏障功能。血-视网膜屏障的破坏会引发视网膜疾病。本研究证实，Netrin-1/Unc5b与Norrin/Lrp5信号通路共同构建了分区化的内皮细胞基因表达程序，以此调控血-视网膜屏障的完整性。我们对出生后具备血-视网膜屏障功能的小鼠视网膜内皮细胞（endothelial cells, ECs）开展单细胞RNA测序（single-cell RNA sequencing, scRNA-seq），分析了超过100个编码Wnt信号通路组分、紧密连接蛋白以及离子与营养转运蛋白的血-视网膜屏障相关基因。研究发现，血-视网膜屏障基因的表达在动脉、毛细血管与静脉中呈现分区化特征，并受到视网膜动脉与静脉之间Netrin-1受体Unc5b与Lrp5-β-连环蛋白（β-catenin）信号的反向梯度调控。缺乏Ntn1或Unc5b的小鼠在血管动脉端表现出更为严重的血-视网膜屏障渗漏，而Lrp5功能缺失则主要引发静脉端的血-视网膜屏障渗漏。对Ntn1与Unc5b突变型内皮细胞的单细胞RNA测序结果显示，β-连环蛋白信号与血-视网膜屏障基因的表达均出现下调，而通过过度激活Ctnnb1可挽救该表型，并恢复血-视网膜屏障的完整性。机制层面，我们证实Netrin-1与Norrin可通过Discs大同源物1（Discs large homologue 1, Dlg1）支架蛋白，协同增强β-连环蛋白的转录活性与Lrp5的磷酸化水平，且内皮细胞Lrp5与Unc5b的功能在保护毛细血管血-视网膜屏障完整性的过程中具有协同效应。上述发现阐明了血-视网膜屏障中动静脉分区化模式的建立与维持机制，并揭示了血-视网膜屏障基因的表达可在内皮细胞亚型层面受到调控。实验设计：对突变型小鼠与对应野生型对照小鼠在出生后第6天至第8天（P6-P8）注射他莫昔芬（TAM），并于出生后第12天（P12）处死小鼠。分离视网膜组织，按基因型分组混合，随后通过内皮细胞富集方法分离目标细胞。