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 identify >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.

中文翻译：

---

Netrin-1 和 Norrin 之间的相互作用控制血液的动静脉分区-视网膜屏障完整性


血液-视网膜屏障 （BRB） 的完整性对光转导和视觉至关重要，因为它严格限制了分子在血液和周围神经元细胞之间的运输。BRB 的崩溃导致视网膜疾病的发展。在这里，我们表明 Netrin-1/Unc5b 和 Norrin/Lrp5 信号转导建立了一个控制 BRB 完整性的分区内皮细胞基因表达程序。使用出生后 BRB 能力小鼠视网膜内皮细胞 （EC） 的单细胞 RNA 测序 （scRNA-seq），我们鉴定了编码 Wnt 信号成分、紧密连接蛋白以及离子和营养转运蛋白的 >100 BRB 基因。我们发现 BRB 基因表达在动脉、毛细血管和静脉中分区，并受视网膜小动脉和小静脉之间 Netrin-1 受体 Unc5b 和 Lrp5-β-catenin 信号传导的相反梯度调节。缺乏 Ntn1 或 Unc5b 的小鼠在脉管系统的动脉端显示更多的 BRB 渗漏，而 Lrp5 功能丧失主要导致静脉 BRB 渗漏。Ntn1 和 Unc5b 突变 ECs 的 ScRNA-seq 揭示了 Ctnnb1 过度激活以及 BRB 完整性所拯救的下调 β-catenin 信号转导和 BRB 基因表达。从机制上讲，我们证明 Netrin-1 和 Norrin 通过 Discs 大同源物 1 （Dlg1） 支架蛋白加增性增强 β-catenin 转录活性和 Lrp5 磷酸化，并且内皮 Lrp5-Unc5b 功能收敛以保护毛细血管 BRB 完整性。这些发现解释了 BRB 中动静脉分区是如何建立和维持的，并揭示了 BRB 基因表达在内皮亚型水平上受到调节。