Furtado Jessica, Geraldo Luiz Henrique, Leser Felipe Saceanu, Bartkowiak Bartlomiej, Poulet Mathilde, Park Hyojin, Robinson Mark, Pibouin-Fragner Laurence, Eichmann Anne, Boyé Kevin
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06511.
Cardiovascular Research Center, Department of Internal Medicine, Yale University School of Medicine, New Haven CT 06511.
Proc Natl Acad Sci U S A. 2024 Dec 24;121(52):e2408674121. doi: 10.1073/pnas.2408674121. Epub 2024 Dec 18.
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 or display more BRB leakage at the arterial end of the vasculature, while loss of function causes predominantly venular BRB leakage. ScRNA-seq of and mutant ECs reveals down-regulated β-catenin signaling and BRB gene expression that is rescued by 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 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.
血视网膜屏障（BRB）的完整性对于光转导和视觉至关重要，它通过严格限制血液与周围神经元细胞之间的分子运输来实现。BRB的破坏会导致视网膜疾病的发生。在此，我们表明Netrin-1/Unc5b和Norrin/Lrp5信号通路建立了一种分区的内皮细胞基因表达程序，该程序控制着BRB的完整性。通过对出生后具有BRB功能的小鼠视网膜内皮细胞（ECs）进行单细胞RNA测序（scRNA-seq），我们鉴定出超过100个编码Wnt信号通路成分、紧密连接蛋白以及离子和营养转运蛋白的BRB基因。我们发现BRB基因表达在动脉、毛细血管和静脉之间呈分区状态，并受视网膜小动脉和小静脉之间Netrin-1受体Unc5b和Lrp5-β-连环蛋白信号通路相反梯度的调节。缺乏 或 的小鼠在血管的动脉端表现出更多的BRB渗漏，而 功能丧失主要导致静脉BRB渗漏。对 和 突变ECs的scRNA-seq显示β-连环蛋白信号通路和BRB基因表达下调，而 的过度激活可挽救这种下调以及BRB的完整性。从机制上讲，我们证明Netrin-1和Norrin通过盘状大同源物1（Dlg1）支架蛋白加成增强β-连环蛋白的转录活性和Lrp5磷酸化，并且内皮 功能在保护毛细血管BRB完整性方面汇聚。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基因表达是在内皮细胞亚型水平上受到调节的。
