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

Abstract

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.

Keywords: Netrin-1-Unc5b; WNT-b-catenin; blood brain barrier; blood retina barrier; single cell sequencing.

Fig. 1.

Arteriovenous zonation of BRB regulatory genes. (A) ScRNA-seq experimental design. (B) UMAP of EC subclusters from P12 retinas. (C) Dot plot of expression levels and frequency of selected genes in P12 EC subclusters. Color scale: yellow, high expression; dark blue, low expression. The percent expression is indicated by the sizes of the dots. (D) Dot plot of expression levels and frequency of selected BRB genes in P12 EC subclusters. Color scale: yellow, high expression; dark blue, low expression. The percent expression is indicated by the sizes of the dots. (E) Confocal scan of superficial P12 retina layer stained with the indicated antibodies. A: arteriole, C: capillary, V: venule.

Fig. 2.

Netrin-1/Unc5b and Norrin/Lrp5 regulate BRB integrity along the arteriovenous axis. (A) Immunofluorescence of whole-mount P12 retinas stained with indicated antibodies after TAM treatment at P6-8. (B) Immunofluorescence of whole-mount P12 retinas, 1 h after i.p. injection with sulfo-NHS-biotin. (C and D) Immunofluorescence of whole-mount P12 retinas stained with indicated antibodies, after TAM treatment at P6-8 and 1 h after i.p. injection with sulfo-NHS-biotin. (E) Transmission electron microscopy on indicated P12 genotypes after TAM treatment at P6-8. (F) Quantification of the number of vesicles (purple arrows in E) and gaps between tight junctions (green arrows in E). Each dot corresponds to one TEM image. (G and H) Immunofluorescence of whole-mount P12 retinas stained with indicated antibodies, after TAM treatment at P6-8 and 1 h after i.p. injection with sulfo-NHS-biotin. All data are shown as mean ± SEM. A two-sided Mann–Whitney U test was performed for statistical analysis. A: Artery, V: Vein

Fig. 3.

Netrin-1 and Unc5b regulate arterial and capillary BRB stabilization. (A) Volcano plot of differentially expressed genes between P12 Ntn1iKO and Control ECs. (B) Normalized enrichment scores of specified gene sets comparing transcripts from P12 Ntn1iKO versus Control ECs. P adjusted values are shown. (C) Dot plot of expression levels and frequency of selected Wnt target genes between Ntn1iKO and Control EC subclusters. Color scale: yellow, high expression; dark blue, low expression. (D) Volcano plot of differentially expressed genes between P12 Unc5biECKO and Control ECs. (E) Normalized enrichment scores of specified gene sets comparing transcripts from P12 Unc5biECKO versus Control ECs. P adjusted values are shown. (F) Dot plot of expression levels and frequency of selected Wnt target genes between Unc5biECKO and Control EC subclusters. Color scale: yellow, high expression; dark blue, low expression.(G) Western blot and (H) quantification of retina protein lysates extracted from Ntn1iKO and littermate controls. (I) Western blot and (J) quantification of retina protein lysates extracted from Unc5biECKO mice and littermate controls. (K–M) Immunofluorescence of whole-mount P12 retinas stained with the indicated antibodies, 1 h after i.p. injection with sulfo-NHS-biotin. (N) Volcano plot of differentially expressed genes between P12 Unc5biECKO;Ctnnb1^flex3/+ and Unc5biECKO ECs. (O) Normalized enrichment scores of specified gene sets comparing transcripts from P12 Unc5biECKO;Ctnnb1^flex3/+ and Unc5biECKO ECs. P adjusted values are shown. (P) Dot plot of expression level and frequency of selected Wnt target genes in P12 Unc5biECKO;Ctnnb1^flex3/+ and Unc5biECKO EC subclusters. Color scale: yellow, high expression; dark blue, low expression. All data are shown as mean ± SEM. A two-sided Mann–Whitney U test was performed for statistical analysis. Data points and western blot lanes represent one animal. A: Artery, V: Vein.

Fig. 4.

Netrin-1 and Norrin cooperate to regulate β-catenin signaling and BRB integrity in capillaries. (A) Gene deletion strategy. (B) Western blot and (C) quantification of retina protein lysates extracted from Ntn1iKO mice and littermate controls. Western blot lanes and data points represent one animal. (D) Western blot and (E) quantification of retina protein lysates extracted from Unc5biECKO mice and littermate controls. Western blot lanes and data points represent one animal. (F) Western blot of protein extracts from C57BL/6 mouse primary retinal ECs treated for the indicated times (min or h) with Netrin-1 (250 ng/ml), Norrin (250 ng/ml), or both. (G) Quantification of F, n = 3 independent experiments. P-values were calculated based on values obtained 8 h after stimulation (H) TOPflash or FOPflash activity of transfected retina ECs stimulated as indicated, each dot represents one independent experiment. (I) CTRL IgG and Unc5b immunoprecipitation from cultured ECs, and western blot probed for indicated proteins. (J) CTRL IgG and Unc5b immunoprecipitation from cultured ECs treated with Netrin-1 for 30 min or 8 h, probed by western blot with antibodies against the indicated proteins, and (K) quantification of the amount of Dlg1 bound to the immunoprecipitated Unc5b, each dot represents one independent experiment. (L) CTRL IgG and Unc5b immunoprecipitation of CTRL or Dlg1 siRNA-treated ECs, probed with antibodies against the indicated proteins, and (M) quantification of the amount of Lrp5 bound to the immunoprecipitated Unc5b, each dot represents one independent experiment. (N) Western blot and (O) quantification of siRNA-transfected ECs treated with Netrin-1 (250 ng/ml), n = 4 independent experiment. P-values were calculated based on values obtained 60’ after stimulation. P-value comparing siUnc5b to siCtrl is written in blue; P-value comparing siDlg1 to siCtrl is written in red (P) Experimental design for (Q), whole-mount P12 retinas stained as indicated. All data are shown as mean ± SEM. ANOVA followed by Bonferroni’s multiple comparisons test (G, H, K, and O). A two-sided Mann–Whitney U test was performed for statistical analysis between two groups (C, E, and M). A: Artery, V: Vein.

Fig. 5.

Netrin-1, Unc5b, and Lrp5 regulation of BRB genes. (A–C) GSEA plots of BRB gene sets comparing transcripts from P12 mutant and Ctrl ECs, as indicated. P adjusted values are shown. (D) Number of significantly differentially expressed genes in indicated comparisons. (E) Heat map of BRB genes in controls from Fig. 1A and mutants as indicated. Relative gene expression differences are shown by Z-score. Color code: dark blue high expression, light blue low expression. (F–S) Confocal scans of superficial P12 retinal layers stained with the indicated antibodies, and corresponding quantifications. All data are shown as mean ± SEM. ANOVA followed by Tukey’s multiple comparisons test was performed for statistical analysis between multiple groups. A: Artery, V: Vein.