Figure 2. Pdcd10 deletion induces specific transcriptional profiles in distinct endothelial cell subpopulations.

(A) Numbers of significant differentially expressed genes (DEGs) (padj <0.05) in each cluster, showing up-regulation (red) and down-regulation (blue). (B) Average log fold changes of Klf4 (orange) and Klf2 (gray) expression in each cluster (Pdcd10-ko vs. Pdcd10-wt; padj <0.05). (C) Heatmap of selected known CCM lesion markers, as average logFC (padj <0.05) of Pdcd10-ko versus Pdcd10-wt cells (see also Supplementary file 2). (D) Enrichment of CCM-associated genes (source: Rare Diseases GeneRIF Library) among DEGs (Pdcd10-ko vs. Pdcd10-wt) for each cluster (see Materials and methods). X-axis indicates the number of DEGs identified as CCM-associated genes in each cluster. Asterisks show the significance of enrichment: *p<0.05; **p<0.01. (E) Over-representated Molecular Signatures Databases hallmark gene sets in DEGs (average |logFC| ≥ 0.3; padj <0.05, see Materials and methods). The sizes of the dots reflect the proportion (%) of overlap between the DEGs and the reference gene sets, while the intensities of the colors show the -log10(q-value), color coded in red for up-regulated gene sets and in blue for down-regulated gene sets. Gene sets in italics have not been described previously for CCM. (F) Representative confocal microscopy of IgG leakage in Pdcd10-wt (left) and Pdcd10-ko (right) brain sections. Bottom images: higher magnification of the cerebellum (light blue boxed areas at top). Higher magnification of the hippocampus and cortex are shown in Figure 2—figure supplement 1. Scale bars: 1 mm. Bottom panel: Quantification of IgG leakage (mean ± SEM; **p<0.01; ANOVA followed by Sidak multiple comparisons). Pdcd10-wt, n = 6; Pdcd10-ko, n = 7. (G) Heatmap of log fold expression changes of selected genes (padj <0.05) important for BBB formation and maturation (pink), and typical BBB transporters (green) between Pdcd10-ko and Pdcd10-wt. (H) Representative confocal microscopy of the venous marker COUP-TFII (encoded by the Nr2f2 transcript, white), Podocalyxin (pan-endothelial, green) and DAPI (blue) of a Pdcd10-wt vessel (top) and a Pdcd10-ko lesion (bottom), both in the cerebellum. Arrows, COUP-TFII–positive endothelial nuclei. Scale bars: 25 μm.

Figure 2—figure supplement 1. Pdcd10 deletion increases vessel permebility and impairs endothelial cell-to-cell junction organization.

(A) Representative confocal microscopy for IgG leakage in Pdcd10-wt (top) and Pdcd10-ko (bottom) brain sections, as in Figure 2F. Higher magnifications of hippocampus (red box) and cortex (black box) are shown on the right. Quantification of IgG leakage is shown in Figure 2F. (B) Heatmap of average logFC (Pdcd10-ko vs. Pdcd10-wt; p<0.05) of junctional molecule expression in the endothelial clusters (as indicated). (C–E) Representative confocal microscopy of adherens and tight junction markers VE-cadherin (C; encoded by Cdh5; right), Claudin-5 (D; right) and Cingulin (E; right) in Pdcd10-wt vessels and Pdcd10-ko lesions in the cerebellum. PECAM-1 staining is shown on left of each panel. Yellow arrowheads, regular junctions in Pdcd10-wt vessels; light blue arrows, altered junctions in Pdcd10-ko. Scale bars: 25 μm.