Figure 4. Pdcd10-ko cells of C9 express the same top mitotic phenotype as overt cavernomas.

(A) Plot of fold-change in numbers of Pdcd10-ko versus Pdcd10-wt cells in each cluster. (B) Plots of fold-change in the percentages (Pdcd10-ko vs. Pdcd10-wt) of cells positive for Mki67, Plk1, Mxd3, Birc5, and Ube2c in each cluster (as indicated). Dashed lines in (A) and (B) show upper limit of 95% confidence interval (CI), calculated from the mean fold-changes among the clusters. Red lines, clusters with fold-change >95% CI upper limit. (C) Heatmap of average logFC (padj <0.05) in the Pdcd10-ko vs. Pdcd10-wt for selected mitotic markers (as indicated). (D) Representative confocal microscopy of Pdcd10-wt and Pdcd10-ko mouse cerebellum at P8. Upper panels: Immunostained for PECAM-1 (pan-endothelial, blue), ERG (pan-endothelial nuclei, red) and Ki-67 (encoded by Mki67 transcript; mitotic nuclei, green). Central panels: Immunostained for ERG (green) with outlines of the segmented nuclei (magenta). Lower panels: Results of the filtering procedure showing EC-specific immunostaining of Ki-67 (ECs Ki-67, light blue) and PECAM-1 (red) (see Materials and methods). Arrows, mitotic ECs. Scale bars: 100 μm. (E) Quantification of mitotic ECs in the cerebellum, as normal vessels in Pdcd10-wt, Pdcd10-ko pseudo-normal vessels, and Pdcd10-ko lesions (mean ± SEM; *p<0.01; ANOVA followed by Tukey’s multiple comparisons). Pdcd10-wt, n = 6; Pdcd10-ko, n = 6. (F) Visium analysis showing spots positive for ECs (yellow) and lesion EC markers (red), identified as indicated, with co-expression of Mki67. Proliferating ECs (light blue) and proliferating lesion ECs (green) containing spots are shown. Higher magnifications of boxed areas are also shown. (G) Quantification of the percentages (%) of proliferating ECs (light blue) and proliferating lesion ECs (green) containing spots as in (F) (Mean; *p<0.01 Fisher’s exact test).

Figure 4—figure supplement 1. Validation of Visium analysis and identification of spots co-expressing endothelial and CCM lesion markers.

(A) Quality control of Visium analysis showing identification of the distinct areas of the representative Pdcd10-wt and Pdcd10-ko brain sections, as indicated. Identification was carried out using the Allen Brain Atlas (http://atlas.brain-map.org/) (see Material and methods for details). With the exception of Pdcd10-wt mouse #1 (second row), 9 (K-means) Visium clusters were necessary to obtain correct identification. Color coding of Visium clusters is shown superimposed on hematoxylin and eosin staining. (B) Representative Visium analysis for positive spots for ECs (yellow) and lesion ECs (red), identified at low (left) and high magnification (boxed areas), as indicated. Green dashed line, lesions in hematoxylin and eosin staining (right panel). (C) Quantification of proportions (%) of endothelial cell positive spots co-expressing lesion endothelial cell markers, as illustrated in (B).