Figure 1. CCMs are composed of clonally dominant ECs labeled with a single confetti color.
(A) Experimental mice contain three transgenes: endothelial-specific, tamoxifen-inducible cre recombinase with platelet derived growth factor b promoter (PDGFb-iCreERT2), loxP-flanked and null Ccm3 alleles (Ccm3fl/KO), and R26R-Confetti reporter allele. (B) Cre recombination of a single R26R-Confetti allele leads to fluorescent labeling with one of four different constructs: nuclear GFP, cytoplasmic YFP, cytoplasmic RFP, and membrane-bound CFP. (C) Experimental design of a single, low dose (2μg) of tamoxifen injected into neonatal pups on postnatal days 3, 4, or 5. (D) Ccm3fl/KO, PDGFb-iCreERT2, R26R-Confettifl/wt experimental mice develop a modest CCM burden in the cerebellum and cerebral hemispheres (n=3, scale bar: 2mm). (E) A representative 80-μm thick brain slice with discrete CCMs dispersed throughout the cerebellum as indicated by arrows (scale bar: 1 mm). (F-I) 3D reconstructions of Z-series confocal images acquired across multiple serial brain slices to visualize the entire volume of CCMs in Ccm3fl/KO, PDGFb-iCreERT2, R26R-Confettifl/wt mice. Gray translucent surfaces have been added to the images to aid in visualizing the vascular lumens of each CCM. Representative CCMs for (F) YFP, (G) nGFP, (H) mCFP, and (I) RFP (scale bars: 100μm). (J) Number of ECs expressing each confetti color in serial brains slices of non-CCM control mice (PDGFb-iCreERT2, R26R-Confettifl/wt, n=1) and CCM mice (Ccm3fl/KO, PDGFb-iCreERT2, R26R-Confettifl/wt, n=3). (K) Number of each confetti color expressed by ECs lining individual CCMs (n=42). CCMs #40, #13, #18, and #s 5–8 are the CCMs shown in panels F, G, H, and I respectively. (L) Percentage of the clonally dominant ECs, labeled with the most common confetti color within a CCM, when considering all confetti labeled ECs within the CCM (94+9 (mean + SD), n = 42).