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. 2022 Nov 28;220(2):e20211390. doi: 10.1084/jem.20211390

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© 2022 Nielsen et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure S1. — Additional characterizations of brain AVM established by P16 and P21. (A and B) Postnatal time is depicted along the timeline. (A) Constitutive activation of Notch4*^tetEC from birth led to features of brain AVM by P16 and to moribundity/death by P36. (B) Endothelial deletion of Rbpj from P16 attenuated features of Notch4*^tetEC induced brain AVM and increased the time to moribundity/death. (C) Normal AV connections were defined as <12.5 μm diameter (white box); moderately enlarged AV connections were defined as 12.5–30 μm diameter (gray box); severely enlarged AV connection were defined as >30 μm diameter (black box). In P16 control brains, 100% of AV connections were of normal diameter (N = 3 mice, 88 connections). In P16 Notch4*^tetEC brains, 72.6 and 8.8% of AV connection diameters were moderately and severely enlarged, respectively (N = 4 mice, 113 connections). Two independently repeated experiments. (D) Percentage of heart weight/total body weight was calculated for control (1.00 ± 0.3%, N = 3 mice) and Notch4*^tetEC (1.03 ± 0.15%, N = 3 mice) mice at P16, following Notch4*^tetEC activation from birth. No significant difference was seen among cohorts, suggesting that cardiomegaly did not develop by P16. P = 0.8796. Two independently repeated experiments. (E and F) Tie2-tTA perfused whole brain did not show evidence for cerebrovascular hemorrhages (N = 5) or vessel tortuosities (N = 3) at P21. (G) Flat-mount immunostaining against endothelial marker CD31 and smooth muscle cell marker αSMA showed normal αSMA expression restricted to arteries and absent from AV connections (arrowheads; N = 5). (H and I) Hemorrhages (arrowheads; N = 6) and enlarged and tortuous vessels (N = 3) were present in perfused Tie2-tTA;TRE-Notch4* brains at P21. (J) CD31 and αSMA immunostaining showed AV shunting (arrowheads) and veins with αSMA expression in P21 cortex from Tie2-tTA;TRE-Notch4* brains (N = 6). (K) Average diameter of AV connections was higher in Tie2-tTA;TRE-Notch4* mice (N = 6) at P21 as compared to Tie2-tTA negative controls (N = 5). P < 0.0001. (L) Body weight was decreased in Tie2-tTA;TRE-Notch4* mice (N = 20) at P21, as compared to Tie2-tTA controls (N = 22). P = 0.0002. (M) Percent of heart weight/body weight was higher in Tie2-tTA;TRE-Notch4* mice (N = 4) at P21, as compared to Tie2-tTA negative controls (N = 6). P = 0.0045. a, artery; v, vein. E–J, K, and M from four independently repeated experiments and L from 14 independently repeated experiments. Scale bars: 2 mm in E, F, H, and I, 100 μm in G and J. **P<0.01; ***P<0.001; ****P<0.0001.