Extended Data Fig. 2: Ectopic Notch activation is compatible with vein development and impairs coronary artery development.

a, Genetic construct used to generate mice with Cre-dependent conditional expression of MbTomato, H2B-GFP and N1ICDP in Tie2-Cre⁺ coronary vessels which results in increased expression of canonical Notch target genes. b, Bright-field images of E12.5 and E15.5 embryos and hearts showing the appearance of microcirculatory defects at E15.5. c, Statistical analysis of mouse survival rate per stage indicates that most mutant mice do not pass embryonic stage E16.5. d, Cardiovascular development defects in N1ICDP^Tie2-cre hearts may result in tissue hypoxia and the observed upregulation of Vegf mRNA. e, f, Myocardial EC proliferation analysis. g, h, CD31 staining of control and N1ICDP^Tie2-cre heart sections showing vascular defects and thinner myocardial wall (inset) in mutant hearts. i–m, Analysis of the heart surface coronary vein diameter and plexus development and density. n, Confocal images showing immunostaining of the heart surface venous plexus for the arterial marker CX40. ECs with high Notch activity (Tomato⁺) do not express CX40 and are able to form veins. o, Confocal images of E14.5 heart veins showing that Tomato/N1ICDP⁺ cells (yellow arrowheads) express EPHB4 like wild-type cells (white arrowheads). p, Confocal images of E14.5 heart veins showing that GFP⁺/N1ICDP⁺ cells (yellow arrowheads) express COUP-TFII like GFP⁻/wild-type cells (white arrowheads). q, Chart showing the quantification of COUP-TFII immunosignals intensity in 280 GFP⁺ and 280 GFP⁻ cells present in the veins of 3 independent N1ICDP^Tie2-cre hearts (2 veins per heart were quantified). r, Schematic of the mouse postnatal retina vascular development, in which tamoxifen was induced at P1, when the vessels start to grow, and analysis was carried at P6. s–v, Cells with induced high Notch activity (MbTomato⁺ or H2B-GFP⁺, yellow arrowheads) can differentiate and form veins like wild-type cells (white arrowheads). The Tomato⁺/N1ICDP⁺ cells found in veins do not express the arterial marker CX40, and instead express the venous markers VEGFR3, COUP-TFII and EPHB4. w, Quantification of signals (in pictures like shown in s–v) indicate no change in the expression of arteriovenous genes. x, Analysis of coronary artery development. y, z, Confocal images of heart sections and quantifications of immunosignals in the myocardium showing an increase in CX40, DLL4 and SM22α in N1ICDP^Tie2-cre myocardium vessels. Data shown as mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 100 μm. For statistics, see Supplementary Data 1.

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