Extended Data Fig. 9: iFlp^{MTomato-cre/MYfp} genetic mosaics reveal the long term fate of Rbpj^KO and Rbpj/Myc^DKO ECs in different organs.

a, Schematic of the Apln-FlpO and iFlp^{MTomato-cre/MYfp} alleles used to induce full loss-of-function genetic mosaics in the mouse heart, retina, liver and aorta. b, Lineage qRT–PCR analysis of Tomato⁺ and YFP⁺ cells isolated from Apln-FlpO;iFlp^{MTomato-cre/MYfp};Rbpj^f/f and Apln-FlpO; iFlp^{MTomato-cre/MYfp};Rbpj^f/f;Myc^f/f E15.5 hearts. Note that Apln-FlpO recombines the alleles at an early stage and that mutant cells differentially segregate throughout development, leading to gene expression changes at E15.5 that may reflect not only the regulation by Rbpj and Myc (shown in Extended Data Fig. 8) but also their segregational arteriovenous fate. c, Apln-FlpO;iFlp^{MTomato-cre/MYfp};Rbpj^f/f;Myc^f/f mouse survival rate. d, Expression of Apln-HA-FlpO in retina vessels at P6 shows the localization of HA-FlpO in the nuclei of angiogenic front ECs, and not in arteries. e, Schematic of recombination of the Apln-FlpO and iFlp^{MTomato-cre/MYfp} alleles in the retina angiogenic front at P2 and distribution of MbTomato⁺ and MbYFP⁺ cells at P6. f–h, Representative confocal images of P6 retinas from mice with the indicated genotypes, and immunostained for isolectinB4, Tomato/Dsred and YFP. Arteries are easily distinguishable from veins by their relatively stronger IsolectinB4 signal and morphology/diameter. i, Quantifications shown are complementary to those in Fig. 4g and allow the direct comparison of significance in the differential distribution of Tomato⁺ cells only within arteries, or only within capillaries, or only within veins, without taking into account their distinct capillary frequencies and developmental proliferation bias. The results indicate that Rbpj/Myc^DKO ECs are less frequently found in capillaries, and therefore proliferate less, but have a normal ability of forming arteries, unlike Rbpj^KO ECs, that occur at a relatively high frequency in capillaries and at very low frequency in arteries (see also log₂-transformed fold change indicated in Fig. 4g). j, k, Sectional analysis of control and mutant genetic mosaics of embryonic, newborn and adult hearts showing the very low frequency or absence of Tomato⁺/mutant cells in coronary arteries of Rbpj^flox/flox hearts, and their presence in the coronary arteries of control and Rbpj^flox/flox/Myc^flox/flox hearts. For quantifications of adult hearts data, see Fig. 4e. l, Sectional analysis of control and mosaic mutant adult livers showing portal arteries (PA) and portal veins (PV) and respective quantifications in these oxygen-rich (DLL4⁺ and EFNB2⁺) liver portal vessels. m, Confocal imaging of the inner surface of aortas, stained for CD144 (VE-cadherin), showing the relative occurrence of control and mutant cells and chart with the respective quantifications. 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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