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Fig. 6 Notch signaling protects ISVs from transforming into veins. All images are representative from at least three independent experiments. Panels in (a), (b), (d), (e) and (g) show lateral images of zebrafish embryos with anterior side facing left. Embryos in (a), (b), (e) and (g) are 60 hpf. Red arrows point to arterial ISVs and red brackets highlight regions of venous ISVs without arterial ECs. Orange arrows indicate the direction of blood flow. Scale bars are 25 μm. All numbers are averages ± SEM from at least three independent experiments with a minimum of n = 25 animals per conditions per experiment. P < 0.001. a Inhibition of Notch signaling at different time points of the development. Venous ECs are labelled with mCitrine and arterial ECs are labelled with mCitrine and tdTomato. Red brackets highlight regions of venous ISVs without arterial ECs. b Ectopic expression of the Notch Intracellular Domain (NICD) specifically in arterial ECs. Venous ECs are labelled with lifeactCherry and arterial ECs are labelled with lifeactGFP and lifeactCherry. c The percentage of venous ISVs in embryos with perturbed Notch signaling. (At least three independent experiments with a minimum of n = 25 animals per conditions per experiment). d The inhibition of Notch does not affect EC migration in an arterial ISV. All ECs express the photo-convertible (green-to-red) fluorescent protein DENDRA2. Red brackets highlight ECs with photo-converted DENDRA2. The photo-conversion was done at 30 hpf in an arterial ISV. e The effect of inhibition of Notch on the number of ECs in ISVs. Arterial ECs express mCitrine, tdTomato and nuclear-Cherry, and venous ECs express mCitrine and nuclear-Cherry. Red brackets depict venous ECs in venous ISVs. f Number of arterial and venous ECs in arterial and venous ISVs in experiments with inhibition of Notch. g Notch inhibition does not affect EC polarization in ISVs. All ECs express nuclear-GFP and mCherry-fused marker of the Golgi. Planar polarization of ECs in ISVs is measured by a vector connecting the nucleus with Golgi. h Quantification of EC planar polarization in venous and arterial ISVs in experiments with inhibition of Notch