Blood flow controls vascular remodeling of the trunk. Panels in (b) and (d) show lateral images of zebrafish embryos at 60 hpf with anterior side facing left. Venous ECs are labelled with mCitrine and arterial ECs are labelled with mCitrine and tdTomato. Orange arrows indicate the direction of blood flow, red arrows point to arterial ISVs and red brackets highlight regions of venous ISVs without arterial ECs. Scale bars are 25??m. The numbers are averages ± SEM from at least three independent experiments with a minimum of n?=?25 animals per conditions per experiment. P?

Displacement of arterial ECs by venous ECs in venous ISVs. Lateral images of zebrafish embryos with anterior side facing left. Orange arrows indicate the direction of blood flow through the ISVs. Scale bars are 25??m. a?c Representative images from four independent experiments. Venous ECs are labelled with lifeactCherry and arterial ECs are labelled with lifeactGFP and lifeactCherry. a Stills from Supplementary Movie 2. Red arrows point at an arterial EC migrating in a venous ISV, ventral part. b Stills from Supplementary Movie 2. Red arrows point at an arterial EC migrating in a venous ISV, dorsal part. c Stills from Supplementary Movie 4. Red arrows point at an arterial EC in an arterial ISV, ventral part. d?f Representative images from three independent experiments. 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 the posterior cardinal vein (PCV) (d), a venous ISV (e) or an arterial ISV (f)

Upstream polarization and migration of ECs under flow. a Lateral images of zebrafish embryos with anterior side facing left. Orange arrows indicate the direction of blood flow through the ISVs. All ECs express nuclear-GFP and mCherry-fused marker of the Golgi. Planar polarization of ECs in ISVs is measured by the vector connecting the nucleus with the Golgi. Scale bar is 25??m. b Quantification of EC planar polarization in venous and arterial ISVs (n?=?10 embryos). c Positions of microtubule organization complexes (MTOCs) and nuclei in individual HUVECs and instantaneous velocities of HUVECs in a microfluidic perfusion chamber were monitored for 300?min after the exposure to flow with a shear stress of 7.2?dyn/cm2. Blue dashes show the values of the polarization angle, ?, with 90° corresponding to polarization against the flow and ??=??90°?polarization along the flow. Red circles show the values of the migration angle, ?*, with 90° corresponding to migration against the flow and ??90°?migration along the flow. Grey line (ordinate on the right) show the average cell migration velocity in the upstream direction. d Phase images from Supplementary Movie 6 showing confluent HUVECs after 10?h under laminar flow with shear stresses of 0.23 and 14.5?dyn/cm2. Scale bar is 100?µm. e Average velocities of upstream migration for HUVECs exposed to different shear stresses as functions of time after the inception of shear flow (n?=?250 to 600 for individual shear stresses). f Average velocities of upstream migration as functions of time after the inception of shear flow (n?=?250). Arrows at the bottom (colors correspond to those of the velocity data points) indicate the time points at which the migration of cells against the flow becomes statistically significant (average upstream velocity becomes positive with p?

Blood flow promotes EC migration in veins but not in arteries. Panels (c), and (e) show lateral images of zebrafish embryos with anterior side facing left. Orange arrows indicate the direction of blood flow. a Velocity of erythrocytes as a function of time within one heart beat (n?=?16 vessels per condition; average of large number of heart beats per vessel). b Schematic representation of the dynamics of blood flow in the intersegmental vasculature. c All ECs express nuclear-localized GFP. Intersegmental vessels (yellow arrows) serve as reference points for determining the location of tracked arterial (red ovals) and venous (blue ovals) ECs. Scale bar is 30??m. d Average displacements of ECs in the PCV (blue, n?=?42, 6 embryos) and DA (red, n?=?25, 6 embryos) as functions of time. Displacement of ECs was analyzed with at least 6 venous and 4 arterial ECs per embryo. Displacement is considered positive, if the cell migrates upstream. e All ECs express photo-convertible (green-to-red) fluorescent protein DENDRA2. The photo-conversion was done at 28 hpf in the DA and PCV. Red arrows point to an arterial EC and blue arrows point to a venous EC. Scale bar is 25??m

Arterial blood flow activates Notch signaling in ISVs. Panels (a?d) show lateral images of zebrafish embryos with anterior side facing left. ECs express lifeact-mCherry. Notch signaling is reported by the expression of destabilized GFP (d2GFP) under the control of 12xCSL Notch responsive elements. Red arrows point to arterial ISVs. Orange arrows indicate the direction of blood flow. White arrowheads highlight ISVs without blood flow. Scale bars are 25??m. The numbers are averages ± SEM. P?

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??m. All numbers are averages ± SEM from at least three independent experiments with a minimum of n?=?25 animals per conditions per experiment. P?

Summary Model. This model explains how blood flow-induced Notch signaling and endothelial migration enable the remodeling of all-arterial intersegmental vasculature into a network with ~1:1 ratio of arteries and veins