Fig. 1: Tertiary sprouts emanate from veins, and consist of a single lumenized endothelial cell that remodels into an anastomotic connection. | Nature Communications

Fig. 1: Tertiary sprouts emanate from veins, and consist of a single lumenized endothelial cell that remodels into an anastomotic connection.

From: Parenchymal cues define Vegfa-driven venous angiogenesis by activating a sprouting competent venous endothelial subtype

Fig. 1

a Confocal images of Tg(−0.8flt1:RFP;flt4:mCitrine) showing overview and high magnification of trunk vasculature. Dotted arrows indicate flow direction; Dotted box shows ROI. b Trunk vasculature in WT, flt1−/− and vhl−/− mutants. Arrowheads indicate ectopic sprouts. Dotted line indicates the spinal cord. c Confocal images showing blood vessels (green) and spinal cord neuronal cells (red) in WT (left) and flt1−/− mutant (middle). 3D reconstruction of a confocal stack in flt1−/− mutant (right). Arrowheads indicate tertiary sprouts. d Tertiary sprout branching from vISV during initiation (left), elongation (middle) and upon anastomosis formation (right). Arrowheads indicate nuclei. e FITC-Dextran labelled blood plasma shows lumenized tertiary sprout at initiation stage (left) and upon anastomosis formation (right). f Confocal imaging of Tg(fli1:PECAM1a-EGFP) upon flt1 loss of function (LOF). g Endothelial junctional morphology in the lumenized endothelial tip cell. Arrowheads indicate finger-like structures. h Finger-like structure number per sprout upon flt1 LOF. Percentage (top) and Mean±s.e.m. (bottom); n = 10 sprouts/embryos. i–k Confocal images of pericyte (green) distribution surrounding blood vessels (blue arrowheads) during tertiary sprout initiation (i), elongation (j) and anastomosis formation (k) in flt1−/− mutant. Yellow arrowhead indicates pericytes at the anastomotic-aISV connection. l Confocal imaging of actin reporter in WT and flt1−/− mutant, arrowhead indicates tertiary sprout. m Endothelial surface area for indicated scenario. Violin plot shows median and interquartiles; two-sided Mann–Whitney U test, WT: n = 74 cells (13 embryos), flt1−/− vISV: n = 116 cells (13 embryos), flt1−/− sprout: n = 28 cells (13 embryos). p < 0.0001 for all comparison. n, o Confocal imaging of Tg(kdrl:Hsa.HRAS-mcherry;fli1:PECAM1a-EGFP), segmentation and 3D rendering of primary sprout tip cell (n) and tertiary sprout volume (o). p Cell volume and surface area for indicated scenario. Mean ± s.e.m., two-sided Mann–Whitney U test, n = 7 tip cells/embryos and 6 L-Tip cells/embryos. p(volume) = 0.0221, p(surface) = 0.0350. Scale bars indicate 50 µm in b, c; 20 µm in d, f, g, i–l; 25 µm in e, n, o. DA dorsal aorta, SC spinal cord, DLAV dorsal lateral anastomotic vessel, aISV arterial intersegmental vessel, vISV venous intersegmental vessel, PCV posterior cardinal vein, dpf days post fertilization, LOF loss of function, EC endothelial cell, PS primary sprouting, TS tertiary sprouting. Source data are provided as a Source Data file.

Back to article page