Fig. 3: Tertiary sprouting upon Vegf gain of function requires aplnra.

a Confocal images showing trunk vasculature in flt1^−/− and flt1^−/−; aplnra^−/− double mutant at 4 dpf. Dotted box indicates ROI; arrowheads indicate ectopic vessels. b Quantification of branching complexity for indicated scenario. Violin plot shows median and interquartiles; two-sided Unpaired t test with Welch’s correction, flt1^−/−: n = 36 and flt1^−/−; aplnra^−/−: n = 26; p = 0.0001. c Arterial to venous ISV ratio for indicated genotype. Mean±s.e.m.; two-sided Fisher’s exact test. flt1^−/−: n = 36, flt1^−/−; aplnra^−/−: n = 26. d Confocal images showing trunk vasculature in vhl^−/− mutant injected with control or aplnra targeting morpholino at 4 dpf. Dotted box indicates ROI, Arrowheads indicate ectopic vessels. e Branching complexity for indicated scenario. Violin plot showing median and interquartiles; two-sided Unpaired t test, control MO: n = 14; aplnra MO: n = 28; p < 0.0001. f Single cell sequencing pipeline. GFP+ cells were sorted from Tg(kdrl:EGFP) embryos, captured, bar-coded and sequenced. g UMAP plot of GFP+ sorted cells. Square indicates endothelial cell clusters. h UMAP plot of GFP+ sorted cells, color coded by expression level for tie1 and cdh5. i UMAP plot of EC subclusters. j Violin plot showing the expression of marker genes used to identify the individual EC subclusters. k Dot-plot showing aplnra and aplnrb expression for indicated endothelial subclusters. l Imaging of trunk vasculature in flt1^−/− and flt1^−/− injected with flt4-GFP-p2A-PTXS1. Dotted box indicates ROI; arrowheads indicate ectopic vessels. m Quantification of ectopic sprouts for indicated scenario. Mean±s.e.m., two-sided Mann-Whitney U test, flt1^−/−: n = 30; flt1^−/−+flt4-GFP-p2A-PTXS1: n = 37; p = 0.0006. n Arterial to venous ISV ratio for indicated scenario. Mean±s.e.m. Two-sided fisher’s exact test. flt1^−/−: n = 30; flt1^−/−+flt4-GFP-p2A-PTXS1: n = 37. o Confocal images showing trunk vasculature in flt1^−/− mutant treated with DMSO or with Dasatinib at 3 dpf. p Quantification of ectopic sprouts for the indicated scenario. Mean±s.e.m.; two-sided Mann-Whitney U test, flt1^−/− + DMSO: n = 8, flt1^−/− + Dasatinib: n = 11; p = 0.0393. Scale bars indicate 50 µm in a, d, l, o. MO morpholino, EC endothelial cells, LEC lymphatic endothelial cell, vEC venous endothelial cell, cap. venous capillaries venous, cap. arterial capillaries arterial, aEC arterial endothelial cell, pEC proliferating EC, EdC endocardial cell, PTXS1 Pertussis-Toxin subunit 1, DA dorsal aorta, PCV posterior cardinal vein. Source data are provided as a Source Data file.