RCAN1.4 regulates endothelial cell sprouting and vascular formation in zebrafish. a In vitro sprouting angiogenesis assay. HDMECs were transfected with control non-targeting siRNA (N.S. siRNA) or RCAN1 siRNA prior to incubation with cell tracker dye green or orange. Cells were mixed 1:1 and incubated with carboxymethycellulose in ULA 96-well plates for 18 h prior to embedding in collagen matrix. Cells were stimulated with VEGF (50 ng/ml) for 18 h and fixed in 2% PFA prior to fluorescence imaging. b Quantification of endothelial tip and stalk cell formation. Sprouting tubes from VEGF treated spheroids were quantified (mean ± S.D., n = 3). ***P < 0.001 (unpaired Student’s t test). c Transgenic fli1-GFP zebrafish embryos were injected with mismatch or RCAN1a-4 morpholino oligonucleotides and imaged at 48 hpf. Scale bar represents 500 μm. d Analyses of total vessel length and junction number, n = 14 for mismatch, n = 15 for RCAN morpholino. Asterisk indicates P < 0.01 (unpaired student’s t test)