Inhibition of endothelial PKA increases the motility of endothelial cells and their probability to become a tip cell. (A) Tg(fli1a:eGFP)^y1 zebrafish embryos were injected with a plasmid encoding RFP (as control) or PKI-mCherry fusion protein (left). Localization of the cells expressing red fluorescent protein was determined at 29 hpf. Mosaic ISVs (containing both transfected and untransfected cells) were used to identify whether transfected cells were in a tip or stalk cell position. RFP and PKI-mCherry signals are in black and white on the left panel and in red on the overlay image (middle); GFP signal is green on the overlay image. Ratio of tip-to-stalk cells for RFP-expressing (control) and PKI-mCherry-expressing (PKI) cells (right). For RFP and PKI, 15 and 26 embryos were analyzed, respectively. The number of analyzed cells is indicated on the figure (Chi-square test: P<0.05). (B) Filopodia in endothelial cells expressing control fluorescent protein or PKI-mCherry fusion were analyzed in fish embryos. Shown are representative images of cells expressing control fluorescent protein (RFP; left) or PKI-mCherry (PKI; middle) and quantification of filopodia number per cell (right). For RFP and PKI, 20 and 29 embryos were analyzed, respectively. The number of analyzed cells is indicated on the figure. Boxes encompass values between the 25th and 75th percentiles, centered lines represent median, mean is indicated by +, whiskers and outliers are calculated using the Tukey method (*P<0.05). (C) Tg(Kdrl:Ras-mCherry)^s916 embryos were injected with plasmid expressing PKI-GFP fusion under the control of endothelial fli1ep promoter and vascular development was monitored from 29 to 42 hpf. Membrane-bound mCherry fluorescent protein (black in left panel and red on the right) depicts higher mobility and excessive filopodia formation in PKI-GFP-expressing cells (green) compared with the wild-type GFP-negative cells. The approximate area imaged is depicted on the insert showing the zebrafish embryo.