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Fig. 2 ECs within the posterior circulatory loop of the fin vasculature become arterialized and activate cxcr4a and Notch signalling. (A) Still images at indicated time points of ECs sprouting from the PCV in Tg(fli1a:nEGFP)y7; Tg(-0.8flt1:RFP)hu5333 double-transgenic wild-type control animal, labelling EC nuclei (green) and arterial ECs (red). White arrowheads with numbers indicate individual ECs. Time-encoded migration tracks of marked ECs. t0=52 hpf. Schematic representation of sprout formation; white arrows indicate the direction of blood flow. Scale bar: 50 μm. (B) Still images at indicated time points of ECs sprouting from PCV in Tg(fli1a:nEGFP)y7; Tg(-0.8flt1:RFP)hu5333 double-transgenic cxcr4aum20 mutants. White arrowheads with numbers indicate individual ECs. Schematic representation of observed phenotype in cxcr4a mutants. Black arrowheads indicate clustering of arterial ECs at the tip of the sprout. Scale bar: 50 μm. (C) Maximum intensity projections of confocal z-stacks of Tg(fli1a:nEGFP)y7; Tg(-0.8flt1:RFP)hu5333; Tg(Tp1:Venus-Pest)s940 triple-transgenic fish labelling all EC nuclei (blue), arterial ECs (red) and cells with Notch pathway activation (green) in lateral views; anterior towards the left. White arrowheads indicate arterial ECs with activated Notch signalling. Boxed regions are enlarged. Scale bars: 20 μm (top row); 4 μm (middle and bottom rows). (D) Still images at indicated time points of ECs sprouting from PCV in Tg(fli1a:nEGFP)y7; Tg(cxcr4a:YFP)mu104; Tg(-0.8flt1:RFP)hu5333 triple-transgenic embryos labelling all EC nuclei (blue), arterial ECs (red) and cxcr4a-positive cells (green) in lateral views; anterior towards the left. White arrowheads indicate ECs that express cxcr4a and show onset of arterial specification. Boxed regions are enlarged. Scale bars: 20 μm (top row); 4 μm (middle and bottom rows).