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Fig. s2
The amount of VE-cadherin at the junctions is tightly regulated and under acto-myosin dependent tension (a) Representative image a wild-type (ubs8+/+) Tg(ve-cad:ve-cadTS) animal (top) and a vecadherin mutant (ubs8-/-) Tg(ve-cad:ve-cadTS) animal showing that mutants are viable and morphologically indistinguishable from genotypic wild-type animals. Scale bar = 1 cm.(b) Quantification of the number of pixels within groups separated by increments of Venus intensity levels of 1000. Each data point is the number of pixels in each group. Pixels numbers are compared between heterozygous ubs8+/- (black, pixels in n=47 junctions selected from n=9 embryos) and homozygous ubs8-/- (grey, pixels in n=52 junctions selected from n=10 embryos) showing an improved detection range in the homozygous mutants. (c) Schematic representation of VE-cadherin-TS (top) and VE-cadherin-TL protein (bottom) which lacks the β-catenin binding domain (β-catenin=red, α-catenin=green, actomyosin= blue). (d) Heatmap image of ratio-metric FRET values in junctions expressing VE-cadherin-TS versus VE-cadherin-TL at 2 dpf. Colors range from blue (=low FRET index/high tension) to red (=high FRET index/low tension). Scale bar = 5μm. (e) Ratio-metric FRET values of VE-cadherin-TS (n=22 junctions from n=6 embryos) versus VE-cadherin-TL (n=21 junctions from n=6 embryos) at 2 dpf. Error bars represent mean ± s.d.; ****p<0.0001, from unpaired two-sided t-test. (f) Venus expression (grey) showing representative examples of junctional expression of VEcadherin- TS (left, black arrowheads) and aggregating VE-cadherin-TL expression (right, black arrowheads) in the DA upon DNA injections. Scale bar = 10μm. (g) Quantification of junctional versus non-junctional expression in cells expressing VEcadherin- TS (n=72 cells analysed) and VE-cadherin-TL (n=32 cells analysed) at 2 dpf. (h) Junctional morphology of ECs in the DA (Venus, grey) at 3 dpf in 1%DMSO and Y-27632 (45μM/1%DMSO) treated embryos. Scale bar = 10μm. (i) Junctional morphology of ECs in DA (Venus, grey) at 3 dpf in 0.5%DMSO and ML-141 (50μM/0.5%DMSO, Cdc42 inhibitor) treated embryos. Scale bar = 10μm. (j) Heatmap image of ratio-metric FRET values in junctions of 0.5%DMSO and ML-141 (50μM/0.5%DMSO) treated embryos at 3 dpf. Colors range from blue (=low FRET index/high tension) to red (=high FRET index/low tension). Scale bar = 5μm.(k) Ratio-metric FRET values in junctions from 0.5%DMSO treated controls (n=81 junctional ROIs from n=9 embryos) and ML-141 (50μM/0.5%DMSO) treated embryos (n=46 junctional ROIs from n=5 embryos) at 3 dpf. Error bars represent mean ± s.d.; DMSO – ML-141 ****p<0.0001, from unpaired two-sided t-test.