Live imaging molecular changes in junctional tension upon VE-cadherin in zebrafish

Abstract

Forces play diverse roles in vascular development, homeostasis and disease. VE-cadherin at endothelial cell-cell junctions links the contractile acto-myosin cytoskeletons of adjacent cells, serving as a tension-transducer. To explore tensile changes across VE-cadherin in live zebrafish, we tailored an optical biosensor approach, originally established in vitro. We validate localization and function of a VE-cadherin tension sensor (TS) in vivo. Changes in tension across VE-cadherin observed using ratio-metric or lifetime FRET measurements reflect acto-myosin contractility within endothelial cells. Furthermore, we apply the TS to reveal biologically relevant changes in VE-cadherin tension that occur as the dorsal aorta matures and upon genetic and chemical perturbations during embryonic development.

Fig. 1

VE-cadherin-TS is localized to cell-cell junctions and reports a differential FRET signature. a Schematic representation of the tension sensor (TS) module. The Donor fluorescent protein, Teal, is separated from the acceptor, Venus, by a stretchable linker peptide (top). When the module is under tension (bottom), energy transfer from Teal to Venus will decrease. b Schematic of zebrafish VE-cadherin-TS cDNA recombined into the ve-cadherin BAC clone. c Venus expression from Tg(ve-cad:ve-cadTS) throughout the blood vasculature at 2 dpf. Scale bar = 150 μm. d Maximum projection of the dorsal aorta (DA) at 3 dpf showing expression of Teal (top) and Venus (bottom) localized at cell–cell junctions. Scale bar = 10 μm. e Co-expression (Merge, bottom) of Venus (yellow, top) from Tg(ve-cad:ve-cadTS) and Tg(fli1ep:lifeact-mCherry), labeling F-actin (purple, middle), in endothelial junctions at 3 dpf. Scale bar = 5 μm. f–g Fluorescence intensity of Venus (f) and Tg(fli1ep:lifeact-mCherry), labeling F-actin (g), along a single 3 μm line-region in a single Z-section (shown in e). h RAW FRET signal at endothelial junctions at 2 dpf. Scale bar = 5 μm. i Ratio-metric FRET signal at 2 dpf in endothelial junctions (shown in h). Scale bar = 5 μm. j Variable ratio-metric FRET values along a single 10 μm line-region in a single Z-section in a proportion of the junction (boxed in i)

Fig. 2

VE-cadherin-TS is functional and under acto-myosin controlled tension. a Schematic representation of genetic cross to test functionality of VE-cadherin-TS. b Wild-type (top) and ve-cadherin mutant (bottom) phenotype at 2 dpf (arrowhead indicates cardiac oedema in mutant). Scale bar = 1 mm. c Phenotypic scoring all embryos (n = 430) collected from cross in a, showing 23% phenotypic mutants (n = 49/217) in TS-negative population (n = 217) and 0% (n = 0/213) in TS-positive siblings (n = 213). d Phenotypic scoring of n = 286 embryos from a cross between a Tg(ve-cad:ve-cadTS)^+/−, cdh5 ^ubs8−/− mutant and a non-transgenic cdh5 ^ubs8+/− animal. In the TS negative population 52% (n = 76/146) displayed the mutant phenotype whilst there were no phenotypic mutants in the TS positive clutch (n = 141). e Schematic representation of Y-27632 (ROCK inhibitor) acto-myosin inhibition. f Heatmap image of ratio-metric FRET values in junctions of 1%DMSO and Y-27632 (45 μM/1%DMSO) treated embryos. Colors range from blue ( = low FRET index/high tension) to red ( = high FRET index/low tension). Scale bar = 5 μm. g Quantification of ratio-metric FRET values in junctions of 1 % DMSO (n = 4) versus Y-27632 (45 μM/1%DMSO) (n = 5) treated embryos. Each data point represents a junctional region of interest (ROI), n = 33 junctions selected from DMSO control embryos and n = 40 selected in Y-27632 treated embryos. Treatment for 5 h prior to imaging. Error bars represent mean ± s.d.; ****p < 0.0001 from unpaired two-sided Mann–Whitney test. h Heatmap image of Teal lifetime values in junctions of 1%DMSO (top) and Y-27632 (bottom, 45 μM/1%DMSO)- treated embryos. Colors range from blue ( = low lifetime/low tension) to red ( = high lifetime/high tension). Scale bar = 5μm. i Quantification of Teal lifetime values (nano-sec) in junctions of 1 % DMSO (n = 4) versus Y-27632 (45 μM/1%DMSO) (n = 4) treated embryos. Each data point represents a single junctional ROI, n = 25 junctions segmented from DMSO control embryos and n = 33 from Y-27632 treated embryos. Treatment for 5 h prior to imaging. Error bars represent mean ± s.d.; **p = 0.0060 from unpaired two-sided t test

Fig. 3

Cellular morphology and VE-cadherin tensile changes occur during artery maturation. a Junctional morphology of ECs in the dorsal aorta (DA) (Venus, grey) at 2 dpf (top), 3 dpf (middle) and 4 dpf (bottom). Scale bar = 10 μm. b Junctional linearity index over time at 2 dpf (n = 37 junctions from n = 4 embryos), 3 dpf (n = 37 junctions from n = 5 embryos) and 4 dpf (n = 51 junctions from n = 4). Error bars represent mean ± s.d.; 2–3 dpf **p = 0.0010; 3–4 dpf ****p < 0.0001, from unpaired two-sided Mann–Whitney test. c Junctional angle relative to the direction of blood flow over time at 2 dpf (n = 53 junctions from n = 4 embryos), 3 dpf (n = 89 junctions from n = 5 embryos) and 4 dpf (n = 42 junctions from n = 4). Error bars represent mean ± s.d.; 2–3 dpf **p = 0.0010; 3dpf– 4dpf ****p < 0.0001 from unpaired two-sided Mann–Whitney test. d Heatmap image of ratio-metric FRET values in junctions at 2 dpf (top), 3 dpf (middle) and 4 dpf (bottom). Colors range from blue ( = low FRET index/high tension) to red ( = high FRET index/low tension). Scale bar = 5 μm. e Quantification of ratio-metric FRET values in junctions at 2 dpf (n = 54 junctional ROIs from n = 4 embryos), 3 dpf (n = 79 junctional ROIs from n = 5 embryos) and 4 dpf (n = 71 junctional ROIs from n = 4 embryos). Error bars represent mean ± s.d.; 2–3 dpf ****p < 0.0001, from unpaired two-sided t test; 3–4 dpf ****p < 0.0001 from unpaired two-sided Mann–Whitney test. f Quantification of Teal lifetime values (nano-sec) comparing n = 43 junctional ROIs segmented from 2 dpf embryos (n = 8) and n = 54 junctional ROIs from 4 dpf embryos (n = 10). Error bars represent mean ± s.d.; ****p < 0.0001 from unpaired two-sided Mann–Whitney test. g Heatmap image of Teal lifetime values in junctions of 2 dpf (left) versus 4 dpf (right) embryos. Colors range from blue ( = low lifetime/low tension) to red ( = high lifetime/high tension). Scale bar = 5 μm

Fig. 4

Inhibiting Vegf signaling impairs linearisation and VE-cadherin tension changes during arterial maturation. (a, b) Junctional morphology of ECs in the DA (Venus, grey) in uic (top) and kdr/kdrl morphants (bottom) at 2 dpf (a) and 3 dpf (b). Scale bar = 10 μm. c Number of ECs quantified at 2 dpf (uic n = 9, kdr/kdrl MO n = 6 embryos) and 3 dpf (uic n = 10, kdr/kdrl MO n = 7 embryos). Error bars represent mean ± s.d.; 2 dpf uic–2 dpf kdr/kdrl MO ****p < 0.0001 from unpaired two-sided t test, 3 dpf uic–3 dpf kdr/kdrl MO **p = 0.0068 from unpaired two-sided Mann–Whitney test. d Junctional linearity index of junctions in uic and kdr/kdrl morphants at 2 dpf and 3 dpf. Error bars represent mean ± s.d.; 2dpf uic–3dpf uic ****p < 0.0001; 2 dpf kdr/kdrl MO–3dpf kdr/kdrl MO, p = 0.0862 from unpaired two-sided Mann–Whitney test. e, f Ratio-metric FRET values of uic and kdr/kdrl morphants at 2 dpf e (uic n = 74 junctional ROIs from n = 9 embryos, kdr/kdrl MO n = 86 junctional ROIs from n = 6 embryos) and 3 dpf f (uic n = 62 junctional ROIs from n = 10 embryos, kdr/kdrl MO n = 58 junctional ROIs from n = 7 embryos). Error bars represent mean ± s.d.; ***p = 0.0007; *p = 0.0391 from unpaired two-sided t test. g Ratio-metric FRET values from 1% DMSO controls (n = 48 junctional ROIs from n = 6 embryos) versus SL327 (4 μM/1% DMSO)-treated embryos (n = 66 junctional ROIs from n = 10 embryos). Treatment from 20 hpf to 3 dpf. Error bars represent mean ± s.d.; ****p < 0.0001 from unpaired two-sided t test. h Junctional linearity index in 1% DMSO treated controls (n = 32 junctional ROIs from n = 6 embryos) versus SL327 (4 μM/1% DMSO)-treated embryos (n = 56 junctional ROIs from n = 10 embryos). Error bars represent mean ± s.d.; **p = 0.0045 from unpaired two-sided Mann–Whitney test. i Junctional morphology of ECs in the DA (Venus, grey) at 3 dpf in 1% DMSO and SL327 (4 μM/1% DMSO) treated embryos at 3 dpf. Scale bar = 10 μm. j Ratio-metric FRET values from uic (n = 45 junctional ROIs from n = 8 embryos) versus kdr/kdrl morphants incubated in 1% DMSO as a control (n = 43 junctional ROIs from n = 9 embryos) and kdr/kdrl Y-27632-treated morphants (50 μM/1% DMSO, n = 40 junctional ROIs from n = 9 embryos) at 2 dpf. Error bars represent mean ± s.d.; uic–kdr/kdrlMO + DMSO ****p < 0.0001; kdr/kdrlMO + DMSO – kdr/kdrlMO + Y-27632 ***p = 0.0005 from unpaired two-sided t test

Fig. 5

Calcium signaling maintains lower VE-cadherin tension during artery maturation. a Heatmap image showing average intensity projection of fli1a-driven GCaMP5G expression (indicating calcium) in the DA at 2 dpf (left) and 4 dpf (right). Colors range from black ( = low calcium) to white ( = high calcium). Scale bar = 10 μm. b Increase in fluorescence intensity index ( = fli1a driven GCaMP5G/ fli1a-driven RFP) in the dorsal wall of the DA of n = 9 embryos imaged at 2 and 4 dpf. **p = 0.0012, from paired t test. c Junctional morphology of ECs in the DA (Venus, grey) of a 0.5% DMSO, a 15 μM/0.5% DMSO Nifedipine and a 25 μM/0.5% DMSO Nifedipine treated embryo at 4 dpf. Treatment from 2 to 4 dpf. Scale bar = 10 μm. d Ratio-metric FRET values in junctions of 0.5% DMSO (n = 42 junctional ROIs from n = 5 embryos), 15 μM Nifedipine (n = 54 junctional ROIs from n = 6 embryos) and 25 μM Nifedipine (n = 59 junctional ROIs from n = 6 embryos) treated embryos. Treatment was from 2 to 4 dpf. Error bars represent mean ± s.d.; DMSO – 15 μM Nifedipine **p = 0.0068, DMSO – 25 μM Nifedipine ****p < 0.0001, from unpaired two-sided Mann–Whitney test.e Junctional linearity index in 0.5% DMSO treated controls (n = 5) versus 25 μM Nifedipine-treated embryos (n = 6). Each data point represents a junction, n = 18 control junctions measured and n = 18 junctions from Nifedipine-treated embryos. Error bars represent mean ± s.d.; not significant (ns) p = 0.8147, from unpaired two-sided Mann–Whitney test. f Ratio-metric FRET values quantified from 1% DMSO-treated controls (n = 39 junctional ROIs from n = 6 embryos), BAPTA-AM-treated (100 μM for 1 h, n = 58 junctional ROIs from n = 8 embryos), Nifedipine-treated (50 μM for 30 min, n = 27 junctional ROIs from n = 5 embryos) and Nifedipine + Y-27632-treated embryos (n = 47 junctional ROIs from n = 8 embryos). Error bars represent mean ± s.d.; 1% DMSO – 100 μM BAPTA-AM *p = 0.0498; 1% DMSO – 50 μM Nifedipine ****p < 0.0001; 50 μM Nifedipine - 50 μM Nifedipine + 50 μM Y-27632 ***p = 0.0008, from unpaired two-sided t test