Double transgenic visualizing endothelial membrane (red) and endothelial nuclei (green). Endothelial nuclei (arrowhead) were observed close to but never within the kugel (unfilled arrowhead).
Double transgenic showing endothelial membrane (red) and endothelial F‐actin (green). F‐actin was found to localize at the neck of the
kugeln (arrowhead; see also:
Movie EV3).
Double transgenic showing endothelial membrane (red) and endothelial cytoplasm (green). The cytoplasmic reporter was visible in the parent vessel but not in the kugel (unfilled arrowhead).
Triple transgenic showing endothelial membrane (red), neurons (green) and erythrocytes (red). Surrounding neurons were excluded from the volume of the kugel (unfilled arrowhead), and erythrocytes were not observed inside kugeln (arrowhead).
Treatment with the inhibitor of actin polymerization Latrunculin B statistically significantly increased number of kugeln per embryo (100 nM 1 h; **P = 0.0041; control n = 21 embryos 8.05 ± 1.76 (mean ± s.e.m.); Latrunculin n = 21 embryos 17.19 ± 2.93 (mean ± s.e.m.); 4 dpf; three experimental repeats; Mann–Whitney U‐test).
Latrunculin B treatment statistically significantly reduced kugel diameter (*P = 0.0164; control n = 169 kugeln from 21 embryos 7.56 ± 2.25 (mean ± s.e.m.); Latrunculin n = 361 kugeln from 21 embryos 5.91 ± 1.78 (mean ± s.e.m.); 4 dpf; three experimental repeats; Student's t‐test).
Treatment with the Myosin II inhibitor Blebbistatin statistically significantly reduced number of kugeln per embryo (25 μM 1 h; ****P < 0.0001; control n = 22 embryos 3.77 ± 0.56 (mean ± s.e.m.), Blebbistatin n = 24 embryos 1.08 ± 0.22 (mean ± s.e.m.); 3 dpf; three experimental repeats; Mann–Whitney U‐test).
Blebbistatin treatment had no effect on kugel diameter (P = 0.3731; control n = 83 kugeln from 22 embryos 6.27 ± 0.72 (mean ± s.e.m.), Blebbistatin n = 26 kugeln from 24 embryos 7.02 ± 0.89 (mean ± s.e.m.); 3 dpf; three experimental repeats; Mann–Whitney U‐test).
