Fig. 5: Migrating myotubes need cell–cell contact to achieve directionality.

a–g Isolation of a single nascent myotube by laser ablation. a Overview of a testis after laser ablation (33 h APF). htl-Gal4 drives UAS-LifeAct-EGFP expression. Scale bar, 100 µm. b Close-up on the ablation site. c Same site as in b, before ablation. Scale bar in c and c’: 20 µm. The dashed line represents the area affected by laser ablation. c’ Behavior of the isolated cell from b after ablation. The isolated cell (yellow asterisk) shows no forward motion if it has no contact to adjacent cells (upper row). After contact is established, it moves along in the migrating sheet (bottom row). d, e To quantify the directionality of the isolated cell, cell motion was tracked using the Imaris software. The isolated cell before contacting to the migrating sheet is depicted in red, after contacting it is depicted in green. As a control, adjacent cells were tracked. They are showed in blue. f, g As a measurement tool, we used the biased angle to x-axis. The mean angle (0–180°) of every track is blotted. When isolated, cells lose their directionality, but regain it after establishing contact to adjacent cells. N = 5 testes with 38 cells (adjacent cells), 5 cells (after ablation, before contact), and 5 cells (after ablation, after contact). Boxplot center: median. Bounds: 25th and 75th percentiles. Whiskers: minimum/maximum values. *P ≤ 0.05, **P  ≤ 0.01, ***P  ≤ 0.001, ****P  ≤ 0.0001. Statistical testing: one-way ANOVA. The color code is the same as in e, f N = 5 testes. h–j Isolation of two adjacent myotubes by laser ablation. h Overview of a testis after laser ablation (33 h APF). htl-Gal4 drives LifeAct-EGFP (gray) and Myr-RFP (magenta) expression. The dashed line represents the area affected by laser ablation. Scale bar, 100 µm. h’ Behavior of the two isolated myotubes from h after ablation. Scale bar in c and c’: 20 µm. i Rose plot shows the distribution of the biased angle to x-axis. j Measurement of the distance between two myotubes over time.