Filopodia-based contact stimulated collective migration drives tissue morphogenesis

Cells migrate collectively to form tissues and organs during morphogenesis. Contact inhibition of locomotion (CIL) drives collective migration by inhibiting lamellipodial protrusions at cell-cell contacts and promoting polarization at the leading edge. Here, we report on a CIL-related collective cell behavior of myotubes that lack lamellipodial protrusions, but instead use filopodia to move as a cohesive cluster in a formin-dependent manner. Genetic, pharmacological and mechanical perturbation analyses reveal essential roles of Rac2, Cdc42 and Rho1 in myotube migration. They differentially control not only protrusion dynamics but also cell-matrix adhesion formation. Here, active Rho1 GTPase localizes at retracting free edge filopodia. Rok-dependent actomyosin contractility does not mediate a contraction of protrusions at cell-cell contacts but likely plays an important role in the constriction of supracellular actin cables. We propose that contact-dependent asymmetry of cell-matrix adhesion drives directional movement, whereas contractile actin cables contribute to the integrity of the migrating cell cluster.

APF, myotubes cover the whole pupal testis as a thin muscular sheet 22 . 137 To better understand how myotubes cover the testis, we established a protocol for ex 138 vivo organ cultivation and long-term imaging (7 h) of isolated 33h APF pupal testes 139 ( Figure 1c). We used the muscle-specific mef2-Gal4 or the heartless-Gal4 (htl-Gal4) 140 driver to express a UAS-LifeAct-EGFP transgene either in myotubes or in both, S2g). Thus, the Arp2/3-WRC pathway promotes motility, but seems to be 204 dispensable for directed migration of myotubes.      such as WAVE and the Rac-effector Sra-1, resulted into more moderate 353 morphological defects compared to rac2 or cdc42 depletion. Adult testes deficient for 354 Arp3, WAVE and Sra-1 still had about 1.5 to 2 coils, however many myotubes also 355 did not reach the testis apex resulting into bulky tips (Supplementary figure S1h-j).   In this study, we established a new model system for studying collective cell 440 migration in organ culture that allows high-resolution long-term live-imaging 441 microscopy combined with genetic, pharmacological, and mechanical perturbation 442 analysis. Our data implies that a contact-dependent migration mechanism acts as a 443 driving force to polarize Drosophila myotubes and to promote their directional 444 movement along the testes. A contact-stimulated migration has been already 445 observed in cultured cells many years ago, but the molecular mechanisms underlying 446 this phenomenon has been never analyzed in more detail 45 . Thomas

Microscopy/4D live cell imaging of testicular nascent myotubes 591
Fixed pupal testes were embedded in Fluoromount-G (SouthernBiotech) and imaged 592 on object slides. Adult testes were imaged in live-culture dishes in PBS, to maintain 593 their natural shape. Light micrographs were taken with a Leica M165 FC stereo 594 microscope equipped with a Leica DFC7000 T CCD camera. All fluorescent 595 microscopic stills were taken with a Leica TCS SP8 with a HC PL APO CS2 20x/0.75 596 dry objective. 4D live cell imaging was performed on developing testes of 33 h APF 597 pupae. Prepupae were collected and timed as described elsewhere 30 . Life imaging 598 of pupal testes was performed like on egg chambers, as described before 62 . Images 599 were taken on a Zeiss Observer.Z1 with a Yokogawa CSU-X1 spinning disc scanning 600 unit and an Axiocam MRm CCD camera (6.45 µm x 6.45 µm). Long-term imaging 601 was performed using a LD LCI Plan-Apochromat 25x/0.8 Imm Korr DIC oil-immersion 602 objective over 7 h, with a z-stack every 5 min. Close-ups were taken with a C Plan-603 Apochromat 63x/1.4 oil-immersion objective over 2 h, with a z-stack every 2 min. 604 Laser ablation of single cells on the testis was performed with a Rapp TB 355 laser. 605 606

Chemical inhibitors 607
Live imaging experiments with chemical inhibitors were performed exactly as 608

Data processing and quantification of 4D life image stacks 639
Manual tracking of migrating myotubes was performed using the spots-module in the 640 Imaris 9.3 software. For drift correction, the reference frame module was used. The 641 x-axis was positioned as axis from the genital disc to the testis hub. Excel was used 642 for all processing and quantification. Distance on X is defined as the difference 643 between the x-Values of the same track at t=0 and t=7 h on unprojected and 644 unsmoothed 3D-data. It was used as a measuring tool instead of speed, as 645 fluctuations in manual tracking strongly affects velocity especially in slow cells.

Neighbor permanency is defined as
Neighbors 647 are defined as the 6 closest cells to a given cell at t=0. A value of 1 means, that all 648 neighbors were kept.
A new y-coordinate is generated using the formula: Fig. S2 C-E).  Fig. S3 E-F). 676 For the very first point the formula is: For all further points : Track speed mean was measured in motility lab using smoothed tracking data, in 680 order not to quantify manual tracking inaccuracies. 681 Biased angle to x-axis. The usual "biased angle" method measures the bias 682 towards a predefined point. As myotubes do not migrate towards a point, but along a 683 defined axis, we measured the angle-distribution to the x-axis to analyze myotube 684 directionality. As angles get strongly affected by speed, this method can only 685 compare cells with the same "distance on x" value (summarized in Fig. S3 F). Rose 686 plots were generated in R using the ggplot2 package. formula. The median for all tracks on the testis was calculated. We thank the Bloomington Stock Center and VDRC for fly stocks. We thank Thomas 733 Lecuit for providing the Rho1-biosensor. We thank Susanne Önel for providing the 734 To assess, how far cells were able to migrate on the testis, the difference of x values 898 (x-axis = defined as the axis from base to apex) of testis myotubes at t = 0 min and t= 899 420 min was calculated. The mean of each testis was compared. Upon N-Cadherin 900 reduction, myotubes come as far as in WT. Source data are provided as a Source 901 Data file. p. As a tool for directionality, biased angle in regard to the testis axis was 902 measured. Datasets were smoothed and Mercator-projected before (see also 903 supplementary figure S3). The mean angle (0-180°) of every track is blotted. N-904 Cadherin reduction causes myotubes to migrate less directional. The same is true 905 using a second RNAi line. Source data are provided as a Source Data file. q. 906 Quantification of track speed mean in µm/sec. RNAi line #1 is subject to wider 907 fluctuation but not significantly faster. RNAi line #2 is significantly faster than wild 908 type (WT). Source data are provided as a Source Data file. d, e. To quantify the directionality of the isolated cell, cell motion was tracked using 920 the Imaris software. The isolated cell before contacting to the migrating sheet is 921 depicted in red, after contacting it is depicted in green. As a control, adjacent cells 922 were tracked. They are showed in blue. Source data are provided as a Source Data 923 file. f, g. As a measurement tool, we used the biased angle to x-axis. The mean 924 angle (0-180°) of every track is blotted. When isolated, cells lose their directionality, 925 but regain it after establishing contact to adjacent cells. The color code is the same 926 as in e, f. n= 5 testes. Source data are provided as a Source Data file. h-j. Isolation