Dissecting Collective Cell Behavior in Migrating Testis Myotubes in Drosophila.

Collective cell migration has a key role in tissue morphogenesis, wound healing, tissue regeneration, and cancer invasion. In recent years, different animal models have been established to analyze how chemical and mechanical stimuli shape the behavior of single cells into tissues and organs. At present, there are still only a few model systems that allow to genetically dissect underlying molecular mechanisms driving cell motility during tissue morphogenesis at high resolution in real time. Here, we provide a detailed protocol and toolbox for ex vivo culturing of Drosophila testes for 4D live imaging of myotube collective migration, which allows to genetically address a wide range of developmental and cell biological questions regarding modes of filopodia-based protrusion/locomotion, cell-cell adhesion, cytoskeletal modes of collective decision-making, and collective closure processes. Additionally, this protocol has been successfully used in combination with laser-induced single-cell ablation and pharmacological treatments, but it can also be used with confocal microscopy after tissue fixation.

Cell biology: Keeping the epithelium together when your neighbor divides.

The dramatic cell-shape changes involved in mitosis and cell division challenge the integrity of epithelial tissues. A new study reveals a surprising role for atypical protein kinase C in keeping apical contractility in balance and thus preventing epithelial disruption.

Collective cell migration driven by filopodia-New insights from the social behavior of myotubes.

Collective migration is a key process that is critical during development, as well as in physiological and pathophysiological processes including tissue repair, wound healing and cancer. Studies in genetic model organisms have made important contributions to our current understanding of the mechanisms that shape cells into different tissues during morphogenesis. Recent advances in high-resolution and live-cell-imaging techniques provided new insights into the social behavior of cells based on careful visual observations within the context of a living tissue. In this review, we will compare Drosophila testis nascent myotube migration with established in vivo model systems, elucidate similarities, new features and principles in collective cell migration.

Filopodia-based contact stimulation of cell 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 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. We perform genetic, pharmacological and mechanical perturbation analyses to reveal the essential roles of Rac2, Cdc42 and Rho1 in myotube migration. These factors differentially control protrusion dynamics and cell-matrix adhesion formation. We also show that active Rho1 GTPase localizes at retracting free edge filopodia and that 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. Based on these findings, 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.

Serpent/dGATAb regulates Laminin B1 and Laminin B2 expression during Drosophila embryogenesis.

Transcriptional regulation of Laminin expression during embryogenesis is a key step required for proper ECM assembly. We show, that in Drosophila the Laminin B1 and Laminin B2 genes share expression patterns in mesodermal cells as well as in endodermal and ectodermal gut primordia, yolk and amnioserosa. In the absence of the GATA transcription factor Serpent, the spatial extend of Laminin reporter gene expression was strongly limited, indicating that Laminin expression in many tissues depends on Serpent activity. We demonstrate a direct binding of Serpent to the intronic enhancers of Laminin B1 and Laminin B2. In addition, ectopically expressed Serpent activated enhancer elements of Laminin B1 and Laminin B2. Our results reveal Serpent as an important regulator of Laminin expression across tissues.

Myotube migration to cover and shape the testis of Drosophila depends on Heartless, Cadherin/Catenin, and myosin II.

During Drosophila metamorphosis, nascent testis myotubes migrate from the prospective seminal vesicle of the genital disc onto pupal testes and then further to cover the testes with multinucleated smooth-like muscles. Here we show that DWnt2 is likely required for determination of testis-relevant myoblasts on the genital disc. Knock down of fibroblast growth factor receptor (FGFR) heartless by RNAi and a dominant-negative version revealed multiple functions of Heartless, namely regulation of the amount of myoblasts on the genital disc, connection of seminal vesicles and testes, and migration of muscles along the testes. Live imaging indicated that the downstream effector Stumps is required for migration of testis myotubes on the testis towards the apical tip. After myoblast fusion, myosin II is needed for migration of nascent testis myotubes, in which Thisbe-dependent fibroblast growth factor (FGF) signaling is activated. Cadherin-N is essential for connecting these single myofibers and for creating a firm testis muscle sheath that shapes and stabilizes the testis tubule. Based on these results, we propose a model for the migration of testis myotubes in which nascent testis myotubes migrate as a collective onto and along the testis, dependent on FGF-regulated expression of myosin II.