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.