Studies of embryogenesis conducted with genetic model organisms have proved invaluable to our understanding of how cells are assembled into tissues. Studies of Drosophila melanogaster, in particular, have led to the identification of key genes and biological principles that direct the morphogenesis and development of organisms from fertilization to adulthood1,2,3. In parallel to the genetic research conducted with Drosophila, cultured cell lines derived from fruit fly tissues have also emerged as a powerful system to address a wide range of molecular and cell biological questions4,5,6. Drosophila tissue culture cells have minimal requirements for maintenance, as they are cultured at room temperature and without CO2. As such, they are amenable to high-resolution imaging, and they have a high degree of susceptibility to gene inhibition by RNAi6,7. Many groups have used Drosophila tissue culture cells as a tool to discover genes involved in defining cell shape, cytoskeletal dynamics, viability, phagocytosis, and signal transduction pathways4,8,9,10,11. When employed as a model alongside the whole animal, cultured Drosophila cell lines offer a set of very complementary approaches that accelerate the identification of molecules important for the development and provide a framework in which to determine their mechanistic roles12. Here, we describe a cell-based assay to study the signaling pathways that trigger apical constriction via the folded-gastrulation (Fog) pathway13,14. This cell-based contractility assay allows researchers to investigate both the Fog signaling pathway and the molecular mechanisms that regulate non-muscle myosin II contractility.
Gastrulation of the early Drosophila embryo has been studied for many years as a genetic model for the epithelial morphogenesis and the cellular transition from epithelial to mesenchymal cell identity. One of the key events of gastrulation is the morphogenesis of a subset of epithelial cells along the embryonic ventral midline from columnar to pyramidal in shape15,16,17,18. This simple cell shape change results in the internalization of the presumptive mesodermal cells and is driven by the motor activity of non-muscle myosin II constricting the actin network16,19,20. Decades of genetic research has identified the molecular components of this pathway and has sequentially placed them in the following order: 1) Fog is secreted from the apical domain of the epithelial cells at the ventral midline; 2) Fog binds to the G-protein-coupled co-receptors, Mist and Smog, and signals through a heterotrimeric G-protein complex containing the Gα12/13 subunit Concertina (Cta), which is chaperoned by the non-canonical GEF Ric-8; 3) Cta activates a guanine nucleotide exchange factor, RhoGEF2, which in turn activates the small G-protein Rho1; 4) Rho1 activates Rho kinase (Rok); 5) Rok activates non-muscle myosin II contractility at the apical domain through phosphorylation of the regulatory light chain (RLC), thus producing apical constriction (Figure 1)15,21,22,23,24,25,26,27,28,29. Mutations in several of these components interfere with the normal gastrulation and other morphogenetic movements, including the formation of the wing disc and salivary glands, indicating that this pathway is used at several stages of Drosophila embryogenesis30,31,32. The Fog pathway is one of the best-studied models for epithelial shaping and has provided important insights into how tissue-level morphogenesis is regulated from gene transcription to cytoskeleton-driven cell movements14,15,21.
We developed a cell-based contractility assay that recapitulates many of the cellular responses downstream of Fog that have been observed in developing fly embryos17. We engineered a stable S2 cell line that expresses Fog tagged at its C-terminus with myc under the control of an inducible metallothionine promoter that can be harvested upon the addition of copper sulfate (CuSO4) to the medium. When Fog-conditioned media is applied ectopically to S2 Receptors+ (S2R+) cells, which are a subline of S2 cells distinguished by their differential expression of receptors such as Frizzled and integrin subunits, the cells undergo a reorganization of the cytoskeleton highly reminiscent of apical constriction12,17,27,33. These changes can be observed by phase-contrast microscopy in which Fog treatment leads to the appearance of phase-dark ruffles indicative of a radial increase in non-muscle myosin II contractility, or by fluorescence microscopy where Fog treatment leads to the formation of non-muscle myosin II rings in cells expressing EGFP-tagged RLC34. These rings contain a myosin-phosphorylated regulatory light chain (pRLC) visible via immunostaining23,34,35. This Fog-induced response required Cta, RhoGEF2, Rho, and Rok; thus, using recombinant Fog-Myc and S2R+ cells, we have established a means to investigate Fog-induced constriction in a tissue-culture-based system24,25,34.