Background

While Drosophila embryonic muscles have long been an established model system for the study of muscle development (Volk, 1999; Chen and Olson, 2004; Abmayr et al., 2008; Richardson et al., 2008) the adult Drosophila indirect flight muscles (IFMs), which form during pupal stages, have emerged in recent years as a complementary system to address cell-biological processes during myogenesis (Dutta, 2006; Oas et al., 2014; Weitkunat et al., 2014; Shwartz et al., 2016). Their large size, ample fusion events, structural similarity to vertebrate muscles, and amenability to powerful genetic techniques of the Drosophila system make the IFMs an attractive system to study muscle development. Historically, study of IFM development has been limited compared to embryonic muscle for several reasons. First, classic genetic approaches are difficult to implement in IFMs due to functional requirements earlier in development for many of the genes potentially involved in muscle development in the adult and to the syncytial nature of muscles, which restricts the usefulness of clonal analysis. Relatively recent advances in the available tools utilizing the GAL4-UAS system allow circumvention of these limitations, by expressing RNAi in a tissue specific manner. The power of this approach was demonstrated in a comprehensive screen for genes involved in pupal myogenesis (Schnorrer et al., 2010), and has been successfully implemented in several recent studies of myoblast fusion in IFMs (Mukherjee et al., 2011; Gildor et al., 2012; Dhanyasi et al., 2015; Segal et al., 2016). In addition, the technical challenges associated with dissection, accessibility, and visualization of IFMs have been overcome by advances in techniques and technology (Weitkunat and Schnorrer, 2014; Segal et al., 2016). This protocol is an expanded version of the methods used in the manuscript by (Segal et al., 2016), and is intended to contribute to the growing repertoire of techniques for study of IFMs. Here we describe methods for live imaging of IFMs, both in intact pupae, and in isolated IFMs ex-vivo. While previous work focused on stages of myotube growth via fusion (18-22 h after puparium formation [APF], 25 °C), these methods are readily applicable to other stages of IFM myogenesis, starting at 12 h APF onwards. Imaging of intact pupae can be suitable for studies of developmental processes which span several hours, while imaging of ex-vivo cultures is intended to better visualize finer structural details and dynamic behaviors over shorter time periods (e.g., 1 h). Parts of this protocol are variations on (Weitkunat and Schnorrer, 2014).