Live Imaging of Drosophila Myoblast Fusion

There are 30 individual muscles per hemisegment of the Drosophila embryo. Formation of these individual body wall muscles depends on the specification and fusion of two myoblast cell types, founder cells (FCs) and fusion-competent myoblasts (FCMs) (discussed in greater detail in Chapter X). Each FC contains the necessary information to direct the formation of a specific muscle. FCs can be identified by expression of identity genes, such as the transcriptional regulators even-skipped, apterous, slouch and Kruppel. The combination of identity genes expressed by a particular FC is thought to regulate the final morphology of the specific muscle. FCMs, in contrast, are thought to be naive cells. Upon fusion to an FC, FCMs become reprogrammed to the FC’s specific developmental program, as witnessed by each newly incorporated FCM nucleus expressing the FC’s particular combination of identity genes (1–5). Myoblast fusion is a reiterative process; depending on the particular muscle, body wall muscles in Drosophila embryos arise from between 2 and 25 fusion events (6). Fusion occurs during stages 12 to 15 [7.5–13 hrs after egg laying (AEL)]. FCs/myotubes and FCMs are arranged in multiple cell layers prior to and during the fusion process [Figure 1; (7)]. As fusion commences, the mesoderm is arranged with the FCs occupying both the most external and internal positions with multiple layers of FCMs found in between (Figure 1A). As germband retraction and dorsal closure proceed during stages 13 and 14, the ventral FCs/myotubes and FCMs move externally to lie underneath the epidermis and central nervous system (Figure 1B–C). While some FCMs contact the FCs/myotubes and are responsible for the initial fusion events, the remaining FCMs are located more internally and must migrate to find their fusion partners. An appreciation of these cell arrangements and movements is essential for the analysis of myoblast fusion. Figure 1 FC and FCM arrangements and fusion profile of individual muscles Fusion does not start concurrently in all muscles [Figure 1D; (7)]. For example, fusion begins during stage 12 (7.5–9.5 hrs AEL) in the dorsal DA1 muscle, but does not begin until stage 13 (9.5–10.5 hrs AEL) in the ventral VA2 muscle. However, for all muscles examined to date, the majority of fusion events occur during stage 14 (10.5–11.5 hrs AEL; Figure 1D), making this a particularly useful stage for the analysis of the cellular biology underlying myoblast fusion. While genetic analysis has revealed a number of genes required for the fusion process (reviewed in Chapter x), their precise function during fusion has been hampered by the lack of direct, cellular assays. For example, while many of the known genes encode regulators of the actin cytoskeleton (8), the impact of cytoskeletal rearrangements on fusion were unclear. Furthermore, the arrangements of FCs and FCMs (7) have implicated a critical role of migration in the fusion process that remains to be analyzed. Lastly, the site of fusion has only been implied by localization of proteins implicated in fusion and not directly located. To address these issues, we developed live imaging techniques in Drosophila to further our understanding of the myoblast fusion process. This chapter deals with the methodology and considerations underlying this approach including collection and mounting of the embryos for live imaging, as well as the imaging itself and techniques for processing and presenting the data.