Dissection of <em>Drosophila melanogaster</em> Indirect Flight Muscles for Microscopy Approaches

The indirect flight muscles (IFMs) of Drosophila melanogaster are a powerful genetic model to explore foundational principles of myogenesis. The contractile mechanism and many sarcomere components are conserved from flies to vertebrates, enabling the study of cellular processes from transcriptional regulation and RNA processing to metabolism and mechanobiology that direct and fine-tune muscle development. Many of these cellular pathways are altered in human myopathies, and IFM studies provide relevant insight into the molecular etiology of muscle disease. In particular, flies are well-suited for microscopy to analyze myofiber and sarcomere morphology and function across the entire process of IFM myogenesis, from myoblast specification to myofibril formation, maturation, and maintenance. Here, a protocol is presented for the dissection of D. melanogaster IFMs at pupal and adult timepoints for microscopy approaches. Illustrated protocols are provided for hemithorax dissection of late pupal and adult IFMs, as well as open-book dissection of early pupal IFMs. Fixation, staining, and sample mounting procedures and common dissection artifacts are described, with representative data demonstrating the compatibility of IFM dissection with different fixation reagents. These protocols are applied to study the hypomorphic Smn^E33 allele of RNA-binding protein survival motor neuron (Smn)at 26 h after puparium formation (APF), 72 h APF, and in adult IFMs, providing new insight into a Spinal Motor Atrophy (SMA) model and illustrating the general utility of this protocol. This detailed protocol facilitates access to the IFM model system and acquisition of high-quality microscopy data to investigate principles of myogenesis.