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

Abstract

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (<48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (>48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis. Video Link The video component of this article can be found at https://www.jove.com/video/60309/ Drosophila melanogaster is another well-established genetic model organism. The structure of the sarcomere as well as individual sarcomere components are highly conserved from flies to vertebrates 4,9,10 , and the indirect flight muscles (IFMs) have become a powerful model to study multiple aspects of muscle development 11,12. First, the fibrillar flight muscles are functionally and morphologically distinct from tubular body muscles 11,13 , allowing investigation of muscle-type specific developmental mechanisms. Transcription factors including Spalt major (Salm) 14 , Extradenticle (Exd), and Homothorax (Hth) 15 have been identified as fibrillar fate regulators. Additionally, downstream of Salm, the CELF1 homolog Bruno1 (Bru1, Aret) directs a fibrillar-specific splicing program 16,17. Second, IFMs are an important model for understanding the process of myogenesis itself, from myoblast fusion and myotube attachment to myofibrillogenesis and sarcomere maturation 9,18,19. Third, Drosophila genetics permits investigation of contributions by individual proteins, protein domains, and protein isoforms to sarcomere formation, function, and biophysical properties 20,21,22,23. Lastly, IFM models have been developed for the study of...

Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals

“…To place the eMIC splicing program within the broader AS landscape of Drosophila , we reanalysed published transcriptomic datasets 38 (Table S1) using vast-tools , and searched for AS exons with tissue-level regulation. Similar to previous reports 42, 48, 49 , we found that neural samples showed the highest prevalence of both tissue-enriched and -depleted exons (Supp. Figure 4E).…”

Natural variation in the regulation of neurodevelopmental genes modifies flight performance inDrosophila

“…Lasp (human ortholog LASP1), is the only nebulin family gene in Drosophila, and shown to modify sarcomere and thin filament length, and myofibril diameter [88]. We also identified bruno 1 (bru1 or aret; human homolog CLEF1 and CLEF2), a transcription factor that controls alternative splicing of myofibrils in the IFM [9,89], among other developmental processes. bru1…”

“…Annotations and unreferenced descriptors of genes' functions, expression profiles, and orthologs were gathered from autogenerated summaries on FlyBase [34,35]. These summaries and descriptors were compiled from data supplied by the Gene Ontology Consortium [36,37], the Berkeley Drosophila Genome Project [38], FlyAtlas [39], The Alliance of Genome Resources Consortium [40], modENCODE [34], PAINT [41], the DRSC Integrative Ortholog Prediction Tool (DIOPT) [42], and several transcriptomics and proteomic datasets [9,12,39,[43][44][45][46]. threshold (gray line).…”

“…In an evolutionary context, purifying selection would act on flight-critical genes more strongly than flight-important genes, meaning flight-important genes can harbor natural variants that might otherwise vary the flight phenotype. These genes are found across systems, including metabolism [8], muscle function [9], neuronal function [10,11], and anatomical development [12,13]. Genes filling multiple roles across systems are pleiotropic, and those with sufficient natural variation are likely to contribute to complex traits and disease [14,15].…”

Natural variation in the regulation of neurodevelopmental genes modifies flight performance in Drosophila

“…Genes in the network are connected by edges representing various interaction types (gene-gene, protein-protein, epistatic, etc.). In the case of flight performance, for example, central genes like Wingless [8] and Act88F [9] are essential for wing and indirect flight muscle development, respectively, while peripheral genes would have more subtle effects on flight from systems like metabolism [10], muscle function [11], neuronal function [12,13], and anatomical development [14,15]. Peripheral genes are less likely to experience the same degree of purifying selection as central genes, meaning they are more likely to harbor natural variants that can have subtle effects on phenotype.…”