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Schönbauer, Cornelia (2013): Genetic analysis of Drosophila adult muscle type specification. Dissertation, LMU München: Faculty of Chemistry and Pharmacy

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Muscles of all higher animals comprise different muscle types adapted to perform distinct functions in the body. These express different sets of genes controlled by distinct combinations of transcriptional programs and extracellular signals, and thus differ in their myofibrillar organization and contractile properties. Despite major progress in our understanding of myogenesis, the genetic pathways controlling the formation and function of different muscle types are still largely uncharacterized. Flying insects possess specialized flight muscles enabling wing oscillations with frequencies of up to 1000 Hz together with high power outputs of 80 W per kg muscle. To achieve these parameters, flight muscles contain stretch-activated myofibrils with a unique fibrillar organization, whereas all other, more slowly contracting muscles, such as leg muscles, display a tubular morphology. To delineate the genetic regulation of muscle development and function, and, in particular, muscle type specification, we performed a genome-wide RNA interference (RNAi) screen in Drosophila, in which we systematically inactivate genes exclusively in muscle tissue. We uncovered more than 2000 genes with putative roles in muscles, many of which we were able to assign to specific functions in muscle, myofibril or sarcomere organization by phenotypic characterization. Muscle-specific knockdown of 315 genes resulted in viable, but completely flightless animals, indicating a specific function of those genes in fibrillar flight muscles. Detailed morphological analysis of these 315 genes revealed a striking phenotype upon knockdown of the zinc finger transcription factor spalt major (salm): the fibrillar flight muscles are switched to tubular muscles, whereas tubular leg muscles are wild type, demonstrating that salm is a key determinant of fibrillar muscle fate. We could show that the transcription factor vestigial (vg) acts upstream of salm to induce its expression specifically in fibrillar flight muscles. Importantly, salm is not only required but also sufficient to induce the fibrillar muscle fate upon ectopic expression in other muscle types. Microarray analysis, comparing mRNA expression from adult wild-type flight and leg muscles to salm knockdown flight muscles, indicates that salm instructs most features of fibrillar muscles by regulating both gene expression as well as alternative splicing. Remarkably, we could show that spalt’s function in programming stretch-activated fibrillar muscles is conserved in insect species separated by 280 million years of evolution. Interestingly, in mouse two of the four spalt-like (sall) genes are expressed in heart, a stretch-activated muscle, sharing some features with insect fibrillar flight muscles. Since heart abnormalities observed in patients suffering from the Towns-Brocks syndrome are caused by a mutation in SALL1, it is possible that Spalt’s function to determine a fibrillar, stretch-modulated muscle type is conserved to vertebrates.