A guide to study Drosophila muscle biology
Introduction
Muscles enable higher animals to move actively. Muscle contractions can be very strong and long lasting, as is the case for the adductor muscles that close the shells of a clam, or can be very fast and accurate as in rapidly manoeuvring bats. Despite these functional differences, the principle organisation of the contractile apparatus in muscles of higher animals is evolutionarily conserved. Thus, muscle development can be studied in model organisms like Drosophila to better understand and eventually treat human myopathies.
Drosophila is a powerful genetic model to functionally investigate muscle development. It allows the study of all major developmental steps during muscle morphogenesis and offers a rich repertoire of functional muscle tests. Here, we provide a comprehensive collection of experimental protocols, as well as genetic and molecular markers to study morphogenesis and function of both, Drosophila embryonic and adult body muscles.
Section snippets
Histology
A powerful technique to investigate muscle morphogenesis is immunohistochemistry, which can be applied at all stages of the fly’s life cycle. In the following, we provide an overview of the most commonly used preparation methods and introduce selected marker proteins that can be used to visualise key steps of muscle development.
Live imaging of muscle development
Muscle morphogenesis is a highly dynamic process. Thus, live imaging of muscle development can be very informative, particularly if wild type and mutant animals can be compared. Many of the endogenous GFP fusions listed in Table 1 can be used for live imaging, however it is often more practical to use GAL4 induced overexpression of GFP effector molecules due to the resulting increase in brightness. Useful GAL4 lines and GFP effectors are listed in Table 2. One should be aware that any imaging
Muscle function
Larvae use their body muscles mainly for locomotion, either spontaneously or oriented towards a food attractant. Adult flies use their muscles to walk, jump or fly. Any of these behaviours can be used as a measure for muscle function. Moreover, the large IFMs or the jump muscles can be dissected from adults and used for direct mechanical measurements in a force meter. The researcher should be aware that any behavioural experiment needs to be carefully controlled. For example genetic background,
Muscle genetics
All Drosophila tissues, including muscle, can be functionally investigated with the large plethora of genetic tools available in the fly. Classical chemically induced zygotic loss of function screens identified various key regulators of muscle development. To efficiently screen for muscle morphogenesis mutants, it is convenient to use living embryos with fluorescently labelled balancer chromosomes, such as CyO, Kr-GAL4, UAS-GFP and a muscle-specific GFP marker, like Mhc-TAU-GFP [40], [54].
Concluding remarks
Drosophila is a very powerful model to mechanistically investigate all aspects of muscle morphogenesis. Recent developments of imaging and mechanical measurement techniques, together with systematic genetics using RNAi libraries should transform Drosophila into a leading model for muscle research in the 21st century. As many aspects of muscle biology are conserved from insects to vertebrates, mechanistic insights in Drosophila should be important for vertebrate muscle biology.
Acknowledgements
We thank Belinda Bullard and Cornelia Schönbauer for the myofibril dissection protocol. We are grateful to Aynur Kaya-Çopur, Cornelia Schönbauer and Maria Spletter for insightful comments on this manuscript. This work was supported by the Max Planck Society, the European Research Council and a Career Development Award by the Human Frontier Science Programme to F.S.
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