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Myogenesis pp 331-348 | Cite as

RNA Interference Screening for Genes Regulating Drosophila Muscle Morphogenesis

  • Aynur Kaya-Çopur
  • Frank SchnorrerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1889)

Abstract

RNA interference (RNAi) is the method of choice to systematically test for gene function in an intact organism. The model organism Drosophila has the advantage that RNAi is cell autonomous, meaning it does not spread from one cell to the next. Hence, RNAi can be performed in a tissue-specific manner by expressing short or long inverted repeat constructs (hairpins) designed to target mRNAs from one specific target gene. This achieves tissue-specific knock-down of a target gene of choice. Here, we detail the methodology to test gene function in Drosophila muscle tissue by expressing hairpins in a muscle-specific manner using the GAL4-UAS system. We further discuss the systematic RNAi resource collections available which also permit large scale screens in a muscle-specific manner. The full power of such screens is revealed by combination of high-throughput assays followed by detailed morphological assays. Together, this chapter should be a practical guide to enable the reader to either test a few candidate genes, or large gene sets for particular functions in Drosophila muscle tissue and provide first insights into the biological process the gene might be important for in muscle.

Key words

Drosophila Muscle Sarcomere RNAi GAL4-UAS Genetic screens Off-target effects 

References

  1. 1.
    Mohr SE, Perrimon N (2011) RNAi screening: new approaches, understandings, and organisms. WIREs RNA 3:145–158.  https://doi.org/10.1002/wrna.110CrossRefPubMedGoogle Scholar
  2. 2.
    Boutros M, Ahringer J (2008) The art and design of genetic screens: RNA interference. Nat Rev Genet 9:554–566.  https://doi.org/10.1038/nrg2364CrossRefPubMedGoogle Scholar
  3. 3.
    Kaya-Copur A, Schnorrer F (2016) A guide to genome-wide in vivo RNAi applications in Drosophila. Methods Mol Biol 1478:117–143.  https://doi.org/10.1007/978-1-4939-6371-3_6CrossRefPubMedGoogle Scholar
  4. 4.
    Roignant J-Y, Carré C, Mugat B, Szymczak D, Lepesant J-A, Antoniewski C (2003) Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila. RNA 9:299–308CrossRefGoogle Scholar
  5. 5.
    Dietzl G, Chen D, Schnorrer F, Su K-C, Barinova Y, Fellner M et al (2007) A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448:151–156.  https://doi.org/10.1038/nature05954CrossRefPubMedGoogle Scholar
  6. 6.
    Ni J-Q, Zhou R, Czech B, Liu L-P, Holderbaum L, Yang-Zhou D et al (2011) A genome-scale shRNA resource for transgenic RNAi in Drosophila. Nat Methods 8:405–407.  https://doi.org/10.1038/nmeth.1592CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Mohr SE, Smith JA, Shamu CE, Neumüller RA, Perrimon N (2014) RNAi screening comes of age:improved techniques andcomplementary approaches. Nat Rev Mol Cell Biol 15:591–600.  https://doi.org/10.1038/nrm3860CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    McGuire SE, Le PT, Osborn AJ, Matsumoto K, Davis RL (2003) Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302:1765–1768.  https://doi.org/10.1126/science.1089035CrossRefPubMedGoogle Scholar
  9. 9.
    Pospisilik JA, Schramek D, Schnidar H, Cronin SJF, Nehme NT, Zhang X et al (2010) Drosophila genome-wide obesity screen reveals hedgehog as a determinant of brown versus white adipose cell fate. Cell 140:148–160.  https://doi.org/10.1016/j.cell.2009.12.027CrossRefPubMedGoogle Scholar
  10. 10.
    Schnorrer F, Schönbauer C, Langer CCH, Dietzl G, Novatchkova M, Schernhuber K et al (2010) Systematic genetic analysis of muscle morphogenesis and function in Drosophila. Nature 464:287–291.  https://doi.org/10.1038/nature08799CrossRefPubMedGoogle Scholar
  11. 11.
    Handler D, Meixner K, Pizka M, Lauss K, Schmied C, Gruber FS et al (2013) The genetic makeup of the Drosophila piRNA pathway. Mol Cell 50:762–777.  https://doi.org/10.1016/j.molcel.2013.04.031CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Reim G, Hruzova M, Goetze S, Basler K (2014) Protection of armadillo/β-catenin by armless, a novel positive regulator of wingless signaling. PLoS Biol 12:e1001988.  https://doi.org/10.1371/journal.pbio.1001988CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Neumüller RA, Richter C, Fischer A, Novatchkova M, Neumüller KG, Knoblich JA (2011) Genome-wide analysis of self-renewal in Drosophila neural stem cells by transgenic RNAi. Cell Stem Cell 8:580–593.  https://doi.org/10.1016/j.stem.2011.02.022CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Roy S, VijayRaghavan K (1998) Patterning muscles using organizers: larval muscle templates and adult myoblasts actively interact to pattern the dorsal longitudinal flight muscles of Drosophila. J Cell Biol 141:1135CrossRefGoogle Scholar
  15. 15.
    Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415Google Scholar
  16. 16.
    Bryantsev AL, Duong S, Brunetti TM, Chechenova MB, Lovato TL, Nelson C et al (2012) Extradenticle and homothorax control adult muscle fiber identity in Drosophila. Dev Cell 23:664–673.  https://doi.org/10.1016/j.devcel.2012.08.004CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Menon SD, Chia W (2001) Drosophila rolling pebbles: a multidomain protein required for myoblast fusion that recruits D-Titin in response to the myoblast attractant Dumbfounded. Dev Cell 1:691–703CrossRefGoogle Scholar
  18. 18.
    Spletter ML, Barz C, Yeroslaviz A, Zhang X, Lemke SB, Bonnard A et al (2018) A transcriptomics resource reveals a transcriptional transition during ordered sarcomere morphogenesis in flight muscle. elife 7:1361.  https://doi.org/10.7554/eLife.34058CrossRefGoogle Scholar
  19. 19.
    Ranganayakulu G, Zhao B, Dokidis A, Molkentin JD, Olson EN, Schulz RA (1995) A series of mutations in the DMEF2 transcription factor reveal multiple functions in larval and adult myogenesis in Drosophila. Dev Biol 171:169–181.  https://doi.org/10.1006/dbio.1995.1269CrossRefPubMedGoogle Scholar
  20. 20.
    Klein P, Müller-Rischart AK, Motori E, Schönbauer C, Schnorrer F, Winklhofer KF et al (2014) Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants. EMBO J 33:341–355.  https://doi.org/10.1002/embj.201284290CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Schönbauer C, Distler J, Jährling N, Radolf M, Dodt H-U, Frasch M et al (2011) Spalt mediates an evolutionarily conserved switch to fibrillar muscle fate in insects. Nature 479:406–409.  https://doi.org/10.1038/nature10559CrossRefPubMedGoogle Scholar
  22. 22.
    Kocherlakota KS, Wu J-M, McDermott J, Abmayr SM (2008) Analysis of the cell adhesion molecule sticks-andstones reveals multiple redundant functional domains, protein-interaction motifs and phosphorylated tyrosines that direct myoblast fusion in Drosophila melanogaster. Genetics 178:1371–1383.  https://doi.org/10.1534/genetics.107.083808CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Usui K, Pistillo D, Simpson P (2004) Mutual exclusion of sensory bristles and tendons on the notum of dipteran flies. Curr Biol 14:1047–1055.  https://doi.org/10.1016/j.cub.2004.06.026CrossRefPubMedGoogle Scholar
  24. 24.
    Morin X, Daneman R, Zavortink M, Chia W (2001) A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc Natl Acad Sci U S A 98:15050–15055.  https://doi.org/10.1073/pnas.261408198CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Orfanos Z, Sparrow JC (2013) Myosin isoform switching during assembly of the Drosophila flight muscle thick filament lattice. J Cell Sci 126:139–148.  https://doi.org/10.1242/jcs.110361CrossRefPubMedGoogle Scholar
  26. 26.
    Orfanos Z, Leonard K, Elliott C, Katzemich A, Bullard B, Sparrow J (2015) Sallimus and the dynamics of sarcomere assembly in drosophila flight muscles. J Mol Biol 427:2151–2158.  https://doi.org/10.1016/j.jmb.2015.04.003CrossRefPubMedGoogle Scholar
  27. 27.
    Katzemich A, Liao KA, Czerniecki S, Schöck F (2013) Alp/enigma family proteins cooperate in Z-disc formation and myofibril assembly. PLoS Genet 9:e1003342.  https://doi.org/10.1371/journal.pgen.1003342.s007CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Klapholz B, Herbert SL, Wellmann J, Johnson R, Parsons M, Brown NH (2015) Alternative mechanisms for talin to mediate integrin function. Curr Biol 25:847–857.  https://doi.org/10.1016/j.cub.2015.01.043CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Sarov M, Barz C, Jambor H, Hein MY, Schmied C, Suchold D et al (2016) A genome-wide resource for the analysis of protein localisation in Drosophila. elife 5:e12068.  https://doi.org/10.7554/eLife.12068CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Richardson B, Beckett K, Nowak S, Baylies M (2007) SCAR/WAVE and Arp2/3 are crucial for cytoskeletal remodeling at the site of myoblast fusion. Development 134:4357CrossRefGoogle Scholar
  31. 31.
    Chen E, Olson E (2001) Antisocial, an intracellular adaptor protein, is required for myoblast fusion in Drosophila. Dev Cell 1:705–715CrossRefGoogle Scholar
  32. 32.
    Millard TH, Martin P (2008) Dynamic analysis of filopodial interactions during the zippering phase of Drosophila dorsal closure. Development 135:621–626.  https://doi.org/10.1242/dev.014001CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Dutta D, Bloor JW, Ruiz-Gómez M, VijayRaghavan K, Kiehart DP (2002) Real-time imaging of morphogenetic movements in Drosophila using Gal4-UAS-driven expression of GFP fused to the actin-binding domain of moesin. Genesis 34:146–151.  https://doi.org/10.1002/gene.10113CrossRefPubMedGoogle Scholar
  34. 34.
    Hatan M, Shinder V, Israeli D, Schnorrer F, Volk T (2011) The Drosophila blood brain barrier is maintained by GPCR-dependent dynamic actin structures. J Cell Biol 192:307–319.  https://doi.org/10.1083/jcb.201007095CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Lee T, Luo L (1999) Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22:451–461CrossRefGoogle Scholar
  36. 36.
    Spletter ML, Barz C, Yeroslaviz A, Schönbauer C, Ferreira IRS, Sarov M et al (2015) The RNA-binding protein Arrest (Bruno) regulates alternative splicing to enable myofibril maturation in Drosophila flight muscle. EMBO Rep 16:178–191.  https://doi.org/10.15252/embr.201439791CrossRefPubMedGoogle Scholar
  37. 37.
    Lemke SB, Schnorrer F (2018) In Vivo imaging of muscle-tendon morphogenesis in Drosophila pupae. J Vis Exp:e57312–e57312.  https://doi.org/10.3791/57312
  38. 38.
    Lakey A, Labeit S, Gautel M, Ferguson C, Barlow DP, Leonard K et al (1993) Kettin, a large modular protein in the Z-disc of insect muscles. EMBO J 12:2863–2871CrossRefGoogle Scholar
  39. 39.
    Saide J, Chin-Bow S, Hogan-Sheldon J, Busquets-Turner L, Vigoreaux J, Valgeirsdottir K et al (1989) Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster. J Cell Biol 109:2157CrossRefGoogle Scholar
  40. 40.
    Qiu F, Brendel S, Cunha P, Astola N, Song B, Furlong E et al (2005) Myofilin, a protein in the thick filaments of insect muscle. J Cell Sci 118:1527CrossRefGoogle Scholar
  41. 41.
    Bullard B, Leonard K, Larkins A, Butcher G, Karlik C, Fyrberg E (1988) Troponin of asynchronous flight muscle. J Mol Biol 204:621–637CrossRefGoogle Scholar
  42. 42.
    Wilcox M, Brower DL, Smith RJ (1981) A position-specific cell surface antigen in the drosophila wing imaginal disc. Cell 25:159–164CrossRefGoogle Scholar
  43. 43.
    Brower DL, Wilcox M, Piovant M, Smith RJ, Reger LA (1984) Related cell-surface antigens expressed with positional specificity in Drosophila imaginal discs. Proc Natl Acad Sci U S A 81:7485–7489CrossRefGoogle Scholar
  44. 44.
    Brown NH, Gregory SL, Rickoll WL, Fessler LI, Prout M, White RAH et al (2002) Talin is essential for integrin function in Drosophila. Dev Cell 3:569–579CrossRefGoogle Scholar
  45. 45.
    Razzaq A, Robinson I, McMahon H, Skepper J, Su Y, Zelhof A et al (2001) Amphiphysin is necessary for organization of the excitation–contraction coupling machinery of muscles, but not for synaptic vesicle endocytosis in Drosophila. Genes Dev 15:2967CrossRefGoogle Scholar
  46. 46.
    Atreya K, Fernandes J (2008) Founder cells regulate fiber number but not fiber formation during adult myogenesis in Drosophila. Dev Biol 321:123–140CrossRefGoogle Scholar
  47. 47.
    Schmid A, Sigrist SJ (2008) Analysis of neuromuscular junctions: histology and in vivo imaging. Methods Mol Biol 420:239–251.  https://doi.org/10.1007/978-1-59745-583-1_14CrossRefPubMedGoogle Scholar
  48. 48.
    Budnik V, Gorczyca M, Prokop A (2006) Selected methods for the anatomical study of Drosophila embryonic and larval neuromuscular junctions. Int Rev Neurobiol 75:323–365.  https://doi.org/10.1016/S0074-7742(06)75015-2CrossRefPubMedGoogle Scholar
  49. 49.
    Weitkunat M, Schnorrer F (2014) A guide to study Drosophila muscle biology. Methods 68:2–14.  https://doi.org/10.1016/j.ymeth.2014.02.037CrossRefPubMedGoogle Scholar
  50. 50.
    Weitkunat M, Kaya-Copur A, Grill SW, Schnorrer F (2014) Tension and force-resistant attachment are essential for myofibrillogenesis in Drosophila flight muscle. Curr Biol 24:705–716.  https://doi.org/10.1016/j.cub.2014.02.032CrossRefPubMedGoogle Scholar
  51. 51.
    Starz-Gaiano M, Cho NK, Forbes A, Lehmann R (2001) Spatially restricted activity of a Drosophila lipid phosphatase guides migrating germ cells. Development 128:983–991PubMedGoogle Scholar
  52. 52.
    Langer CCH, Ejsmont RK, Schönbauer C, Schnorrer F, Tomancak P (2010) In vivo RNAi rescue in Drosophila melanogaster with genomic transgenes from Drosophila pseudoobscura. PLoS One 5:e8928.  https://doi.org/10.1371/journal.pone.0008928CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Aix Marseille Univ, CNRS, IBDMMarseilleFrance

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