Advertisement

Cell Fusion pp 299-314 | Cite as

A Genomic Approach to Myoblast Fusion in Drosophila

  • Beatriz Estrada
  • Alan M. Michelson
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 475)

Summary

We have developed an integrated genetic, genomic, and computational approach to identify and characterize genes involved in myoblast fusion in Drosophila. We first used fluorescence-activated cell sorting to purify mesodermal cells both from wild-type embryos and from 12 variant genotypes in which muscle development is perturbed in known ways. Then, we obtained gene expression profiles for the purified cells by hybridizing isolated mesodermal RNA to Affymetrix GeneChip arrays. These data were subsequently compounded into a statistical metaanalysis that predicts myoblast subtype-specific gene expression signatures that were later validated by in situ hybridization experiments. Finally, we analyzed the myogenic functions of a subset of these myoblast genes using a double-stranded RNA interference assay in living embryos expressing green fluorescent protein under control of a muscle-specific promoter. This experimental strategy led to the identification of several previously uncharacterized genes required for myoblast fusion in Drosophila.

Key Words

Cell–cell fusion myoblast mesoderm myogenesis muscle development Drosophila genomics gene expression profiling 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

We thank Stephen S. Gisselbrecht and Sung E. Choe for comments on the manuscript. This work was funded by the Howard Hughes Medical Institute and the National Institutes of Health.

References

  1. 1.
    Chen, E. H. and Olson, E. N. (2005) Unveiling the mechanisms of cell–cell fusion. Science 308(5720), 369–373.CrossRefPubMedGoogle Scholar
  2. 2.
    Horsley, V. and Pavlath, G. K. (2004) Forming a multinucleated cell: molecules that regulate myoblast fusion. Cells Tissues Organs 176(1–3), 67–78.CrossRefPubMedGoogle Scholar
  3. 3.
    Pomerantz, J. and Blau, H. M. (2004) Nuclear reprogramming: a key to stem cell function in regenerative medicine. Nat. Cell. Biol. 6(9), 810–816.CrossRefPubMedGoogle Scholar
  4. 4.
    Chen, E.H. and Olson, E. N. (2004) Towards a molecular pathway for myoblast fusion in Drosophila. Trends Cell Biol. 14(8), 452–460.CrossRefPubMedGoogle Scholar
  5. 5.
    Dworak, H. A. and Sink, H. (2002) Myoblast fusion in Drosophila. BioEssays 24, 591–601.CrossRefPubMedGoogle Scholar
  6. 6.
    Estrada, B., Choe, S. E., Gisselbrecht, S. S., et al. (2006) An integrated strategy for analyzing the unique developmental programs of different myoblast subtypes. PLoS Genet. 2(2), e16.CrossRefPubMedGoogle Scholar
  7. 7.
    Baylies, M. K. and Michelson, A. M. (2001) Invertebrate myogenesis: looking back to the future of muscle development. Curr. Opin. Genet. Dev. 11, 431–439.CrossRefPubMedGoogle Scholar
  8. 8.
    Halfon, M. S., Gisselbrecht, S., Lu, J., Estrada, B., Keshishian, H., and Michelson, A. M. (2002) New fluorescent protein reporters for use with the Drosophila Gal4 expression system and for vital detection of balancer chromosomes. Genesis 34, 135–138.CrossRefPubMedGoogle Scholar
  9. 9.
    Greig, S. and Akam, M. (1993) Homeotic genes autonomously specify one aspect of pattern in the Drosophila mesoderm. Nature 362, 630–632.CrossRefPubMedGoogle Scholar
  10. 10.
    Queenan, A. M., Ghabrial, A., and Schüpbach, T. (1997) Ectopic activation of torpedo/Egfr, a Drosophila receptor tyrosine kinase, dorsalizes both the eggshell and the embryo. Development 124, 3871–3880.PubMedGoogle Scholar
  11. 11.
    Michelson, A. M., Gisselbrecht, S., Buff, E., and Skeath, J. B. (1998) Heartbroken is a specific downstream mediator of FGF receptor signalling in Drosophila. Development 125, 4379–4389.PubMedGoogle Scholar
  12. 12.
    Vincent, S., Wilson, R., Coelho, C., Affolter, M., and Leptin, M. (1998) The Drosophila protein Dof is specifically required for FGF signaling. Mol. Cell 2, 515–525.CrossRefPubMedGoogle Scholar
  13. 13.
    Carmena, A., Gisselbrecht, S., Harrison, J., Jiménez, F., and Michelson, A. M. (1998) Combinatorial signaling codes for the progressive determination of cell fates in the Drosophila embryonic mesoderm. Genes Dev. 12, 3910–3922.CrossRefPubMedGoogle Scholar
  14. 14.
    Halfon, M. S., Carmena, A., Gisselbrecht, S., et al. (2000) Ras pathway specificity is determined by the integration of multiple signal-activated and tissue-restricted transcription factors. Cell 103(1), 63–74.CrossRefPubMedGoogle Scholar
  15. 15.
    Nellen, D., Burke, R., Struhl, G., and Basler, K. (1996) Direct and long-range action of a DPP morphogen gradient. Cell 85(3), 357–368.CrossRefPubMedGoogle Scholar
  16. 16.
    Pai, L.-M., Orsulic, S., Bejsovec, A., and Peifer, M. (1997) Negative regulation of Armadillo, a Wingless effector in Drosophila. Development 124, 2255–2266.PubMedGoogle Scholar
  17. 17.
    Lieber, T., Kidd, S., Alcamo, E., Corbin, V., and Young, M. W. (1993) Antineurogenic phenotypes induced by truncated Notch proteins indicate a role in signal transduction and may point to a novel function for Notch in nuclei. Genes Dev. 7, 1949–1965.CrossRefPubMedGoogle Scholar
  18. 18.
    Duan, H., Skeath, J. B., and Nguyen, H. T. (2001)Drosophila Lame duck, a novel member of the Gli superfamily, acts as a key regulator of myogenesis by controlling fusion-competent myoblast development. Development 128, 4489–4500.PubMedGoogle Scholar
  19. 19.
    Nose, A., Isshiki, T., and Takeichi, M. (1998) Regional specification of muscle progenitors in Drosophila: the role of the msh homeobox gene. Development 125, 215–223.PubMedGoogle Scholar
  20. 20.
    Chen, E. H. and Olson, E. N. (2001) Antisocial, an intracellular adaptor protein, is required for myoblast fusion in Drosophila. Dev. Cell 1, 705–715.CrossRefPubMedGoogle Scholar
  21. 21.
    Brand, A. H. and Perrimon, N. (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415.PubMedGoogle Scholar
  22. 22.
    Donady, J. J. and Fyrberg, E. A. (1977) Mass culturing of Drosophila embryonic cells in vitro. Tissue Culture Assoc. Manual 3, 685–687.CrossRefGoogle Scholar
  23. 23.
    Singleton, K. and Woodruff, R. I. (1994) The osmolarity of adult Drosophila hemolymph and its effect on oocyte-nurse cell electrical polarity. Dev. Biol. 161(1), 154–167.CrossRefPubMedGoogle Scholar
  24. 24.
    Choe, S. E., Boutros, M., Michelson, A. M., Church, G. M., and Halfon, M. S. (2005) Preferred analysis methods for Affymetric GeneChips revealed by a wholly-defined control dataset. Genome Biol. 6, R16.CrossRefPubMedGoogle Scholar
  25. 25.
    Baldi, P. and Long, A. D. (2001) A Bayesian framework for the analysis of microarray expression data: regularized t -test and statistical inferences of gene changes. Bioinformatics 17(6), 509–519.CrossRefPubMedGoogle Scholar
  26. 26.
    Ruiz-Gomez, M., Coutts, N., Suster, M. L., Landgraf, M., and Bate M. (2002) Myoblasts incompetent encodes a zinc finger transcription factor required to specify fusion-competent myoblasts in Drosophila. Development 129(1), 133–141.PubMedGoogle Scholar
  27. 27.
    Carmena, A., Buff, E., Halfon, M. S., et al. (2002) Reciprocal regulatory interactions between the Notch and Ras signaling pathways in the Drosophila embryonic mesoderm. Dev. Biol. 244, 226–242.CrossRefPubMedGoogle Scholar
  28. 28.
    Bour, B. A., Chakravarti, M., West, J. M., and Abmayr, S. M. (2000)Drosophila SNS, a member of the immunoglobulin superfamily that is essential for myoblast fusion. Genes Dev. 14, 1498–1511.PubMedGoogle Scholar
  29. 29.
    Ma, Y., Creanga, A., Lum, L., and Beachy, P. A. (2006) Prevalence of off-target effects in Drosophila RNA interference screens. Nature 443(7109), 359–363.CrossRefPubMedGoogle Scholar
  30. 30.
    Kulkarni, M. M., Booker, M., Silver, S. J., et al. (2006) Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays. Nat. Methods 3(10), 833–838.PubMedGoogle Scholar
  31. 31.
    Kennerdell, J. R. and Carthew, R. W. (1998) Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95, 1017–1026.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Beatriz Estrada
    • 1
  • Alan M. Michelson
    • 1
  1. 1.Division of Genetics, Department of MedicineBrigham and Women's Hospital and Harvard Medical SchoolBoston

Personalised recommendations

Cite protocol

Buy options