Visualizing Mitosis in Whole-Mount Larval Brains

  • Daryl S. Henderson
Part of the Methods in Molecular Biology book series (MIMB, volume 247)


The central nervous system (CNS) of the third instar larva is a tissue of choice for studying conventional mitotic cycles in Drosophila. For example, squash preparations of the larval CNS are routinely used to investigate chromosome structural and numerical anomalies in late larval lethal mutants (e.g., refs. 1,2; also see  Chapters 16 18), to study heterochromatin (e.g., refs. 3, 4, 5), and to localize chromosomal proteins by immunostaining (see  Chapter 19). Mitotic chromosomes are not unduly harmed upon squashing, and for many experimental purposes it is advantageous to have them flat and well spread. However, the same cannot be said of the mitotic spindle, which is distorted or destroyed in squash preparations. A simple method for live analysis of mitosis in larval brain cells involves “pulverizing” dissected brain tissue with fine scalpel blades to produce a monolayer of cells for short-term (approx 1 h) study (6). The method can be used to visualize any mitotic proteins/structures for which green fluorescent protein (GFP)-expressing strains are available, and both wild type and mutants can be studied with equal facility. However, a potential drawback is that both the mechanical disrupting of tissue and nonphysiological culture medium used could have adverse effects on mitosis. Moreover, information about relative spindle geometry in a developmental context (e.g., ref. 7) is lost. A complementary approach to the above methods is to use whole-mount preparations of fixed brains to obtain a three-dimensional (albeit static) view of mitosis.


Propidium Iodide Imaginal Disc Optic Lobe Squash Preparation Propidium Iodide Solution 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Baker, B. S., Smith, D. A., and Gatti, M. (1982) Region specific effects on chromosome integrity of mutations at essential loci in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 79, 1205–1209.PubMedCrossRefGoogle Scholar
  2. 2.
    Gatti, M. and Baker, B. S. (1989) Genes controlling essential cell-cycle functions in Drosophila melanogaster. Genes Dev. 3, 438–453.PubMedCrossRefGoogle Scholar
  3. 3.
    Bonaccorsi, S. and Lohe, A. (1991) Fine mapping of satellite DNA sequences along the Y chromosome of Drosophila melanogaster: relationships between satellite sequences and fertility factors. Genetics 129, 177–189.PubMedGoogle Scholar
  4. 4.
    Lohe, A. R., Hilliker, A. J., and Roberts, P. A. (1993) Mapping simple repeated DNA sequences in heterochromatin of Drosophila melanogaster. Genetics 134, 1149–1174.PubMedGoogle Scholar
  5. 5.
    Csink, A. and Henikoff, S. (1996) Genetic modification of heterochromatic association and nuclear organization in Drosophila. Nature 381, 529–531.PubMedCrossRefGoogle Scholar
  6. 6.
    Savoian, M. S. and Rieder, C. L. (2002) Mitosis in primary cultures of Drosophila melanogaster. J. Cell Sci. 115, 3061–3072.PubMedGoogle Scholar
  7. 7.
    Ceron, J., Gonzalez, C., and Tejedor, F. J. (2001) Patterns of cell division and expression of asymmetric cell fate determinants in postembryonic neuroblast lineages of Drosophila. Dev. Biol. 230, 125–138.PubMedCrossRefGoogle Scholar
  8. 8.
    Truman, J. W. (1990) Metamorphosis of the central nervous system of Drosophila. J. Neurobiol. 21, 1072–1084.PubMedCrossRefGoogle Scholar
  9. 9.
    Truman, J. W. and Bate, M. (1988) Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster. Dev. Biol. 125, 145–157.PubMedCrossRefGoogle Scholar
  10. 10.
    Ito, K., Urban, J., and Technau, G. M. (1995) Distribution, classification, and development of Drosophila glial cells in the late embryonic and early larval ventral nerve cord. Roux’s Archiv Dev. Biol. 204, 284–307.CrossRefGoogle Scholar
  11. 11.
    Datta, S. (1995) Control of proliferation activation in quiescent neuroblasts of the Drosophila central nervous system. Development 121, 1173–1182.PubMedGoogle Scholar
  12. 12.
    Audibert, A., Debec, A., and Simonelig, M. (1996) Detection of mitotic spindles in third-instar imaginal discs of Drosophila melanogaster. Trends Genet. 12, 452–453.PubMedCrossRefGoogle Scholar
  13. 13.
    Schilstra, M. J., Bayley, P. M., and Martin, S. R. (1992) The effect of solution composition on microtubule dynamic instability. Biochem. J. 277, 839–847.Google Scholar
  14. 14.
    Basu, J., Bousbaa, H., Logarihino, E., et al. (1999) Mutations in the essential spindle checkpoint gene bub1 cause chromosome missegregation and fail to block apoptosis in Drosophila. J. Cell Biol. 146, 13–28.PubMedGoogle Scholar
  15. 15.
    Donaldson, M. M., Tavares, A. A. M., Ohkura, H., Deak, P., and Glover, D. M. (2001) Metaphase arrest with centromere separation in polo mutants of Drosophila. J. Cell Biol. 153, 663–675.PubMedCrossRefGoogle Scholar
  16. 16.
    Scaërou, F., Starr, D. A., Piano, F., Papoulas, O., Karess, R. E., and Goldberg, M. L. (2001) The ZW10 and Rough Deal checkpoint proteins function together in a large, evolutionarily conserved complex targeted to the kinetochore. J. Cell Sci. 114, 3103–3114.PubMedGoogle Scholar
  17. 17.
    Williams, B. C. and Goldberg, M. L. (1994) Determinants of Drosophila zw10 protein localization and function. J. Cell Sci. 107, 785–798.PubMedGoogle Scholar
  18. 18.
    Bousbaa, H., Correira, L., Gorbsky, G. J., and Sunkel, C. E. (1997) Mitotic phosphoepitopes are expressed in Kc cells, neuroblasts and isolated chromosomes of Drosophila melanogaster. J. Cell Sci. 110, 1979–1988.PubMedGoogle Scholar
  19. 19.
    Barbosa, V., Yamamoto, R. R., Henderson, D. S., and Glover, D. M. (2000) Mutation of a Drosophila gamma tubulin ring complex subunit encoded by discs degenerate-4 differentially disrupts centrosomal protein localization. Genes Dev. 14, 3126–3139.PubMedCrossRefGoogle Scholar
  20. 20.
    Cullen, C. F., Deak, P., Glover, D. M., and Ohkura, H. (1999) mini spindles: a gene encoding a conserved microtubule-associated protein required for the integrity of the mitotic spindle in Drosophila. J. Cell Biol. 146, 1005–1018.PubMedCrossRefGoogle Scholar
  21. 21.
    Daum, J. R., Tugendreich, S., Topper, L. M., et al. (2000) The 3F3/2 antiphosphoepitope antibody binds the mitotically phosphorylated anaphase-promoting complex/cyclosome. Curr. Biol. 10, R850–R852.PubMedCrossRefGoogle Scholar
  22. 22.
    Rasch, E. M. (1970) DNA cytophotometry of salivary gland nuclei and other tissue systems in dipteran larvae, in Introduction to Quantitative Cytochemistry (Weid, G. W. and Bahr, G. F., eds.), Academic, New York, Vol. 2, pp. 357–397.Google Scholar
  23. 23.
    Prokop, A. and Technau, G. M. (1994) BrdU incorporation reveals DNA replication in non dividing glial cells in the larval abdominal CNS of Drosophila. Roux’s Arch. Dev. Biol. 204, 54–61.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Daryl S. Henderson
    • 1
  1. 1.Department of Pharmacological SciencesState University of New York at Stony Brook

Personalised recommendations