Skip to main content

Studying Nuclear Organization in Embryos Using Antibody Tools

  • Protocol
Drosophila Cytogenetics Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 247))

Abstract

One of the major foci in cell biology is to understand the process of nuclear division. In each cell cycle, the chromosomes must be faithfully replicated and the complex nuclear structure has to be duplicated and reorganized (14). Our understanding of the cell cycle and mitosis has increased dramatically in the last several years, as a result of cross-disciplinary approaches combining molecular, cell biological, and genetic techniques (reviewed in refs. 59). An organism offering a particularly advantageous model system for such studies of mitosis is the early embryo of Drosophila melanogaster. The cytoskeleton and mitotic spindle are large and easily visualized, thus facilitating structural analysis. The embryo undergoes 13 rapid and nearly synchronous nuclear divisions giving rise to about 5000 nuclei before cell boundaries form after 3 h of development (1012). This syncytial organization of nuclei affords excellent accessibility for experimental perturbations (e.g., using antibodies [1317] or pharmacological tools [15,1820]).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Laskey, Fairman, R. A., M. P., and Blow, J. J. (1989) S Phase of the cell cycle. Science 246, 603–608.

    Article  Google Scholar 

  2. McIntosh, J. R. and Koonce, M. P. (1989) Mitosis. Science 246, 603–608.

    Article  Google Scholar 

  3. Pines, J. (1999) Four-dimensional control of the cell cycle. Nature Cell Biol. 1, E73–E79.

    Article  PubMed  CAS  Google Scholar 

  4. Hinchcliffe, E. H. and Sluder, G. (2001) “It Takes Two to Tango”: understanding how centrosome duplication is regulated throughout the cell cycle. Genes Dev. 15, 1167–1181.

    Article  PubMed  CAS  Google Scholar 

  5. Cross, F., Roberts, J., and Weintraub, H. (1989) Simple and complex cell cycles. Annu. Rev. Cell Biol. 5, 341–395.

    Article  PubMed  CAS  Google Scholar 

  6. Norbury, C. and Nurse, P. (1992) Animal cell cycles and their control. Annu. Rev. Biochem. 61, 441–470.

    Article  PubMed  CAS  Google Scholar 

  7. Vidwans, S. J. and Su, T. T. (2001) Cycling through development in Drosophila and other metazoa. Nature Cell Biol. 3, E35–E39.

    Article  PubMed  CAS  Google Scholar 

  8. Pines, J. and Rieder, C. L. (2001) Re-staging mitosis: a contemporary view of mitotic progression. Nature Cell Biol. 3, E3–E6.

    Article  PubMed  CAS  Google Scholar 

  9. Scholey, J. M., Rogers, G. C., and Sharp, D. J. (2001) Mitosis, microtubules, and the matrix. J. Cell Biol. 154, 261–266.

    Article  PubMed  CAS  Google Scholar 

  10. Zalokar, M. and Erk, I. (1976) Division and migration of nuclei during early embryogenesis of Drosophila melanogaster. J. Microsc. Biol. Cell. 25, 97–106.

    Google Scholar 

  11. Foe, V. E. and Alberts, B. M. (1983) Studies of nuclear and cytoplasmic behavior during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis. J. Cell Sci. 61, 31–70.

    PubMed  CAS  Google Scholar 

  12. Foe, V. E. (1989) Mitotic domains reveal early commitment of cells in Drosophila embryos. Development 107, 1–22.

    PubMed  CAS  Google Scholar 

  13. Warn, R. M., Flegg, L., and Warn, A. (1987) An investigation of microtubule organization and functions in living Drosophila embryos by injection of a fluorescently labeled antibody against tyrosinated α-tubulin. J. Cell Biol. 105, 1721–1730.

    Article  PubMed  CAS  Google Scholar 

  14. Kiehart, D. P., Ketchum, A., Young, P., et al. (1990) Contractile proteins in Drosophila development. Ann. NY Acad. Sci. 582, 233–251.

    Article  PubMed  CAS  Google Scholar 

  15. Buchenau, P., Saumweber, H., and Arndt-Jovin, D. J. (1993) Consequences of topoisomerase II inhibition in early embryogenesis of Drosophila revealed by in vivo confocal laser scanning microscopy. J. Cell Sci. 104, 1175–1185.

    PubMed  CAS  Google Scholar 

  16. Mermall, V. and Miller, K. G. (1995) The 95F unconventional myosin is required for proper organization of the Drosophila syncytial blastoderm. J. Cell Biol. 129, 1575–1588.

    Article  PubMed  CAS  Google Scholar 

  17. Johansen, K. M., Johansen, J., Baek, K.-H., and Jin, Y. (1996) Remodeling of nuclear architecture during the cell cycle in Drosophila embryos. J. Cell. Biochem. 63, 268–279.

    Article  PubMed  CAS  Google Scholar 

  18. Edgar, B., Odell, G. M., and Schubiger, G. (1987) Cytoarchitecture and the patterning of fushi tarazu expression in the Drosophila blastoderm. Genes Dev. 1, 1226–1237.

    Article  PubMed  CAS  Google Scholar 

  19. Raff, J. W., Whitfield, W. G., and Glover, D. M. (1990) Two distinct mechanisms localize cyclin B transcripts in syncytial Drosophila embryos. Development 110, 1249–1261.

    PubMed  CAS  Google Scholar 

  20. Planques, V., Warn, A., and Warn, R. M. (1991) The effects of microinjection of rhodamine-phalloidin on mitosis and cytokinesis in early stage Drosophila embryos. Exp. Cell. Res. 192, 557–566.

    Article  PubMed  CAS  Google Scholar 

  21. Jin, Y., Wang, Y., Walker, D. L., et al. (1999) JIL-1: a novel chromosomal tandem kinase implicated in transcriptional regulation in Drosophila. Mol. Cell 4, 129–135.

    Article  PubMed  CAS  Google Scholar 

  22. Wang, Y., Zhang, W., Jin, Y., Johansen, J., and Johansen, K.M. (2001) The JIL-1 tandem kinase mediates histone H3 phosphorylation and is required for maintenance of chromatin structure in Drosophila. Cell 105, 433–443.

    Article  PubMed  CAS  Google Scholar 

  23. Walker, D. L., Wang, D., Jin, Y., et al. (2000) Skeletor, a novel chromosomal protein that redistributes during mitosis provides evidence for the formation of a spindle matrix. J. Cell Biol. 151, 1401–1411.

    Article  PubMed  CAS  Google Scholar 

  24. Zalokar, M. and Erk, I. (1977) Phase-partition fixation and staining of Drosophila eggs. Stain Technol. 52, 89–95.

    PubMed  CAS  Google Scholar 

  25. Mitchison, T. J. and Sedat, J. (1983) Localization of antigenic determinants in whole Drosophila embryos. Dev. Biol. 99, 261–264.

    Article  PubMed  CAS  Google Scholar 

  26. Theurkauf, W. E. (1994) Immunofluorescence analysis of the cytoskeleton during oogenesis and early embryogenesis. Methods Cell Biol. 44, 489–505.

    Article  PubMed  CAS  Google Scholar 

  27. Patel, N. H. (1994) Imaging neuronal subsets and other cell types in whole-mount Drosophila embryos and larvae using antibody probes. Methods Cell Biol. 44, 445–487.

    Article  PubMed  CAS  Google Scholar 

  28. Rothwell, W. F. and Sullivan, W. (2000) Fluorescent analysis of Drosophila embryos, in Drosophila Protocols (Sullivan, W., Ashburner, M., and Hawley, R. S., eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 141–157.

    Google Scholar 

  29. Rebay, I. and Fehon, R.G. (2000) Generating antibodies against Drosophila proteins, in Drosophila Protocols (Sullivan, W., Ashburner, M., and Hawley, R.S., eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 389–411.

    Google Scholar 

  30. Spector, D. L, Goldman, R. D., and Leinwand, L. A. (1998) Immunofluorescence methods for Drosophila tissues, in Cells: A Laboratory Manual (Spector, D. L, Goldman, R. D., and Leinwand, L. A., eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N pp. 107.1–107.9.

    Google Scholar 

  31. Humason, G.L. (1997) Animal Tissue Techniques, W.H. Freeman, San Francisco.

    Google Scholar 

  32. Horobin, R. W. (1982) Histochemistry, Gustav Fischer Verlag, New York.

    Google Scholar 

  33. Santamaria, P. (1986) Injecting eggs, in Drosophila: A Practical Approach. (Roberts, D.B., ed.), IRL, Oxford, pp. 159–173.

    Google Scholar 

  34. Spradling, A. C. (1986) P element-mediated transformation, in Drosophila: A Practical Approach. (Roberts, D. B., ed.), IRL, Oxford, pp. 175–197.

    Google Scholar 

  35. Baek, K.-H. and Ambrosio, L. (1995) An efficient method for microinjection of mRNA into Drosophila embryos. BioTechniques 17, 1024–1026.

    Google Scholar 

  36. Lee, G. M. (1989) Measurement of volume injected into individual cells by quantitative fluorescence microscopy. J. Cell Sci. 94, 443–447.

    PubMed  Google Scholar 

  37. Kiehart, D. P., Crawford, J. M., and Montague, R. A. (2000) Quantitative micro-injection of Drosophila embryos, in Drosophila Protocols (Sullivan, W., Ashburner, M., and Hawley, R. S., eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 345–359.

    Google Scholar 

  38. Campos-Ortega, J. A. and Hartenstein, V. (1997) Stages of Drosophila embryo-genesis, in The Embryonic Development of Drosophila melanogaster. Springer-Verlag, New York, pp. 9–102.

    Google Scholar 

  39. Foe, V.E. and Alberts, M.M. (1985) Reversible chromosome condensation induced in Drosophila embryos by anoxia: visualization of interphase nuclear organization. J. Cell Biol. 100, 1623–1636.

    Article  PubMed  CAS  Google Scholar 

  40. Johansen, K. M., Kopp, D. M., Jellies, J., and Johansen, J. (1992) Tract formation and axon fasiculation of molecularly distinct peripheral neuron subpopulations during leech embryogenesis. Neuron 8, 559–272.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Johansen, K.M., Johansen, J. (2004). Studying Nuclear Organization in Embryos Using Antibody Tools. In: Henderson, D.S. (eds) Drosophila Cytogenetics Protocols. Methods in Molecular Biology, vol 247. Humana Press. https://doi.org/10.1385/1-59259-665-7:215

Download citation

  • DOI: https://doi.org/10.1385/1-59259-665-7:215

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-050-2

  • Online ISBN: 978-1-59259-665-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics