Abstract
Live-imaging of cells has been an excellent technique to provide us with highly accurate and valuable information about cell cycle checkpoint regulation and DNA damage responses. Early stage Drosophila embryos have several advantages to be studied by live-imaging. Fly embryos are much tougher than cultured cells and stand up to relatively rough manipulation, such as protein/chemical microinjection followed by time-lapse imaging. Cell cycles in the embryonic cleavage stage progress rapidly (9–20 min/cycle) and nuclear divisions are synchronous, allowing observation of multiple nuclei/cell cycles in a short period of time. Somatic precursor nuclei form a monolayer at the cortex of the embryo during the syncytial blastoderm stage (cell cycles 10–13). Thus the nuclei in this stage are particularly accessible by various microscopic techniques (Sullivan and Theurkauf, 1995, Curr. Opin. Cell Biol. 7, 18–22). Live-imaging of embryos complements the versatility of the Drosophila embryonic system, in which we can utilize various approaches, including genetics and biochemistry, to obtain comprehensive understanding of biological processes. In this chapter, we will describe basic methods of microinjection and live-imaging during early embryogenesis by differential interference contrast (DIC) or confocal microscopy, and the use of such methods to study cell cycle checkpoints.
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References
Sullivan, W., and Theurkauf, W.E. (1995). The cytoskeleton and morphogenesis of the early Drosophila embryo. Curr. Opin. Cell Biol. 7, 18–22.
Foe, V., Odell, G., and Edgar, B. (1993). Mitosis and Morphogenesis in the Drosophila Embryo: Point and Counterpoint. In The Development of Drosophila melanogaster, M. Bate and A. Martinez Arias, eds. (New York, NY: Cold Spring Harbor Laboratory Press), pp. 149–300.
Liu, Q., Guntuku, S., Cui, X.S., Matsuoka, S., Cortez, D., Tamai, K., Luo, G., Carattini-Rivera, S., DeMayo, F., Bradley, A., Donehower, L.A., and Elledge, S.J. (2000). Chk1 is an essential kinase that is regulated by Atr and required for the G2/M DNA damage checkpoint. Genes Dev. 14, 1448–1459.
Feijoo, C., Hall-Jackson, C., Wu, R., Jenkins, D., Leitch, J., Gilbert, D.M., and Smythe, C. (2001). Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing. J.Cell Biol. 154, 913–923.
Brown, E.J., and Baltimore, D. (2000). ATR disruption leads to chromosomal fragmentation and early embryonic lethality. Genes Dev. 14, 397–402.
Takai, H., Tominaga, K., Motoyama, N., Minamishima, Y.A., Nagahama, H., Tsukiyama, T., Ikeda, K., Nakayama, K., and Nakanishi, M. (2000). Aberrant cell cycle checkpoint function and early embryonic death in Chk1(-/-) mice. Genes Dev. 14, 1439–1447.
Sibon, O.C., Stevenson, V.A., and Theurkauf, W.E. (1997). DNA replication checkpoint control at the Drosophila midblastula transition. Nature 388, 93–97.
Sibon, O.C., Laurencon, A., Hawley, R., and Theurkauf, W.E. (1999). The Drosophila ATM homologue Mei-41 has an essential checkpoint function at the midblastula transition. Curr. Biol. 9, 302–312.
Takada, S., Kwak, S., Koppetsch, B., and Theurkauf, W.E. (2007). grp(chk1) replication-checkpoint mutations and DNA damage trigger a Chk2-dependent block at the Drosophila midblastula transition. Development 134, 1737–1744.
Abraham, R.T. (2001). Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev. 15, 2177–2196.
Sibon, O.C., Kelkar, A., Lemstra, W., and Theurkauf, W.E. (2000). DNA-replication/DNA-damage-dependent centrosome inactivation in Drosophila embryos. Nat. Cell Biol. 2, 90–95.
Takada, S., Kelkar, A., and Thuerkauf W.E. (2003). Drosophila checkpoint kinase 2 couples centrosome function and spindle assembly to genomic integrity. Cell 113, 87–99.
Bartek, J., Falck, J., and Lukas, J. (2001). CHK2 kinase–a busy messenger. Nat. Rev. Mol. Cell Biol. 2, 877–886.
Musacchio, A., and Hardwick, K. G. (2002). The spindle checkpoint: structural insights into dynamic signalling. Nat. Rev. Mol. Cell. Biol. 3, 731–741.
Pérez-Mongiovi1, D., Malmanche1, N., Bousbaa, H., and Sunkel1, C. (2005). Maternal expression of the checkpoint protein BubR1 is required for synchrony of syncytial nuclear divisions and polar body arrest in Drosophila melanogaster. Development 132, 4509–4520.
Weigmann, K., Klapper, R., Strasser, T., Rickert, C., Technau, G., Jäckle, H., Janning, J., and Klämbt, C. (2003). FlyMove – a new way to look at development of Drosophila. Trends Genet. 19, 310. http://flymove.uni-muenster.de
Clarkson, M., and Saint, R. (1999). A His2AvDGFP fusion gene complements a lethal His2AvD mutant allele and provides an in vivo marker for Drosophila chromosome behavior. DNA Cell Biol. 18, 457–462.
Rogers, S.L., Rogers, G.C., Sharp, D.J., and Vale, R.D. (2002). Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle. J. Cell Biol. 158, 873–884.
Sullivan, W., Fogarty, P., and Theurkauf, W. (1993). Mutations affecting the cytoskeletal organization of syncytial Drosophila embryos. Development 118, 1245–1254.
Chou, T.B., and Perrimon, N. (1996). The autosomal FLP-DFS technique for generating germline mosaics in Drosophila melanogaster. Genetics 144, 1673–1679.
Acknowledgments
The authors would like to thank Professor William Theurkauf for providing us with good opportunities and environment to learn live-imaging techniques in flies, and MaryJane O’Connor, Aidan Peterson, and Hideki Aihara for critical reading of the manuscript.
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Takada, S., Cha, B.J. (2011). In Vivo Live-Analysis of Cell Cycle Checkpoints in Drosophila Early Embryos. In: Li, W. (eds) Cell Cycle Checkpoints. Methods in Molecular Biology, vol 782. Humana Press. https://doi.org/10.1007/978-1-61779-273-1_7
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DOI: https://doi.org/10.1007/978-1-61779-273-1_7
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