In vivo live-analysis of cell cycle checkpoints in Drosophila early embryos.

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.