Confocal Microscopy of Drosophila Embryos

The genetically tractable organism Drosophila melanogaster is proving to be an excellent model system for cell biological analysis in the context of the whole organism. The relative ease with which embryos can be obtained in large numbers and processed for highresolution light microscopy has facilitated many recent advances at the interface between cell and developmental biology. Fine subcellular structures previously impossible to visualise by conventional fluorescence microscopy, on account of high noise resulting from out-of-focus signals, are revealed with clarity on a confocal microscope. There are several reasons why scientists who have not used Drosophila before may wish to use Drosophila embryos for the analysis of protein localisation and expression. The embryo contains representatives of each cell type and is small enough (500 x 100 ~tm) to fit within the field of view of a 20X objective lens. The embryos are nearly transparent, permitting visualisation of all cells in whole mount preparations. These features allow one to assay the tissue distribution of a particular protein in a single specimen. The tissues have a relatively simple structure, with the epithelia being made up of a single layer of cells. In general, there are fewer copies of each protein encoded by the genome compared with vertebrates, e.g., one c~-actinin rather than four, further simplifying the analysis of the distribution of a particular kind of protein. Injection of double-stranded RNA can be an effective way to knock down protein expression, provided the bulk of the protein in the embryo comes from new synthesis. Sophisticated manipulation of the proteins is possible using the powerful molecular genetic techniques available in this organism. While Drosophila has many advantages for cell biological analysis, it also has some drawbacks. The cells are small: an embryonic epidermal cell, for example, has dimensions of only 2 x 5 ~tm compared to a vertebrate epidermal cell of 10 x 20~tm, which can make it difficult to resolve different intracellular compartments. The embryo is the only stage where the whole animal can be stained in its entirety; at late stages of development, antibody penetration is blocked by the secreted exoskeleton [although the use of proteins tagged with green fluorescent protein (GFP) circumvents this problem]. Therefore, the most easily generated samples for analysis are the embryo or tissues, that are easily dissected from the larva or adult, such as the imaginal discs and ovaries. Only a small number of Drosophila cell lines are available for in vitro culture and experimentation. These represent just a few cell types and are also small. Finally, antibodies raised against vertebrate proteins rarely bind to Drosophila orthologues, even when they are highly conserved. Therefore, new antibodies need to be raised to see the distribution of a given protein in Drosophila. An important exception to this are antibodies raised to specific motifs of proteins, such as those recognising particular phosphorylated residues. A growing number of antibodies against Drosophila proteins are available, either from the Developmental Studies Hybridoma Bank or directly from the laboratory that has generated them; few are available commercially. This article describes the techniques used to look at cell junctions and cytoskeletal structures in the developing embryo, using both antibodies against