Chapter 21 Looking at Drosophila Mitotic Chromosomes

Abstract

The repertoire of cytological procedures described in the present paper permits full analysis of brain neuroblast chromosomes. Moreover, if brains are cultured for 13 hr in the presence of 5-bromo-2'-deoxy-uridine, our fixation and Hoechst staining protocols allow visualization of sister chromatid differentiation and the scoring of sister chromatid exchanges (Gatti et al., 1979). Finally, we note that our cytological procedures can be successfully employed for preparation and staining of gonial cells of both sexes and male meiotic chromosomes (Ripoll et al., 1985; our unpublished results). Good chromosome preparations of female meiosis are obtained with the procedure described by Davring and Sunner (1977, 1979), Nokkala and Puro (1976), and Puro and Nokkala (1977). In this chapter, we have focused on the organization and behavior of Drosophila mitotic chromosomes, describing a repertoire of cytological techniques for neuroblast chromosome preparations. We have not considered the numerous excellent cytological procedures for embryonic chromosome preparations (for an example, see Foe and Alberts, 1985; Foe, 1989), because these chromosomes are usually less clearly defined than those of larval neuroblasts. In addition, we have not included the whole-mount and squashing techniques that allow chromosome visualization and spindle immunostaining of neuroblast cells (Axton et al., 1990; Gonzalez et al., 1990), male meiotic cells (Casal et al.. 1990; Cenci et al., 1994), and female meiotic cells (Theurkauf and Hawley. 1992), because the fixation methods used in these procedures alter chromosome morphology. Fixation methods for antibody staining result in poorly defined chromosomes, whereas the methanol/acetic acid fixation techniques, such as those described here, preserve very well chromosome morphology but remove a substantial fraction of chromosomal proteins. Thus, one of the major technical breakthroughs in Drosophila mitotic cytology will be the development of fixation procedures that maximize chromosomal quality with minimal removal of proteins. This will be particularly useful for precise immunolocalization of heterochromatic proteins, including those associated with the centromere.