Three-dimensional light microscopy for the analysis of chromosome organization in Drosophila embryo

Abstract

We are interested in the three-dimensional physical relationships among chromosomes in mitotically active nuclei. Using high-resolution, three-dimensional optical sectioning microscopy incorporating a charge-coupled device (CCD), we have analyzed the spatial arrangements of chromosomes in nuclei from Drosophila melanogaster embryos. During early stages of nuclear division, these embryos exist as syncytial blastoderms, with up to 5000 nuclei forming a single layer near the embryo's surface and dividing essentially synchronously. This greatly facilitates the analysis of chromosome organization because of the simple monolayer arrangement of nuclear structures at defined mitotic stages.From prophase to anaphase, three-dimensional paths of condensed chromosomes stained with a DNA-specific fluorescent dye can be traced using an interactive computer program. We have used this method to ask the specific biological question of whether pairs of homologous chromosomes have a consistent, ordered arrangement in diploid nuclei. Our results have shown that homologous chromosomes are not associated with each other from prophase to anaphase in syncytial blastoderm stage embryos.