CONSEQUENCES OF TOPOISOMERASE-II INHIBITION IN EARLY EMBRYOGENESIS OFDROSOPHILA REVEALED BY INVIVO CONFOCAL LASER SCANNING MICROSCOPY

The regulation of DNA topology by topoisomerase II from Drosophila mel anogaster has been studied extensively by biochemical methods but litt le is known about its roles in vivo. We have performed experiments on the inhibition of topoisomerase II in living Drosophila blastoderm emb ryos. We show that the enzymatic activity can be specifically disrupte d by microinjection of anti-topoisomerase II antibodies as well as the epipodophyllotoxin VM26, a known inhibitor of topoisomerase II in vit ro. By labeling the chromatin of live embryos with tetramethylrhodamin e-coupled histones, the effects of inhibition on nuclear morphology an d behaviour was followed in vivo using confocal laser scanning microsc opy. Both the antibodies and the drug prevented or hindered the segreg ation of chromatin daughter sets at the anaphase stage of mitosis. In addition, high concentrations of inhibitor interfered with the condens ation of chromatin and its proper arrangement into the metaphase plate . The observed effects yielded non-functional nuclei, which were drawn into the inner yolk mass of the embryo. Concurrently, undamaged nucle i surrounding the affected region underwent compensatory division, lea ding to the restoration of the nuclear population, and thereby demonst rating tbe regulative capacity of Drosophila blastoderm embryos.