INTRODUCTION Ctenophores have a single body axis (oral-aboral) with two perpendicular planes of symmetry dividing the embryo into four quadrants. Each quadrant derives from one blastomere at the 4-cell stage. These blastomeres undergo two unequal divisions to give rise to two types of macromere and two types of micromere at the 16-cell stage. These all have dif- ferent fates and develop autonomously in classical mosaic fashion. Two of the eight characteristic rows of ciliated comb-plates of ctenophores derive from one micromere in each quadrant (see Reverberi, 1971; Freeman, 1979; Sardet et al., 1990 for reviews). The high degree of organisation in cleavage stage embryos becomes established entirely after fertilisation, the egg having no pre-determined devel- opmental axis. The position of the oral pole coincides with the site of initiation of the first cleavage furrow, and not to the ‘animal pole’ where the polar bodies are emitted (Free- man, 1977). The site of first cleavage depends in turn on the position of sperm entry, which can be anywhere over the egg surface (Freeman, 1977; Carré and Sardet, 1984). The female pronucleus migrates to join a stationary male pronucleus, ‘choosing’ one in cases where polyspermy has occurred (Carré and Sardet, 1984). At this site, the zygote nucleus forms, first mitosis occurs and the first cleavage furrow starts, cleavage being unipolar. The planes of sym- metry of the larvae can be predicted by the orientation of the first cleavage, and may be related to the path of migra- tion of the female pronucleus (Carré et al., 1991). Pronu- clear migration, mitosis and all other cytoplasmic events occur in a 5-50 μm thick peripheral layer of ‘ectoplasm’ that surrounds a central ball of yolky ‘endoplasm’. This 75 Development 117, 75-87 (1993) Printed in Great Britain © The Company of Biologists Limited 1993 The single axis (oral-aboral) and two planes of symme- try of the ctenophore Beroe ovata become established with respect to the position of zygote nucleus formation and the orientation of first cleavage. Bisection of Beroe eggs at different times revealed that differences in egg organisation are established in relation to the pre- sumptive oral-aboral axis before first cleavage. Lateral fragments produced after but not before the time of first mitosis developed into larvae lacking comb-plates on one side. Time-lapse video demonstrated that waves of cytoplasmic reorganisation spread through the layer of peripheral cytoplasm (ectoplasm) of the egg during the 80 minute period between pronuclear fusion and first cleavage, along the future oral-aboral axis. These waves are manifest as the progressive displacement and dis- persal of plaques of accumulated organelles around supernumerary sperm nuclei, and a series of surface movements. Their timing and direction of propagation suggest they may be involved in establishing cytoplas- mic differences with respect to the embryonic axis. Inhibitor experiments suggested that the observed cytoplasmic reorganisation involves microtubules. Noco- dazole and taxol, which prevent microtubule turnover, blocked plaque dispersal and reduced surface move- ments. The microfilament-disrupting drug cytochalasin B did not prevent plaque dispersal but induced abnor- mal surface contractions. We examined changes in microtubule organisation using immunofluorescence on eggs fixed at different times and in live eggs following injection of rhodamine-tubulin. Giant microtubule asters become associated with each male pronucleus after the end of meiosis. Following pronuclear fusion they disappear successively, those nearest the zygote nucleus shrinking first, to establish gradients of aster size within single eggs. Regional differences in micro- tubule behaviour around the time of mitosis were revealed by brief taxol treatment, which induced the for- mation of small microtubule asters in the region of the nucleus or spindle during both first and second cell cycles. The observed wave of change may thus reflect the local appearance and spreading of mitotic activity as the zygote nucleus approaches mitosis. Key words: cleavage, ctenophore, embryonic axis, microtubule, mitosis, aster SUMMARY Axis establishment and microtubule-mediated waves prior to first cleavage in Beroe ovata Evelyn Houliston, Danièle Carré, Jennifer A. Johnston 1 and Christian Sardet Unité de Biologie Cellulaire Marine (URA 671 CNRS/Université Paris VI), Observatoire de Villefranche-sur-mer, Station Zoologique, 06230 Villefranche-sur-mer, France 1 Department of Biological Science, Dartmouth College, 306 Gilman, Hanover, NH 03755-1893, USA