Worm Breeder's Gazette 9(3): 85
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Recently, we have been determining the time microfilaments (MFs) are needed to generate and maintain proper asymmetry during the first cell cycle of Caenorhabditis We can disrupt the MF system using the MF inhibitor cytochalasin D (CD). As an indication of asymmetry, we have been observing the behaviour of the zygote during pseudocleavage, pronuclear migration, segregation of germ-cell- specific granules (P granules), and first cell cleavage, all of which clearly show some indication of asymmetry. As has already been reported, when embryos are made permeable to CD early in the first cell cycle all manifestations of asymmetry are destroyed: pseudocleavage disappears, the egg and sperm yronuclei meet in the center of the embryo, P granules fail to segregate posteriorly and the first mitotic spindle sets up and remains symmetrically placed throughout karyokinesis. If one-cell embryos are treated with CD after the pronuclei have met and P granules have been segragated, P granules remain posterior but the mitotic spindle sets up and remains symmetric. This suggests that CD affects those events which have not yet taken place, but does not reverse the asymmetries already generated. It also suggests that MFs are not required to hold P granules in the posterior of the embryo once they have been segregated there. They may be anchored to non-MF components or may not be free to diffuse due to the nature of the cytoplasm. Zygotes can be pulsed with CD by introducing drug and then washing the drug out of the zygotes at specific times. Control experiments show that CD disrupts MFs within one minute of drug treatment. The minimum time zygotes were treated with drug during experiments was two minutes. Based on three types of CD pulse experiments (see diagram), it appears that MFs are not required prior to pseudocleavage and pronuclear migration but are required from pronuclear meeting until cleavage to generate proper asymmetry. l)If the embryo is treated very early, during pronuclear formation or early pseudocleavage, and the drug is washed out two minutes later, the embryo will resume pseudocleaving and behave like a normal embryo with respect to all observable aspects of asymmetry. 2)If CD is added to the embryo prior to pronuclear migration and washed out shortly after the pronuclei reach the center of the embryo, the embryo is able to recover enough to form a cleavage furrow and carry out cytokinesis. However, the proper asymmetry of the cleavage is lost. Most often the embryo divides into two equal-sized daughters, although a small percentage of the time the furrow is formed to give either a slightly larger AB-cell or a slightly larger Pcell. The P granules are not able to recover from the treatment and remain localized in the center of the embryo as they do in a continuously cytochalasin-treated embryo. 3) Preliminary results show that embryos treated shortly after pronuclear migration and P granule segregation and then washed several minutes later also undergo symmetric cleavage. However, in these embryos, P granules are segregated to the posterior cell. We conclude from these experiments that proper MF structure is needed at a critical time, during late pseudocleavage and pronuclear migration, in order for pronuclei to meet properly and P granules to segregate properly. After this time, microfilaments are needed in order to generate proper asymmetry in the first cell division itself but are dispensible with respect to events which have already occurred (P granule localization). Further experiments will be done with various cytochalasin-pulsed embryos to examine how pulses affect the developmental potential of daughter cells. {Figure 1}