Worm Breeder's Gazette 9(3): 85

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The Critical Times Microfilaments are Needed to Generate Asymmetries in the First Cell Division of C. elegans

D. Hill and S. Strome

Figure 1

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}

Figure 1