Worm Breeder's Gazette 8(1): 21
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.
We have developed a technique to extrude either considerable amounts of cytoplasm without nuclei or individual nuclei with small amounts of cytoplasm from early embryos through holes produced by a laser microbeam in the egg shell. Using these techniques in conjunction with laser-induced cell fusion (Schierenberg, Dev. Biol., in press) we have performed various experiments to test cytoplasmic influences on cell cycle timing. We summarize some of the experimental results below. 1) Nuclei in a cleavage-blocked blastomere divide synchronously. Their division cycle is faster than the mean division cycle of the progeny cells that would normally arise from that blastomere. To obtain these results, we blocked 1-, 2-, and 4-cell embryos with cytochalasin D, after making them permeable with a laser-induced hole in the egg shell, and then timed several cycles of nuclear division. 2) Enucleated 1-cell embryos cycle with a period close to that of nuclei in cleavage-arrested 1-cell embryos. When both pronuclei (with or without centrioles) are extruded from embryos prior to first cleavage, the remaining cytoplasts undergo cycles of contraction (with little cytoplasmic streaming) and expansion (with strong cytoplasmic streaming, forming temporary pseudopodia). 3) Enucleated blastomeres cycle with different periods. When AB cells and P1 cells are enucleated and their cycling periods recorded, P1 cytoplasts cycle more slowly (similarly to cleavage- blocked P1 cells) than AB cytoplasts (similarly to cleavage-blocked AB cells). Small extruded partial cytoplasts containing AB or P1 cytoplasm exhibit cycling periods close to those of the respective whole cytoplasts. 4) Cell cycle periods are only marginally influenced by cell size. The normal AB:P1 volume ratio is about 60:40. When cytoplasm is extruded from the AB cell so that it becomes smaller than P1 (ratio about 40:60), the AB cell cycle becomes slightly slower, but remains faster than that of any P1 descendant. When ABa is enucleated and fused to a granddaughter of ABp, increasing the volume of the latter several fold, the subsequent cell cycles of the enlarged cell and its descendants are only marginally accelerated. 5) Cell cycle periods are not (much) influenced by DNA content. Extrusion of the maternal pronucleus gives a haploid embryo which shows apparently normal early cleavage but arrests at several hundred cells without visible morphogenesis. When haploid, normal diploid, and tetraploid embryos (of a tetraploid N2 strain obtained from R. Herman) were compared, the cell cycle periods were approximately the same in all three. 6) Cell cycle periods can be strongly influenced by cytoplasm from a different lineage. When ABp is enucleated and fused to P2 the cell cycles of P cells and D cells are always considerably accelerated. In addition, the P4 descendants undergo extra divisions, and C cells often show accelerated cycles. When P2 is enucleated and fused to ABp the subsequent cycles of the fused cell are retarded only slightly. 7) Short-term effects of cytoplasm differ with time in the cell cycle. When one of two adjacent cells at different stages in the cell cycle (1) is enucleated and then fused to the other (2), the time at which the hybrid cleaves is different from the time at which cell (2) would have cleaved: sooner if cell (1) was closer to its next mitosis than cell (2), and later if cell (1) was further from its next mitosis. For example, if ABp is enucleated and fused just after prophase to P2, which is then in interphase, the subsequent division of P2 is delayed, even though the cell cycle in P2 descendants is accelerated (see 6). 8) Cytoplasmic changes during the cell cycle occur in the absence of a nucleus. When ABp is enucleated during interphase and fused to P2 immediately, the subsequent division of P2 occurs sooner than normally. However, when ABp is enucleated during interphase and fused only several minutes later (after the division of ABa, which normally divides synchronously with ABp), the division of P2 is delayed. These observations are consistent with the presence of cytoplasmic substances which 1) are partitioned at different concentrations to different blastomeres, 2) oscillate from an active to an inactive form during the cell cycle to initiate or prevent mitosis, and 3) have a period of oscillation that depends upon their concentrations.