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.

Cytoplasmic Cell Cycle Control in Embryos of C. elegans

E. Schierenberg, W.B. Wood

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 
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 
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 
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.