Worm Breeder's Gazette 10(3): 161

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.

Centrifugation of Early C. elegans Embryos

Petra Schlicht and Einhard Schierenberg

Individual embryos in the pronuclear stage have been attached to a 
polylysine-coated microscope slide and centrifuged in a predetermined 
orientation at 16.000g.  For this the slide is fixed in the slot of a 
plastic container which fits exactly into the metal bucket of a swing-
out rotor.  Under these conditions the eggs reliably stick to the 
glass surface and slides do never break.  At a starting temperature of 
80 C the eggs are centrifuged for 15 min.  During this time they 
usually cleave once.  Longer centrifugation at lower temperatures can 
suppress cleavage but obviously not replication of centrioles because 
frequently tetrapolar cleavage takes place after transfer to room 
temperature.  Using the parameters stated above, eggs were centrifuged 
in three different orientations: a.  from posterior to anterior, b.  
from anterior to posterior, c.  perpendicular to the centrifugal axis. 
In all cases three distinct cytoplasmic layers formed during 
centrifugation: at the centrifugal pole a densely packed area of yolk 
granules, a granula-free area in the middle and lipid droplets 
concentrated at the centrifugal pole.  Neither yolk granules nor lipid 
droplets appear to be essential for cleavage.  Those cells which had 
received essentially clear cytoplasm nevertheless were found to cleave 
with the same cell cycle periods as others and appear to produce a 
comparable number of cells.  Centrifugation from the posterior to the 
anterior pole of the egg results in an unequal cleavage, AB always 
being too large relative to P1.  Consequently, abnormal cell cycle 
rhythms are expressed such that in AB descendants they are accelerated 
and P1 descendants they are retarded, leading to an altered sequence 
of cleaving cell groups and early pattern defects.  After 
centrifugation from the anterior to the posterior pole, different 
types of 2-cell stages result.  These include an enlarged posterior 
cell with faster cell cycle periods than the smaller anterior cell or, 
alternatively, the generation of two cells of similar size and 
behavior.  None of them expresses germline quality.  After a-p 
centrifugation often a cytoplast containing most of the yolk granules 
forms at the posterior pole which does not participate in cleavage.  
Consequently, monsters with several hundred cells develop which may 
express visible signs of differentiation (e.g gut birefringency and 
muscle twitching).  Centrifugation perpendicular to the long axis of 
the egg gave results similar to centrifugation from anterior to 
posterior.  Attempts to obtain two P1-like cells (like in Boveri's 
experiments with Ascaris eggs ) have been unsuccessful so far.  
Analysis of P granule distribution and of development after pulsed 
application of cytoskeletal inhibitors prior to centrifugation are