Worm Breeder's Gazette 3(2): 18

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.

A Permeable Egg-shell Mutant of C. elegans and its use in Development of a Culture Medium for Embryonic Cells

J. Laufer

C.  elegans eggs are permeable to oxygen and water, but impermeable 
to most solutes.  When dissected from a gravid adult into distilled 
water, they will develop and hatch normally.  One of the recessive 
lethal mutations we have recently isolated (ct11r1), characterized 
originally as a maternal egg-lethal, appears to have a more permeable 
egg shell.  This mutant was isolated and maintained as CT-H11, a 
triple heterozygote of genotype dpy-10 (e128) 11/ct11 unc-4 (e120).  
Analysis of the fertile UNC segregants of CT-H11 indicates that ct11 
maps to the left of dpy-10, about 3% from unc-4.  CT-H11 gives UNC 
wild-type and DPY progeny in the ratio 1:2:1 indicating that the + 
maternal allele is sufficient to rescue embryos homozygous for ct11.  
Most of the UNC progeny of CT-H11 are homozygous for ct11 and lay eggs 
that fail to hatch.  The mutant embryos undergo some cleavages inside 
the adult or in vitro in a buffered salt solution, but die immediately 
in distilled water.  The defective ct11 unc-4/ct11 
e up methylene blue and cytochalasin B, which 
exhibits the classical effect of blocking cytokinesis but not nuclear 
division.  They also incorporate radioactive amino acids into acid-
precipitable material.
As an approach toward developing a culture medium for embryonic 
cells, we are attempting to find conditions under which the permeable 
embryos will develop normally to hatching.  In a mammalian tissue 
culture medium (medium 199), defective embryos develop further and 
more consistently than in buffered saline.  Addition of a number of 
mammalian serum supplements slightly improves the results.  However, 
addition of Ascaris des coelomic fluid (collected 
using a hypodermic syringe from live Ascaris obtained from pig 
intestines) allows the permeable embryos to consistently continue 
cleavage to a late stage of embryogenesis.  Many of the embryos twitch,
and some undergo limited morphogenesis to form a normal appearing 
comma stage.  In fact, the use of Ascaris coelomic fluid obviates 
altogether the need for the tissue culture medium.  Our best results 
to date have been obtained with 25% Ascaris coelomic fluid in a simple 
phosphate-buffered saline solution (7.5 mM sodium phosphate pH 7.2, 
100 mM NaCl, 4 mM KCl, 0.75 mM MgSO4, and 0.75 mM CaCl2).  We plan 
fractionation experiments to partially characterize the active 
components in the coelomic fluid.