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.