Worm Breeder's Gazette 15(4): 42 (October 1, 1998)

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.

mev-1 and rad-8 embryos undergo massive apoptosis under high oxygen

Phil Hartman1, Naoaki Ishii2, Nanami Senoo-Matsuda2, Asako Sugimoto3

1 Department of Biology, Texas Christian University, Fort Worth, TX
2 Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa 259-11
3 Department of Biophysics and Biochemistry, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113

           We have studied mev-1 and rad-8 extensively owing to our
interest in free radical damage and its effects on aging.1-3  Mutations
in both genes render animals hypersensitive to high concentrations of
oxygen as well as cause precocious aging.  We now report that oxygen
also triggers  massive apoptosis in rad-8 and mev-1 embryos.  Evidence
includes the following:

        1) mev-1 and rad-8 embryos contain many cells that appear
apoptotic when viewed under Nomarski DIC.   The two mutations are
different in terms of the kinetics of apoptosis.   Specifically, rad-8
early embryos (<8 cell) will arrest- usually before the 16 cell
stage--if exposed to high oxygen and not produce apoptotic nuclei. 
Conversely, apoptosis is readily evident if rad-8  embryos are exposed
to high oxygen at the 50-300 cells stage.  On the other hand, apoptosis
in mev-1 embryos is most prevalent if early embryos exposed immediately
to high oxygen.  Apoptotic nuclei are not widespread in either mutant
until 24 hours after oxygen presentation.  The number of apoptotic
nuclei per embryo varies and approaches 100% with extended incubation. 
Such embryos are indistinguishable from ced-9(gf) embryos, which serve
as nice positive controls. 

        2) DAPI stained mev-1 and rad-8 embryos contain nuclei that have
a punctate appearance.  The kinetics correlate with the Nomarski
observations mentioned above.  In addition, these embryos appear
indistinguishable from DAPI-stained ced-9(gf) embryos, which are known
to undergo massive apoptosis.

        3) DNA extracted from mev-1 and rad-8 embryos yields a
nucleosomal ladder upon gel electrophoresis.  As with the other two
criteria, the ladder becomes apparent after 24-48 hours of embryo
incubation in high oxygen.

        To elucidate the relationship between this phenotype and that
controlled by the ced genes, we have constructed double mutants using lf
mutations in ced-3 and ced-9 with rad-8 and mev-1.   Under atmospheric
oxygen ced-3 suppresses the increased number of apoptotic nuclei caused
by the mev-1 mutation; specifically, the number of apoptotic nuclei per
embryo were 3.8, 8.4 and 0.1 for unc-50, unc-50 mev-1 and unc-50 mev-1;
ced-3 mutants, respectively.  In addition, ced-3 renders mev-1 animals
slightly less hypersensitive to high oxygen concentrations.  These
interactions are under continued investigation.

        1Ishii et al., (1990) Mutat. Res. 237, 165; 

        2Ishii et al. (1993) Mech. Ag. Develop. 68, 1; 

        3Ishii et al. (1998) Nature 394,694.