Worm Breeder's Gazette 11(2): 102
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.
In C. elegans hermaphrodites, 131 cells undergo programmed cell death during development. Whereas mutations in ced-3 and ced-4 block almost all of these deaths, mutations in ces-1 and ces-2 allow the survival of only a few cells, including the pharyngeal NSM sister cells, which normally undergo programmed cell death (Ellis and Horvitz, WBG, 10, 1, p106). While screening for more mutations that allow the survival of the NSM sisters, we isolated six new mutations that prevent all programmed cell deaths. Five of these mutations are new recessive alleles of ced-3 and ced-4. The sixth mutation, n1950, is a semidominant suppressor of cell death, and displays a significant maternal effect. We have quantified the degree of cell survival by counting the number of extra cells found in the anterior pharynx, a physically well delimited and easily scored region of the worm. Homozygous n1950/n1950 mutants possess an average of 13 extra cells in the anterior pharynx; approximately the same number is found in ced-3 and ced-4 homozygotes (13 and 14 extra cells, respectively). n1950/+ heterozygotes from +/+ mothers have an average of four extra cells in the anterior pharynx, while heterozygotes from n1950/+ or n1950/n1950 mothers have an average of 11 extra cells. We mapped n1950sd to the right arm of chromosome III, very close (< 0.2 m.u.) to and right of unc-69. The n1950 mutation defines a new gene, ced-9, that affects programmed cell death. To test whether ced-9(n1950) affects programmed cell deaths outside the pharynx, we constructed a ced-9( n1950) III; ced-5(n1812) IV double mutant. Like ced-1 and ced-2 mutants, ced-5 worms are defective in cell corpse engulfment, and ced- 5 L1 larvae contain large numbers of undergraded corpses throughout the worm. The double mutants show a greatly reduced number of cell corpses in all regions we examined. In addition, n1950 suppresses egl- 1 mutations. The HSN neurons inappropriately undergo programmed cell death in egl-1 hermaphrodites, leading to an Egl phenotype. The ced-9( n1950); utant is non-Egl, indicating that n1950 allows the HSNs to survive, differentiate, and function properly. The semidominance of n1950 suggests that this allele does not represent the ced-9 loss-of-function phenotype. This hypothesis is supported by the fact that n1950-like mutations appear to be rare (one allele found out of a total of 27,000 haploid genomes screened). We therefore sought loss-of-function alleles of ced-9 by reverting n1950 using an egl-1 suppression scheme, taking advantage of the fact that while most egl-1(n487sd)/+ V heterozygotes are Egl, n1950/+; egl-1/+ animals are non-Egl. We mated egl-1 males with EMS-mutagenized unc-69 xol-1 hrodites, and screened for Egl F1 cross- progeny. From a screen of 1,000 haploid genomes, we isolated one suppressor of n1950, which is tightly linked to ced-9. Homozygous n1950 n2077 animals derived from heterozygotes are viable, but produce only a few eggs, which never hatch. Since n1950 results in a gain of ced-9 function and blocks cell death, we reasoned that the loss of ced- 9 function might be lethal because it leads to ectopic cell death. This model predicts that ced-3 or ced-4 mutations could suppress the lethality of ced-9(n1950 n2077). Indeed, both unc-69 50n2077); unc-69 50 n2077) worms generate viable and fertile progeny. These experiments demonstrate that ced-3 and ced-4 function are essential for the expression of the ced-9(n1950 n2077) lethal phenotype. An interesting possibility is that the ced-9 product usually acts to prevent ced-3 and ced-4 from acting in cells destined to survive, thus protecting these cells from programmed, cell death. In cells destined to die, the ced-9 gene product is inactivated, thus allowing the ced-3 and ced-4 activities to kill the cell. We plan to test this hypothesis by further genetic experiments and by molecular cloning of ced-9.