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.

ced-9: A New Gene that Determines if Cells Live or Die

Michael Hengartner, Ronald Ellis and Bob Horvitz

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.