Development 112, 591-603 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

591

Two C. elegans genes control the programmed deaths of specific cells in

the pharynx

RONALD E. ELLIS* and H. ROBERT HORVITZ

Howard Hughes Medical Institute, Department of Biology, M.l.T. Cambridge, MA 02139, USA

•Current address: Laboratory of Molecular Biology, University of Wisconsin, 1525 Linden Drive, Madison, WI 53706, USA

Summary

The genes ces-1 and ces-2 control the decisions of twocells in the nematode Caenorhabditis elegans to undergoprogrammed cell death. Mutations that cause a gain ofces-1 function or a reduction of ces-2 function preventthese cells, the sisters of the two pharyngeal NSMneurons, from dying. These mutations do not affect most

other cell deaths. Genetic studies indicate that ces-1 andces-2 affect the fates of the NSM sisters by regulating thegenes required for all programmed cell deaths to occur.

Key words: cell death, C. elegans, nematode, neuron,serotonin, ces-1, ces-2, ced-3, ced-4.

Introduction

During the development of both vertebrates andinvertebrates, many cells are born that neither dividenor form part of the adult, but instead die (Saunders,1966; Cowan et al. 1984; Truman, 1984). Programmedcell death removes cells that do not function or thatfunction only transiently (Finlayson, 1956; Sulston et al.1983), influences morphogenesis (e.g. Hinchliffe, 1981;Steinberg and Horvitz, 1981), regulates the size ofneuronal populations (Hamburger and Oppenheim,1982) and helps create sexual dimorphism (e.g. Hinch-liffe, 1981; Sulston et al. 1983). One animal in whichprogrammed cell death has been studied in detail is thenematode Caenorhabditis elegans. Of the 1090 somaticcells generated during the normal development of a C.elegans hermaphrodite, 131 die. The same cells die inevery animal, each at its own precise time, and all showsimilar changes in morphology as they undergo celldeath (Sulston and Horvitz, 1977; Sulston et al. 1983).

Genetic studies of C. elegans have denned ten genesthat function in the programmed deaths of these 131cells. Eight of these genes are required for thedegradation of dead cells. Mutations in the genes ced-1,ced-2, ced-5, ced-6, ced-7, ced-8 and ced-10 preventdead cells from being engulfed and degraded by theirneighbors (ced=cell death; Hedgecock et al. 1983; Ellisetal. 1991), and mutations in the gene nuc-1 prevent theDNA of a dead cell from being digested (nuc=nuclease;Sulston, 1976; Hedgecock et al. 1983). Two geriesTced-3and ced-4, are required for the deaths of all 131 cells(Ellis and Horvitz, 1986). Mutations in either of thesegenes prevent all of these cell deaths from occurring.Mosaic analyses indicate that ced-3 and ced-4 act within

dying cells, possibly as part of a cellular suicide program(Yuan and Horvitz, 1990). These results suggest thatced-3 and ced-4 either encode or regulate the expressionof cytotoxic substances.

How are the genes that act in programmed cell deathregulated? Cell deaths occur throughout C. elegansdevelopment (Sulston and Horvitz, 1977; Sulston et al.1983), and cells that are prevented from dying bymutations in ced-3 or ced-4 develop into many differentcell types (Ellis and Horvitz, 1986; Avery and Horvitz,1987). Because of the diversity in origin and cell typeamong dying cells, there could well be regulatory genesthat control the fates of some dying cells but not ofothers. Such regulatory genes might choose betweenlife and death for specific cells by controlling theactivities of ced-3 and ced-4 in specific cell types. Tounderstand how cell death is initiated in the correct cellsduring development, it is important to identify suchregulatory genes and determine how they interact withthe genes that cause cells to die.

We have identified and characterized two genes thatcontrol the decision by specific cells to live or die: ces-1and ces-2 (ces=cell death specification). Mutations inthese genes can~~prevent specific cell deaths withoutaffecting the deaths of other cells. We show that thesegenes act together to determine the fates of two cells inthe pharynx, the sisters of the NSM neurons, probablyby regulating the cell death genes ced-3 and ced-4.

Materials and methods

General methods and strain maintenanceTechniques for culturing C. elegans are described by Brenner

592 R. E. Ellis and H. R. Horvitz

(1974). All strains were grown at 20 °C unless otherwiseindicated. The wild-type parent of all strains described here isC. elegans variety Bristol strain N2 (Brenner, 1974). Geneticnomenclature is described by Horvitz et al. (1979).

Genetic markersWe used the following mutations as genetic markers: LGI,dpy-5(e61), dpy-14(el88), unc-13(e51), unc-13(elO91), lin-10(el439), daf-8(el393), fer-l(hcl), sup-17 (nl258), unc-29(elO72), ced-l(e!752), unc-59(el005), lev-10(xl7), unc-54(elO92), let-208(el719), let(e2000); LGIII, ced-4(nll62);LGIV, ced-2(el752), him-8(el489), unc-31(e928), ced-3(n717); LGV, him-5(el490). We used the following chromo-somal rearrangements: nDf23 I, nDf24 I, nDf25 I, eDf3 I,eDf4 I, eDf6 I, eDf9 I, eDflO I, eDfl3 I, eDfl4 I,nDp4(I;unknown), sDpl(I;f). The mutations unc-54(elO92)and let(e2000) (previously known as let(r202)), are describedby Anderson and Brenner (1984), both sup-17(nl258) andnDp4 have been characterized by J. Thomas (personalcommunication), the mutations unc-13(elO91) and unc-31(e928) are from the strain collection established by Brenner(1974), and the other mutations are described by Hodgkin etal. (1988).

Serotonin stainingTechniques for staining worms with anti-serotonin antiserumare described by Desai et al. (1988).

Nomarski microscopyProcedures for using Nomarski microscopy to observe livinganimals are described by Sulston and Horvitz (1977). Becauseit is difficult to count cells in the pharynx of an animal that ismoving and feeding, we anesthetized the worms by mountingthem in a drop of M9 salt solution (Sulston and Brenner, 1974)containing 30 ITIM NaN3 (Avery and Horvitz, 1987). Weusually studied the survival of cells in worms during the L2, L3or L4 larval stages, although occasionally we scored adults orLI larvae.

Identification o/ces-l(n703) and ces-2(n732)The mutations n703 and n732 were isolated by N. Tsung andC. Trent (Trent, 1982), who used the technique of formal-dehyde-induced fluorescence (Sulston et al. 1975; Horvitz etal. 1982) to identify animals that had unusual patterns ofserotonin expression from among the broods of 4594 F2worms isolated after mutagenesis of the wild type with ethylmethanesulfonate.

Mutagenesis experimentsWe used the technique for mutagenesis with ethyl methane-sulfonate (EMS) described by Brenner (1974). In thereversion of ces-l(n703), the number of F] animals scoredequals the number of haploid genomes screened. However, inthe screen for new ces mutants, we screened 21000 F2 animalsfrom among the descendants of about 27000 Ft animals froman EMS mutagenesis, which corresponds to 54000 mutanthaploid genomes. Some of the chromosomes from Fj animalswould have been homozygous in more than one F2 animalscored, so we estimated the probability of scoring oneparticular haploid genome present among the Fj to bel-(53,999/54000)loS°°=0.18. Thus we screened(54000)(0.18)=9720 haploid genomes for recessive mu-tations. Similar considerations indicate that we screenedabout 24000 haploid genomes for dominant mutations.

Mapping ces-l(gf) allelesL. Avery and H. Ellis (personal communications) had

observed linkage of ces-l(n703) to markers on LGI. Thefollowing cross confirms this linkage, and demonstrates thatces-l(n703) is located to the left of sup-17: 30/34 Unc non-Suprecombinant progeny of unc-13(e51) sup-17/ ces-l(n703)heterozygotes segregated ces-l(n703). The other dominantalleles of ces-1 also map to the left of sup-17: 12/13 Unc non-Sup recombinant progeny of unc-13(e51) sup-17/ ces-1 (n!895)heterozygotes segregated ces-1 (n!895), and 11/12 Unc non-Sup recombinant progeny of unc-13(e51) sup-17/ ces-1 (nl896)segregated ces-1 (nl896). The following cross demonstratesthat ces-1 (n703) is probably located to the right of both daf-8and fer-1, and to the left of unc-29: 7/8 homozygous Daf non-Fer Unc recombinant progeny from daf-8 ces-1 (n703)/ lin-10fer-1 unc-29 hermaphrodites carried ces-1 (n703). The mu-tations ces-1 (nl895) and ces-1 (nl896) are also located to theright of daf-8 and to the left of unc-29: 0/1 Unc-29 Fer non-Daf non-Unc-13 recombinant progeny of ces-1 (nl895)/ unc-13(e51) daf-8 fer-1 unc-29 heterozygotes segregated ces-I(nl895) and 2/2 Unc-29 non-Fer non-Daf non-Unc-13recombinants segregated ces-1 (n!895). From an equivalentexperiment using ces-1 (n!896), we found 0/2 Unc-29 Fer non-Daf non-Unc-13 recombinant progeny segregated ces-I(nl896), and 6/6 Unc-29 non-Fer non-Daf non-Unc-13progeny segregated ces-1 (n!896).

Mapping ces-l(lf) allelesWe mapped the location of ces-1 (n703 n!434) by examininghomozygous recombinant F2 progeny of daf-8 fer-1 unc-29/lin-10 ces-1 (n703 nl434) heterozygotes: 0/8 Lin Daf Fer Uncrecombinants, 0/1 Lin non-Daf Fer Unc recombinants, and0/2 Lin non-Daf non-Fer Unc recombinants had survivingNSM sisters. Because ces-l(n703) maps much closer to fer-1than it does to unc-29 (only 1/33 recombination events in thisinterval separated fer-1 and ces-1 (see above, also unpub-lished data)), one or both of the two lin-10 daf-8(+) fer-1 (+)unc-29 recombinant chromosomes probably carried the ces-l(n703) mutation. Neither of these animals showed the Ces-l(n703) phenotype, so the suppressor mutation nl434 musteither map to the left of n703, or near to n703 but on its right.We also tested one of the lin-10 daf-8 fer-1 unc-29chromosomes, the lin-10 daf-8(+) fer-1 unc-29 chromosome,and the lin-10 daf-8(+) fer-1 (+) unc-29 chromosomes forsuppression of ces-2. Only the two lin-10 daf-8(+) fer-l(+)unc-29 recombinants carried a suppressor of ces-2, presum-ably the nl434 mutation. These results show that nl434 islocated to the right of daf-8. Overall, these experiments showthat nl434 maps very near to n703.

Similarly, to map the location of n!406, we examinedhomozygous recombinant F2 progeny of daf-8 fer-1 unc-29/lin-10 ces-l(n703 nl406) heterozygotes: 0/6 Lin Daf Fer Uncand 0/3 Lin non-Daf non-Fer Unc recombinants hadsurviving NSM sisters. We also tested each of the three lin-10daf-8(+) fer-1 (+) unc-29 chromosomes for suppression of ces-2(n732), and all three chromosomes carried a suppressor ofces-2, presumably nl406. These results indicate that, if nl406is located to the right of n703, it must be close to the n703mutation and that, if nl406 is located to the left of n703, itmust lie in the small region that extends from just left of thegene daf-8 to ces-1 (n703).

Mapping ces-2(n732)In preliminary experiments, ces-2(n732) showed weak linkageto the gene dpy-5 on LGI (data not shown). The followingcross confirms this assignment: 16/16 Ced non-Unc recombi-nant progeny of ces-2/ ced-1 unc-54 heterozygotes segregatedces-2(n732).• Furthermore, ces-2 is located to the right of unc-54 and possibly to the right of let-208: 4/4 Unc non-Let

C. elegans genes that control specific cell deaths 593

recombinant progeny of ces-2/unc-54 let-208 heterozygotessegregated ces-2(n732). To complete the mapping of ces-2, weexamined male progeny from the following crosses involvingdeletions in this region: ces-2 males mated with eDf9/let(e2000) hermaphrodites yielded 13/20 Ces cross progeny,ces-2 males mated with eDf6/Iet(e2000) hermaphroditesyielded 8/16 Ces cross progeny, ces-2 males mated witheDf4/tet(e2000) hermaphrodites yielded 18/35 Ces crossprogeny, ces-2 males mated with eDfl3/let(e2000) her-maphrodites yielded 1/36 Ces cross progeny, and ces-2 malesmated with eDfl4/let(e2000) hermaphrodites yielded 1/22Ces cross progeny. Note that deletions that fail to comp-lement ces-2(n732) show a semidominant effect on thesurvival of the NSM sisters (see below), as does ces-2(n732)itself; however, the NSM sisters survive much less often inthese heterozygotes than in ces-2(n732)/Df animals. Becauseof the semidominant behavior of n732 and the deficiencies,these complementation tests are not conclusive, but indicatethat ces-2 probably lies in the region deleted by eDf4, eDf6,and eDf9, and does not lie in the region deleted by eithereDfl3 or eDfl4.

Mapping ces(nl952)The mutation nl952 maps to a different location than doesces-1. From lin-10 ces-l(n703 nl406) unc-29/+ + +;ces(nl952)/+ heterozygotes, 2/6 Ces(nl952) progeny segre-gated Lin Unc animals. We tested one of these two strains bycrossing with the wild type, and recovered the ces(nl952)mutation from this lin-10 ces-1 (n703 nl406) unc-29; ces(nl952)strain, which confirmed its genotype. These results indicatethat ces(nl952) is not located between lin-10 and unc-29, andso cannot be an allele of ces-1, and suggest that ces(n!952)may not be linked to ces-1.

The mutation nl952 is also not an allele of ces-2. Becauseces-2(n732) is tightly linked to the unc-54 gene, we testedces(n!952) for linkage to unc-54. We examined at 25°C fourCes(nl952) progeny of unc-54/+; ces(nl952)/+; him-8/+heterozygotes. One of these progeny was unc-54/unc-54, onewas unc-54/+, and two were +/+ . These results indicate thatces(nl952) is not closely linked to unc-54, and thus is notclosely linked to ces-2 either.

Complementation testsTo determine if ces-1 (n703 nl406) and ces-1 (n703 nl434) failto complement for the suppression of ces-2, we mated lin-10ces-l(n703 n!434) ced-1 ces-2/+ + + ces-2 males with unc-13(e51) lin-10 ces-1 (n703nl406) ced-1 ces-2 hermaphrodites at25°C. Among the non-Unc cross progeny, only 1/12 Linworms showed the Ces-2 phenotype of NSM sister survival(these animals were ces-l(n703 nl406)/ces-1 (n703 nl434)),but 16/26 non-Lin worms snowed the Ces-2 phenotype (theseanimals were ces-l(n703 nl406)/+). Therefore ces-l(n703nl406) and ces-1 (n703 nl434) fail to complement for thesuppression of ces-2.

To test ces-2(n732) and ces(nl952) for complementation,we mated ces-2(n732) males with ces(n!952) hermaphroditesat 25°C. Among the male cross progeny, only 1/17 wormsshowed the Ces phenotype, approximately the frequency atwhich ces-2/+ animals show this phenotype.

Triple mutants with the ces genes and ced-1; ced-2In ced-1 or ced-2 mutants, some cell corpses are not engulfed(Hedgecock et al. 1983), and, in ced-1; ced-2 double mutants,the number of unengulfed corpses increases significantly (Elliset al. 1991). For example, the NSM sister corpses are found ina ced-1; ced-2 double mutant, but not in ced-1 or ced-2 singlemutants (Ellis et al. 1991). We therefore constructed ces-

l(n703) ced-1; ced-2 and ced-1 ces-2(n732); ced-2 strains byselecting for recombinant progeny from the appropriate tripleheterozygotes in which (1) the extra neurons characteristic ofces-1 and ces-2 mutants were present, and (2) the number ofcell corpses was otherwise characteristic of the ced-1; ced-2double mutant. The presence of both ced-1 and ced-2 in theces-1 ced-1; ced-2 strain was verified by failure to complementced-1 and ced-2, and the presence of both ced-1 and ced-2 inthe ced-1 ces-2; ced-2 strain was verified by the presence ofNSM sister corpses at 15 °C (at which temperature the NSMsisters die in ces-2 animals, as in the wild type).

Gene dosage studiesIn all gene dosage studies, animals were anesthetized withNaN3 and examined using Nomarski microscopy to determinehow often the NSM sisters and the 12 sisters survive. Thelocation of the NSM sisters at the rear of the anterior bulb isdistinctive, but the 12 sisters are located extremely close toother cells that also die. As noted in the text, the number ofpharyngeal cells varies slightly in the wild type, so we firstdetermined the variability in the number of cells near the 12neurons to determine if this variability would introducesignificant errors into our assay for 12 sister survival. Incontrol wild-type animals at 15 CC, neither NSM sistersurvived in 95 animals scored, but an extra neuron was foundnear an 12 neuron in 21 of these animals. In 20 of the 21 cases,the extra cell was found on the left side. Most pharyngealdevelopment is left/right symmetrical, and this asymmetrysuggests that the extra cell might be the surviving sister ofMCL, since the sister of the right-side cell MCR normallysurvives, whereas the sister of the left-side cell MCL normallydies (Sulston et al. 1983). At 20°C the NSM sisters again alldied, but in 19/205 animals an extra cell was present near an12 neuron. Similarly, at 25°C the NSM sisters all died, but in7/100-animals an extra cell was found near an 12 neuron. Inboth cases, extra cells were found more often on the left sidethan on the right side. The extra cells found near the 12neurons in the wild type could be (1) surviving 12 sisters, (2)other surviving cells, such as II sisters or 12 aunts or, whenfound on the left side of the pharynx, MCL sisters, or (3)neurons generated by extra cell divisions. In the wild type, thecold-sensitive survival of the MCL sister, and the rare survivalof unknown cells occur infrequently, and so should notintroduce significant error into the measurement of 12 sistersurvival in the various ces strains that we have examined.

To determine if a deletion of the ces-1 gene results in thedominant phenotype caused by ces-1 (n703), we examined 20unc-13(elO91) lin-11/ nDf23 animals, and 20 unc-13(elO91)

• lin-11 / nDf24 animals. The NSM sisters always died in theseanimals, and 79/80 12 sisters clearly died. An extra neuronwas located near the right 12 neuron in one animal; as notedabove, an extra cell is sometimes found in this position in thewild type.

To measure the effects of altering ces-1 gene dosage, weexamined animals of several genotypes in order to determinehow often the 12 sisters and NSM sisters survived. We onlypresent data for the 12 sisters because in all strains with onecopy of ces-1 (n703) the NSM sisters survive about 90 % of thetime, so that NSM sister survival cannot be used to measurethe effects of small changes in gene dosage (Table 1, Fig. 4,data not shown). The three deficiencies of the ces-1 genebehave similarly in trans to ces-1 (n703), consistent with thegenetic map data (Fig. 1), which indicates that each deficiencyshould completely eliminate ces-1 function. Furthermore, inthe ces-1 (n703)/+ animals, it appears to make no difference ifthe ces-1 mutation is derived from the mother or from thefather, and the results also show little dependence on which

594 R. E. Ellis and H. R. Horvitz

Table 1. ces-1 gene dosage experiments

Live 12 sisters/total 12 sisters

Genotype

ces-1 (n703)/ces-l(n703)ces-1 (n703)Total

ces-l(n703)/+dpy-14+ces-l(n703)/+unc-13(e51) ++ +/ces-1 (n703) unc-29ces-1 (n703)+/ + unc-29+ ces-1 (n703)/unc-13(e51) ++ +/ unc-13(e!091) ces-1 (n703)+ Iin-10 ces-1 (n703)/unc-13 + +; him-5/+Total

ces-1 (n 703) / ces-1 (n 703nl434)+ Hn-10 ces-](n703nl434)/unc-13(el091)+ces-l(n703); him-5/+Total

ces-1 (n 703)/ces-1 (n 703nl406)+ + ces-1 (n703)/unc-13(e51) hn-10 ces-1 (n703nl406)lel(nl407) + Hn-10 ces-1(n703nl406)/+ unc-13 + ces-l(n703)Total

ces-1 (n703)/Df

dpy-14 ces-1 (n703)/nDf23dpy-14 ces-1 (n703)/nDf24unc-13(elO91) ces-1 (n703)/nDJ25dpy-14 ces-1 (n703)/nDf25Total

'Same', the genotype of the mother was identical to that of the animal being scored.'N.D.', not determined. The mutation nl407 is an uncharacterized lethal mutation on LGI that we identified dunng our reversion of ces-

l(n703) (data not shown). Fig. 4 summarizes the results of these experiments.

Maternalgenotype

same

sameces-1 unc-29

unc-29unc-13

unc-13 ces-1unc-13

unc-13 ces-1

unc-13 Hn-10 ces-1 (n703nl406),unc-13 ces-1

samesamesamesamesame

15 °C

96/11196/111

N.D.N.D.N.D.N.D.

111/200N.D.

111/200

58/14258/142

53/140N.D.

53/140

58/1%13/86N.D.N.D.N.D.

71/282

20 °C

194/211194/211

114/20354/9748/97

192/459N.D.

246/520654/1376

42/13642/136

62/19312/38

74/231

37/20010/72

31/2129/84

16/152103/720

25 °C

135/160135/160

N.D.N.D.N.D.N.D.

50/200N.D.

50/200

24/15024/150

20/140N.D.

20/140

10/10012/100N.D.N.D.N.D.

22/200

marker mutations were used (Table 1). That ces-l(n703)/+animals have more surviving 12 sisters than do ces-1 (n703)/Dfanimals indicates that the ces-1 (+) allele enhances the effectof one copy of ces-1 (n703).

We also examined animals carrying a wild-type copy of theces-1 gene on a duplication. The attached duplication nDp4covers the ces-1 gene. Animals homozygous for nDp4 are verysick and can be identified easily, and animals with a singlecopy oinDp4 are defective in egg-laying (J. Thomas, personalcommunication). In the nDp4/ + animals that we examined,which had three wild-type copies of the ces-1 gene, 30/30NSM sisters died and, in nDp4/nDp4 animals, which had fourcopies of ces-1, 20/20 NSM sisters died. The free duplicationsDpl also covers ces-1, and 12/12 NSM sisters died in dpy-5unc-13(e51)/ dpy-5 unc-13(e51)/ sDpl animals, which havethree copies of ces-1. These results suggest that increasing ces-1 gene expression even two-fold is not enough to cause theNSM sister survival seen in ces-1 (gf) mutants.

In addition, we used the attached duplication nDp4 to studythe survival of the 12 sisters in animals with two copies of ces-l(n703) and one copy of ces-l(+). Among the progeny of ces-l(n703) males crossed with unc-13(elO91) ces-l(n703); nDp4homozygotes, 32/72 12 sisters survived, and among thehealthy non-Unc progeny of unc-13(elO91) ces-1 (n703);nDp4/+ heterozygotes, 75/107 12 sisters survived. We alsostudied animals with two copies of ces-1 (+) and one copy ofces-1 (n703). Among the progeny of wild-type males crossedwith unc-13(elO91) ces-1 (n703); nDp4 homozygotes, 5/32 12sisters survived, and among the progeny of ces-1 (n703) malescrossed with unc-13(e51); nDp4 homozygotes, 11/4112 sisterssurvived. Thus, the duplication nDp4 appears to lower, not

enhance, the effect of two copies of ces-1 (n703) on thesurvival of the 12 sisters. Experiments involving these largeduplications are difficult, because they cause general sicknessin animals, and the pharynges are sometimes distorted anddifficult to score. Furthermore, we cannot prove that theeffect caused by these duplications on 12 sister survival is aconsequence of the extra copy of ces-l(+) that they contain.The fact that with one copy of ces-1 (n703) the ces-l(+) alleleenhances the effect of the ces-l(n703) mutation, whereas withtwo copies of ces-1 (n703) the ces-1 (+) allele appears tosuppress ces-1 (n703), suggests that the regulation of ces-1activity is complicated. Perhaps the ces-1 (+) product bothincreases ces-1 (n703) expression by a trans-acting auto-regulation, and also competes with ces-l(n703) product forsome limiting molecule necessary for ces-1 function. In thiscase, the overall effect of increasing ces-1 (+) activity coulddepend upon the level of ces-1 (n703) activity.

The mutation ces-2(n732) is not completely recessive. Froma cross of ces-2(n732) males with unc-13(e51) lev-10 her-maphrodites at 25 °C, 4 % of the cross progeny had a survivingNSM sister (n = 100 animals), and from a cross of wild-typemales with ces-2(n732); unc-31 hermaphrodites at 25°C, 7%of the cross progeny had a surviving NSM sister (n=100animals). Similarly, the NSM sisters sometimes survive inanimals heterozygous for a deletion of ces-2. At 25 °C, theNSM sisters survived in 12/30 eDf4/let(e2000) animals (a totalof 13/60 cells survived), and the NSM sisters survived in 9/18eDf9/let(e2000) animals (13/36 cells survived). By contrast,the nearby deletions eDfl3 and eDfl4 do not remove ces-2,and in both eDfl3/let(e2000) and eDfl4/let(e2000) animals at25°C all of the NSM sisters died («=30 animals each). As

C. elegans genes that control specific cell deaths 5951 m. u.

II 1 I I

ces-1 ces-2i i r \ I

C1 J'I

nDf23

nD/24,nD/25 \-

nDp4, sDpl :

-\ eDfl3/cDfl4

\'Df4

-\'DJ9

-\eD/6

I 0.6 m. u. 2 m. u. J

Fig. 1. A partial genetic map of Linkage Group I. The mapping of ces-1 and ces-2 is described in Materials and methods,m.u., map units.

noted above, the NSM sisters always die in the wild type aswell. (The deficiency eDflO might show a partial failure tocomplement ces-2(n732), and eDf3/eDflO heterozygotes havea low rate of NSM sister survival; data not shown).

ces-l(dm) trans-heterozygotesWe examined animals carrying different ces-1 dominantalleles in trans. Among the cross progeny of ces-1 (n703) malesmated with unc-13(e51) ces-1 (nl895) hermaphrodites 161/20012 sisters survived, among the cross progeny of ces-1 (n703)males mated with unc-13(e51) ces-l(nl896) hermaphrodites159/200 12 sisters survived, among the cross progeny of ces-I(nl896) males mated with unc-13(e51) ces-1 (nl896) her-maphrodites 168/22012 sisters survived, and among the crossprogeny of ces-1 (nl895) males mated with unc-13(e51) ces-I(nl895) hermaphrodites 160/200 12 sisters survived. Finally,among the cross progeny of ces-1 (n!895) males mated withunc-13(e51) ces-1 (nl896) 193/234 12 sisters survived, andamong the cross progeny of ces-1 (nl896) males mated withunc-13(e51) ces-1 (nl895) hermaphrodites 214/300 12 sisterssurvived. These values are all similar.

Phenotype of ces-l(lf) mutantsPreliminary observations using Nomarski microscopy sugges-ted that both ces-1 (n703 n!4O6) and ces-1 (n703 nl434) animals(n=10) appear wild-type. We further examined ces-l(n703nl406) animals in the following ways. At 25CC, two unc-13(e51) lin-10 ces-1 (n703 nl406) animals each had 80 cells inthe pharynx, the number found in the wild-type. Further-more, at 25°C, nine lin-10 ces-l(n703 n!406) unc-29 animals,examined less thoroughly, all had the wild-type number ofneurons and epithelial cells in the pharynx, and at 20°C, 19/20

lin-10 ces-1 (n703 nl406) unc-29 animals had the wild-typenumber of neurons and epithelial cells in the anteriorpharynx, and 1/20 had a single extra neuron near the 12 cell.We also examined the Unc male progeny of lin-10 ces-1 (n703nl406) unc-29/ + + + males crossed with nDf24/ unc-13(elO91) lin-11 hermaphrodites. At25°C, 3/4 such lin-10 ces-l(n703 nl406) unc-29'/ nDf24 animals had the wild-typenumber of neurons and epithelial cells in the pharynx, and 1/4may have been missing one neuron in the posterior pharynx;at 20°C, 6/6 such animals appeared wild-type. Thus, itappears that nl406 and nl434 mutants may indeed have awild-type phenotype; even ces-1 (n703 n!406)/Df worms at25 °C, which should have very little ces-1 activity, appear wild-type.

Construction of double mutantsBecause ces-1 (If) mutations result in a wild-type phenotype,we used ces-1 (If) mutations closely linked to markermutations in the constructions described in this section.Furthermore, we studied several isolates of most of thesestrains to ensure that the ces-1 (If) allele had not been lost by arare recombination event in any particular construction. Allmarkers segregated at expected frequencies in these crosses(data not shown), so we do not believe that ces-1 (If) alleleshave any lethal interactions with the other genes that we used.

Double mutants between the ces-1 (If) mutations and ced-3or ced-4 mutations were built as follows. From lin-10 ces-l(n703 nl434)/++; ced-3/+ heterozygotes we isolated Cedprogeny and, from these Ced progeny, we isolated Linoffspring. These animals are lin-10; ced-3 based on theirphenotypes, and probably are homozygous for ces-1 (n703nl406), which is tightly linked to lin-10. In four separate Lin

596 R. E. Ellis and H. R. Horvitz

Ced-3 isolates the NSM sisters always survived. A lin-10 ces-l(n703 nl406); ced-4 strain was constructed similarly, andthree separate isolates all showed the same phenotype. Webuilt unc-13(e51) lin-10 ces-l(n703 nl406); ced-3 using anequivalent procedure, and tested three separate Unc Lin Cedisolates for NSM sister survival. The strain unc-13(e51) lin-10ces-l(n703 nl406); ced-4 was built similarly, and one isolatetested. The survival of the NSM sisters in the above strainswas tested using Nomarski optics for all isolates, and withanti-serotonin staining for one isolate of each strain.

To construct the lin-10 ces-l(n703 nl434) ced-1 ces-2 strain,we isolated Lin Ced recombinant progeny from a lin-10 ces-l(n703 n!434) + +/+ + ced-1 ces-2 heterozygote. Our resultsindicated that ces-2 is suppressed by ces-1 (If), so to ensure thatces-2 was present on the lin-10 ced-1 chromosome, we matedces-2 males into the Lin Ced strain and selected progeny inwhich both NSM sisters survived. Because ces-2 is recessivefor this trait, these animals must be ces-2 homozygotes. Wethen re-isolated Lin Ced worms from among the progeny ofthe putative lin-10 ces-l(n703 nl434) ced-1 ces-2/'+ + + ces-2heterozygotes. The NSM sisters die in these Lin Ced animals,which indicates that the ces-l(lf) allele is also homozygous inthese worms. Genetic mapping (see above) proves that theactivity that suppresses ces-2 is tightly linked to ces-1, and so isnot caused by one of the other markers in this strain.Therefore, this strain must be of genotype lin-10 ces-l(n703nl406) ced-1 ces-2. As a final test that ces-2 is homozygous inthis strain, we showed that 8/8 putative lin-10 ces-l(n703nl406) ced-1 ces-2 animals behave as ces-2 homozygotes whentested for complementation with ces-2.

To construct an unc-13(e51) lin-10 ces-1 (n703 nl406) ced-1ces-2 strain, we isolated three Unc Lin Ced recombinantsfrom unc-13(e51) lin-10 ces-1 (n703 nl406) + + /+ + + ced-1ces-2 heterozygotes. The NSM sisters die in all three of theserecombinant strains, so if ces-2 is homozygous, the ces-1 (If)allele must also be homozygous so that it suppresses ces-2. Weshowed that 5/5 putative unc-13 lin-10ces-l(n703 n!406) ced-1ces-2 worms from each of these three strains behave as ces-2homozygotes when used in complementation tests with ces-2,so we conclude that each strain is homozygous for ces-2. Thuseach strain is of the genotype unc-13 lin-10 ces-l(n703 nl406)ced-1 ces-2.

To construct an unc-13(e51) lin-10 ces-1 (n703 nl406) ced-1ces-2; ced-3 strain, we isolated an Unc Lin Ced-3 animal froman unc-13(e51) lin-10 ces-l(n703 nl406) ced-1 ces-2/+ + + +ces-2; ced-31+ heterozygote. The ces-1 (If) allele is stillpresent in this strain because, in a cross of ces-2 males with theputative unc-13(e51) lin-10 ces-l(n703 nl406) ced-1 ces-2; ced-3 hermaphrodites, the F : animals segregate Unc animals inwhich the NSM sisters die (except those F2 worms homo-zygous for ced-3, in which all cells, including the NSM sisters,live).

Finally, we constructed a lin-10 ces-l(n703 nl406) unc-29;n!952 strain by first isolating animals in which one or bothNSM sisters survived from among the progeny of lin-10 ces-1unc-291 + + +; nl952/+ heterozygotes. From one of thesenl952 homozygotes, we isolated Lin Unc progeny. The deathsof the NSM sisters in this strain were confirmed usingNomarski optics.

Results

Identification o/ces-l(n703) and ces-2(n732)The mutations n703 and n732 were isolated by N. Tsungand C. Trent (Trent, 1982). In the pharynges of wild-type animals only the two bilaterally symmetric NSM

neurons contain serotonin (Horvitz et al. 1982); bycontrast, in the pharynges of both n703 and n732animals, there are four serotonergic cells (Trent, 1982).Nomarski microscopy revealed that there are two extraneurons in this region of the pharynx (H. Ellis, personalcommunication). We mapped the n703 and n732mutations, and showed that each defines a new gene onlinkage group I (Materials and methods, Fig. 1).Because these genes appear to be involved in thespecification of which cells live and which cells die (seebelow), we have named them ces-1 (n703) and ces-2(n732), where ces stands for cell death specification.

ces-l(n703) and ces-2(n732) prevent the deaths ofspecific cellsIn wild-type animals, the two NSM cells differentiateinto serotonergic neurons, and the sisters of the NSMneurons die. By contrast, in ced-3 mutants, the sisters ofthe NSM neurons along with many other cells fail todie, and there are four cells in the pharynx that containserotonin (Ellis and Horvitz, 1986). Some cells that areprevented from dying by a mutation in ced-3 adopt thefate of a near relative, which suggests that the two extraserotonergic cells in ced-3 animals are the survivingNSM sisters (Ellis and Horvitz, 1986). The NSM sistersmight similarly survive in ces-1 and ces-2 mutants,which would account for the two extra neurons and thetotal of four serotonergic cells in the pharynges of theseanimals.

To see if the NSM sisters fail to die in ces-l(n703) andces-2(n732) animals, we examined mutant larvae usingNomarski microscopy. We observed in both ces-1 andces-2 mutants an extra neuron just posterior and dorsalto each NSM neuron (Fig. 2). This position is exactlythat of the NSM sisters in wild-type animals before theydie (Sulston et al. 1983). Furthermore, staining withanti-serotonin antisera (Desai et al. 1988) revealed thatthe extra serotonergic cells in ces-1 and ces-2 mutantsstrongly resemble those found in ced-3 animals, inwhich cell deaths do not occur; in all three mutants, thenerve processes of these cells are similar in morphologyto those of the NSM neurons (data not shown). Whileviewing ces-1 mutants with Nomarski microscopy, wealso identified two additional extra neurons locatedanterior to the 12 neurons, one on each side of thepharynx (Fig. 2). These results suggest that the NSMsisters indeed fail to die in ces-1 and ces-2 animals, andthat two additional cells (possibly the 12 sisters) fail todie in ces-1 worms.

It remained possible that these extra neurons resultedfrom extra cell divisions, rather than from the survivalof cells that normally die. To explore this possibility, wedirectly examined cells that die in this region of thepharynx. In ced-1; ced-2 double mutants, the corpses ofdead cells are not quickly degraded and instead persistfor hours; these corpses can be assayed reliably(Hedgecock et al. 1983; Ellis et al. 1991). (Note that inC. elegans genetic nomenclature a semicolon separatesgenes located on different chromosomes; Horvitz et al.1979). In particular, two specific corpses, which byposition are likely to be the dead NSM sisters, are easily

C. elegans genes that control specific cell deaths 597

Fig. 2. Extra neurons in the pharynx of ces-1 animals.Nomarski photomicrographs of the pharynges of (A) wild-type and (B) ces-l(n703) L3 larvae. In the ces-l(n703)animal, the extra neurons are indicated with arrows; thecell anterior to the 12 neuron is the surviving 12 sister, andthe cell posterior to the NSM neuron is the surviving NSMsister. Anterior is to the left, and ventral is down.

visible in ced-1; ced-2 double mutants (Fig. 3A). If theNSM sisters do not die in ces-1 and ces-2 animals, thenthe corpses of the NSM sisters should be missing in ces-l(n703) ced-1; ced-2 and in ced-1 ces-2(n732); ced-2triple mutants.

We constructed the appropriate triple mutants (seeMaterials and methods) and examined newly hatchedanimals to see which cell corpses were present andwhich were missing. The putative NSM sister corpsefound posterior to each NSM neuron in ced-1; ced-2animals is present 100-fold less often in ces-1 (n703) ced-1; ced-2 animals (Table 2, Fig. 3B). Often a cell corpseanterior to the 12 neuron is also missing in ces-1 (n703)ced-1; ced-2 worms, which suggests that the extraneuron anterior to the 12 in ces-1 animals is also a cellthat fails to die. Thus, two pairs of corpses are missingin ces-1 pharynges, and these corpses correspond inposition to the two pairs of extra cells found in thesemutants (Fig. 3).

In ced-1 ces-2; ced-2 mutants, only the putative NSMsister corpse is affected by the presence of the ces-2mutation. This corpse is present about seven timesmore often in ced-1; ced-2 worms than in ced-1 ces-2(n732ts); ced-2 animals raised at 25°C (Table 2).Furthermore, these data reveal that the ces-2(n732)mutation is strongly temperature-sensitive. Thus, inces-2 mutants at 25 °C, one pair of pharyngeal corpses ismissing, and these missing corpses correspond inposition to the extra pair of serotonergic neurons foundin these animals.

These results strongly suggest that the NSM sisters donot die in ces-1 and ces-2 mutants, and that twoadditional cells, located anterior to the 12 neurons, fail

Fig. 3. The corpses of the NSM and 12 sisters in ced-1;ced-2 animals. Nomarksi photomicrographs of thepharynges of (A) ced-1; ced-2 and (B) ces-l(n703) ced-1;ced-2 LI larvae. In the ces-l(+) animal, the corpseindicated anterior to the 12 neuron is the 12 sister corpse,and the corpse indicated posterior to the NSM neuron isthe NSM sister corpse. In the ces-1 (n703) animal, the cellindicated anterior to the 12 neuron is the surviving 12sister, and the cell indicated posterior to the NSM neuronis the surviving NSM sister. Additional corpses outside thepharynx are visible in both animals. Anterior is the left,and ventral is down.

to die in ces-1 animals. Direct observation of the celllineage of developing ces-1 embryos by J. Sulston(personal communication) has confirmed this hypoth-esis: in ces-1 embryos the NSM sisters and the 12 sistersfail to die. Although the cell lineages of ces-2 embryoshave not been directly observed, the two extra cells inces-2 mutants resemble the surviving NSM sisters in ces-1 animals in position, morphology, and the ability toproduce serotonin; so we feel confident that they alsoare NSM sisters that fail to die.

ces-l(n703) and ces-2(n732) do not prevent most celldeathsAlthough mutations in ces-1 and ces-2 prevent somecells from undergoing cell death, these mutations donot affect most of the cells that die during C. elegansdevelopment. First, none of the other 18 cells thatnormally die during the development of the pharynx(Sulston et al. 1983) seems affected by these twomutations. We used Nomarski optics to determine thetotal number of cells in the pharynges of three wild-type, three ces-1, three ces-2 (25°C) and three ced-3larvae. Although the wild-type animals studied bySulston et al. (1983) had 80 nuclei in the pharynx, weobserved a small amount of natural variability amongthe three wild-type animals that we scored (80, 81, and83 cells, for an average of 81). Some of this variabilityappears to be caused by variable survival of the

598 R. E. Ellis and H. R. Horvitz

Table 2. ces-1 and ces-2 mutations prevent specific cells from dyingFrequency of undegraded

corpses at hatching

Genotype

ced-1; ced-2

ces-1 (n703) ced-1; ced-2+ ced-1

ces-1 (n703) ced-1; ced-2

ced-1 ces-2(n732ts); ced-2ced-1 ces-2 (n732is); ced-2ced-1 ces-2(n732ts); ced-2

Temperature

20°C

20°C

20°C

15 °C20°C25 °C

NSM region

0.50'

0.06

0.004

0.570.380.08

12 region

0.90

0.53

0.23

0.750.700.65

No.animals

165

160

240

625155

The number of cell corpses that persist until hatching in ces-1 and ces-2 strains containing mutations in both ced-1 and ced-2. Newlyhatched larvae were scored for undegraded corpses near the 12 and NSM neurons using Nomarski microscopy. The ces-l(n703)heterozygotes scored were the progeny of ces-l(n703) ced-1; ced-2; him-5 males mated with either unc-13 ced-1; ced-2 or ced-1 unc-54; ced-2 hermaphrodites. (A him-5 mutation was also present in some of the ces-l(n703) ced-1; ced-2 and some of the ced-1; ced-2 animals thatwe scored.) Each value represents the frequency that a corpse was found near a single NSM or a single 12 neuron (these values are theaverage of results obtained by scoring both the left and right halves of the pharynx). Although the NSM sisters are the only cells that diein the region just posterior to the NSM neurons, several other cells die in the region of the 12 neurons (Sulston et al. 1983). The data inthis table indicate that corpses from these other cell deaths are present infrequently, because in the ces-1 ced-1; ces-2 animals few corpsesare found in the 12 region.

pharyngeal MCL sister (see Materials and methods). Inaddition, in the ces-2 pharynges, one or both NSMsisters survived (81, 81, and 82 pharyngeal cells, for anaverage of 81) and, in all three ces-1 pharynges, both 12sisters and both NSM sisters survived (85, 86, and 84cells, for an average of 85). By contrast, when all celldeaths are prevented by a mutation in ced-3, thepharynx has 20-21 extra cells (101 cells observed in allthree animals). Based on the cell lineage, if all cells thatform the pharynx lived, there would be 22 extra cellspresent, for a total of 102 cells (Sulston et al. 1983).

A second observation also indicates that ces-1 andces-2 mutations do not affect all dying cells. In ces-1 ced-1; ced-2 and ced-1 ces-2; ced-2 triple mutants only thecorpses of the NSM sisters (and of the 12 sisters in ces-1animals) are missing; all other cell corpses appear to bepresent. In particular, we have observed that the ninecell deaths in the ventral nervous system (Sulston andHorvitz, 1977) occur in ces-1 and ces-2 mutants, andthat many cell corpses are found in the heads of ces-1ced-1; ced-2 mutants and ced-1 ces-2; ced-2 mutants,just as they are in ced-1; ced-2 animals. Thus, mutationsin ces-1 and ces-2 prevent specific cell deaths in thepharynx, but do not affect any other dying cells we haveexamined.

The mutation n703 results in a gain of ces-1 functionTo learn how the ces-1 mutation n703 prevents thedeaths of specific cells, we studied the effects ofdifferent doses of the ces-1 gene on the survival of the 12sisters, which are much more sensitive to changes in ces-1 dosage than are the NSM sisters. We used Nomarskimicroscopy to directly count surviving cells, and threedifferent deficiencies - nDf23, nDf24, and nDf25(Fig. 1) - to decrease the level of ces-1 activity.

Two experiments show that none of these deletions ofthe ces-1 gene behaves like the dominant ces-1 (n703)mutation, which suggests that n703 does not cause a loss

100%-,

50%-

0%

n703ln703

n703/+n703ln703nI434n703ln703nl4O6K703IDJ

/

15° C 20°C 25° C

Fig. 4. 12 sister survival in animals with different doses ofthe ces-1 gene. The survival of the 12 sisters was scored bydirect observation using Nomarski microscopy. The datafrom which this figure is derived are presented in Table 1.As discussed in Materials and methods, in the wild typeother cells very near the 12 sisters occasionally fail to die;such surviving cells are included in the values for the 12sisters, but should not alter them significantly.

of ces-1 function. First, in ces-l(n703)/+ animals, the 12sisters survive 48% of the time (n=688 animals), andthe NSM sisters survive 91 % of the time (n=226animals). By contrast, these four cells always die innDf23/+ animals (n=20) and nDf24/+ animals(n=20), just as they do in the wild type (n=100).Second, we performed gene dosage experiments usingdifferent alleles of ces-1 in trans to the mutation n703, asshown in Fig. 4. The frequency that the 12 sisterssurvive in these strains decreases in the order: ces-1 (n703)/ces-1 (n703)>ces-l (n703)/+>ces-l (n703)/Df.The n703 mutation is stronger than the wild-type alleleof ces-1, whereas deletions of the ces-1 gene are weakerthan the wild-type allele. These observations show thatn703 has a gain of ces-1 function.

Experiments using the duplication nDp4, whichcontains the ces-1 gene, indicate that n703 does not act

by causing overexpression of ces-1. Although the 12sisters survive 92% of the time in ces-1 (n703)/ces-l(n703) animals («=212 cells), they only survive 60%of the time in ces-1 (n703)/ces-1 (n703)/+ worms(n=179 cells) (see Materials and methods, Fig. 4 andTable 1). Similarly, the 12 sisters survive 48% of thetime in ces-1 (n703)/+ heterozygotes (n=1376 cells),but only 22% of the time in ces-l(n703)/+/+ animals(n=74 cells). Thus in some circumstances extra wild-type copies of ces-1 antagonize the n703 mutation. Thisresult indicates that n703 does not simply cause higherlevels of ces-1 gene expression or ectopic ces-1expression, and suggests that n703 results in a novel ces-1 function.

Loss-of-function alleles of ces-1Because n703 results in a gain of ces-1 gene function, wesought mutations that cause a loss of ces-1 function byisolating suppressors of n703. A second mutation withinthe ces-1 gene could suppress the dominant effects ofn703 by eliminating ces-1 function. To find such newces-1 aUeles, we mutagenized ces-1 (n703) males markedwith a closely linked lin-10 mutation, and mated themwith non-ces-1 animals marked with a closely linkedunc-13 mutation (Fig. 5). Because +/nDf23, +/nDf24,and +/nDf25 animals live and appear wild-type (seeabove), we know that this screen can recover mutationsthat completely eliminate ces-1 function, just as thesedeficiencies do. We screened about 9600 Fi progeny,using Nomarski optics to determine if the NSM and 12sisters were alive or dead in each animal. From thisscreen, we isolated two mutations that are cis-dominantsuppressors of ces-1 (n703). These mutations are called

us

Iin-10 ces-l(n703); him-5 <f x unc-13

+ lin-10 us-l(mO3) him-S

unc-13 + + +

+ lin-10 ces-I(n703 *) him-5

unc-13 + + +

Common class: someNSM and 12 sisters live

Rare class: all NSMand 12 sisters die

Fig. 5. The protocol for isolating mutations that cause aloss of ces-1 function, lin-10 ces-1(n703); him-5 males weremutagenized with EMS and mated with unc-13(e51)hermaphrodites. The non-Unc cross progeny wereexamined using Nomarski optics for rare animals in whichthe two NSM sisters and the two 12 sisters all died. Theseanimals might contain a second mutation (marked as * inthe figure) within the ces-1 gene that causes a loss of ces-1function. Two animals were isolated that segregated Linnon-Ces progeny but no Lin Ces progeny. The Lin non-Ces strains obtained from these animals each carried a newces-1 loss-of-function mutation.

C. elegans genes that control specific cell deaths 599

nl406 and nl434. Complementation tests show thatthese mutations are allelic (see Materials and methods).

We believe that these suppressors are located withinthe ces-1 gene for two reasons. First, both mutations arecis-dominant suppressors of ces-1 (n703). Second, bothrevertant mutations are tightly linked to ces-1 (n703) -each of these mutations is located within 0.1 map unitsof ces-1 (n703), and we have not recovered the n.703allele from either revertant chromosome (see Materialsand methods). These mutations suppress ces-2(n732) aswell (see below), and genetic mapping shows that thissuppressor activity is also located in the intervalbetween the genes daf-8 and unc-29, where ces-1 islocated. Measurements of the ces-1 activities of therevertant chromosomes show that both behave as ifthey have reduced levels of ces-1 function (Fig. 4). At15° and 20 °C the revertant chromosomes appear tohave more ces-1 activity than deficiencies have, sonl406 and nl434 might not entirely eliminate ces-1 genefunction at these temperatures.

In ces-1 (n703 nl406) and ces-1 (n703 nl434) mutants,the NSM and 12 sisters die, as they do in wild-typeanimals. There are no apparent differences from thewild type elsewhere in the pharynx, at either 20° or25°C. Furthermore, in these mutants serotonin isproduced by the NSM neurons at apparently normallevels (data not shown). In addition, ces-1 (n703 n!406)/nDf24 animals are also phenotypically wild-type, bothin pharyngeal anatomy and in the appearance andbehavior of these animals as viewed with a dissectingmicroscope (see Materials and methods). Thus we donot know what function, if any, the ces-1 gene plays inthe normal development of the animal.

A reduction of ces-2 function causes the NSM sistersto surviveWe believe that n732 acts by lowering but noteliminating ces-2 gene function at higher temperatures.In two different experiments, n732 has effects similar tobut weaker than those of a deletion of the ces-2 gene.First, both ces-2(n732) and deletions of the ces-2 geneshow semi-dominance for the survival of the NSMsisters. These cells always die in the wild type (n=800cells), but at 25°C, 2.3% of them survive in ces-2(n732)/+ animals (n=400 cells) and 27% survive inDf/+ animals (n=% cells, see Materials and methods).Second, in both ces-2(n732)/ces-2(n732) animals andces-2(n732)/eDf6 animals at 25 °C the NSM sistersusually survive (Fig. 6). However, the NSM sisterssurvive more often in ces-2(n732)/eDf6 animals than inces-2(n732) homozygotes, which suggests that n732does not completely eliminate ces-2 function, even athigh temperatures.

Isolation of additional ces mutationsTo isolate more mutations that affect the decision of theNSM sisters to live or die, we developed a generalscreen. Looking for animals with extra serotonergiccells, as was done to isolate ces-1 (n703) and ces-2(n732),is time-consuming. Such a screen requires the examin-ation of fixed and stained animals, and mutants must

600 R. E. Ellis and H. R. Horvitz

1 0 0 * -i 100%H

o*n732l+

15° C 20°C 2J°C

Fig. 6. NSM sister survival in animals with different dosesof the ces-2 gene. The survival of the NSM sisters wasscored by direct observation using Nomarksi microscopy.Each point represents the frequency of NSM sister survivaldetermined by scoring 100 animals of one genotype at aparticular temperature, except for the unc-59 ces-2/eDf6points, for which 37 animals were scored at 15°, 92 at 20°,and 99 at 25°C. The ces-2(n732)/+ animals were the crossprogeny of ces-2(n732) males mated with unc-13 lev-10hermaphrodites. The error bars represent 95 % confidencelimits as determined by the binomial distribution [%Error=±196(x(l-x)/n)0-5, where x=(% NSM sisterssurviving)/100 and n=number of cells].

therefore be recovered from among previously clonedsiblings. Instead, we used Nomarski optics to examineliving worms, seeking mutants in which the NSM sisterssurvived. We screened 21000 F2 worms, which rep-resents about 9700 haploid genomes scored for recess-ive mutations (see Materials and methods).

From this mutagenesis, we recovered three new cesmutations - two ces-1 dominant alleles, nl895 andnl896, and one recessive, temperature-sensitive mu-tation, n.1952. We also isolated six mutations thatprevent the deaths of not only the NSM sisters, but ofall other dying cells as well. These six mutations will bedescribed elsewhere.

The penetrance of NSM sister survival is low for therecessive mutation ces(nl952): 25 % of the animals hadat least one surviving NSM sister at 25 °C, and only 2 %at 20°C (AJ=100 animals in each case). Because of itslow penetrance, we have not yet fully characterized thismutation, but preliminary results suggest that nl952might define a new gene specifically involved in thedeaths of the NSM sisters. First, nl952 complementsces-2(n732) and is not linked to ces-1 or ces-2 (seeMaterials and methods). Second, the only abnormalitythat we have observed in these animals is the presenceof two extra pharyngeal cells. These extra cells areserotonergic neurons, and based upon their positionsappear to be the surviving sisters of the NSM neurons(data not shown).

We believe the two new dominant mutations arealleles of ces-1 for three reasons. First, the ces-1dominant mutation n703 and the new mutations nl895arid nl896 result in the same phenotype: the NSM and12 sisters fail to die, but other cell deaths appearunaffected (data not shown). Second, all three mu-

u3

in2

c/5

50%-

mJm mJ+ mllf mJnD/23

H n703 • nl895 g | n!896

Fig. 7. 12 sister survival in animals with different doses ofces-1 (n703), ces-1 (nl895), and ces-1 (nl896). The survival ofthe 12 sisters was scored by direct observation usingNomarski microscopy. Each bar represents the frequencyof 12 sister survival determined by scoring at least 100animals of a given genotype at 20°C. In the figure, 'm'represents n703, nl895, or nl896, as indicated. The m/+animals were the cross progeny of ces-1(m) males matedwith unc-13(e51) hermaphrodites, the m/lf animals werethe non-Unc progeny of either unc-13(elO91) ces-l(n703)/nDf23, or unc-13(e51) ces-1 (nl895)/nDf23, or unc-13 (e51) ces-1 (nl896)/nDf23 hermaphrodites. The error barsrepresent 95 % confidence limits as determined by thebinomial distribution [% Error=±196(x(l—x)/n) , wherex=(%12 sisters surviving)/100 and n=number of animals].

tations map between the genes daf-8 and sup-17 (seeMaterials and methods), which places them all withinan interval of about 0.1 map units. Third, all transheterozygotes involving these mutations appear ident-ical: between 76% and 8 1 % of the 12 sisters survive(n>=200 cells in all cases, see Materials and methods);furthermore, all three mutations behave similarly instudies of ces-1 gene dosage (Fig. 7).

Loss of ces-1 function suppresses ces-2(n732)The NSM sisters die in the two ces-1 loss-of-functionmutants, whereas they live in ces-2, ced-3 and ced-4mutants. This difference in phenotype allowed us tostudy the interactions between ces-1 and these othergenes that affect the deaths of the NSM sisters. Weconstructed double mutants between ces-1 (n703 nl406)and mutations in each of the other genes, anddetermined if the NSM sisters lived or died.

In animals carrying ces-1 (n703 nl406) and a mutationin either ced-3 or ced-4, the NSM sisters live, just asthey do in ced-3 and ced-4 mutants. These resultsindicate that ces-1 function is not required for NSMsister survival in ced-3 or ced-4 animals. Since loss-of-function mutations in ces-1 and these ced genes result inopposite effects on the NSM sisters (death vs. life,respectively), it seems likely that ces-1 and the cedgenes do not control sequential steps in a pathway {e.g.of biosynthesis) but rather that one negatively regulatesthe other. If so, our results imply that ces-1 acts beforeced-3 and ced-4 to decide whether the NSM sistersshould live or die. By contrast, the NSM sisters die in

C. elegans genes that control specific cell deaths 601

ces-1 (n703 nl406) ces-2 and ces-1 (n703 nl406); nl952animals, just as they do in ces-1 (n703 nl406) mutants.These results show that ces-1 function is probablyrequired for ces-2(n732) or ces(n!952) to prevent thedeaths of the NSM sisters, suggesting that ces-1 actsafter ces-2 and ces(nl952). However, since neither n732nor nl952 results in a complete loss-of-function, thisconclusion must be regarded as tentative.

We repeated several of these experiments using thesecond ces-1 loss-of-function allele. In each case, ces-l(n703 nl434) behaved like ces-1 (n703 nl406). Specifi-cally, ces-1 (n703 nl434) was suppressed by ced-3 or ced-4 mutations, and suppresses the ces-2 mutation. We alsoconstructed the mutant ces-1 (n703 nl406) ces-2; ced-3.In this animal, the NSM sisters survive, which showsthat these cells die in ces-1 (If) ces-2 animals by thenormal process of programmed cell death, whichdepends on ced-3 function.

Life or death is decided independently by each cellWe examined a group of 200 ces-1 (n703)/+ animals at20°C, in which 93 % of the NSM sisters survived. Ifmutations in ces-1 act independently on each NSMsister, then (93 %)2=86.5 % of the worms should havetwo surviving NSM sisters, 2(93 %)(7 %)=13 % of theworms should have only a single NSM sister surviving,and (7%)2=0.5% of the worms should have no NSMsisters surviving. Among these 200 animals, we saw87.5% with two NSM sisters, 11% with one NSMsister, and 1.5% with no NSM sisters. This resultsuggests that ces-1 (n703) might act independently onthese two cells. Similar data indicate the independenceof the 12 sister deaths in ces-1 (n703)/+ animals and ofthe NSM sister deaths in ces-2 animals (all of theseexperiments were done at 15°, 20° and 25 °C; data notshown). In each case, the data fit the hypothesis thatthese two genes act independently on each of theaffected cells.

Discussion

We have identified two genes, ces-1 and ces-2, thataffect the decisions of specific cells to live or die. Gain-of-function mutations in ces-1 and loss-of-functionmutations in ces-2 (both n732 and deficiencies) preventthe sisters of the NSM neurons from dying. These ces-1mutations also prevent the sisters of the 12 neuronsfrom dying. However, other cell deaths in thesemutants occur normally, and there are no other obviousabnormalities in phenotype. In particular, the othercells that form the pharynx, many of which are closelyrelated to the NSM sisters and the 12 sisters, all appearnormal. Thus mutations in the ces-1 and ces-2 genes canaffect the fates of the NSM and 12 sisters in the pharynxwithout changing other aspects of development.

Our observations indicate that a normal function ofboth the ces-1 and ces-2 genes is to control the deaths ofthe NSM sisters. First, since a loss of ces-2 functionprevents the NSM sisters from dying, ces-2 normallycauses these cells to die. Second, since a loss of ces-1

function results in the NSM sisters dying in a ces-2mutant animal, ces-1 can cause the NSM sisters to live.

Only one other C. elegans gene, egl-1, is known toaffect specifically the decisions of particular cells toundergo programmed cell death (Trent et al. 1983; Ellisand Horvitz, 1986). In hermaphrodites, the two HSNneurons control egg-laying, whereas in males these cellsundergo programmed cell death. Mutations in egl-1cause the HSN neurons to die in hermaphrodites as wellas in males, possibly by transforming the sexual identityof these cells. It is conceivable that egl-1 acts in theprocess of sex determination rather than in the directspecification of cell death. This reservation does notapply to the functions of ces-1 and ces-2, which actsimilarly in both sexes.

Two observations suggest that ces-1 and ces-2 controlthe decision of the NSM sisters to live or die bycontrolling genes that act in all cell deaths. First, theNSM sisters die in ces-1 (If) animals, but in ces-1 (If); ced-3 or ces-1 (If); ced-4 animals these cells live. As wediscuss above, this result indicates that ces-1 acts priorto ced-3 and ced-4 and is consistent with models inwhich ces-1 decides if the NSM sisters should live or dieand ced-3 and ced-4 are then required to kill these cells.Second, the NSM sisters die in ces-1 (If) ces-2 animals,suggesting that ces-2 acts with or through ces-1 toregulate the genes involved in all programmed celldeaths. The fact that in ces-1 (If) ces-2; ced-3 animals theNSM sisters live demonstrates that in ces-1 (If) ces-2mutants these cells die by normal programmed celldeath, which requires ced-3 function.

Mutations in the ces genes could affect the activitiesof ced-3 and ced-4 directly, by controlling the initiationof cell death, or indirectly, by transforming the NSMsisters into cells that normally live, presumably theNSM neurons themselves. It seems unlikely that ces-1normally acts to determine NSM identity: although ces-1 gain-of-function mutations cause the NSM sisters tosurvive and develop like NSM neurons, ces-1 loss-of-function mutations eliminate only the surviving NSMsisters and not the NSM neurons themselves. In fact, inmutants with a loss of ces-1 function, the NSM neuronsappear to survive, differentiate and produce serotoninnormally. Mutations in ces-2 also have no known effectson the NSM neurons, and genetic results (see above)indicate that ces-2 acts via ces-1. Thus, these genesprobably control the initiation of programmed celldeath rather that the acquisition of the NSM cell fate.When the deaths of the NSM sisters are blocked, as inced-3 or ced-4 animals, these cells appear identical tothe surviving NSM sisters found in the ces mutants,consistent with the hypothesis that the ces genes areinvolved in directly controlling the deaths of these twocells.

What are the phenotypes of animals with a completeloss of function for ces-1 or ces-2? It is possible that acomplete loss of ces-1 function results in a wild-typephenotype, because the two ces-1 alleles that weisolated in a screen for mutations that reduce oreliminate ces-1 activity both result in a wild-typephenotype. Both alleles resemble deficiencies in sev-

602 R. E. Ellis and H. R. Horvitz

A -Drc

ces-2 1 ces-1 1 ced-3,ced-4

I OFF*LIVE

oNy

OFFX

B

ces-2 ces-1 ced-3, ced-4

ONy

OFFX

DIE

LIVE

Fig. 8. Two models of the control of cell death in thesisters of the NSM neurons. (A) ces-2 and ces-1 act in aprocess that can inhibit ced-3 and ced-4 activity and soprevent the deaths of the NSM sisters. An unknownprocess activates ced-3 and ced-4. (B) ces-2 and ces-1 actthrough an unknown gene that activates ced-3 and ced-4function. —•, activation; —I, inhibition.

eral, although not all, of our gene dosage tests. Bycontrast, only one allele of ces-2 exists. The n732mutation is extremely temperature-sensitive, and genedosage experiments suggest that this mutation reducesonly partially the activity of the ces-2 gene. It is possiblethat a complete loss of ces-2 function would affect otheraspects of development besides the deaths of the NSMsisters. Screens for new mutations in this gene shouldhelp determine the phenotype that results from acomplete loss of ces-2 function.

If a loss of ces-1 function results in a wild-typephenotype, there must be other genes that cause theNSM sisters to decide to die. One possibility is thatthere are two regulatory pathways, each of whichcontrols the deaths of the NSM sisters (Fig. 8A). Oneprocess acts to prevent cell death and includes ces-2 andces-1; cell death is prevented effectively only in mutantswith a gain of ces-1 function or a loss of ces-2 function,whereas in animals with wild-type or inactive ces-1genes the second pathway successfully initiates celldeath. Alternatively, ces-2 and ces-1 might both actupstream of an unknown regulatory gene that directlyregulates ced-3 and ced-4 to initiate the deaths of theNSM sisters (Fig. 8B).

In some organisms, hormonal signals play animportant role in deciding if or when a cell should die.For example, in the moth Manduca sexta, a decrease inthe level of ecdysteroids initiates many programmedcell deaths (Truman and Schwartz, 1982). Cells thatwould normally die continue to live if provided withecdysteroids, and when ecdysteroid levels decline thesecells die; since other cells do not die, factors other thanhormone levels must specify which cells are capable ofdying. Are the genes ces-1 and ces-2 involved in asimilar system? Our data indicate that the decision tolive or die of each of the cells affected by ces-1 and ces-2mutations appears to be independent of that of theother cells. These observations suggest that ces-1 and

ces-2 do not act to control the level of a systemichormonal factor. Although it remains possible thatthese genes act in response to such a factor or in asystem involving signalling between adjacent cells,several lines of evidence suggest that in general in C.elegans the decision to die occurs within dying cells ortheir parents (reviewed by Yuan and Horvitz, 1990).We therefore suspect that ces-1 and ces-2 encode factorsthat act in a pathway operating entirely within the NSMsisters to control the deaths of these cells.

We thank Nancy Tsung and Carol Trent for providing themutations n703 and n732, John Sulston for his generous helpin studying the embryonic lineage of ces-l(n703) animals, PhilAnderson and Jonathan Hodgkin for providing strains, andHilary Ellis and Leon Avery for sharing unpublishedobservations. We are also grateful to Erik Jorgensen, PatriciaKuwabara and Eric Lambie for suggestions concerning thismanuscript. This work was supported by US Public HealthResearch Grants GM24663 and GM24943. R. E. E. wassupported by a National Science Foundation GraduateFellowship, and by a National Institutes of Health TrainingGrant. H. R. H. is an Investigator of the Howard HughesMedical Institute.

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(Accepted 8 March 1991)