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591 Development 112, 591-603 (1991) Printed in Great Britain © The Company of Biologists Limited 1991 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 two cells in the nematode Caenorhabditis elegans to undergo programmed cell death. Mutations that cause a gain of ces-1 function or a reduction of ces-2 function prevent these cells, the sisters of the two pharyngeal NSM neurons, from dying. These mutations do not affect most Introduction During the development of both vertebrates and invertebrates, many cells are born that neither divide nor form part of the adult, but instead die (Saunders, 1966; Cowan et al. 1984; Truman, 1984). Programmed cell death removes cells that do not function or that function only transiently (Finlayson, 1956; Sulston et al. 1983), influences morphogenesis (e.g. Hinchliffe, 1981; Steinberg and Horvitz, 1981), regulates the size of neuronal populations (Hamburger and Oppenheim, 1982) and helps create sexual dimorphism (e.g. Hinchliffe, 1981; Sulston et al. 1983). One animal in which programmed cell death has been studied in detail is the nematode Caenorhabditis elegans. Of the 1090 somatic cells generated during the normal development of a C. elegans hermaphrodite, 131 die. The same cells die in every animal, each at its own precise time, and all show similar changes in morphology as they undergo cell death (Sulston and Horvitz, 1977; Sulston et al. 1983). Genetic studies of C. elegans have denned ten genes that function in the programmed deaths of these 131 cells. Eight of these genes are required for the degradation of dead cells. Mutations in the genes ced-1, ced-2, ced-5, ced-6, ced-7, ced-8 and ced-10 prevent dead cells from being engulfed and degraded by their neighbors (ced=cell death; Hedgecock et al. 1983; Ellis etal. 1991), and mutations in the gene nuc-1 prevent the DNA of a dead cell from being digested (nuc=nuclease; Sulston, 1976; Hedgecock et al. 1983). Two geriesTced-3 and ced-4, are required for the deaths of all 131 cells (Ellis and Horvitz, 1986). Mutations in either of these genes prevent all of these cell deaths from occurring. Mosaic analyses indicate that ced-3 and ced-4 act within other cell deaths. Genetic studies indicate that ces-1 and ces-2 affect the fates of the NSM sisters by regulating the genes 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. dying cells, possibly as part of a cellular suicide program (Yuan and Horvitz, 1990). These results suggest that ced-3 and ced-4 either encode or regulate the expression of cytotoxic substances. How are the genes that act in programmed cell death regulated? Cell deaths occur throughout C. elegans development (Sulston and Horvitz, 1977; Sulston et al. 1983), and cells that are prevented from dying by mutations in ced-3 or ced-4 develop into many different cell types (Ellis and Horvitz, 1986; Avery and Horvitz, 1987). Because of the diversity in origin and cell type among dying cells, there could well be regulatory genes that control the fates of some dying cells but not of others. Such regulatory genes might choose between life and death for specific cells by controlling the activities of ced-3 and ced-4 in specific cell types. To understand how cell death is initiated in the correct cells during development, it is important to identify such regulatory genes and determine how they interact with the genes that cause cells to die. We have identified and characterized two genes that control the decision by specific cells to live or die: ces-1 and ces-2 (ces=cell death specification). Mutations in these genes can~~prevent specific cell deaths without affecting the deaths of other cells. We show that these genes act together to determine the fates of two cells in the pharynx, the sisters of the NSM neurons, probably by regulating the cell death genes ced-3 and ced-4. Materials and methods General methods and strain maintenance Techniques 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 otherwise indicated. The wild-type parent of all strains described here is C. elegans variety Bristol strain N2 (Brenner, 1974). Genetic nomenclature is described by Horvitz et al. (1979). Genetic markers We used the following mutations as genetic markers: LGI, dpy-5(e61), dpy-14(el88), unc-13(e51), unc-13(elO91), lin10(el439), daf-8(el393), fer-l(hcl), sup-17 (nl258), unc29(elO72), ced-l(e!752), unc-59(el005), lev-10(xl7), unc54(elO92), let-208(el719), let(e2000); LGIII, ced-4(nll62); LGIV, ced-2(el752), him-8(el489), unc-31(e928), ced3(n717); LGV, him-5(el490). We used the following chromosomal 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 described by Anderson and Brenner (1984), both sup-17(nl258) and nDp4 have been characterized by J. Thomas (personal communication), the mutations unc-13(elO91) and unc31(e928) are from the strain collection established by Brenner (1974), and the other mutations are described by Hodgkin et al. (1988). Serotonin staining Techniques for staining worms with anti-serotonin antiserum are described by Desai et al. (1988). Nomarski microscopy Procedures for using Nomarski microscopy to observe living animals are described by Sulston and Horvitz (1977). Because it is difficult to count cells in the pharynx of an animal that is moving and feeding, we anesthetized the worms by mounting them in a drop of M9 salt solution (Sulston and Brenner, 1974) containing 30 ITIM NaN3 (Avery and Horvitz, 1987). We usually studied the survival of cells in worms during the L2, L3 or L4 larval stages, although occasionally we scored adults or LI larvae. Identification o/ces-l(n703) and ces-2(n732) The mutations n703 and n732 were isolated by N. Tsung and C. Trent (Trent, 1982), who used the technique of formaldehyde-induced fluorescence (Sulston et al. 1975; Horvitz et al. 1982) to identify animals that had unusual patterns of serotonin expression from among the broods of 4594 F2 worms isolated after mutagenesis of the wild type with ethyl methanesulfonate. Mutagenesis experiments We used the technique for mutagenesis with ethyl methanesulfonate (EMS) described by Brenner (1974). In the reversion of ces-l(n703), the number of F] animals scored equals the number of haploid genomes screened. However, in the screen for new ces mutants, we screened 21000 F2 animals from among the descendants of about 27000 F t animals from an EMS mutagenesis, which corresponds to 54000 mutant haploid genomes. Some of the chromosomes from Fj animals would have been homozygous in more than one F2 animal scored, so we estimated the probability of scoring one particular haploid genome present among the Fj to be l-(53,999/54000)loS°°=0.18. Thus we screened (54000)(0.18)=9720 haploid genomes for recessive mutations. Similar considerations indicate that we screened about 24000 haploid genomes for dominant mutations. Mapping ces-l(gf) alleles L. Avery and H. Ellis (personal communications) had observed linkage of ces-l(n703) to markers on LGI. The following cross confirms this linkage, and demonstrates that ces-l(n703) is located to the left of sup-17: 30/34 Unc non-Sup recombinant progeny of unc-13(e51) sup-17/ ces-l(n703) heterozygotes segregated ces-l(n703). The other dominant alleles of ces-1 also map to the left of sup-17: 12/13 Unc nonSup recombinant progeny of unc-13(e51) sup-17/ ces-1 (n!895) heterozygotes segregated ces-1 (n!895), and 11/12 Unc nonSup recombinant progeny of unc-13(e51) sup-17/ ces-1 (nl896) segregated ces-1 (nl896). The following cross demonstrates that ces-1 (n703) is probably located to the right of both daf-8 and fer-1, and to the left of unc-29: 7/8 homozygous Daf nonFer Unc recombinant progeny from daf-8 ces-1 (n703)/ lin-10 fer-1 unc-29 hermaphrodites carried ces-1 (n703). The mutations ces-1 (nl895) and ces-1 (nl896) are also located to the right of daf-8 and to the left of unc-29: 0/1 Unc-29 Fer nonDaf non-Unc-13 recombinant progeny of ces-1 (nl895)/ unc13(e51) daf-8 fer-1 unc-29 heterozygotes segregated cesI(nl895) and 2/2 Unc-29 non-Fer non-Daf non-Unc-13 recombinants segregated ces-1 (n!895). From an equivalent experiment using ces-1 (n!896), we found 0/2 Unc-29 Fer nonDaf non-Unc-13 recombinant progeny segregated cesI(nl896), and 6/6 Unc-29 non-Fer non-Daf non-Unc-13 progeny segregated ces-1 (n!896). Mapping ces-l(lf) alleles We mapped the location of ces-1 (n703 n!434) by examining homozygous recombinant F2 progeny of daf-8 fer-1 unc-29/ lin-10 ces-1 (n703 nl434) heterozygotes: 0/8 Lin Daf Fer Unc recombinants, 0/1 Lin non-Daf Fer Unc recombinants, and 0/2 Lin non-Daf non-Fer Unc recombinants had surviving NSM sisters. Because ces-l(n703) maps much closer to fer-1 than it does to unc-29 (only 1/33 recombination events in this interval separated fer-1 and ces-1 (see above, also unpublished data)), one or both of the two lin-10 daf-8(+) fer-1 (+) unc-29 recombinant chromosomes probably carried the cesl(n703) mutation. Neither of these animals showed the Cesl(n703) phenotype, so the suppressor mutation nl434 must either 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-29 chromosomes, the lin-10 daf-8(+) fer-1 unc-29 chromosome, and the lin-10 daf-8(+) fer-1 (+) unc-29 chromosomes for suppression of ces-2. Only the two lin-10 daf-8(+) fer-l(+) unc-29 recombinants carried a suppressor of ces-2, presumably the nl434 mutation. These results show that nl434 is located to the right of daf-8. Overall, these experiments show that nl434 maps very near to n703. Similarly, to map the location of n!406, we examined homozygous recombinant F2 progeny of daf-8 fer-1 unc-29/ lin-10 ces-l(n703 nl406) heterozygotes: 0/6 Lin Daf Fer Unc and 0/3 Lin non-Daf non-Fer Unc recombinants had surviving NSM sisters. We also tested each of the three lin-10 daf-8(+) fer-1 (+) unc-29 chromosomes for suppression of ces2(n732), and all three chromosomes carried a suppressor of ces-2, presumably nl406. These results indicate that, if nl406 is located to the right of n703, it must be close to the n703 mutation and that, if nl406 is located to the left of n703, it must lie in the small region that extends from just left of the gene daf-8 to ces-1 (n703). Mapping ces-2(n732) In preliminary experiments, ces-2(n732) showed weak linkage to the gene dpy-5 on LGI (data not shown). The following cross confirms this assignment: 16/16 Ced non-Unc recombinant progeny of ces-2/ ced-1 unc-54 heterozygotes segregated ces-2(n732).• Furthermore, ces-2 is located to the right of unc54 and possibly to the right of let-208: 4/4 Unc non-Let C. elegans genes that control specific cell deaths recombinant progeny of ces-2/unc-54 let-208 heterozygotes segregated ces-2(n732). To complete the mapping of ces-2, we examined male progeny from the following crosses involving deletions 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) hermaphrodites yielded 8/16 Ces cross progeny, ces-2 males mated with eDf4/tet(e2000) hermaphrodites yielded 18/35 Ces cross progeny, ces-2 males mated with eDfl3/let(e2000) hermaphrodites yielded 1/36 Ces cross progeny, and ces-2 males mated with eDfl4/let(e2000) hermaphrodites yielded 1/22 Ces cross progeny. Note that deletions that fail to complement ces-2(n732) show a semidominant effect on the survival of the NSM sisters (see below), as does ces-2(n732) itself; however, the NSM sisters survive much less often in these heterozygotes than in ces-2(n732)/Df animals. Because of the semidominant behavior of n732 and the deficiencies, these complementation tests are not conclusive, but indicate that ces-2 probably lies in the region deleted by eDf4, eDf6, and eDf9, and does not lie in the region deleted by either eDfl3 or eDfl4. 593 l(n703) ced-1; ced-2 and ced-1 ces-2(n732); ced-2 strains by selecting for recombinant progeny from the appropriate triple heterozygotes in which (1) the extra neurons characteristic of ces-1 and ces-2 mutants were present, and (2) the number of cell corpses was otherwise characteristic of the ced-1; ced-2 double mutant. The presence of both ced-1 and ced-2 in the ces-1 ced-1; ced-2 strain was verified by failure to complement ced-1 and ced-2, and the presence of both ced-1 and ced-2 in the ced-1 ces-2; ced-2 strain was verified by the presence of NSM sister corpses at 15 °C (at which temperature the NSM sisters die in ces-2 animals, as in the wild type). Gene dosage studies In all gene dosage studies, animals were anesthetized with NaN3 and examined using Nomarski microscopy to determine how often the NSM sisters and the 12 sisters survive. The location of the NSM sisters at the rear of the anterior bulb is distinctive, but the 12 sisters are located extremely close to other cells that also die. As noted in the text, the number of pharyngeal cells varies slightly in the wild type, so we first determined the variability in the number of cells near the 12 neurons to determine if this variability would introduce Mapping ces(nl952) significant errors into our assay for 12 sister survival. In control wild-type animals at 15 CC, neither NSM sister The mutation nl952 maps to a different location than does ces-1. From lin-10 ces-l(n703 nl406) unc-29/+ + +; survived in 95 animals scored, but an extra neuron was found near an 12 neuron in 21 of these animals. In 20 of the 21 cases, ces(nl952)/+ heterozygotes, 2/6 Ces(nl952) progeny segrethe extra cell was found on the left side. Most pharyngeal gated Lin Unc animals. We tested one of these two strains by development is left/right symmetrical, and this asymmetry crossing with the wild type, and recovered the ces(nl952) mutation from this lin-10 ces-1 (n703 nl406) unc-29; ces(nl952) suggests that the extra cell might be the surviving sister of MCL, since the sister of the right-side cell MCR normally strain, which confirmed its genotype. These results indicate that ces(nl952) is not located between lin-10 and unc-29, and survives, whereas the sister of the left-side cell MCL normally dies (Sulston et al. 1983). At 20°C the NSM sisters again all so cannot be an allele of ces-1, and suggest that ces(n!952) died, but in 19/205 animals an extra cell was present near an may not be linked to ces-1. 12 neuron. Similarly, at 25°C the NSM sisters all died, but in The mutation nl952 is also not an allele of ces-2. Because ces-2(n732) is tightly linked to the unc-54 gene, we tested 7/100-animals an extra cell was found near an 12 neuron. In both cases, extra cells were found more often on the left side ces(n!952) for linkage to unc-54. We examined at 25°C four than on the right side. The extra cells found near the 12 Ces(nl952) progeny of unc-54/+; ces(nl952)/+; him-8/+ neurons in the wild type could be (1) surviving 12 sisters, (2) heterozygotes. One of these progeny was unc-54/unc-54, one other surviving cells, such as II sisters or 12 aunts or, when was unc-54/+, and two were + / + . These results indicate that found on the left side of the pharynx, MCL sisters, or (3) ces(nl952) is not closely linked to unc-54, and thus is not neurons generated by extra cell divisions. In the wild type, the closely linked to ces-2 either. cold-sensitive survival of the MCL sister, and the rare survival of unknown cells occur infrequently, and so should not Complementation tests To determine if ces-1 (n703 nl406) and ces-1 (n703 nl434) fail introduce significant error into the measurement of 12 sister survival in the various ces strains that we have examined. to complement for the suppression of ces-2, we mated lin-10 ces-l(n703 n!434) ced-1 ces-2/+ + + ces-2 males with uncTo determine if a deletion of the ces-1 gene results in the 13(e51) lin-10 ces-1 (n703nl406) ced-1 ces-2 hermaphrodites at dominant phenotype caused by ces-1 (n703), we examined 20 25°C. Among the non-Unc cross progeny, only 1/12 Lin unc-13(elO91) lin-11/ nDf23 animals, and 20 unc-13(elO91) worms showed the Ces-2 phenotype of NSM sister survival • lin-11 / nDf24 animals. The NSM sisters always died in these (these animals were ces-l(n703 nl406)/ces-1 (n703 nl434)), animals, and 79/80 12 sisters clearly died. An extra neuron but 16/26 non-Lin worms snowed the Ces-2 phenotype (these was located near the right 12 neuron in one animal; as noted animals were ces-l(n703 nl406)/+). Therefore ces-l(n703 above, an extra cell is sometimes found in this position in the nl406) and ces-1 (n703 nl434) fail to complement for the wild type. suppression of ces-2. To measure the effects of altering ces-1 gene dosage, we To test ces-2(n732) and ces(nl952) for complementation, examined animals of several genotypes in order to determine we mated ces-2(n732) males with ces(n!952) hermaphrodites how often the 12 sisters and NSM sisters survived. We only at 25°C. Among the male cross progeny, only 1/17 worms present data for the 12 sisters because in all strains with one showed the Ces phenotype, approximately the frequency at copy of ces-1 (n703) the NSM sisters survive about 90 % of the which ces-2/+ animals show this phenotype. time, so that NSM sister survival cannot be used to measure the effects of small changes in gene dosage (Table 1, Fig. 4, Triple mutants with the ces genes and ced-1; ced-2 data not shown). The three deficiencies of the ces-1 gene In ced-1 or ced-2 mutants, some cell corpses are not engulfed behave similarly in trans to ces-1 (n703), consistent with the (Hedgecock et al. 1983), and, in ced-1; ced-2 double mutants, genetic map data (Fig. 1), which indicates that each deficiency should completely eliminate ces-1 function. Furthermore, in the number of unengulfed corpses increases significantly (Ellis the ces-1 (n703)/+ animals, it appears to make no difference if et al. 1991). For example, the NSM sister corpses are found in a ced-1; ced-2 double mutant, but not in ced-1 or ced-2 single the ces-1 mutation is derived from the mother or from the father, and the results also show little dependence on which mutants (Ellis et al. 1991). We therefore constructed ces- 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-29 ces-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 Maternal genotype 15 °C 20 °C 25 °C same 96/111 96/111 194/211 194/211 135/160 135/160 same N.D. N.D. N.D. N.D. 111/200 N.D. 111/200 114/203 54/97 48/97 192/459 N.D. 246/520 654/1376 N.D. N.D. N.D. N.D. 50/200 N.D. 50/200 unc-13 ces-1 58/142 58/142 42/136 42/136 24/150 24/150 unc-13 Hn-10 ces-1 (n703nl406), unc-13 ces-1 53/140 N.D. 53/140 62/193 12/38 74/231 20/140 N.D. 20/140 same same same same same 58/1% 13/86 N.D. N.D. N.D. 71/282 37/200 10/72 31/212 9/84 16/152 103/720 10/100 12/100 N.D. N.D. N.D. 22/200 ces-1 unc-29 unc-29 unc-13 unc-13 ces-1 unc-13 ces-1 (n703)/Df dpy-14 ces-1 (n703)/nDf23 dpy-14 ces-1 (n703)/nDf24 unc-13(elO91) ces-1 (n703)/nDJ25 dpy-14 ces-1 (n703)/nDf25 Total '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 cesl(n703) (data not shown). Fig. 4 summarizes the results of these experiments. marker mutations were used (Table 1). That ces-l(n703)/+ animals have more surviving 12 sisters than do ces-1 (n703)/Df animals indicates that the ces-1 (+) allele enhances the effect of one copy of ces-1 (n703). We also examined animals carrying a wild-type copy of the ces-1 gene on a duplication. The attached duplication nDp4 covers the ces-1 gene. Animals homozygous for nDp4 are very sick and can be identified easily, and animals with a single copy oinDp4 are defective in egg-laying (J. Thomas, personal communication). In the nDp4/ + animals that we examined, which had three wild-type copies of the ces-1 gene, 30/30 NSM sisters died and, in nDp4/nDp4 animals, which had four copies of ces-1, 20/20 NSM sisters died. The free duplication sDpl also covers ces-1, and 12/12 NSM sisters died in dpy-5 unc-13(e51)/ dpy-5 unc-13(e51)/ sDpl animals, which have three copies of ces-1. These results suggest that increasing ces1 gene expression even two-fold is not enough to cause the NSM sister survival seen in ces-1 (gf) mutants. In addition, we used the attached duplication nDp4 to study the survival of the 12 sisters in animals with two copies of cesl(n703) and one copy of ces-l(+). Among the progeny of cesl(n703) males crossed with unc-13(elO91) ces-l(n703); nDp4 homozygotes, 32/72 12 sisters survived, and among the healthy non-Unc progeny of unc-13(elO91) ces-1 (n703); nDp4/+ heterozygotes, 75/107 12 sisters survived. We also studied animals with two copies of ces-1 (+) and one copy of ces-1 (n703). Among the progeny of wild-type males crossed with unc-13(elO91) ces-1 (n703); nDp4 homozygotes, 5/32 12 sisters survived, and among the progeny of ces-1 (n703) males crossed with unc-13(e51); nDp4 homozygotes, 11/4112 sisters survived. Thus, the duplication nDp4 appears to lower, not enhance, the effect of two copies of ces-1 (n703) on the survival of the 12 sisters. Experiments involving these large duplications are difficult, because they cause general sickness in animals, and the pharynges are sometimes distorted and difficult to score. Furthermore, we cannot prove that the effect caused by these duplications on 12 sister survival is a consequence of the extra copy of ces-l(+) that they contain. The fact that with one copy of ces-1 (n703) the ces-l(+) allele enhances the effect of the ces-l(n703) mutation, whereas with two copies of ces-1 (n703) the ces-1 (+) allele appears to suppress ces-1 (n703), suggests that the regulation of ces-1 activity is complicated. Perhaps the ces-1 (+) product both increases ces-1 (n703) expression by a trans-acting autoregulation, and also competes with ces-l(n703) product for some limiting molecule necessary for ces-1 function. In this case, the overall effect of increasing ces-1 (+) activity could depend upon the level of ces-1 (n703) activity. The mutation ces-2(n732) is not completely recessive. From a cross of ces-2(n732) males with unc-13(e51) lev-10 hermaphrodites at 25 °C, 4 % of the cross progeny had a surviving NSM sister (n = 100 animals), and from a cross of wild-type males with ces-2(n732); unc-31 hermaphrodites at 25°C, 7% of the cross progeny had a surviving NSM sister (n=100 animals). Similarly, the NSM sisters sometimes survive in animals heterozygous for a deletion of ces-2. At 25 °C, the NSM sisters survived in 12/30 eDf4/let(e2000) animals (a total of 13/60 cells survived), and the NSM sisters survived in 9/18 eDf9/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 at 25°C all of the NSM sisters died («=30 animals each). As C. elegans genes that control specific cell deaths 595 1 m. u. II 1 I I ces-2 ces-1 i i r C1 nDf23 \ I I J' -\ eDfl3/cDfl4 \'Df4 nD/24,nD/25 \- -\'DJ9 -\eD/6 nDp4, sDpl : 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 as well. (The deficiency eDflO might show a partial failure to complement ces-2(n732), and eDf3/eDflO heterozygotes have a low rate of NSM sister survival; data not shown). lin-10 ces-1 (n703 nl406) unc-29 animals had the wild-type number of neurons and epithelial cells in the anterior pharynx, and 1/20 had a single extra neuron near the 12 cell. We also examined the Unc male progeny of lin-10 ces-1 (n703 nl406) unc-29/ + + + males crossed with nDf24/ unc13(elO91) lin-11 hermaphrodites. At25°C, 3/4 such lin-10 cesces-l(dm) trans-heterozygotes l(n703 nl406) unc-29'/ nDf24 animals had the wild-type We examined animals carrying different ces-1 dominant alleles in trans. Among the cross progeny of ces-1 (n703) males number of neurons and epithelial cells in the pharynx, and 1/4 mated with unc-13(e51) ces-1 (nl895) hermaphrodites 161/200 may have been missing one neuron in the posterior pharynx; 12 sisters survived, among the cross progeny of ces-1 (n703) at 20°C, 6/6 such animals appeared wild-type. Thus, it males mated with unc-13(e51) ces-l(nl896) hermaphrodites appears that nl406 and nl434 mutants may indeed have a wild-type phenotype; even ces-1 (n703 n!406)/Df worms at 159/200 12 sisters survived, among the cross progeny of cesI(nl896) males mated with unc-13(e51) ces-1 (nl896) her- 25 °C, which should have very little ces-1 activity, appear wildtype. maphrodites 168/22012 sisters survived, and among the cross progeny of ces-1 (nl895) males mated with unc-13(e51) cesI(nl895) hermaphrodites 160/200 12 sisters survived. Finally, Construction of double mutants among the cross progeny of ces-1 (n!895) males mated with Because ces-1 (If) mutations result in a wild-type phenotype, unc-13(e51) ces-1 (nl896) 193/234 12 sisters survived, and we used ces-1 (If) mutations closely linked to marker among the cross progeny of ces-1 (nl896) males mated with mutations in the constructions described in this section. unc-13(e51) ces-1 (nl895) hermaphrodites 214/300 12 sisters Furthermore, we studied several isolates of most of these survived. These values are all similar. strains to ensure that the ces-1 (If) allele had not been lost by a rare recombination event in any particular construction. All Phenotype of ces-l(lf) mutants markers segregated at expected frequencies in these crosses (data not shown), so we do not believe that ces-1 (If) alleles Preliminary observations using Nomarski microscopy suggested that both ces-1 (n703 n!4O6) and ces-1 (n703 nl434) animals have any lethal interactions with the other genes that we used. Double mutants between the ces-1 (If) mutations and ced-3 (n=10) appear wild-type. We further examined ces-l(n703 or ced-4 mutations were built as follows. From lin-10 cesnl406) animals in the following ways. At 25CC, two unc13(e51) lin-10 ces-1 (n703 nl406) animals each had 80 cells in l(n703 nl434)/++; ced-3/+ heterozygotes we isolated Ced progeny and, from these Ced progeny, we isolated Lin the pharynx, the number found in the wild-type. Furthermore, at 25°C, nine lin-10 ces-l(n703 n!406) unc-29 animals, offspring. These animals are lin-10; ced-3 based on their phenotypes, and probably are homozygous for ces-1 (n703 examined less thoroughly, all had the wild-type number of nl406), which is tightly linked to lin-10. In four separate Lin neurons and epithelial cells in the pharynx, and at 20°C, 19/20 596 R. E. Ellis and H. R. Horvitz Ced-3 isolates the NSM sisters always survived. A lin-10 cesl(n703 nl406); ced-4 strain was constructed similarly, and three separate isolates all showed the same phenotype. We built unc-13(e51) lin-10 ces-l(n703 nl406); ced-3 using an equivalent procedure, and tested three separate Unc Lin Ced isolates for NSM sister survival. The strain unc-13(e51) lin-10 ces-l(n703 nl406); ced-4 was built similarly, and one isolate tested. The survival of the NSM sisters in the above strains was tested using Nomarski optics for all isolates, and with anti-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 cesl(n703 n!434) + +/+ + ced-1 ces-2 heterozygote. Our results indicated that ces-2 is suppressed by ces-1 (If), so to ensure that ces-2 was present on the lin-10 ced-1 chromosome, we mated ces-2 males into the Lin Ced strain and selected progeny in which both NSM sisters survived. Because ces-2 is recessive for this trait, these animals must be ces-2 homozygotes. We then re-isolated Lin Ced worms from among the progeny of the putative lin-10 ces-l(n703 nl434) ced-1 ces-2/'+ + + ces-2 heterozygotes. The NSM sisters die in these Lin Ced animals, which indicates that the ces-l(lf) allele is also homozygous in these worms. Genetic mapping (see above) proves that the activity that suppresses ces-2 is tightly linked to ces-1, and so is not caused by one of the other markers in this strain. Therefore, this strain must be of genotype lin-10 ces-l(n703 nl406) ced-1 ces-2. As a final test that ces-2 is homozygous in this strain, we showed that 8/8 putative lin-10 ces-l(n703 nl406) ced-1 ces-2 animals behave as ces-2 homozygotes when tested for complementation with ces-2. To construct an unc-13(e51) lin-10 ces-1 (n703 nl406) ced-1 ces-2 strain, we isolated three Unc Lin Ced recombinants from unc-13(e51) lin-10 ces-1 (n703 nl406) + + /+ + + ced-1 ces-2 heterozygotes. The NSM sisters die in all three of these recombinant strains, so if ces-2 is homozygous, the ces-1 (If) allele must also be homozygous so that it suppresses ces-2. We showed that 5/5 putative unc-13 lin-10ces-l(n703 n!406) ced-1 ces-2 worms from each of these three strains behave as ces-2 homozygotes when used in complementation tests with ces-2, so we conclude that each strain is homozygous for ces-2. Thus each 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-1 ces-2; ced-3 strain, we isolated an Unc Lin Ced-3 animal from an unc-13(e51) lin-10 ces-l(n703 nl406) ced-1 ces-2/+ + + + ces-2; ced-31+ heterozygote. The ces-1 (If) allele is still present in this strain because, in a cross of ces-2 males with the putative unc-13(e51) lin-10 ces-l(n703 nl406) ced-1 ces-2; ced3 hermaphrodites, the F : animals segregate Unc animals in which the NSM sisters die (except those F2 worms homozygous 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 both NSM sisters survived from among the progeny of lin-10 ces-1 unc-291 + + +; nl952/+ heterozygotes. From one of these nl952 homozygotes, we isolated Lin Unc progeny. The deaths of the NSM sisters in this strain were confirmed using Nomarski optics. Results Identification o/ces-l(n703) and ces-2(n732) The mutations n703 and n732 were isolated by N. Tsung and C. Trent (Trent, 1982). In the pharynges of wildtype animals only the two bilaterally symmetric NSM neurons contain serotonin (Horvitz et al. 1982); by contrast, in the pharynges of both n703 and n732 animals, there are four serotonergic cells (Trent, 1982). Nomarski microscopy revealed that there are two extra neurons in this region of the pharynx (H. Ellis, personal communication). We mapped the n703 and n732 mutations, and showed that each defines a new gene on linkage group I (Materials and methods, Fig. 1). Because these genes appear to be involved in the specification of which cells live and which cells die (see below), we have named them ces-1 (n703) and ces2(n732), where ces stands for cell death specification. ces-l(n703) and ces-2(n732) prevent the deaths of specific cells In wild-type animals, the two NSM cells differentiate into serotonergic neurons, and the sisters of the NSM neurons die. By contrast, in ced-3 mutants, the sisters of the NSM neurons along with many other cells fail to die, and there are four cells in the pharynx that contain serotonin (Ellis and Horvitz, 1986). Some cells that are prevented from dying by a mutation in ced-3 adopt the fate of a near relative, which suggests that the two extra serotonergic cells in ced-3 animals are the surviving NSM sisters (Ellis and Horvitz, 1986). The NSM sisters might similarly survive in ces-1 and ces-2 mutants, which would account for the two extra neurons and the total of four serotonergic cells in the pharynges of these animals. To see if the NSM sisters fail to die in ces-l(n703) and ces-2(n732) animals, we examined mutant larvae using Nomarski microscopy. We observed in both ces-1 and ces-2 mutants an extra neuron just posterior and dorsal to each NSM neuron (Fig. 2). This position is exactly that of the NSM sisters in wild-type animals before they die (Sulston et al. 1983). Furthermore, staining with anti-serotonin antisera (Desai et al. 1988) revealed that the extra serotonergic cells in ces-1 and ces-2 mutants strongly resemble those found in ced-3 animals, in which cell deaths do not occur; in all three mutants, the nerve processes of these cells are similar in morphology to those of the NSM neurons (data not shown). While viewing ces-1 mutants with Nomarski microscopy, we also identified two additional extra neurons located anterior to the 12 neurons, one on each side of the pharynx (Fig. 2). These results suggest that the NSM sisters indeed fail to die in ces-1 and ces-2 animals, and that two additional cells (possibly the 12 sisters) fail to die in ces-1 worms. It remained possible that these extra neurons resulted from extra cell divisions, rather than from the survival of cells that normally die. To explore this possibility, we directly examined cells that die in this region of the pharynx. In ced-1; ced-2 double mutants, the corpses of dead cells are not quickly degraded and instead persist for hours; these corpses can be assayed reliably (Hedgecock et al. 1983; Ellis et al. 1991). (Note that in C. elegans genetic nomenclature a semicolon separates genes located on different chromosomes; Horvitz et al. 1979). In particular, two specific corpses, which by position are likely to be the dead NSM sisters, are easily C. elegans genes that control specific cell deaths Fig. 2. Extra neurons in the pharynx of ces-1 animals. Nomarski photomicrographs of the pharynges of (A) wildtype and (B) ces-l(n703) L3 larvae. In the ces-l(n703) animal, the extra neurons are indicated with arrows; the cell anterior to the 12 neuron is the surviving 12 sister, and the cell posterior to the NSM neuron is the surviving NSM sister. Anterior is to the left, and ventral is down. visible in ced-1; ced-2 double mutants (Fig. 3A). If the NSM sisters do not die in ces-1 and ces-2 animals, then the corpses of the NSM sisters should be missing in cesl(n703) ced-1; ced-2 and in ced-1 ces-2(n732); ced-2 triple mutants. We constructed the appropriate triple mutants (see Materials and methods) and examined newly hatched animals to see which cell corpses were present and which were missing. The putative NSM sister corpse found posterior to each NSM neuron in ced-1; ced-2 animals is present 100-fold less often in ces-1 (n703) ced1; ced-2 animals (Table 2, Fig. 3B). Often a cell corpse anterior to the 12 neuron is also missing in ces-1 (n703) ced-1; ced-2 worms, which suggests that the extra neuron anterior to the 12 in ces-1 animals is also a cell that fails to die. Thus, two pairs of corpses are missing in ces-1 pharynges, and these corpses correspond in position to the two pairs of extra cells found in these mutants (Fig. 3). In ced-1 ces-2; ced-2 mutants, only the putative NSM sister corpse is affected by the presence of the ces-2 mutation. This corpse is present about seven times more often in ced-1; ced-2 worms than in ced-1 ces2(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, in ces-2 mutants at 25 °C, one pair of pharyngeal corpses is missing, and these missing corpses correspond in position to the extra pair of serotonergic neurons found in these animals. These results strongly suggest that the NSM sisters do not die in ces-1 and ces-2 mutants, and that two additional cells, located anterior to the 12 neurons, fail 597 Fig. 3. The corpses of the NSM and 12 sisters in ced-1; ced-2 animals. Nomarksi photomicrographs of the pharynges of (A) ced-1; ced-2 and (B) ces-l(n703) ced-1; ced-2 LI larvae. In the ces-l(+) animal, the corpse indicated anterior to the 12 neuron is the 12 sister corpse, and the corpse indicated posterior to the NSM neuron is the NSM sister corpse. In the ces-1 (n703) animal, the cell indicated anterior to the 12 neuron is the surviving 12 sister, and the cell indicated posterior to the NSM neuron is the surviving NSM sister. Additional corpses outside the pharynx are visible in both animals. Anterior is the left, and ventral is down. to die in ces-1 animals. Direct observation of the cell lineage of developing ces-1 embryos by J. Sulston (personal communication) has confirmed this hypothesis: in ces-1 embryos the NSM sisters and the 12 sisters fail to die. Although the cell lineages of ces-2 embryos have not been directly observed, the two extra cells in ces-2 mutants resemble the surviving NSM sisters in ces1 animals in position, morphology, and the ability to produce serotonin; so we feel confident that they also are NSM sisters that fail to die. ces-l(n703) and ces-2(n732) do not prevent most cell deaths Although mutations in ces-1 and ces-2 prevent some cells from undergoing cell death, these mutations do not affect most of the cells that die during C. elegans development. First, none of the other 18 cells that normally die during the development of the pharynx (Sulston et al. 1983) seems affected by these two mutations. We used Nomarski optics to determine the total number of cells in the pharynges of three wildtype, three ces-1, three ces-2 (25°C) and three ced-3 larvae. Although the wild-type animals studied by Sulston et al. (1983) had 80 nuclei in the pharynx, we observed a small amount of natural variability among the three wild-type animals that we scored (80, 81, and 83 cells, for an average of 81). Some of this variability appears 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 dying Frequency of undegraded corpses at hatching Temperature NSM region 12 region No. animals ced-1; ced-2 20°C 0.50' 0.90 165 ces-1 (n703) ced-1; ced-2 + ced-1 ces-1 (n703) ced-1; ced-2 20°C 0.06 0.53 160 20°C 0.004 0.23 240 ced-1 ces-2(n732ts); ced-2 ced-1 ces-2 (n732is); ced-2 ced-1 ces-2(n732ts); ced-2 15 °C 20°C 25 °C 0.57 0.38 0.08 0.75 0.70 0.65 62 51 55 Genotype 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. Newly hatched 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; ced2 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 that we scored.) Each value represents the frequency that a corpse was found near a single NSM or a single 12 neuron (these values are the average of results obtained by scoring both the left and right halves of the pharynx). Although the NSM sisters are the only cells that die in 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 in this table indicate that corpses from these other cell deaths are present infrequently, because in the ces-1 ced-1; ces-2 animals few corpses are found in the 12 region. pharyngeal MCL sister (see Materials and methods). In addition, in the ces-2 pharynges, one or both NSM sisters survived (81, 81, and 82 pharyngeal cells, for an average of 81) and, in all three ces-1 pharynges, both 12 sisters and both NSM sisters survived (85, 86, and 84 cells, for an average of 85). By contrast, when all cell deaths are prevented by a mutation in ced-3, the pharynx has 20-21 extra cells (101 cells observed in all three animals). Based on the cell lineage, if all cells that form the pharynx lived, there would be 22 extra cells present, for a total of 102 cells (Sulston et al. 1983). A second observation also indicates that ces-1 and ces-2 mutations do not affect all dying cells. In ces-1 ced1; ced-2 and ced-1 ces-2; ced-2 triple mutants only the corpses of the NSM sisters (and of the 12 sisters in ces-1 animals) are missing; all other cell corpses appear to be present. In particular, we have observed that the nine cell deaths in the ventral nervous system (Sulston and Horvitz, 1977) occur in ces-1 and ces-2 mutants, and that many cell corpses are found in the heads of ces-1 ced-1; ced-2 mutants and ced-1 ces-2; ced-2 mutants, just as they are in ced-1; ced-2 animals. Thus, mutations in ces-1 and ces-2 prevent specific cell deaths in the pharynx, but do not affect any other dying cells we have examined. The mutation n703 results in a gain of ces-1 function To learn how the ces-1 mutation n703 prevents the deaths of specific cells, we studied the effects of different doses of the ces-1 gene on the survival of the 12 sisters, which are much more sensitive to changes in ces1 dosage than are the NSM sisters. We used Nomarski microscopy to directly count surviving cells, and three different deficiencies - nDf23, nDf24, and nDf25 (Fig. 1) - to decrease the level of ces-1 activity. Two experiments show that none of these deletions of the ces-1 gene behaves like the dominant ces-1 (n703) mutation, which suggests that n703 does not cause a loss 100%-, n703ln703 50%n703/+ n703ln703nI434 n703ln703nl4O6 K703IDJ / 0% 15° C 20°C 25° C Fig. 4. 12 sister survival in animals with different doses of the ces-1 gene. The survival of the 12 sisters was scored by direct observation using Nomarski microscopy. The data from which this figure is derived are presented in Table 1. As discussed in Materials and methods, in the wild type other cells very near the 12 sisters occasionally fail to die; such surviving cells are included in the values for the 12 sisters, but should not alter them significantly. of ces-1 function. First, in ces-l(n703)/+ animals, the 12 sisters survive 48% of the time (n=688 animals), and the NSM sisters survive 91 % of the time (n=226 animals). By contrast, these four cells always die in nDf23/+ animals (n=20) and nDf24/+ animals (n=20), just as they do in the wild type (n=100). Second, we performed gene dosage experiments using different alleles of ces-1 in trans to the mutation n703, as shown in Fig. 4. The frequency that the 12 sisters survive in these strains decreases in the order: ces1 (n703)/ces-1 (n703)>ces-l (n703)/+>ces-l (n703)/Df. The n703 mutation is stronger than the wild-type allele of ces-1, whereas deletions of the ces-1 gene are weaker than the wild-type allele. These observations show that n703 has a gain of ces-1 function. Experiments using the duplication nDp4, which contains the ces-1 gene, indicate that n703 does not act C. elegans genes that control specific cell deaths 599 nl406 and nl434. Complementation tests show that these mutations are allelic (see Materials and methods). We believe that these suppressors are located within the ces-1 gene for two reasons. First, both mutations are cis-dominant suppressors of ces-1 (n703). Second, both revertant mutations are tightly linked to ces-1 (n703) each of these mutations is located within 0.1 map units of ces-1 (n703), and we have not recovered the n.703 allele from either revertant chromosome (see Materials and methods). These mutations suppress ces-2(n732) as well (see below), and genetic mapping shows that this suppressor activity is also located in the interval between the genes daf-8 and unc-29, where ces-1 is located. Measurements of the ces-1 activities of the revertant chromosomes show that both behave as if they have reduced levels of ces-1 function (Fig. 4). At Loss-of-function alleles of ces-1 15° and 20 °C the revertant chromosomes appear to Because n703 results in a gain of ces-1 gene function, we have more ces-1 activity than deficiencies have, so sought mutations that cause a loss of ces-1 function by nl406 and nl434 might not entirely eliminate ces-1 gene isolating suppressors of n703. A second mutation within function at these temperatures. the ces-1 gene could suppress the dominant effects of In ces-1 (n703 nl406) and ces-1 (n703 nl434) mutants, n703 by eliminating ces-1 function. To find such new the NSM and 12 sisters die, as they do in wild-type ces-1 aUeles, we mutagenized ces-1 (n703) males marked with a closely linked lin-10 mutation, and mated them animals. There are no apparent differences from the wild type elsewhere in the pharynx, at either 20° or with non-ces-1 animals marked with a closely linked 25°C. Furthermore, in these mutants serotonin is unc-13 mutation (Fig. 5). Because +/nDf23, +/nDf24, produced by the NSM neurons at apparently normal and +/nDf25 animals live and appear wild-type (see levels (data not shown). In addition, ces-1 (n703 n!406)/ above), we know that this screen can recover mutations nDf24 animals are also phenotypically wild-type, both that completely eliminate ces-1 function, just as these in pharyngeal anatomy and in the appearance and deficiencies do. We screened about 9600 Fi progeny, behavior of these animals as viewed with a dissecting using Nomarski optics to determine if the NSM and 12 microscope (see Materials and methods). Thus we do sisters were alive or dead in each animal. From this not know what function, if any, the ces-1 gene plays in screen, we isolated two mutations that are cis-dominant the normal development of the animal. suppressors of ces-1 (n703). These mutations are called by causing overexpression of ces-1. Although the 12 sisters survive 92% of the time in ces-1 (n703)/cesl(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 and Table 1). Similarly, the 12 sisters survive 48% of the time 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 wildtype copies of ces-1 antagonize the n703 mutation. This result indicates that n703 does not simply cause higher levels of ces-1 gene expression or ectopic ces-1 expression, and suggests that n703 results in a novel ces1 function. us Iin-10 ces-l(n703); him-5 + lin-10 us-l(mO3) unc-13 + + + lin-10 ces-I(n703 *) unc-13 + + him-S + him-5 + <f x unc-13 Common class: some NSM and 12 sisters live Rare class: all NSM and 12 sisters die Fig. 5. The protocol for isolating mutations that cause a loss of ces-1 function, lin-10 ces-1(n703); him-5 males were mutagenized with EMS and mated with unc-13(e51) hermaphrodites. The non-Unc cross progeny were examined using Nomarski optics for rare animals in which the two NSM sisters and the two 12 sisters all died. These animals might contain a second mutation (marked as * in the figure) within the ces-1 gene that causes a loss of ces-1 function. Two animals were isolated that segregated Lin non-Ces progeny but no Lin Ces progeny. The Lin nonCes strains obtained from these animals each carried a new ces-1 loss-of-function mutation. A reduction of ces-2 function causes the NSM sisters to survive We believe that n732 acts by lowering but not eliminating ces-2 gene function at higher temperatures. In two different experiments, n732 has effects similar to but weaker than those of a deletion of the ces-2 gene. First, both ces-2(n732) and deletions of the ces-2 gene show semi-dominance for the survival of the NSM sisters. These cells always die in the wild type (n=800 cells), but at 25°C, 2.3% of them survive in ces2(n732)/+ animals (n=400 cells) and 27% survive in Df/+ animals (n=% cells, see Materials and methods). Second, in both ces-2(n732)/ces-2(n732) animals and ces-2(n732)/eDf6 animals at 25 °C the NSM sisters usually survive (Fig. 6). However, the NSM sisters survive more often in ces-2(n732)/eDf6 animals than in ces-2(n732) homozygotes, which suggests that n732 does not completely eliminate ces-2 function, even at high temperatures. Isolation of additional ces mutations To isolate more mutations that affect the decision of the NSM sisters to live or die, we developed a general screen. Looking for animals with extra serotonergic cells, as was done to isolate ces-1 (n703) and ces-2(n732), is time-consuming. Such a screen requires the examination of fixed and stained animals, and mutants must 600 R. E. Ellis and H. R. Horvitz 1 0 0 * -i 100%H u 3 in 2 50%- c/5 n732l+ o* 15° C 20°C 2J°C Fig. 6. NSM sister survival in animals with different doses of the ces-2 gene. The survival of the NSM sisters was scored by direct observation using Nomarksi microscopy. Each point represents the frequency of NSM sister survival determined by scoring 100 animals of one genotype at a particular temperature, except for the unc-59 ces-2/eDf6 points, for which 37 animals were scored at 15°, 92 at 20°, and 99 at 25°C. The ces-2(n732)/+ animals were the cross progeny of ces-2(n732) males mated with unc-13 lev-10 hermaphrodites. The error bars represent 95 % confidence limits as determined by0 the binomial distribution [% Error=±196(x(l-x)/n) -5, where x=(% NSM sisters surviving)/100 and n=number of cells]. therefore be recovered from among previously cloned siblings. Instead, we used Nomarski optics to examine living worms, seeking mutants in which the NSM sisters survived. We screened 21000 F 2 worms, which represents about 9700 haploid genomes scored for recessive mutations (see Materials and methods). From this mutagenesis, we recovered three new ces mutations - two ces-1 dominant alleles, nl895 and nl896, and one recessive, temperature-sensitive mutation, n.1952. We also isolated six mutations that prevent the deaths of not only the NSM sisters, but of all other dying cells as well. These six mutations will be described elsewhere. The penetrance of NSM sister survival is low for the recessive mutation ces(nl952): 25 % of the animals had at least one surviving NSM sister at 25 °C, and only 2 % at 20°C (AJ=100 animals in each case). Because of its low penetrance, we have not yet fully characterized this mutation, but preliminary results suggest that nl952 might define a new gene specifically involved in the deaths of the NSM sisters. First, nl952 complements ces-2(n732) and is not linked to ces-1 or ces-2 (see Materials and methods). Second, the only abnormality that we have observed in these animals is the presence of two extra pharyngeal cells. These extra cells are serotonergic neurons, and based upon their positions appear to be the surviving sisters of the NSM neurons (data not shown). We believe the two new dominant mutations are alleles of ces-1 for three reasons. First, the ces-1 dominant mutation n703 and the new mutations nl895 arid nl896 result in the same phenotype: the NSM and 12 sisters fail to die, but other cell deaths appear unaffected (data not shown). Second, all three mu- mJm H mJ+ n703 • mllf mJnD/23 nl895 g | n!896 Fig. 7. 12 sister survival in animals with different doses of ces-1 (n703), ces-1 (nl895), and ces-1 (nl896). The survival of the 12 sisters was scored by direct observation using Nomarski microscopy. Each bar represents the frequency of 12 sister survival determined by scoring at least 100 animals 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 mated with unc-13(e51) hermaphrodites, the m/lf animals were the non-Unc progeny of either unc-13(elO91) cesl(n703)/nDf23, or unc-13(e51) ces-1 (nl895)/nDf23, or unc13 (e51) ces-1 (nl896)/nDf23 hermaphrodites. The error bars represent 95 % confidence limits as determined by the binomial distribution [% Error=±196(x(l—x)/n) , where x=(%12 sisters surviving)/100 and n=number of animals]. tations map between the genes daf-8 and sup-17 (see Materials and methods), which places them all within an interval of about 0.1 map units. Third, all trans heterozygotes involving these mutations appear identical: 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 in studies 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-function mutants, whereas they live in ces-2, ced-3 and ced-4 mutants. This difference in phenotype allowed us to study the interactions between ces-1 and these other genes that affect the deaths of the NSM sisters. We constructed double mutants between ces-1 (n703 nl406) and mutations in each of the other genes, and determined if the NSM sisters lived or died. In animals carrying ces-1 (n703 nl406) and a mutation in either ced-3 or ced-4, the NSM sisters live, just as they do in ced-3 and ced-4 mutants. These results indicate that ces-1 function is not required for NSM sister survival in ced-3 or ced-4 animals. Since loss-offunction mutations in ces-1 and these ced genes result in opposite effects on the NSM sisters (death vs. life, respectively), it seems likely that ces-1 and the ced genes do not control sequential steps in a pathway {e.g. of biosynthesis) but rather that one negatively regulates the other. If so, our results imply that ces-1 acts before ced-3 and ced-4 to decide whether the NSM sisters should live or die. By contrast, the NSM sisters die in C. elegans genes that control specific cell deaths ces-1 (n703 nl406) ces-2 and ces-1 (n703 nl406); nl952 animals, just as they do in ces-1 (n703 nl406) mutants. These results show that ces-1 function is probably required for ces-2(n732) or ces(n!952) to prevent the deaths of the NSM sisters, suggesting that ces-1 acts after ces-2 and ces(nl952). However, since neither n732 nor nl952 results in a complete loss-of-function, this conclusion must be regarded as tentative. We repeated several of these experiments using the second ces-1 loss-of-function allele. In each case, cesl(n703 nl434) behaved like ces-1 (n703 nl406). Specifically, ces-1 (n703 nl434) was suppressed by ced-3 or ced4 mutations, and suppresses the ces-2 mutation. We also constructed the mutant ces-1 (n703 nl406) ces-2; ced-3. In this animal, the NSM sisters survive, which shows that these cells die in ces-1 (If) ces-2 animals by the normal process of programmed cell death, which depends on ced-3 function. Life or death is decided independently by each cell We examined a group of 200 ces-1 (n703)/+ animals at 20°C, in which 93 % of the NSM sisters survived. If mutations in ces-1 act independently on each NSM sister, then (93 %)2=86.5 % of the worms should have two surviving NSM sisters, 2(93 %)(7 %)=13 % of the worms should have only a single NSM sister surviving, and (7%) 2 =0.5% of the worms should have no NSM sisters surviving. Among these 200 animals, we saw 87.5% with two NSM sisters, 11% with one NSM sister, and 1.5% with no NSM sisters. This result suggests that ces-1 (n703) might act independently on these two cells. Similar data indicate the independence of the 12 sister deaths in ces-1 (n703)/+ animals and of the NSM sister deaths in ces-2 animals (all of these experiments were done at 15°, 20° and 25 °C; data not shown). In each case, the data fit the hypothesis that these two genes act independently on each of the affected cells. Discussion We have identified two genes, ces-1 and ces-2, that affect the decisions of specific cells to live or die. Gainof-function mutations in ces-1 and loss-of-function mutations in ces-2 (both n732 and deficiencies) prevent the sisters of the NSM neurons from dying. These ces-1 mutations also prevent the sisters of the 12 neurons from dying. However, other cell deaths in these mutants occur normally, and there are no other obvious abnormalities in phenotype. In particular, the other cells that form the pharynx, many of which are closely related to the NSM sisters and the 12 sisters, all appear normal. Thus mutations in the ces-1 and ces-2 genes can affect the fates of the NSM and 12 sisters in the pharynx without changing other aspects of development. Our observations indicate that a normal function of both the ces-1 and ces-2 genes is to control the deaths of the NSM sisters. First, since a loss of ces-2 function prevents the NSM sisters from dying, ces-2 normally causes these cells to die. Second, since a loss of ces-1 601 function results in the NSM sisters dying in a ces-2 mutant animal, ces-1 can cause the NSM sisters to live. Only one other C. elegans gene, egl-1, is known to affect specifically the decisions of particular cells to undergo programmed cell death (Trent et al. 1983; Ellis and Horvitz, 1986). In hermaphrodites, the two HSN neurons control egg-laying, whereas in males these cells undergo programmed cell death. Mutations in egl-1 cause the HSN neurons to die in hermaphrodites as well as in males, possibly by transforming the sexual identity of these cells. It is conceivable that egl-1 acts in the process of sex determination rather than in the direct specification of cell death. This reservation does not apply to the functions of ces-1 and ces-2, which act similarly in both sexes. Two observations suggest that ces-1 and ces-2 control the decision of the NSM sisters to live or die by controlling genes that act in all cell deaths. First, the NSM sisters die in ces-1 (If) animals, but in ces-1 (If); ced3 or ces-1 (If); ced-4 animals these cells live. As we discuss above, this result indicates that ces-1 acts prior to ced-3 and ced-4 and is consistent with models in which ces-1 decides if the NSM sisters should live or die and 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 to regulate the genes involved in all programmed cell deaths. The fact that in ces-1 (If) ces-2; ced-3 animals the NSM sisters live demonstrates that in ces-1 (If) ces-2 mutants these cells die by normal programmed cell death, which requires ced-3 function. Mutations in the ces genes could affect the activities of ced-3 and ced-4 directly, by controlling the initiation of cell death, or indirectly, by transforming the NSM sisters into cells that normally live, presumably the NSM neurons themselves. It seems unlikely that ces-1 normally acts to determine NSM identity: although ces1 gain-of-function mutations cause the NSM sisters to survive and develop like NSM neurons, ces-1 loss-offunction mutations eliminate only the surviving NSM sisters and not the NSM neurons themselves. In fact, in mutants with a loss of ces-1 function, the NSM neurons appear to survive, differentiate and produce serotonin normally. Mutations in ces-2 also have no known effects on the NSM neurons, and genetic results (see above) indicate that ces-2 acts via ces-1. Thus, these genes probably control the initiation of programmed cell death rather that the acquisition of the NSM cell fate. When the deaths of the NSM sisters are blocked, as in ced-3 or ced-4 animals, these cells appear identical to the surviving NSM sisters found in the ces mutants, consistent with the hypothesis that the ces genes are involved in directly controlling the deaths of these two cells. What are the phenotypes of animals with a complete loss of function for ces-1 or ces-2? It is possible that a complete loss of ces-1 function results in a wild-type phenotype, because the two ces-1 alleles that we isolated in a screen for mutations that reduce or eliminate ces-1 activity both result in a wild-type phenotype. Both alleles resemble deficiencies in sev- 602 R. E. Ellis and H. R. Horvitz Drc A ces-2 oNy 1 ces-1 1 ced-3,ced-4 I OFF* OFFX LIVE B ces-2 ONy ces-1 DIE ced-3, ced-4 OFFX LIVE Fig. 8. Two models of the control of cell death in the sisters of the NSM neurons. (A) ces-2 and ces-1 act in a process that can inhibit ced-3 and ced-4 activity and so prevent the deaths of the NSM sisters. An unknown process activates ced-3 and ced-4. (B) ces-2 and ces-1 act through an unknown gene that activates ced-3 and ced-4 function. —•, activation; —I, inhibition. eral, although not all, of our gene dosage tests. By contrast, only one allele of ces-2 exists. The n732 mutation is extremely temperature-sensitive, and gene dosage experiments suggest that this mutation reduces only partially the activity of the ces-2 gene. It is possible that a complete loss of ces-2 function would affect other aspects of development besides the deaths of the NSM sisters. Screens for new mutations in this gene should help determine the phenotype that results from a complete loss of ces-2 function. If a loss of ces-1 function results in a wild-type phenotype, there must be other genes that cause the NSM sisters to decide to die. One possibility is that there are two regulatory pathways, each of which controls the deaths of the NSM sisters (Fig. 8A). One process acts to prevent cell death and includes ces-2 and ces-1; cell death is prevented effectively only in mutants with a gain of ces-1 function or a loss of ces-2 function, whereas in animals with wild-type or inactive ces-1 genes the second pathway successfully initiates cell death. Alternatively, ces-2 and ces-1 might both act upstream of an unknown regulatory gene that directly regulates ced-3 and ced-4 to initiate the deaths of the NSM sisters (Fig. 8B). In some organisms, hormonal signals play an important role in deciding if or when a cell should die. For example, in the moth Manduca sexta, a decrease in the level of ecdysteroids initiates many programmed cell deaths (Truman and Schwartz, 1982). Cells that would normally die continue to live if provided with ecdysteroids, and when ecdysteroid levels decline these cells die; since other cells do not die, factors other than hormone levels must specify which cells are capable of dying. Are the genes ces-1 and ces-2 involved in a similar system? Our data indicate that the decision to live or die of each of the cells affected by ces-1 and ces-2 mutations appears to be independent of that of the other cells. These observations suggest that ces-1 and ces-2 do not act to control the level of a systemic hormonal factor. Although it remains possible that these genes act in response to such a factor or in a system involving signalling between adjacent cells, several lines of evidence suggest that in general in C. elegans the decision to die occurs within dying cells or their parents (reviewed by Yuan and Horvitz, 1990). We therefore suspect that ces-1 and ces-2 encode factors that act in a pathway operating entirely within the NSM sisters to control the deaths of these cells. We thank Nancy Tsung and Carol Trent for providing the mutations n703 and n732, John Sulston for his generous help in studying the embryonic lineage of ces-l(n703) animals, Phil Anderson and Jonathan Hodgkin for providing strains, and Hilary Ellis and Leon Avery for sharing unpublished observations. We are also grateful to Erik Jorgensen, Patricia Kuwabara and Eric Lambie for suggestions concerning this manuscript. This work was supported by US Public Health Research Grants GM24663 and GM24943. R. E. 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