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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. E. was
supported by a National Science Foundation Graduate
Fellowship, and by a National Institutes of Health Training
Grant. H. R. H. is an Investigator of the Howard Hughes
Medical Institute.
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