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The Caenorhabditis elegans genes ced-3 and ced-4 act cell autonomously to cause programmed cell death
1)EVELOPMENTAI.
BIOLOGY
138, 33-41
(1990)
The Caenorhabditis elegans Genes ted-3 and ted-4 Act Cell Autonomously to Cause Programmed Cell Death JUNYING YuAN*~~ AND *Program
of Neurosciences, Department
Harvard
of Biology,
H. ROBERT
HORVITZt
Medical School, Boston, Massachusetts 02115; and THoward Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Accepted Nmember
7, 1989
Mutations in the genes ted-3 and ted-4 prevent almost all of the programmed cell deaths that occur during Caenorhabditis eleguns development. To determine the sites of action of these two genes, we performed genetic mosaic analyses. We generated C. elegans animals that carried a free chromosomal duplication bearing either ted-3(+) or ted-4(+) in an otherwise homozygous ted-3 or ted-4 genetic background. We used other genes on the duplication as markers to identify genetic mosaic animals in which the duplication was present in some but not all cells. The patterns of cell death survivors in these mosaic animals indicated that the products of both ted-3 and ted-4 function within dying cells to cause cell death. 0 1990Academic Press, Inc.
mutant. By knowing the genotype of specific cells, it is possible to determine whether the phenotype of a cell depends upon its own genotype or upon that of other cells. Free chromosomal duplications, which are genetically unstable and can be lost at cell division, are used to generate such mosaic animals. If a free duplication carries the wild-type allele of a gene and the chromosomes are homozygous for a recessive mutant allele of that gene, only cells that have lost the duplication will be mutant in genotype. If the gene in question functions cell autonomously, cells that have lost the duplication will be mutant in phenotype, whereas if the gene acts cell nonautonomously, such cells might not exhibit a mutant phenotype. By using appropriate genetic markers, such mosaic animals can be recognized and their phenotypes can be analyzed to determine whether the phenotypes of particular cells depend upon their own genotypes. Here we describe genetic mosaic analyses of the genes cedd and ted-4. Mosaic animals were isolated using appropriate behavioral and morphological markers, and the survival or death of individual cells was scored using Nomarski optics. The rigidly determined and completely known C. elegans cell lineage (Sulston and Horvitz, 1977; Sulston et al., 1983) allowed us to identify at which cell division duplication loss occurred in each mosaic animal. These genetic mosaic analyses demonstrated that ted-3 and ted-4 most likely act cell autonomously within dying cells.
INTRODUCTION
Programmed cell death is common during animal development (Glticksman, 1951; Saunders, 1966; Oppenheim, 1981; Truman, 1984). The phenomenon of programmed cell death raises a number of interesting questions, such as: What are the mechanisms that determine which cells live and which cells die? What are the mechanisms that cause cells to die? The nematode Caenorhabditis elegans is an appropriate organism with which to address these questions. During C. elegans development, the generation of the 959 somatic cells of the hermaphrodite is accompanied by the generation and subsequent deaths of an additional 131 cells (Sulston and Horvitz, 19’77;Sulston et al., 1983). Mutations in two C. elegans genes, ted-3 and ted-4, prevent almost all of these programmed cell deaths (Ellis and Horvitz, 1986). Cells that die in wild-type animals survive, differentiate, and, in at least some cases, function in ted-3 and ted-4 animals (Ellis and Horvitz, 1986; Avery and Horvitz, 1987). The products of ted-3 and ted-.$ presumably are either cytotoxic killer proteins or control the activities of such killer proteins. Elucidating the mechanisms of action of these two genes would be an important step toward understanding how programmed cell death is regulated and executed in C. elegans, One basic question is whether ted-3 and ted-4 act cell autonomously, i.e., whether these genes act within the cells that die or within other cells that control cell death through cell interactions. The method of genetic mosaic analysis, developed for C. elegans by Herman (1984), can be used to answer this question. In mosaic analysis, animals are generated in which some cells are genotypically mutant and some cells are genotypically non-
MATERIALS
AND
General Methods, Nomenclature,
METHODS
and Strains
Methods for the growth, handling, and general genetic studies of C. elegans have been described by 33
0012-1606/90 Copyright All rights
$3.00
0 1990 by Academic Press. Inc. of reproduction in any form reserved.
34
DEVELOPMENTALBIOLOGYVOLUME138,199O
Brenner (1974). The wild-type strain is N2 (Brenner, 1974). All strains were grown at 20°C. The genetic markers used are as follows: LG I-ted-l(el7’35); LG III-ted-4(nll62), dpy-17(el64), uric-36(e251); LG IVbeen described by Brenner (1974), Ellis and Horvitz (1986), and Hodgkin et al. (1988). Genetic nomenclature follows the conventions of Horvitz et al. (1979).
IV, uric-60(+) V, and uric-46(+) V (DeLong et ah, 1987) while nDp3 does not (data not shown). The frequency of nDp3 loss per cell division can be estimated as the frequency of ABp mosaic animals isolated. Since three ABp mosaic animals were isolated from among 1300 animals screened, the frequency of nDp3 loss in a given cell division is about 0.2%. By contrast, based on the data above, the frequency of yDp1 loss per division is 0.006% or less.
Generation
General Procedures fbr Mosaic Analysis
me-3l(e169), uric-30(e191), ced+?(n717), uric-26(e205), dpy-h(e1166); LG V-egl-l(n487). These markers have
of the Free Duplication
nDp3
Usually lo-15 adult animals carrying the appropriate duplication and markers were placed on a loo-mm petri plate seeded with Escherichia coli OP50 (Brenner, 1974). end of chromosome IV and the left end of chromosome Late L3 and L4 progeny from these animals were V (DeLong et aZ., 1987). Because yDp1 carries the wild- screened for apparent mosaic phenotypes using the distype alleles of uric-31, ted-3, and dpy-4, animals of geno- secting microscope (see Results). When such animals type ted-1; uric-31 ted-3 dpy-4; egl-1; yDp1 are phenotyp- were identified, they were examined for cell death surically wild-type in movement and body shape. (egl-1 vivors using Nomarski optics as described by Sulston confers an egg-laying defect that is not relevant to the and Horvitz (1977). Generally, after one side of an aniresults described in this paper). The progeny of such mal was examined, the animal was remounted with the animals are mainly of two types: Dp+ animals that are other side up and that side was then examined. Animals just like their parents, and Dp- animals that are both were carefully removed from the slide and transferred Uric and Dpy. We attempted to screen for mosaic ani- to a 60-mm petri plate seeded with bacteria. We exammals among the progeny of ted-1; uric-31 ted-3 dpy-4; ined the body shapes and behaviors of these animals egl-1; yDp1 animals. Among 18,000 animals screened, 17 with the dissecting microscope the next day when they Uric non-Dpy mosaic candidates were picked. Of these, had grown into adults. Their progeny were checked 3-4 16 segregated Uric Dpy and Uric non-Dpy progeny, but days later to confirm the presence of duplication-carnot non-Uric non-Dpy progeny, suggesting that they rying animals. carried some altered forms of yDp1, e.g., they might have lost the part of yDp1 that carries uric-31(+), or that RESULTS a recombination event had occurred so that the new yDp1 derivative carried the mutant uric-31 gene. One of To identify mosaic animals generated by the somatic the 16, when examined using Nomarski optics, showed loss of free duplications, we have used marker mutamosaic expression of ted-3, i.e., cell death was absent tions that result in uncoordinated (Uric) movement or among cells derived from the ABp lineage. We sus- dumpy (Dpy) body shape. Each free duplication carries pected that only part of yDp1 might have been main- a wild-type allele of an uric, dpy, and ted gene, while tained in this animal and that the smaller derivative of chromosomes are homozygous for the corresponding yDp1 in this animal might be less stable than yDp1 and, recessive mutant alleles. Animals of genotype uric dpy therefore, more suitable for mosaic analysis. The strain ted; Dp are phenotypically wild-type, because of the carrying this putative new duplication was crossed into presence of the wild-type alleles on the duplication. The a strain carrying ted-3 uric-26 dpy-4. The average per- self-progeny of uric dpy ted; Dp animals are of three centage of Dp- self-progeny of Dp+ hermaphrodites was general classes, as scored with a dissecting microscope 43% for this duplication (447 progeny scored) as op- (the Ced phenotype cannot be scored with a dissecting posed to 34% for yDp1 (DeLong et al, 1987), suggesting microscope): phenotypically wild-type animals (which this derivative of yDp1 was lost more frequently in carry the duplication and are of genotype uric dpy ted; germ line cells than was yDp1. Using DAPI (4’-6-diami- Dp), Uric Dpy animals (which do not carry the duplicadino-2-phenylindole dihydrochloride) to stain chromo- tion and are of genotype uric dpy ted), and rare animals somes (Fixsen, 1985), we found this strain to contain an that are neither wild-type nor Uric Dpy in phenotype. extra chromosomal fragment (data not shown). The This third class, which includes mosaic animals that new duplication, named nDp3, was found to carry wild- have lost the duplication in a subset of their somatic type alleles of uric-30 IV, ted-3 IV, uric-26 IV, dpy-4 IV, cells, can be of any of a variety of phenotypes-e.g., Uric and me-34 V. Both the left and right ends of yDp1 seem non-Dpy, Dpy non-Uric, semi-Dpy non-Uric, and semito have been lost in nDp3, since yDp1 carries uric-31(-b) Dpy semi-Uric,-depending upon the sites of action of nDp3 was generated as a spontaneous breakdown product of yDp1, a free duplication carrying the right
YIJAN
AND
HORVITZ
C.
elegans
the uric and dpy genes. For example, if the duplication is lost in all cells in which the uric gene acts, but in none of the cells in which the dpy gene acts, the animal will be Uric non-Dpy in phenotype. If a duplication loss is appropriately rare, most mosaic animals will be generated as a consequence of a loss at a single cell division. Since the C. eleyans cell lineage is known, if this division is identified, the genotype of every cell in the animal can be established. We can then ask whether the survival or death of particular cells that die in wild-type animals corresponds to their ted genotypes. In other words, if such cells survive only when they are ted in genotype, then the action of the ted gene must be cell autonomous. Of the 131 cell death survivors in ted-3 and ted-4 mutants (Ellis and Horvitz, 1986), we have studied a set of 17 that can be readily recognized in living animals using Nomarski optics. These cells and the early embryonic cell lineages (Sulston et ah, 1983) from which they are derived are shown in Fig. 1. In the experiments described below, our general approach was to identify mosaic animals in the dissecting microscope using the uric and dpy markers and then to examine these animals with Nomarski optics to determine which of the 17 cells had lived and which had died. The patterns of surviving cells were striking: in the vast majority of individuals, all surviving cells were derived from a single branch of the cell lineage. This observation indicated that this branch lacked ted gene activity (i.e., had lost the duplication) and that ted activity was cell autonomous. A specific confirmation of this interpretation was provided by the analysis of a pair of cells (CEMDR and CEMDL, see Fig. 1) that are physically adjacent but derived from quite distinct branches of the cell lineage. That one of these cells often survived while the other died argues against the hypothesis that it is the genotype of some other cell(s) acting at a distance that specifies the cell death phenotypes of CEMDR and CEMDL. Taken together, these studies provide strong evidence for the cell autonomous actions of ted-3 and ted-4, ted-3 Acts Cell Autonomcrusly
We constructed hermaphrodites of genotype uric-30 ted-3 dpy-4; nDpS(IV, V;f), where nDp3 is a free duplication that carries uric-30(+) ted-3(+) dpy-h(f) (see Materials and Methods). nDp3 is fairly stable but can be lost during both somatic and germ line cell divisions. The chance of nDp.3 loss in a given cell division is about 0.2% (see Materials and Methods). uric-30 ted-3 dpy-4; nDp3 animals are wild-type in movement and slightly short in body length as dpy-4 is weakly semidominant. Their progeny are predominantly of two classes: animals like their parents, which carry the duplication, and Dpy Uric
Programmed Cell Death
35
animals, which do not. None of these genes exhibits any detectable maternal effect: the Uric Dpy animals from duplication-bearing parents are indistinguishable from their own progeny, and ted-3 progeny of a cross between ted-3 males and ted-3/+ hermaphrodites exhibit the same Ced-3 phenotype as their own progeny (data not shown). Rare animals with mosaic phenotypes, e.g., Uric non-Dpy, Dpy non-Uric, semi-Dpy semi-Uric, and so on, were sought and then examined for their patterns of cell deaths using Nomarski optics. A total of five animals with obvious mosaic phenotypes were found using the dissecting microscope. These animals are described in categories 1 and 2 of Table 1. The mosaic animals in the first category were completely Uric but only semi-Dpy. They were found to have cell death survivors present only among cells derived from the ABp lineage (cf. Table 1 and Fig. 1). This observation suggests that nDp3 was lost in ABp and that ted-3 acts cell autonomously. That the cells CEMDL and CEMDR, which die in wild-type hermaphrodites, are of opposite phenotypes in category 1 mosaic animals is of particular interest: as noted above, these cells are derived from quite different branches of the cell lineage (ABp and ABa, respectively), but are adjacent physically when they survive in wild-type males and in ted-3 mutant hermaphrodites and before they die in wildtype hermaphrodites (Sulston et al., 1983; J. Sulston, personal communication; H. Ellis and J. White, personal communication). Since CEMDL and CEMDR are in essentially the same local environment, if ted-3 acted nonautonomously, one would expect that these cells would display the same fate. That in category 1 mosaic animals only CEMDL is present strongly supports the conclusion that ted-3 acts cell autonomously. The Uric phenotype of these category 1 mosaic animals indicates that we-30 acts within the ABp lineage. This conclusion is consistent with the observation that the locomotory defect of uric-30 animals is associated with a lack of GABA in the ABp-derived class D motor neurons of the ventral nervous system (S. McIntire and J. White, personal communication). The semi-Dpy phenotype of these animals indicates that dpy-4 acts both within the ABp lineage and elsewhere. The category 2 mosaics were semi-Dpy and non-Uric (although they moved more slowly than did wild-type animals, possibly because uric-30 also acts in a few cells other than those derived from the ABp lineage). These animals were found to have cell death survivors only among cells generated by the ABa lineage. Again, the cells CEMDL and CEMDR are of opposite phenotypes. These results further support the hypothesis that cedd acts cell autonomously. To provide additional evidence that ted-3 acts cell autonomously, we used another behavioral marker,
36
DEVELOPMENTAL BIOLOGY
VOLUME 138,199O
CEMVR CEMDR
Right Posterior
a
lateral
ganglion
R
Anterior
Posterior
Posterior
PTAA-VT. I a2Lais
lateral
ganglion
L
Left
CEMDL
b A%,
1
, ABP
P2
EMS MS
P3 I
Posterior
lateral
C
r-5
P4
A zx
3
-
E
'
J
5x
Pharynx
I
D
I
I-4 “,x rA
I
Germ line
V5Rpaap
ganglia
FIG. 1. Seventeen easily identifiable cell death survivors in ted-3 and ted-4 mutants. (a) The positions of the 17 cell death survivors in a ted-3 or ted-4 mutant are shown in a schematic dorsal view. Cells located within the ventral half of the animal are indicated with dotted lines. The sisters of the cells IZR, 12L, NSMR, NSML, MCL, and mlvR, which die in wild-type animals, are collectively referred to as “pharynx,” because they are located within the ventral part of the anterior bulb of the pharynx. The cells derived from QL and the V5 blast cells that die in the wild-type are collectively referred to as “posterior lateral ganglia,” because when they survive in a ted-3 or ted-4 mutant, they become part of the posterior lateral ganglia. M4sis. g2Rsis, and g2Lsis refer to the sisters of M4, g2R, and g2L. We identified Mdsis, g2Rsis, and g2Lsis on the basis of their positions and their morphological similarities to their sisters. CEMDR, CEMDL, CEMVR, and CEMVL are 4 cells that undergo programmed cell death in the wild-type hermaphrodites but survive in wild-type males or in ted-3 and ted-4 animals. (b) The lineage of the 17 cell death survivors shown in (a) (adapted from Sulston et al,, 1983). The fertilized egg, PO, divides asymmetrically to generate a larger somatic precursor cell AB and a smaller cell Pl. ABa and ABp are the anterior and posterior daughters of AB, respectively. ABpl and ABpr are the left and right daughters of ABp, respectively. P4 is the precursor of the germ line. X, cell that dies in the wild-type but survives in ted-3 and ced.4 animals. The dotted lines indicate that cell divisions are omitted in the figure. Lineages leading to cells that are not scored in our mosaic analyses are not shown in this figure. See Sulston et al. (1983) for the complete embryonic cell lineage and Sulston and Horvitz (1977) for the postembryonic lineages of the QL and V5 cells. For other conventions, see (a).
YIIAN AND H~RVITZ
MOSAIC ANALYSIS OF cdl
C. nlegnns Programmed
TABLE 1 USING ZYGOTES OF GENOTYPE uric-30 ted-3 dpy-4; nDp3 Cell death survivors
Mosaic phenotype Body shape 1 2 3 4 5 6 7
Semi-Dpy Semi-Dpy Slight-Dpy Slight-Dpy Non-Dpy Non-Dpy Non-Dpy
Behavior Uric Non-Uric* Semi-Uric Slight Uric Slight-Uric Non-IJnc* Slight-IJnc
No. animals scored 3 2 2 2 2 1 1
Posterior lateral ganglia
Pharynx R
+
37
Cull Deuth
observed
CEMV
G&is
CEMD
L
R
L
R
L
R
L
R
L
-
+
t
t
t
-
t -
-.
.-
+ ~ + +
f -
~ ~ ~
~ ~ ~
+ -
+ +
-. +
t
-
t
-
t ~
M4sis
-
Dp loss ABP ABa ABpl ABpr ABplap ABarp
ABplap, ABarpa
Note Dpy-dumpy. IJnc-uncoordinated. Pharynx-in N2, there are five cells in the ventral half of the pharynx on each side; in ted-3 animals, there are seven to eight cells due to the absence of programmed cell death. Posterior lateral ganglia, a ganglion located about three-fourths of the way from the nose to the tail. In N2, there are four cells in the right posterior lateral ganglion and six cells in the left. In ccd-d animals, because cell deaths fail to occur in the QL and V5 lineages, there are five cells in the right posterior lateral ganglion and at least seven cells in the left. CEMV and CEMD-ventral and dorsal cephalic companions, respectively. The CEMs die in N2 hermaphrodites and survive in red-:1 animals. g2Lsis, g2Rsis, and M4sis-the sisters of the gland cells g2L and g2R and of the pharyngeal neuron M4 die in N2 but survive and differentiate into cells that look like g2 and M4, respectively, in ted-3 animals. (-) Cell death survivor absent (Ced-3(+) phenotype). (+) Cell death survivor present (Ced-3(p) phenotype). Non-LJnc*-These animals were less active than the wild-type. Wild-type animals move almost constantly, except when molting. These animals move less; but when they do move, they are not uncoordinated. Dp loss-This column indicates our interpretation of the cell in which the duplication was lost in each mosaic animal based upon the pattern of cell death survivors observed. When the duplication may have been lost in either a cell or one of its progeny, the earliest possibility is listed. For example, the category 5 mosaic animals could he caused by loss of the duplication in ABplap or in certain of the progeny of ABplap, e.g., ABplapa or ABplapap, but only ABplap is listed. A total of about 1300 UVLC-30c&-3 dl)y,ll-4;nD@ were screened to obtain the mosaic animals described in this table. The expression of the Ced-3 phenotype as a result of the wd-3(n717) mutation is almost complete. We estimated the expression of crtl-.Kt/717) based upon the presence of the surviving sister of the g2R or g2L cell in cd-X(71717) rhnc-26 dpy-4 animals. Among 100 crd-3 um-26 dj)!/-h animals scored, only in one animal did we fail to see the sister of a g2 cell. Thus, we estimated that there is about a 1% chance that a cell that normally undergoes programmed cell death might still die in a crd-Y(n717) animal. Of the 32 ted-3 mosaic animals described in Tables 1 and 2, only one such cell death survivor seemed to he missing (in an ABp-loss mosaic animal).
uric-26, to do similar analyses. Five obviously mosaic animals are described in categories 1 and 2 of Table 2. The category 1 mosaics are semi-Dpy and Uric. These animals were found to have cell death survivors only among cells generated from the ABp lineage. The category 2 mosaic, with an apparent ABa loss, was semiDpy and non-Uric (although this animal moved more slowly than does a wild-type animal). These results again indicate that ted-3 is cell autonomous and reveal that uric-26, like uric-30, acts in the ABp lineage. One possibility is that uric-26 acts in the ventral nerve cord, because all but one of the ventral cord neurons are generated from the ABp lineage. In these experiments, we have found Uric semi-Dpy and non-Uric semi-Dpy animals but no Uric non-Dpy and Dpy non-Uric animals. In addition, the semi-Dpy phenotype can be caused by duplication loss in either the ABa or ABp lineages. Therefore, we predicted that loss of the duplication from the AB lineage would generate an animal that is both Dpy and Uric. Such animals
would appear the same as their Dp- siblings when viewed with the dissecting microscope, but unlike those Dp- siblings, these mosaics would have cell death survivors (the Ced( -) phenotype) in the AB lineage but not in the Pl lineage. Some of their progeny would carry the duplication and be phenotypically wild-type, since the germ line cells are derived from the Pl lineage. To screen for such animals, Uric Dpy progeny of nDp3-carrying animals were examined using Nomarski optics to identify animals with cell death survivors among cells generated by the AB lineage but not among cells generated by the Pl lineage. Two animals of this phenotype were isolated from among about 200 Dpy Uric animals screened (Table 2, category 3). The presence of the duplication in these animals was confirmed by showing that they generated nDp3-carrying nonDpy non-Uric progeny. This class of mosaics indicates that even when cell death is absent in the AB lineage (which generates 116 of the 131 total cell deaths; Sulston et al, 1983), cells in the Pl lineage still die. This
38
DEVELOPMENTAL BIOLOGY
VOLUME 138,199O
TABLE 2 MOSAIC ANALYSIS OF ted-3 USING ZYGOTES OF GENOTYPE ted-3 uric-26 dpy-4; nDp3 Cell death survivors
Mosaic phenotype Body shape
Behavior
No. animals scored
Posterior lateral ganglia ___
Pharynx ____ R
L -
CEMV ___ L
R
CEMD ~ L
R
G2sis L
R
L
M4sis
+ -
+
+ -
+ -
+
+ -
~ -
-
-
-
+ -
+ + +
+ +
+ + +
+ -
t + +
~
~ -
-
-
+ +
+
+ +
~
-
~ -
-
~
-
-
-
-
+
-
~
-
-
-
-
-
+
-
~
t
t
t
1 2 3 4 5
Semi-Dpy Semi-Dpy DPY* Slight-Dpy Slight-Dpy
Uric Non-Uric* Uric Semi-Uric Semi-Uric
4 1 2 3 1
+ +
+ +
-
6 7
Slight-Dpy Slight-Dpy
Semi-Uric Semi-Uric
1 1
Non-Dpy Non-Dpy Non-Dpy
Slight-Uric Slight-Uric Slight-Uric
2 3 1
8 9 10
R
observed
t
Dp loss ABP ABa AB ABpl ABpl, ABprp ABpr A&r, ABplap ABpra ABplap ABP~P, EMS
Note. Abbreviations and symbols are generally as in Table 1. Non-Uric*-this animal was less active than is the wild-type. Dpy*-this animal was a little longer than a dpy-4 animal. About 1900 ted-3 uric-26 clpy-4; nDp3 animals were examined to obtain the mosaic animals described in this table.
observation further supports the conclusion that ted-3 acts cell autonomously. This result also indicates the site of action of dpy-4: that the loss of nDp3 from the AB lineage is sufficient for these mosaic animals to be Dpy suggests that dpy-4 primarily acts within cells generated by the AB lineage. The experiments described above are concerned only with mosaic animals generated as a result of duplication loss in the first and second divisions of embryonic development. To examine additional classes of mosaic animals, we attempted to identify mosaic animals with more subtle phenotypes. Such mosaic animals are described in categories 3 to 7 in Table 1 and categories 4 to 10 in Table 2. In 16 of the 20 mosaic animals identified in this way, the cell death survivors were derived from a single branch of the cell lineage. These animals further support the conclusion that ted-3 is cell autonomous in its action. In the other four animals, the cell death survivors were derived from two branches of the lineage. Since nDp3 appears to be lost in a given cell division at a frequency of about 0.2%, it is not unreasonable that, given the relatively large number of possible losses that apparently can lead to a weakly mosaic phenotype, double duplication losses would occur in 20% of such weakly mosaic animals. That all of the mosaic phenotypes observed in these experiments can be explained by either single losses (26 eases) or double losses (4 cases) strongly indicates that ted-3 acts cell autonomously.
ted-4 Also Acts Cell Autonomously
We used the same method but a different duplication and different markers to perform mosaic analysis of ted-4. The duplication sDp3(III;f) (Rosenbluth et al., 1985) was known to be mitotically unstable (the chance of sDp3 loss at a given cell division is about 0.15%; see Table 3) and has been used to study the site of action of the gene mab-5 (Kenyon, 1986). The markers dpy-17 and uric-36 also have been used to study the site of mab5 action (Kenyon, 1986). We constructed hermaphrodites of genotype ted-4 dpy-17 uric-36; sDp3. These animals are wild-type in phenotype because sDp3 carries wildtype alleles of all three genes. Most progeny of these animals either carry sDp3 and are phenotypicaly wildtype or have lost sDp3 and are Uric and Dpy. Animals with mosaic phenotypes were identified using the dissecting microscope and examined using Nomarski optics for the presence or absence of particular cell death survivors. Our results are shown in Table 3. The mosaic animals listed in category 1 were Uric and nonDpy. In these animals, cell death survivors were found only among cells generated by the ABp lineage; ABp-derived CEMDL is present, while ABa-derived CEMDR is absent. This result suggests that ted-4, like ted-3, acts within dying cells. This result also indicates that uric-36 acts in cells derived from the ABp lineage, because the loss of sDp3 from the ABp cell is sufficient to make an animal Uric. Kenyon (1986) has previously reported that uric-36 acts within the ABp lineage.
YITAN AND HCJRVITZ
C elegnxs
Progrum
rrwd
Cell
39
llrdl
TABLE 3 MOSAIC ANALYSIS OF ted-4 USING ZKCOTES OF GENOTYPE ted-4 dpy-17 um-36; sDp.9 Cell death survivors Posterior lateral ganglia ___
Mosaic phenotype
Body shape
Behavior
No. animals scored
1 2 3 4 5 6 7
Non-Dpy DPY* Slight-Dpy Non-Dpy Non-Dpy Non-Dpy Non-Dpy
Uric Non-Uric Non-Uric Semi-Uric Semi-Uric Semi-TJnc Semi-Uric
11 6 1 1 1 2 1
8
Non-Dpy
Semi-Uric
1
9
Non-Dpy
Slight-Uric
1
Pharynx ____ R
+
-
-
L
R
L
-
t -.
+++
CEMV
R
observed
CEMD ___ L
R
L
+ .-
+
+ -+-
+
t
+ ~~ -+-
-
+
++-
~
+ -
-
Mlsis
~
+ ~
.~
~ -
+ t
L
-
t +
R -
+ -
~
G2sis
--
-
Dp loss
ABP CC?) ABa ABpl ABpr ABpla ABpr, ABal ABpla, ABpr ABprp
Note. Abbreviations and symbols are generally as in Table 1. The Ced-4 phenotype is the same as the Ced-3 phenotype. Dpy*-These animals are a little longer than dry-17 animals (see text). About 7200 animals were screened to obtain the mosaic animals described in this table. The chance of sDp3 loss at a given cell division is about 0.15%, as estimated from the observation that 11 ABp-loss mosaic animals were isolated from the 7200 animals screened. The expression of the Ced-4 phenotype as a result of the ted-b(nll62) mutation is incomplete. We estimated the in the legend of Table 1. Of 40 ted-4fn1162) dpy-17 uric-36 animals scored, in three we expression of ted-h(nll62) as described for ce&3(n717) failed to see the surviving sister of g2R or g2L. Thus, we estimated that there is about a 7.5% chance for a cell that normally undergoes programmed cell death to die in a ted-4(n1162) animal. Some of our mosaic animals also exhibited incomplete expression of the Ced-4 phenotype. Of the 11 AB-loss mosaic animals described in this table, six seemed to be missing one cell death survivor.
The category 2 mosaic animals were Dpy and nonUric, although these animals were slightly longer than dpy-17 animals. No cell death survivor was found among any of the cells examined; however, the germ line of these animals retained the duplication, since some of their progeny carry the duplication. Thus, these animals must have carried the duplication in the AB, MS, and P4 lineages, but not in the lineage(s) responsible for the Dpy phenotype. In principle, duplication loss could have been within the C, D, or E lineages (Fig. 1). The E lineage, which generates only the intestinal cells, seems highly unlikely. We consider the D lineage also to be unlikely. The D lineage generates 16 cells, all of which are body muscle cells; most body muscle cells are derived from MS. Since no Dpy non-Uric animal was found to have lost the duplication in the MS lineage, body muscle cells seem unlikely to be the site of dpy-17 action. We believe the simplest interpretation of the data is that the duplication was lost within the C lineage. The C lineage generates 12 of the 23 embryonic hyp7 hypodermal nuclei; of the others, five are generated by the ABa lineage and six by the ABp lineage. If the site of action of dpy-I?’ were within hyp7, loss of the duplication in the ABa lineage might cause an animal to be slightly Dpy and non-Uric. By screening for slightly Dpy and non-Uric animals, we identified one such animal
(Table 3, category 3). In this animal, ABa-derived CEMDR is present and ABp-derived CEMDL is absent, as one would expect if ted-4 were cell autonomous in its action. These considerations would also predict that the loss of the duplication in the ABp lineage would result in a slightly Dpy animal. However, as described above, we scored such animals as non-Dpy. We suspect that this apparent inconsistency is caused by effects of the uric-36 mutation, which makes me-36 animals somewhat longer than wild-type animals, and hence could suppress a weak Dpy phenotype. The interpretation that dpy-17 acts within both the C lineage and the AB lineage is consistent with the data of Kenyon (1986). We also identified and characterized additional classes of mosaic animals (Table 3, categories 4-9). Five of the seven animals examined could be explained by cell autonomous ted-4 action and the loss of the duplication at a single cell division; the remaining two appear to have lost the duplication at two cell divisions. In short, all of these data indicate that ted-4 is cell autonomous in its action. ted-3 and ted-4 Are Useful Markers for Future Mosaic Analysis We have used uric and dpy markers, the sites of action of some of which were previously unknown, to perform
40
DEVELOPMENTAL BIOLOGY
mosaic analyses of ted-3 and ted-4. Our results have revealed not only the sites of action of ted-3 and ted-4, but also those of the uric and dpy genes we have used as markers. This identification was possible because cell deaths occur among cells generated from most branches of the cell lineage. The presence or absence of particular cell death survivors in a ted-3 or ted-4 mosaic animal can identify the cell division at which a duplication has been lost. Since the C. elegans cell lineage is invariant and known (Sulston and Horvitz, 1977; Sulston et al., 1983), this information reveals the genotype of every cell in the organism. For this reason, knowing that ted-3 and ted-4 are cell autonomous in their actions allows them to be used for high-resolution mosaic analyses. DISCUSSION
Our genetic mosaic analyses demonstrate that the fate of a cell that normally dies in wild-type C. elegans is correlated with its own genotype in ted-3 or ted-4 mosaic animals rather than with the genotype of cells derived from other branches of the cell lineage. Since we have studied deaths in a number of branches of the lineage, our data establish that neither ted-3 nor ted-4 is acting in a cell (or set of cells) that humorally controls all cell deaths in C. elegans. The simplest interpretation of our data is that both ted-3 and ted-4 act within dying cells to bring about their deaths. However, it remains formally possible that either or both of these genes act not within dying cells but rather within other cells very closely related by lineage. In many mosaic animals (17 ted-3 mosaic animals and 15 ted-.4 mosaic animals) the CEMD cells exhibited opposite Ced phenotypes, i.e., only one of the CEMD cells survived. This result also is most simply interpreted as indicating the cell autonomy of ted-3 and ted-4 action. However, this result is consistent with the hypothesis that a cell or cells physically very close to one of the CEMD cells can induce it to die. Considering these ideas together, it is conceivable that ted-3 and ted-4 are expressed within cells closely related to and adjacent to dying cells (e.g., within their sister cells), and these cells then interact with the dying cells to bring about cell deaths. However, this possibility seems unlikely to be generally true, since J. Sulston (personal communication) has shown that the deaths of cells in the postdeirid and ray lineages occur even if their sisters or other nearby close relatives have been killed with a laser microbeam. Alternatively, perhaps ted-3 and ted-4 act within the ancestors of dying cells (e.g., within their mothers); since dying cells are often smaller than their sisters (Sulston, 1988), certain genes involved in cell death may well act at or before the time of cell division. The apparent cell autonomy of ted-3 and ted-4 action does not indicate that cell death per se is cell autono-
VOLUME 138.1990
mous. For example, these genes might encode receptors or components of a signal transduction system that act within dying cells in response to a signal from other cells. Nonetheless, a number of observations suggest that most of the programmed cell deaths that occur during C. elegans development are cell autonomously determined. First, as noted above, many dying cells are smaller than their sisters at the time of their births, suggesting that their fates have already been specified. Second, most dying cells die within an hour of their births, before any signs of differentiation (Sulston et aZ., 1983), which indicates that these cells are unlikely to be dying as a consequence of a failure to compete for targets (such competition is believed to be the cause of the deaths of certain vertebrate neurons; e.g., Oppenheim, 1981). Third, even though dying cells are engulfed by certain of their neighbors, these engulfing cells are not bringing about the cell deaths, as engulfment can be blocked either by mutation (Hedgecock et a,l., 1983) or by laser-killing of the engulfing cells (J. Sulston, personal communication), and the deaths still occur. Fourth, the killing of neighboring cells in general with a laser microbeam has also failed to rescue dying cells (J. Sulston, personal communication) (except in a few distinctive cases of cell death in the tail of the C. elegans male; Sulston et al., 1980), showing that, at least in those cases tested, cells are not killed by their neighbors. Fifth, as we have shown here, the only genes known to be needed for programmed cell death, ted-3 and ted-4, act within dying cells. Given that ted-3 and ted-4 act within dying cells (or their mothers) to cause cell death, how might the products of these genes function? Either or both could encode cytotoxic gene products. Alternatively, either or both could control the activities of other gene products that are cytotoxic. If ted-3 and ted-.$ encode cytotoxic products, one might expect that they are regulated by other possibly lineage-specific genes. Such lineage-specific genes could control either the expression of ted-3 and ted-4 or the activities of the ted-3 and ted-4 protein products. If ted-3 and ted-4 control the activities of other gene products that are cytotoxic, they might control only one of a number of steps necessary for the activation of a cytotoxic activity. In both cases, the expression of ted-3 and ted-4 could either be specific to dying cells or could be much more general, with specificity conferred by the regulation of other genes in the pathway. Are genes similar to ted-3 and ted-4 likely to be involved in the cell deaths that occur in vertebrates? Some vertebrate cell deaths share certain characteristics with the cell deaths in C. elegans that are controlled by ted-3 and ted-4. For example, up to 14% of the neurons in the chick dorsal root ganglia die immedi-
Y~JAN AND HORVITZ
C. elr~~uns Pro~~rr~m mecl Cdl Deo fh
ately after their births, before any signs of differentiation (Carr and Simpson, 1982). Genes like ted-3 and ted-4 could well function in this class of vertebrate cell death. However, other vertebrate neuronal cell deaths seem quite different in that they appear to be controlled by interactions with target tissues; one hypothesis is that a deprivation of target-derived growth factors is responsible for these cell deaths (Hamburger and Oppenheim, 1982; Thoenen et al., 1987). We believe that even this class of cell death could involve genes like ted-3 and ted-4, as pathways of cell death involving similar genes and mechanisms might be triggered in a variety of ways. Consistent with this idea, the deaths of rat sympathetic neurons in mlfro in response to a deprivation of nerve growth factor (Martin et al., 1988) and of chick motor neurons and neurons in dorsal root ganglia in viva during normal development (Oppenheim and Prevette, 1988) are blocked by inhibitors of mRNA or protein synthesis. Perhaps the genes presumbly induced in these dying vertebrate cells are similar in function and even in structure to the C. elegans genes ted-3 and ted-4. We thank L. DeLong and D. L. Baillie for providing respectively. This work was supported by lJ.S. Public Grants GM24663 and GM24943 and by the Howard Institute. J.Y. was supported by a scholarship from sity and by the Howard Hughes Medical Institute. vestigator of the Howard Hughes Medical Institute.
,yDpl and SD@, Health Research Hughes Medical Harvard UniverH.R.H. is an In-
REFERENCES AVEKY, L., and HORVITZ, H. R. (1987). A cell that dies during wild-type (z fde~J!lnrrs development can function as a neuron in a ted-j! mutant. Cell 51, 1071-1078. BRENNER, S. (1974). The genetics of Ccrerrorhnbditis elqms. Gercrtics 77,71-94. CAKK, V. M., and SIMPSON, S. B., Jr. (1982). Rapid appearance of labeled degenerating cells in the dorsal root ganglia after exposure of chick embryos to tritiated thymidine. Den Bruin Res. 2, 57-162. DELONC;, L., CASSON, L. P., and MF.YF,R, B. J. (1987). Assessment of X chromosome dosage compensation in Cuenorhubdifis elrqcms by phenotgpic analysis of /in-14. Grn&cs 117, 657-670. EI,~,IS, H. M., and HORVITZ, H. R. (1986). Genetic control of programmed cell death in the nematode C. ele.qgn?~s. Cell 44, 817-829. FIXSE:N, W. D. (1985). “The Genetic Control of Hypodermal Cell Lineages during Nematode Development.” Ph.D. thesis, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA.
41
GL~CKSMANN, A. (1951). Cell death in normal vertebrate ontogeny. Bid. Rev. Cambrirlgc~ Ph ilos. Sk 26, 59-86. HAMBIIRGER, V., and OFPENHEIM, R. W. (1982). Naturally occurring neuronal death in vertebrates. NfJurosci Corrc mcnt. 1, 39-55. HEI)C,ECO(:K, E. M., SIILSTON, J. E., and THOMSON, J. N. (1983). Mutations affecting programmed cell death in the nematode Cacnorha~ difis &{JWlS. Sciw~c 220, 1277-1279 HERMAN, R. K. (1984). Analysis of genetic mosaics of the nematode &‘?cc?fics 108, 165-180. (‘cLPl/orhrrMifis &{J!(lnnS. HOIZKIN, J., ED(;LEY, M. L., RII)ULE, D. L., and ALBERTSON, D. G. (1988). Genetics. II/ “The Nemtrfode (:cLP?corh,obditis rlq~ans” (W. B. Wood and the Community of C. rlegans Researchers, Eds.), pp. 491-584. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. HORVITZ, H. R., BK~:NNEK, S., HOI)C;KIN, J., and HE:KMAN, R. K. (1979). A uniform genetic nomenclature for the nematode (:cJr?lorltabdifis C’k,(JU WS. .&fd. (;fW (;W/f,f. 175, 12% 133. KE~YON, C. (1986). A gene involved in the development of the posterior body region of (?. fdqqrrns. Cd 46, 477-487. MARTIN, D. P., SCIIMIII’I’, R. E., DISTEFANO, P. S., LOWRY, 0. H., CAKTEK, J. G., and JOHNSON, E. M., Jr. (1988). Inhibition of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation. J. C& BioL 106, 829-844. OPPF:NHE:IM, R. W. (1981). Neuronal cell death and some related regressive phenomena during neurogenesis: A selective historical review and progress report. 1~ “Studies in Developmental Neurobiology: Essays in Honor of Victor Hamburger” (W. M. Cowan, Ed.), pp. 74-133. Oxford liniv. Press, New York. OPFENHEIM, R. W., and PK~~:v~TT~, D. M. (1988). Reduction of naturally occurring neuronal death ir, ui~o by the inhibition of protein and RNA synthesis. hrc~u,osci. Abstr. 14, 368. ROSENBLIJTH, R., CIIUDEPORD, C., and BAII,I,II+:, D. L. (1985). Mutagenesis in CffPrtorhrthtlifis dfy~cms. II. A spectrum of mutational events induced with 1500 R of y-radiation. Genetics 109,493-511. SA~TNI)EKS,Jr., J. W. (1966). Death in embryonic systems. S’ciexce 154, 604-612. STTLSTON,J. E. (1988). Cell lineage. 1n “The Nematode Cwnwhubditis Researchers, f>/f,qu?!.s”(W. B. Wood and the Community of C. clf:(lrrns Eds.), pp. 123-156. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. SITI,STON, J. E., ALBERTSON, I). G., and THOMSON, J. N. (1980). The (:(/PllOrh(Jbdif is &gu~rs male: Postembryonic development of nongonadal structures. Dw. Bid. 78, 542-576. SITI,STON,J. E., and Ho~v~rz, H. R. (1977). Postembryonic cell lineages of the nematode (‘cJf,?2cnl/trbditis e1qyan.s. DPO. Rid. 56, 110-156. SVLSTON, J. E., SVHIF:RENBER(:, E., WIIITE, J. G., and THOMSON, J. N. (1983). The embryonic cell lineage of the nematode Cawmrhabdifis dfytr t,.s. Del,. Rid. 100, 64-119. TIIOEN~:N, H., BARDE, Y.-A., DAVI~(:S, A. M., and JOI-INSON, J. E. (1987). Nrurotrophic factors and neuronal death. firl “Selective Neuronal Death,” Vol. 126, pp. 82-85. Wiley, New York. TK~IM.&~, J. W. (1984). Cell death in invertebrate nervous systems. Ailrm. &II. ~V~‘/rro.sci. 7, 171-188.
BIOLOGY
138, 33-41
(1990)
The Caenorhabditis elegans Genes ted-3 and ted-4 Act Cell Autonomously to Cause Programmed Cell Death JUNYING YuAN*~~ AND *Program
of Neurosciences, Department
Harvard
of Biology,
H. ROBERT
HORVITZt
Medical School, Boston, Massachusetts 02115; and THoward Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Accepted Nmember
7, 1989
Mutations in the genes ted-3 and ted-4 prevent almost all of the programmed cell deaths that occur during Caenorhabditis eleguns development. To determine the sites of action of these two genes, we performed genetic mosaic analyses. We generated C. elegans animals that carried a free chromosomal duplication bearing either ted-3(+) or ted-4(+) in an otherwise homozygous ted-3 or ted-4 genetic background. We used other genes on the duplication as markers to identify genetic mosaic animals in which the duplication was present in some but not all cells. The patterns of cell death survivors in these mosaic animals indicated that the products of both ted-3 and ted-4 function within dying cells to cause cell death. 0 1990Academic Press, Inc.
mutant. By knowing the genotype of specific cells, it is possible to determine whether the phenotype of a cell depends upon its own genotype or upon that of other cells. Free chromosomal duplications, which are genetically unstable and can be lost at cell division, are used to generate such mosaic animals. If a free duplication carries the wild-type allele of a gene and the chromosomes are homozygous for a recessive mutant allele of that gene, only cells that have lost the duplication will be mutant in genotype. If the gene in question functions cell autonomously, cells that have lost the duplication will be mutant in phenotype, whereas if the gene acts cell nonautonomously, such cells might not exhibit a mutant phenotype. By using appropriate genetic markers, such mosaic animals can be recognized and their phenotypes can be analyzed to determine whether the phenotypes of particular cells depend upon their own genotypes. Here we describe genetic mosaic analyses of the genes cedd and ted-4. Mosaic animals were isolated using appropriate behavioral and morphological markers, and the survival or death of individual cells was scored using Nomarski optics. The rigidly determined and completely known C. elegans cell lineage (Sulston and Horvitz, 1977; Sulston et al., 1983) allowed us to identify at which cell division duplication loss occurred in each mosaic animal. These genetic mosaic analyses demonstrated that ted-3 and ted-4 most likely act cell autonomously within dying cells.
INTRODUCTION
Programmed cell death is common during animal development (Glticksman, 1951; Saunders, 1966; Oppenheim, 1981; Truman, 1984). The phenomenon of programmed cell death raises a number of interesting questions, such as: What are the mechanisms that determine which cells live and which cells die? What are the mechanisms that cause cells to die? The nematode Caenorhabditis elegans is an appropriate organism with which to address these questions. During C. elegans development, the generation of the 959 somatic cells of the hermaphrodite is accompanied by the generation and subsequent deaths of an additional 131 cells (Sulston and Horvitz, 19’77;Sulston et al., 1983). Mutations in two C. elegans genes, ted-3 and ted-4, prevent almost all of these programmed cell deaths (Ellis and Horvitz, 1986). Cells that die in wild-type animals survive, differentiate, and, in at least some cases, function in ted-3 and ted-4 animals (Ellis and Horvitz, 1986; Avery and Horvitz, 1987). The products of ted-3 and ted-.$ presumably are either cytotoxic killer proteins or control the activities of such killer proteins. Elucidating the mechanisms of action of these two genes would be an important step toward understanding how programmed cell death is regulated and executed in C. elegans, One basic question is whether ted-3 and ted-4 act cell autonomously, i.e., whether these genes act within the cells that die or within other cells that control cell death through cell interactions. The method of genetic mosaic analysis, developed for C. elegans by Herman (1984), can be used to answer this question. In mosaic analysis, animals are generated in which some cells are genotypically mutant and some cells are genotypically non-
MATERIALS
AND
General Methods, Nomenclature,
METHODS
and Strains
Methods for the growth, handling, and general genetic studies of C. elegans have been described by 33
0012-1606/90 Copyright All rights
$3.00
0 1990 by Academic Press. Inc. of reproduction in any form reserved.
34
DEVELOPMENTALBIOLOGYVOLUME138,199O
Brenner (1974). The wild-type strain is N2 (Brenner, 1974). All strains were grown at 20°C. The genetic markers used are as follows: LG I-ted-l(el7’35); LG III-ted-4(nll62), dpy-17(el64), uric-36(e251); LG IVbeen described by Brenner (1974), Ellis and Horvitz (1986), and Hodgkin et al. (1988). Genetic nomenclature follows the conventions of Horvitz et al. (1979).
IV, uric-60(+) V, and uric-46(+) V (DeLong et ah, 1987) while nDp3 does not (data not shown). The frequency of nDp3 loss per cell division can be estimated as the frequency of ABp mosaic animals isolated. Since three ABp mosaic animals were isolated from among 1300 animals screened, the frequency of nDp3 loss in a given cell division is about 0.2%. By contrast, based on the data above, the frequency of yDp1 loss per division is 0.006% or less.
Generation
General Procedures fbr Mosaic Analysis
me-3l(e169), uric-30(e191), ced+?(n717), uric-26(e205), dpy-h(e1166); LG V-egl-l(n487). These markers have
of the Free Duplication
nDp3
Usually lo-15 adult animals carrying the appropriate duplication and markers were placed on a loo-mm petri plate seeded with Escherichia coli OP50 (Brenner, 1974). end of chromosome IV and the left end of chromosome Late L3 and L4 progeny from these animals were V (DeLong et aZ., 1987). Because yDp1 carries the wild- screened for apparent mosaic phenotypes using the distype alleles of uric-31, ted-3, and dpy-4, animals of geno- secting microscope (see Results). When such animals type ted-1; uric-31 ted-3 dpy-4; egl-1; yDp1 are phenotyp- were identified, they were examined for cell death surically wild-type in movement and body shape. (egl-1 vivors using Nomarski optics as described by Sulston confers an egg-laying defect that is not relevant to the and Horvitz (1977). Generally, after one side of an aniresults described in this paper). The progeny of such mal was examined, the animal was remounted with the animals are mainly of two types: Dp+ animals that are other side up and that side was then examined. Animals just like their parents, and Dp- animals that are both were carefully removed from the slide and transferred Uric and Dpy. We attempted to screen for mosaic ani- to a 60-mm petri plate seeded with bacteria. We exammals among the progeny of ted-1; uric-31 ted-3 dpy-4; ined the body shapes and behaviors of these animals egl-1; yDp1 animals. Among 18,000 animals screened, 17 with the dissecting microscope the next day when they Uric non-Dpy mosaic candidates were picked. Of these, had grown into adults. Their progeny were checked 3-4 16 segregated Uric Dpy and Uric non-Dpy progeny, but days later to confirm the presence of duplication-carnot non-Uric non-Dpy progeny, suggesting that they rying animals. carried some altered forms of yDp1, e.g., they might have lost the part of yDp1 that carries uric-31(+), or that RESULTS a recombination event had occurred so that the new yDp1 derivative carried the mutant uric-31 gene. One of To identify mosaic animals generated by the somatic the 16, when examined using Nomarski optics, showed loss of free duplications, we have used marker mutamosaic expression of ted-3, i.e., cell death was absent tions that result in uncoordinated (Uric) movement or among cells derived from the ABp lineage. We sus- dumpy (Dpy) body shape. Each free duplication carries pected that only part of yDp1 might have been main- a wild-type allele of an uric, dpy, and ted gene, while tained in this animal and that the smaller derivative of chromosomes are homozygous for the corresponding yDp1 in this animal might be less stable than yDp1 and, recessive mutant alleles. Animals of genotype uric dpy therefore, more suitable for mosaic analysis. The strain ted; Dp are phenotypically wild-type, because of the carrying this putative new duplication was crossed into presence of the wild-type alleles on the duplication. The a strain carrying ted-3 uric-26 dpy-4. The average per- self-progeny of uric dpy ted; Dp animals are of three centage of Dp- self-progeny of Dp+ hermaphrodites was general classes, as scored with a dissecting microscope 43% for this duplication (447 progeny scored) as op- (the Ced phenotype cannot be scored with a dissecting posed to 34% for yDp1 (DeLong et al, 1987), suggesting microscope): phenotypically wild-type animals (which this derivative of yDp1 was lost more frequently in carry the duplication and are of genotype uric dpy ted; germ line cells than was yDp1. Using DAPI (4’-6-diami- Dp), Uric Dpy animals (which do not carry the duplicadino-2-phenylindole dihydrochloride) to stain chromo- tion and are of genotype uric dpy ted), and rare animals somes (Fixsen, 1985), we found this strain to contain an that are neither wild-type nor Uric Dpy in phenotype. extra chromosomal fragment (data not shown). The This third class, which includes mosaic animals that new duplication, named nDp3, was found to carry wild- have lost the duplication in a subset of their somatic type alleles of uric-30 IV, ted-3 IV, uric-26 IV, dpy-4 IV, cells, can be of any of a variety of phenotypes-e.g., Uric and me-34 V. Both the left and right ends of yDp1 seem non-Dpy, Dpy non-Uric, semi-Dpy non-Uric, and semito have been lost in nDp3, since yDp1 carries uric-31(-b) Dpy semi-Uric,-depending upon the sites of action of nDp3 was generated as a spontaneous breakdown product of yDp1, a free duplication carrying the right
YIJAN
AND
HORVITZ
C.
elegans
the uric and dpy genes. For example, if the duplication is lost in all cells in which the uric gene acts, but in none of the cells in which the dpy gene acts, the animal will be Uric non-Dpy in phenotype. If a duplication loss is appropriately rare, most mosaic animals will be generated as a consequence of a loss at a single cell division. Since the C. eleyans cell lineage is known, if this division is identified, the genotype of every cell in the animal can be established. We can then ask whether the survival or death of particular cells that die in wild-type animals corresponds to their ted genotypes. In other words, if such cells survive only when they are ted in genotype, then the action of the ted gene must be cell autonomous. Of the 131 cell death survivors in ted-3 and ted-4 mutants (Ellis and Horvitz, 1986), we have studied a set of 17 that can be readily recognized in living animals using Nomarski optics. These cells and the early embryonic cell lineages (Sulston et ah, 1983) from which they are derived are shown in Fig. 1. In the experiments described below, our general approach was to identify mosaic animals in the dissecting microscope using the uric and dpy markers and then to examine these animals with Nomarski optics to determine which of the 17 cells had lived and which had died. The patterns of surviving cells were striking: in the vast majority of individuals, all surviving cells were derived from a single branch of the cell lineage. This observation indicated that this branch lacked ted gene activity (i.e., had lost the duplication) and that ted activity was cell autonomous. A specific confirmation of this interpretation was provided by the analysis of a pair of cells (CEMDR and CEMDL, see Fig. 1) that are physically adjacent but derived from quite distinct branches of the cell lineage. That one of these cells often survived while the other died argues against the hypothesis that it is the genotype of some other cell(s) acting at a distance that specifies the cell death phenotypes of CEMDR and CEMDL. Taken together, these studies provide strong evidence for the cell autonomous actions of ted-3 and ted-4, ted-3 Acts Cell Autonomcrusly
We constructed hermaphrodites of genotype uric-30 ted-3 dpy-4; nDpS(IV, V;f), where nDp3 is a free duplication that carries uric-30(+) ted-3(+) dpy-h(f) (see Materials and Methods). nDp3 is fairly stable but can be lost during both somatic and germ line cell divisions. The chance of nDp.3 loss in a given cell division is about 0.2% (see Materials and Methods). uric-30 ted-3 dpy-4; nDp3 animals are wild-type in movement and slightly short in body length as dpy-4 is weakly semidominant. Their progeny are predominantly of two classes: animals like their parents, which carry the duplication, and Dpy Uric
Programmed Cell Death
35
animals, which do not. None of these genes exhibits any detectable maternal effect: the Uric Dpy animals from duplication-bearing parents are indistinguishable from their own progeny, and ted-3 progeny of a cross between ted-3 males and ted-3/+ hermaphrodites exhibit the same Ced-3 phenotype as their own progeny (data not shown). Rare animals with mosaic phenotypes, e.g., Uric non-Dpy, Dpy non-Uric, semi-Dpy semi-Uric, and so on, were sought and then examined for their patterns of cell deaths using Nomarski optics. A total of five animals with obvious mosaic phenotypes were found using the dissecting microscope. These animals are described in categories 1 and 2 of Table 1. The mosaic animals in the first category were completely Uric but only semi-Dpy. They were found to have cell death survivors present only among cells derived from the ABp lineage (cf. Table 1 and Fig. 1). This observation suggests that nDp3 was lost in ABp and that ted-3 acts cell autonomously. That the cells CEMDL and CEMDR, which die in wild-type hermaphrodites, are of opposite phenotypes in category 1 mosaic animals is of particular interest: as noted above, these cells are derived from quite different branches of the cell lineage (ABp and ABa, respectively), but are adjacent physically when they survive in wild-type males and in ted-3 mutant hermaphrodites and before they die in wildtype hermaphrodites (Sulston et al., 1983; J. Sulston, personal communication; H. Ellis and J. White, personal communication). Since CEMDL and CEMDR are in essentially the same local environment, if ted-3 acted nonautonomously, one would expect that these cells would display the same fate. That in category 1 mosaic animals only CEMDL is present strongly supports the conclusion that ted-3 acts cell autonomously. The Uric phenotype of these category 1 mosaic animals indicates that we-30 acts within the ABp lineage. This conclusion is consistent with the observation that the locomotory defect of uric-30 animals is associated with a lack of GABA in the ABp-derived class D motor neurons of the ventral nervous system (S. McIntire and J. White, personal communication). The semi-Dpy phenotype of these animals indicates that dpy-4 acts both within the ABp lineage and elsewhere. The category 2 mosaics were semi-Dpy and non-Uric (although they moved more slowly than did wild-type animals, possibly because uric-30 also acts in a few cells other than those derived from the ABp lineage). These animals were found to have cell death survivors only among cells generated by the ABa lineage. Again, the cells CEMDL and CEMDR are of opposite phenotypes. These results further support the hypothesis that cedd acts cell autonomously. To provide additional evidence that ted-3 acts cell autonomously, we used another behavioral marker,
36
DEVELOPMENTAL BIOLOGY
VOLUME 138,199O
CEMVR CEMDR
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V5Rpaap
ganglia
FIG. 1. Seventeen easily identifiable cell death survivors in ted-3 and ted-4 mutants. (a) The positions of the 17 cell death survivors in a ted-3 or ted-4 mutant are shown in a schematic dorsal view. Cells located within the ventral half of the animal are indicated with dotted lines. The sisters of the cells IZR, 12L, NSMR, NSML, MCL, and mlvR, which die in wild-type animals, are collectively referred to as “pharynx,” because they are located within the ventral part of the anterior bulb of the pharynx. The cells derived from QL and the V5 blast cells that die in the wild-type are collectively referred to as “posterior lateral ganglia,” because when they survive in a ted-3 or ted-4 mutant, they become part of the posterior lateral ganglia. M4sis. g2Rsis, and g2Lsis refer to the sisters of M4, g2R, and g2L. We identified Mdsis, g2Rsis, and g2Lsis on the basis of their positions and their morphological similarities to their sisters. CEMDR, CEMDL, CEMVR, and CEMVL are 4 cells that undergo programmed cell death in the wild-type hermaphrodites but survive in wild-type males or in ted-3 and ted-4 animals. (b) The lineage of the 17 cell death survivors shown in (a) (adapted from Sulston et al,, 1983). The fertilized egg, PO, divides asymmetrically to generate a larger somatic precursor cell AB and a smaller cell Pl. ABa and ABp are the anterior and posterior daughters of AB, respectively. ABpl and ABpr are the left and right daughters of ABp, respectively. P4 is the precursor of the germ line. X, cell that dies in the wild-type but survives in ted-3 and ced.4 animals. The dotted lines indicate that cell divisions are omitted in the figure. Lineages leading to cells that are not scored in our mosaic analyses are not shown in this figure. See Sulston et al. (1983) for the complete embryonic cell lineage and Sulston and Horvitz (1977) for the postembryonic lineages of the QL and V5 cells. For other conventions, see (a).
YIIAN AND H~RVITZ
MOSAIC ANALYSIS OF cdl
C. nlegnns Programmed
TABLE 1 USING ZYGOTES OF GENOTYPE uric-30 ted-3 dpy-4; nDp3 Cell death survivors
Mosaic phenotype Body shape 1 2 3 4 5 6 7
Semi-Dpy Semi-Dpy Slight-Dpy Slight-Dpy Non-Dpy Non-Dpy Non-Dpy
Behavior Uric Non-Uric* Semi-Uric Slight Uric Slight-Uric Non-IJnc* Slight-IJnc
No. animals scored 3 2 2 2 2 1 1
Posterior lateral ganglia
Pharynx R
+
37
Cull Deuth
observed
CEMV
G&is
CEMD
L
R
L
R
L
R
L
R
L
-
+
t
t
t
-
t -
-.
.-
+ ~ + +
f -
~ ~ ~
~ ~ ~
+ -
+ +
-. +
t
-
t
-
t ~
M4sis
-
Dp loss ABP ABa ABpl ABpr ABplap ABarp
ABplap, ABarpa
Note Dpy-dumpy. IJnc-uncoordinated. Pharynx-in N2, there are five cells in the ventral half of the pharynx on each side; in ted-3 animals, there are seven to eight cells due to the absence of programmed cell death. Posterior lateral ganglia, a ganglion located about three-fourths of the way from the nose to the tail. In N2, there are four cells in the right posterior lateral ganglion and six cells in the left. In ccd-d animals, because cell deaths fail to occur in the QL and V5 lineages, there are five cells in the right posterior lateral ganglion and at least seven cells in the left. CEMV and CEMD-ventral and dorsal cephalic companions, respectively. The CEMs die in N2 hermaphrodites and survive in red-:1 animals. g2Lsis, g2Rsis, and M4sis-the sisters of the gland cells g2L and g2R and of the pharyngeal neuron M4 die in N2 but survive and differentiate into cells that look like g2 and M4, respectively, in ted-3 animals. (-) Cell death survivor absent (Ced-3(+) phenotype). (+) Cell death survivor present (Ced-3(p) phenotype). Non-LJnc*-These animals were less active than the wild-type. Wild-type animals move almost constantly, except when molting. These animals move less; but when they do move, they are not uncoordinated. Dp loss-This column indicates our interpretation of the cell in which the duplication was lost in each mosaic animal based upon the pattern of cell death survivors observed. When the duplication may have been lost in either a cell or one of its progeny, the earliest possibility is listed. For example, the category 5 mosaic animals could he caused by loss of the duplication in ABplap or in certain of the progeny of ABplap, e.g., ABplapa or ABplapap, but only ABplap is listed. A total of about 1300 UVLC-30c&-3 dl)y,ll-4;nD@ were screened to obtain the mosaic animals described in this table. The expression of the Ced-3 phenotype as a result of the wd-3(n717) mutation is almost complete. We estimated the expression of crtl-.Kt/717) based upon the presence of the surviving sister of the g2R or g2L cell in cd-X(71717) rhnc-26 dpy-4 animals. Among 100 crd-3 um-26 dj)!/-h animals scored, only in one animal did we fail to see the sister of a g2 cell. Thus, we estimated that there is about a 1% chance that a cell that normally undergoes programmed cell death might still die in a crd-Y(n717) animal. Of the 32 ted-3 mosaic animals described in Tables 1 and 2, only one such cell death survivor seemed to he missing (in an ABp-loss mosaic animal).
uric-26, to do similar analyses. Five obviously mosaic animals are described in categories 1 and 2 of Table 2. The category 1 mosaics are semi-Dpy and Uric. These animals were found to have cell death survivors only among cells generated from the ABp lineage. The category 2 mosaic, with an apparent ABa loss, was semiDpy and non-Uric (although this animal moved more slowly than does a wild-type animal). These results again indicate that ted-3 is cell autonomous and reveal that uric-26, like uric-30, acts in the ABp lineage. One possibility is that uric-26 acts in the ventral nerve cord, because all but one of the ventral cord neurons are generated from the ABp lineage. In these experiments, we have found Uric semi-Dpy and non-Uric semi-Dpy animals but no Uric non-Dpy and Dpy non-Uric animals. In addition, the semi-Dpy phenotype can be caused by duplication loss in either the ABa or ABp lineages. Therefore, we predicted that loss of the duplication from the AB lineage would generate an animal that is both Dpy and Uric. Such animals
would appear the same as their Dp- siblings when viewed with the dissecting microscope, but unlike those Dp- siblings, these mosaics would have cell death survivors (the Ced( -) phenotype) in the AB lineage but not in the Pl lineage. Some of their progeny would carry the duplication and be phenotypically wild-type, since the germ line cells are derived from the Pl lineage. To screen for such animals, Uric Dpy progeny of nDp3-carrying animals were examined using Nomarski optics to identify animals with cell death survivors among cells generated by the AB lineage but not among cells generated by the Pl lineage. Two animals of this phenotype were isolated from among about 200 Dpy Uric animals screened (Table 2, category 3). The presence of the duplication in these animals was confirmed by showing that they generated nDp3-carrying nonDpy non-Uric progeny. This class of mosaics indicates that even when cell death is absent in the AB lineage (which generates 116 of the 131 total cell deaths; Sulston et al, 1983), cells in the Pl lineage still die. This
38
DEVELOPMENTAL BIOLOGY
VOLUME 138,199O
TABLE 2 MOSAIC ANALYSIS OF ted-3 USING ZYGOTES OF GENOTYPE ted-3 uric-26 dpy-4; nDp3 Cell death survivors
Mosaic phenotype Body shape
Behavior
No. animals scored
Posterior lateral ganglia ___
Pharynx ____ R
L -
CEMV ___ L
R
CEMD ~ L
R
G2sis L
R
L
M4sis
+ -
+
+ -
+ -
+
+ -
~ -
-
-
-
+ -
+ + +
+ +
+ + +
+ -
t + +
~
~ -
-
-
+ +
+
+ +
~
-
~ -
-
~
-
-
-
-
+
-
~
-
-
-
-
-
+
-
~
t
t
t
1 2 3 4 5
Semi-Dpy Semi-Dpy DPY* Slight-Dpy Slight-Dpy
Uric Non-Uric* Uric Semi-Uric Semi-Uric
4 1 2 3 1
+ +
+ +
-
6 7
Slight-Dpy Slight-Dpy
Semi-Uric Semi-Uric
1 1
Non-Dpy Non-Dpy Non-Dpy
Slight-Uric Slight-Uric Slight-Uric
2 3 1
8 9 10
R
observed
t
Dp loss ABP ABa AB ABpl ABpl, ABprp ABpr A&r, ABplap ABpra ABplap ABP~P, EMS
Note. Abbreviations and symbols are generally as in Table 1. Non-Uric*-this animal was less active than is the wild-type. Dpy*-this animal was a little longer than a dpy-4 animal. About 1900 ted-3 uric-26 clpy-4; nDp3 animals were examined to obtain the mosaic animals described in this table.
observation further supports the conclusion that ted-3 acts cell autonomously. This result also indicates the site of action of dpy-4: that the loss of nDp3 from the AB lineage is sufficient for these mosaic animals to be Dpy suggests that dpy-4 primarily acts within cells generated by the AB lineage. The experiments described above are concerned only with mosaic animals generated as a result of duplication loss in the first and second divisions of embryonic development. To examine additional classes of mosaic animals, we attempted to identify mosaic animals with more subtle phenotypes. Such mosaic animals are described in categories 3 to 7 in Table 1 and categories 4 to 10 in Table 2. In 16 of the 20 mosaic animals identified in this way, the cell death survivors were derived from a single branch of the cell lineage. These animals further support the conclusion that ted-3 is cell autonomous in its action. In the other four animals, the cell death survivors were derived from two branches of the lineage. Since nDp3 appears to be lost in a given cell division at a frequency of about 0.2%, it is not unreasonable that, given the relatively large number of possible losses that apparently can lead to a weakly mosaic phenotype, double duplication losses would occur in 20% of such weakly mosaic animals. That all of the mosaic phenotypes observed in these experiments can be explained by either single losses (26 eases) or double losses (4 cases) strongly indicates that ted-3 acts cell autonomously.
ted-4 Also Acts Cell Autonomously
We used the same method but a different duplication and different markers to perform mosaic analysis of ted-4. The duplication sDp3(III;f) (Rosenbluth et al., 1985) was known to be mitotically unstable (the chance of sDp3 loss at a given cell division is about 0.15%; see Table 3) and has been used to study the site of action of the gene mab-5 (Kenyon, 1986). The markers dpy-17 and uric-36 also have been used to study the site of mab5 action (Kenyon, 1986). We constructed hermaphrodites of genotype ted-4 dpy-17 uric-36; sDp3. These animals are wild-type in phenotype because sDp3 carries wildtype alleles of all three genes. Most progeny of these animals either carry sDp3 and are phenotypicaly wildtype or have lost sDp3 and are Uric and Dpy. Animals with mosaic phenotypes were identified using the dissecting microscope and examined using Nomarski optics for the presence or absence of particular cell death survivors. Our results are shown in Table 3. The mosaic animals listed in category 1 were Uric and nonDpy. In these animals, cell death survivors were found only among cells generated by the ABp lineage; ABp-derived CEMDL is present, while ABa-derived CEMDR is absent. This result suggests that ted-4, like ted-3, acts within dying cells. This result also indicates that uric-36 acts in cells derived from the ABp lineage, because the loss of sDp3 from the ABp cell is sufficient to make an animal Uric. Kenyon (1986) has previously reported that uric-36 acts within the ABp lineage.
YITAN AND HCJRVITZ
C elegnxs
Progrum
rrwd
Cell
39
llrdl
TABLE 3 MOSAIC ANALYSIS OF ted-4 USING ZKCOTES OF GENOTYPE ted-4 dpy-17 um-36; sDp.9 Cell death survivors Posterior lateral ganglia ___
Mosaic phenotype
Body shape
Behavior
No. animals scored
1 2 3 4 5 6 7
Non-Dpy DPY* Slight-Dpy Non-Dpy Non-Dpy Non-Dpy Non-Dpy
Uric Non-Uric Non-Uric Semi-Uric Semi-Uric Semi-TJnc Semi-Uric
11 6 1 1 1 2 1
8
Non-Dpy
Semi-Uric
1
9
Non-Dpy
Slight-Uric
1
Pharynx ____ R
+
-
-
L
R
L
-
t -.
+++
CEMV
R
observed
CEMD ___ L
R
L
+ .-
+
+ -+-
+
t
+ ~~ -+-
-
+
++-
~
+ -
-
Mlsis
~
+ ~
.~
~ -
+ t
L
-
t +
R -
+ -
~
G2sis
--
-
Dp loss
ABP CC?) ABa ABpl ABpr ABpla ABpr, ABal ABpla, ABpr ABprp
Note. Abbreviations and symbols are generally as in Table 1. The Ced-4 phenotype is the same as the Ced-3 phenotype. Dpy*-These animals are a little longer than dry-17 animals (see text). About 7200 animals were screened to obtain the mosaic animals described in this table. The chance of sDp3 loss at a given cell division is about 0.15%, as estimated from the observation that 11 ABp-loss mosaic animals were isolated from the 7200 animals screened. The expression of the Ced-4 phenotype as a result of the ted-b(nll62) mutation is incomplete. We estimated the in the legend of Table 1. Of 40 ted-4fn1162) dpy-17 uric-36 animals scored, in three we expression of ted-h(nll62) as described for ce&3(n717) failed to see the surviving sister of g2R or g2L. Thus, we estimated that there is about a 7.5% chance for a cell that normally undergoes programmed cell death to die in a ted-4(n1162) animal. Some of our mosaic animals also exhibited incomplete expression of the Ced-4 phenotype. Of the 11 AB-loss mosaic animals described in this table, six seemed to be missing one cell death survivor.
The category 2 mosaic animals were Dpy and nonUric, although these animals were slightly longer than dpy-17 animals. No cell death survivor was found among any of the cells examined; however, the germ line of these animals retained the duplication, since some of their progeny carry the duplication. Thus, these animals must have carried the duplication in the AB, MS, and P4 lineages, but not in the lineage(s) responsible for the Dpy phenotype. In principle, duplication loss could have been within the C, D, or E lineages (Fig. 1). The E lineage, which generates only the intestinal cells, seems highly unlikely. We consider the D lineage also to be unlikely. The D lineage generates 16 cells, all of which are body muscle cells; most body muscle cells are derived from MS. Since no Dpy non-Uric animal was found to have lost the duplication in the MS lineage, body muscle cells seem unlikely to be the site of dpy-17 action. We believe the simplest interpretation of the data is that the duplication was lost within the C lineage. The C lineage generates 12 of the 23 embryonic hyp7 hypodermal nuclei; of the others, five are generated by the ABa lineage and six by the ABp lineage. If the site of action of dpy-I?’ were within hyp7, loss of the duplication in the ABa lineage might cause an animal to be slightly Dpy and non-Uric. By screening for slightly Dpy and non-Uric animals, we identified one such animal
(Table 3, category 3). In this animal, ABa-derived CEMDR is present and ABp-derived CEMDL is absent, as one would expect if ted-4 were cell autonomous in its action. These considerations would also predict that the loss of the duplication in the ABp lineage would result in a slightly Dpy animal. However, as described above, we scored such animals as non-Dpy. We suspect that this apparent inconsistency is caused by effects of the uric-36 mutation, which makes me-36 animals somewhat longer than wild-type animals, and hence could suppress a weak Dpy phenotype. The interpretation that dpy-17 acts within both the C lineage and the AB lineage is consistent with the data of Kenyon (1986). We also identified and characterized additional classes of mosaic animals (Table 3, categories 4-9). Five of the seven animals examined could be explained by cell autonomous ted-4 action and the loss of the duplication at a single cell division; the remaining two appear to have lost the duplication at two cell divisions. In short, all of these data indicate that ted-4 is cell autonomous in its action. ted-3 and ted-4 Are Useful Markers for Future Mosaic Analysis We have used uric and dpy markers, the sites of action of some of which were previously unknown, to perform
40
DEVELOPMENTAL BIOLOGY
mosaic analyses of ted-3 and ted-4. Our results have revealed not only the sites of action of ted-3 and ted-4, but also those of the uric and dpy genes we have used as markers. This identification was possible because cell deaths occur among cells generated from most branches of the cell lineage. The presence or absence of particular cell death survivors in a ted-3 or ted-4 mosaic animal can identify the cell division at which a duplication has been lost. Since the C. elegans cell lineage is invariant and known (Sulston and Horvitz, 1977; Sulston et al., 1983), this information reveals the genotype of every cell in the organism. For this reason, knowing that ted-3 and ted-4 are cell autonomous in their actions allows them to be used for high-resolution mosaic analyses. DISCUSSION
Our genetic mosaic analyses demonstrate that the fate of a cell that normally dies in wild-type C. elegans is correlated with its own genotype in ted-3 or ted-4 mosaic animals rather than with the genotype of cells derived from other branches of the cell lineage. Since we have studied deaths in a number of branches of the lineage, our data establish that neither ted-3 nor ted-4 is acting in a cell (or set of cells) that humorally controls all cell deaths in C. elegans. The simplest interpretation of our data is that both ted-3 and ted-4 act within dying cells to bring about their deaths. However, it remains formally possible that either or both of these genes act not within dying cells but rather within other cells very closely related by lineage. In many mosaic animals (17 ted-3 mosaic animals and 15 ted-.4 mosaic animals) the CEMD cells exhibited opposite Ced phenotypes, i.e., only one of the CEMD cells survived. This result also is most simply interpreted as indicating the cell autonomy of ted-3 and ted-4 action. However, this result is consistent with the hypothesis that a cell or cells physically very close to one of the CEMD cells can induce it to die. Considering these ideas together, it is conceivable that ted-3 and ted-4 are expressed within cells closely related to and adjacent to dying cells (e.g., within their sister cells), and these cells then interact with the dying cells to bring about cell deaths. However, this possibility seems unlikely to be generally true, since J. Sulston (personal communication) has shown that the deaths of cells in the postdeirid and ray lineages occur even if their sisters or other nearby close relatives have been killed with a laser microbeam. Alternatively, perhaps ted-3 and ted-4 act within the ancestors of dying cells (e.g., within their mothers); since dying cells are often smaller than their sisters (Sulston, 1988), certain genes involved in cell death may well act at or before the time of cell division. The apparent cell autonomy of ted-3 and ted-4 action does not indicate that cell death per se is cell autono-
VOLUME 138.1990
mous. For example, these genes might encode receptors or components of a signal transduction system that act within dying cells in response to a signal from other cells. Nonetheless, a number of observations suggest that most of the programmed cell deaths that occur during C. elegans development are cell autonomously determined. First, as noted above, many dying cells are smaller than their sisters at the time of their births, suggesting that their fates have already been specified. Second, most dying cells die within an hour of their births, before any signs of differentiation (Sulston et aZ., 1983), which indicates that these cells are unlikely to be dying as a consequence of a failure to compete for targets (such competition is believed to be the cause of the deaths of certain vertebrate neurons; e.g., Oppenheim, 1981). Third, even though dying cells are engulfed by certain of their neighbors, these engulfing cells are not bringing about the cell deaths, as engulfment can be blocked either by mutation (Hedgecock et a,l., 1983) or by laser-killing of the engulfing cells (J. Sulston, personal communication), and the deaths still occur. Fourth, the killing of neighboring cells in general with a laser microbeam has also failed to rescue dying cells (J. Sulston, personal communication) (except in a few distinctive cases of cell death in the tail of the C. elegans male; Sulston et al., 1980), showing that, at least in those cases tested, cells are not killed by their neighbors. Fifth, as we have shown here, the only genes known to be needed for programmed cell death, ted-3 and ted-4, act within dying cells. Given that ted-3 and ted-4 act within dying cells (or their mothers) to cause cell death, how might the products of these genes function? Either or both could encode cytotoxic gene products. Alternatively, either or both could control the activities of other gene products that are cytotoxic. If ted-3 and ted-.$ encode cytotoxic products, one might expect that they are regulated by other possibly lineage-specific genes. Such lineage-specific genes could control either the expression of ted-3 and ted-4 or the activities of the ted-3 and ted-4 protein products. If ted-3 and ted-4 control the activities of other gene products that are cytotoxic, they might control only one of a number of steps necessary for the activation of a cytotoxic activity. In both cases, the expression of ted-3 and ted-4 could either be specific to dying cells or could be much more general, with specificity conferred by the regulation of other genes in the pathway. Are genes similar to ted-3 and ted-4 likely to be involved in the cell deaths that occur in vertebrates? Some vertebrate cell deaths share certain characteristics with the cell deaths in C. elegans that are controlled by ted-3 and ted-4. For example, up to 14% of the neurons in the chick dorsal root ganglia die immedi-
Y~JAN AND HORVITZ
C. elr~~uns Pro~~rr~m mecl Cdl Deo fh
ately after their births, before any signs of differentiation (Carr and Simpson, 1982). Genes like ted-3 and ted-4 could well function in this class of vertebrate cell death. However, other vertebrate neuronal cell deaths seem quite different in that they appear to be controlled by interactions with target tissues; one hypothesis is that a deprivation of target-derived growth factors is responsible for these cell deaths (Hamburger and Oppenheim, 1982; Thoenen et al., 1987). We believe that even this class of cell death could involve genes like ted-3 and ted-4, as pathways of cell death involving similar genes and mechanisms might be triggered in a variety of ways. Consistent with this idea, the deaths of rat sympathetic neurons in mlfro in response to a deprivation of nerve growth factor (Martin et al., 1988) and of chick motor neurons and neurons in dorsal root ganglia in viva during normal development (Oppenheim and Prevette, 1988) are blocked by inhibitors of mRNA or protein synthesis. Perhaps the genes presumbly induced in these dying vertebrate cells are similar in function and even in structure to the C. elegans genes ted-3 and ted-4. We thank L. DeLong and D. L. Baillie for providing respectively. This work was supported by lJ.S. Public Grants GM24663 and GM24943 and by the Howard Institute. J.Y. was supported by a scholarship from sity and by the Howard Hughes Medical Institute. vestigator of the Howard Hughes Medical Institute.
,yDpl and SD@, Health Research Hughes Medical Harvard UniverH.R.H. is an In-
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