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First published online 28 February 2007
doi: 10.1242/dev.02818
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Laboratory of Developmental Genetics, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
* Author for correspondence (e-mail: shaham{at}rockefeller.edu)
Accepted 23 January 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cell death onset, Caspase, ced-3, C. elegans, Tail-spike cell
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Defects in programmed cell death underlie a variety of
pathological processes, including autoimmune and neurodegenerative disorders
and cancer (reviewed in Hanahan and
Weinberg, 2000; Yuan and
Yankner, 2000). The nematode C. elegans has proven to be
instrumental in deciphering the molecular basis of PCD. A genetic pathway
governing the execution of cell death was first identified in C.
elegans (Metzstein et al.,
1998), and the machinery responsible for executing cell death in
this animal is conserved in many metazoans. In most C. elegans cells,
mitochondrially-associated CED-9 protein (similar to vertebrate Bcl-2) binds
to and sequesters CED-4 (similar to vertebrate Apaf-1), thus inhibiting cell
death by blocking CED-4-mediated activation of the CED-3 caspase
(Chen et al., 2000). Release of
CED-4 from the mitochondria allows this protein to promote proteolytic
activation of CED-3, which leads to cellular demise
(Shaham and Horvitz, 1996a;
Chen et al., 2000;
Yan et al., 2004).
Although a conserved cell death execution machinery has been well
characterized, the molecular events that determine the timing of cell death
onset remain poorly understood. In a subset of somatic cells in C.
elegans, egl-1 is believed to be a crucial mediator of the life versus
death decision. egl-1 acts upstream of ced-9 to promote cell
death (Conradt and Horvitz,
1998) by antagonizing the interaction between CED-9 and CED-4,
allowing the release of CED-4 from mitochondria
(Chen et al., 2000). The
transcription of egl-1 is induced in some cells destined to die,
leading to the hypothesis that such transcription may determine the timing of
cell death onset. Several regulators of egl-1 transcription have been
identified, and mutation of these regulators or alteration of their binding
sites in the egl-1 promoter can inhibit or promote cell-specific
death (Conradt and Horvitz,
1999; Ellis and Horvitz,
1991; Metzstein et al.,
1996; Metzstein and Horvitz,
1999; Thellmann et al.,
2003; Hoeppner et al.,
2004; Liu et al.,
2006).
Although control of the timing of egl-1 expression is thought to
be the main avenue regulating the onset of cell death in C. elegans,
transcription of the gene has not been examined at high temporal resolution,
leaving open the possibility that, although egl-1 expression is
necessary for cell death to occur, it may not be the trigger for the process.
Two observations are consistent with the notion that induction of
egl-1 transcription may not be the only temporal cue regulating the
onset of cell death. First, physiological cell death in the C.
elegans germ line occurs independently of egl-1
(Gumienny et al., 1999) or of
the related BH3-only protein CED-13
(Schumacher et al., 2005),
suggesting that the onset of cell death must be regulated by other factors in
this tissue. Second, in double-mutant animals containing a strong
loss-of-function mutation in ced-9 and a very weak loss-of-function
mutation in ced-3, cell death still occurs appropriately in some
cells destined to die, but not in cells destined to live
(Hengartner and Horvitz,
1994). Because egl-1 requires ced-9 to promote
cell death, egl-1 cannot be the determinant of cell death onset in
these cells, which lack ced-9 activity. Thus, other mechanisms
controlling the timing of cell death onset must exist; their nature, however,
is unknown.
Caspases play important roles in programmed cell death as key mediators of
cellular destruction. These proteases are synthesized as pro-proteins with
weak proteolytic activity, and intricate posttranslational regulation ensures
the tight control of their activities (reviewed by
Abraham and Shaham, 2004;
Shi, 2004). Caspases are
expressed in both living and dying cells in C. elegans
(Shaham and Horvitz, 1996a;
Shaham et al., 1999), and this
expression is required for the execution of PCD. Cell-specific regulation of
caspase transcription has been previously described in both vertebrate and
invertebrate systems. For example, during Drosophila larval
metamorphosis, expression of the apical caspase Dronc (also known as Nc -
FlyBase) and of the effector caspase Drice (also known as Ice - FlyBase) is
upregulated in response to the steroid hormone ecdysone
(Cakouros et al., 2002;
Daish et al., 2003;
Cakouros et al., 2004;
Kilpatrick et al., 2005), a
well-known trigger of cell death. Caspase-3 is also transcriptionally
upregulated in peripheral T lymphocytes and in T cell hybridomas following T
cell-receptor signaling (Sabbagh et al.,
2004; Sabbagh et al.,
2005), sensitizing the cells to respond to future death-inducing
insults. In these systems, caspase expression, although crucial for the
execution of cell death, occurs hours to days before cells begin to die, and
cannot, therefore, be the temporal trigger for the onset of cell death.
Here, we report that the death of the C. elegans tail-spike cell
depends only partially on the egl-1 and ced-9 genes,
suggesting that other means of exerting temporal control on the death of this
cell must exist. We demonstrate that the induction of ced-3 caspase
transcription, shortly before morphological features of death are evident,
plays a crucial role in controlling the onset of PCD in the tail-spike cell,
suggesting that ced-3 transcription may provide the temporal cue for
PCD initiation. In a genetic screen for regulators of ced-3
expression, we identified the homeodomain-containing transcription factor
pal-1, which is required for both the expression of ced-3 in
the tail-spike cell and for tail-spike cell death, and in vitro studies
demonstrate that PAL-1 is able to bind the ced-3 promoter at sites
that are crucial for both processes, suggesting that pal-1 directly
promotes ced-3 expression. The PAL-1 protein is similar to the
mammalian protein Cdx2, which promotes intestinal development and, when
mutated, can result in intestinal tumors
(Chawengsaksophak et al., 1997;
Aoki et al., 2003;
Bonhomme et al., 2003). Our
results unveil a novel mechanism for controlling the timing of programmed cell
death by the transcriptional regulation of caspases.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The wild-type strain was C. elegans variety
Bristol strain N2. The following alleles were used: LGI: smg-1(r861);
LGIII: ced-4(n1162), ced-9(n1950, n2812), pal-1(ns114, ns115, ok690), bcIs1;
LGIV: ced-3(n717, n2427, n2436), ced-5(n1812), unc-30(e191);
LGV: egl-1(n1084n3082), bcIs37;
LGX: ced-13(tm536, sv32), nsIs25.
Plasmid constructions
Green fluorescent protein (GFP) reporters were constructed by amplifying
indicated regions of the C. elegans or C. briggsae ced-3
promoters using the polymerase chain reaction (PCR), and cloning them into the
GFP expression vector pPD95.69 (Fire et
al., 1990). For construction of the C. briggsae ced-3
promoter::GFP, we amplified a 0.7 kb DNA fragment from cosmid G45E19 and
ligated the resulting amplicon to pPD95.69 digested with XbaI and
XmaI. To generate the C. elegans ced-3 promoter
(0.35kb)::GFP fusion, we amplified DNA from the C. elegans ced-3
genomic DNA construct pJ40 (Yuan et al.,
1993) and ligated the resulting amplicon to pPD95.69 digested with
HindIII and BamHI. For creation of C. elegans ced-3
promoter (1.5 kb)::GFP, we amplified DNA from pJ40 and ligated the resulting
amplicon to pPD95.69 digested with HindIII and BamHI. The
ced-3 expression reporter used in
Fig. 2 was made as follows: GFP
was amplified by PCR from plasmid pPD95.69 using primers containing
MluI sites followed by the GFP start or stop codons, and was inserted
into the MluI site of plasmid pJ40. This construct encodes a fusion
protein containing 69 amino acids of CED-3 protein fused to GFP. For
construction of the pal-1 genomic-DNA rescue construct, we amplified
a 6 kb DNA fragment from cosmid W05E6 and ligated the resulting amplicon to
the pCR 2.1-TOPO vector (Invitrogen). For construction of ced-3
promoter::ced-3 cDNA, we amplified a 1.5 kb DNA fragment containing
sequences immediately upstream of the ced-3 ATG from pJ40, and
ligated the resulting amplicon to pPD95.69 digested with HindIII and
XbaI. We then amplified a 1.5 kb DNA fragment from the C. elegans
ced-3 cDNA plasmid pS126 (Shaham and
Horvitz, 1996a) and ligated the resulting amplicon to this
ced-3 promoter construct digested with XbaI and
NheI. The deletion constructs described in
Fig. 3 and in Fig. S1B in the
supplementary material, and the mutant B and C constructs described in the
text, were generated using QuikChange XL Site-Directed Mutagenesis kit
(Stratagene).
Transgenic strain constructions
Transformations were performed as previously described
(Mello and Fire, 1995). GFP
reporter constructs were injected into ced-3(n717) animals
at concentrations of 30-40 ng/µl; the plasmid pRF4 (containing the dominant
marker rol-6(su1006)) was used as the transformation marker
(40 ng/µl). The 0.7 kb C. briggsae ced-3 promoter::GFP
extra-chromosomal transgene was stably integrated by treatment with
4,5',8-trimethylpsoralen (TMP). Integrant lines stably transmitting the
transgene to all progeny were isolated and characterized further. The
nsIs25 integrant, mapping to LGX, and the nsIs23 integrant,
not on LGX, were used for the experiments described here. Rescue constructs
containing ced-3 genomic DNA (pJ40) were injected into
ced-3(n717); nsIs25 animals at a concentration of
50 ng/µL, using daf-6::RFP as the co-injection marker.
pal-1 rescue constructs were injected at 25 ng/µL using either
daf-6::RFP or daf-19::NLS-RFP as co-injection markers.
Establishing timing of reporter expression in the tail-spike cell
Individual embryos were collected at the comma stage of embryogenesis and
were allowed to develop for 3 hours at 20°C. Embryos were subsequently
mounted in M9 buffer onto microscope slides containing 0.4 mm agar pads.
Tail-spike cell ced-3::GFP expression and cell morphology were
assessed at 2-4 minute intervals using a fluorescence-equipped compound
microscope. Embryos were followed until visualization of the tail-spike cell
corpse.
Assay for ced-3 reporter expression
Reporter expression in the tail-spike cell was assessed in L2 animals of
lines carrying deletion constructs established as described above. At least 15
transgenic animals were examined per line.
Assay for cell death rescue in ced-3(n717) mutants
To assess rescue of the ced-3 cell death defect, at least two
transgenic lines per rescue construct were examined; 30 transgenic and five
non-transgenic animals were scored per line. L3 animals were assessed for
rescue of both tail-spike and pharyngeal cell death. Cell death in the pharynx
was assessed as previously described
(Hengartner et al., 1992).
Isolation of ns114 and ns115
ced-3(n717); nsIs25 animals were mutagenized
with 30 mM ethyl methanesulfonate (EMS) as described previously
(Sulston and Hodgkin, 1988).
Animals were propagated in 500 ml liquid culture for 5 days. Gravid F1 adults
were harvested, bleached and their progeny incubated overnight in M9 buffer.
F2s were plated onto 9 cm plates and were screened as early larvae for the
absence of tail-spike cell GFP expression. In total, 32,000 F2s were screened
and 621 candidate mutants were isolated. Mutant alleles ns90, ns114,
and ns115 bred true, and were characterized further.
Electrophoretic mobility shift assay
DNA encoding a C-terminal fragment including the PAL-1 homeodomain
(residues 203-270) was cloned into plasmid pGEX-4T-3 (Pharmacia) using the
BamHI and XhoI sites. The resulting plasmid was used to
express the GST-PAL-1HD protein in E. coli BL21. Fusion protein was
isolated by passing supernatant of bacterial sonicate over a Glutathione
Sepharose 4B column (Amersham) and by elution with soluble glutathione. A
total of 30 ng of fusion protein was incubated for 15 minutes at room
temperature with 0.25 ng of double-stranded 32P-labeled
oligonucleotides (B WT: 5'-CATCATAAACTTTTTTTTCCGC-3'; C WT:
5'-GCAATAAACCGGCCAAAAACTT-3'; B MUT:
5'-CATCATCCACTTTTTTTTCCGC-3'; C MUT:
5'-GCAATCCACCGGCCAAAAACTT-3') and unlabeled competitor
oligonucleotides in 20 µl of a solution containing 50 µg/mL bovine serum
albumin, 10 mM Tris (pH 7.5), 1 mM EDTA, 100 mM KCl, 0.1 mM DTT, 5% glycerol
and 100 ng of salmon sperm DNA. Oligonucleotides were end-labeled with
32P
-dATP by incubation with T4 polynucleotide kinase.
Unbound 32P-dATP nucleotide was removed using the Stratagene
NucTrap Probe Purification Column. Binding reactions were run on a 4%
polyacrylamide gel in 0.5xTBE buffer. Each mobility shift assay
described in the paper was repeated at least three times. For
Fig. 6C, the mutant C oligo
used was 5'-GCACGCCCCCGGCCAAAAACTT-3'.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Sulston et al.,
1983), thus facilitating analysis of the kinetics of the onset of
cell death. Whereas most dying cells in C. elegans are
undifferentiated, the bi-nucleate tail-spike cell exhibits extensive
differentiated features before dying, including a posterior filamentous
process that may function as a scaffold for modeling the C. elegans
tail (Sulston et al., 1983)
(Fig. 1A). Thus, precise
control of the timing of tail-spike cell death onset may be important for
C. elegans tail development. In Drosophila and vertebrates,
many cells that die during development exhibit obvious differentiated features
and can live long after they are born
(Meier et al., 2000). Thus,
tail-spike cell death may be more similar to developmental cell deaths that
occur during development in Drosophila and vertebrates than to other
cell deaths that occur in C. elegans.
To study tail-spike cell death, we developed reagents to label the cell. We
noticed that a 1.5 kb region of 5' ced-3 promoter sequences,
when fused to a ced-3 cDNA, could provide ced-3 function to
the tail-spike cell (see below), suggesting that this promoter fragment could
be used to drive reporter gene expression in this cell. Indeed, the same
promoter region was sufficient to drive expression of lacZ (S.S., PhD
thesis, Massachusetts Institute of Technology, 1995)
(Shaham et al., 1999) or GFP
reporter transgenes in the tail-spike cell
(Fig. 1C,D). However, these
transgenes also occasionally blocked tail-spike cell death (see Table S1 in
the supplementary material), limiting their use. A similar transgene
consisting of a 0.7 kb ced-3 promoter fragment from the related
nematode C. briggsae fused to the GFP-encoding gene proved to be a
better reporter because it also marked the tail-spike cell but did not promote
inappropriate cell survival (Table
1A and see Table S1 in the supplementary material).
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). These
corpses do not form if cell death is prevented
(Ellis and Horvitz, 1986).
Consistent with our GFP results, a majority (24 out of 43, 56%) of
ced-5(n1812); egl-1(n1084n3082) first
larval-stage (L1) animals still exhibited a persistent tail-spike cell corpse
(see below), supporting the notion that egl-1 has only a partial role
in promoting tail-spike cell death. Tail-spike cell death was unaffected by
null mutations in the ced-13 gene
(Table 1A), the only other
characterized C. elegans BH3-domain-only gene
(Schumacher et al., 2005), and
ced-13 mutations did not enhance the defects observed in
egl-1 mutants (Table
1A), indicating that the partial block of tail-spike cell death in
egl-1 mutants was not due to redundancy with ced-13.
To determine whether egl-1 is expressed in the tail-spike cell, we
examined the expression of genomically integrated egl-1 promoter::GFP
transgenes (bcIs1 and bcIs37) that have been previously used
to show egl-1 expression in dying cells
(Conradt and Horvitz, 1999;
Thellman et al., 2003; Liu et al.,
2006). We failed to detect GFP expression in 9/9 (bcIs1)
and 6/6 (bcIs37) tail-spike cell corpses
(Fig. 1E), and in 27/27
(bcIs1) and 21/21 (bcIs37) tail-spike cells observed 30-60
minutes before corpse formation. An identical transgene, in which GFP
sequences were replaced with egl-1 coding sequences, fully rescued
the tail-spike cell survival defect in ced-3(n2427);
egl-1(n1084n3082) mutants (147/152 transgenic animals were
rescued in three lines that were observed;
Table 1). These results suggest
that egl-1 expression in the tail-spike cell is low, consistent with
the partial genetic role of egl-1 in tail-spike cell death.
To determine whether the partial pro-apoptotic activity of egl-1
was mediated by the ced-9 gene, we tested whether a ced-9
loss-of-function (lf)-null mutation could suppress the
egl-1(n1084n3082) tail-spike cell death defect. Animals
homozygous for ced-9(lf) alleles do not survive, and this lethality
is rescued by mutations in ced-3
(Hengartner et al., 1992).
Whereas ced-3(weak lf); egl-1(lf) double mutants exhibited a
fully penetrant tail-spike cell death defect, ced-9(lf);
ced-3(weak lf); egl-1(lf) triple mutants showed no defect in
tail-spike cell death (Table
1B). This result demonstrates that, as in other cells destined to
die, EGL-1 exerts its pro-apoptotic function in the tail-spike cell by
inhibiting the anti-apoptotic functions of CED-9.
Previous studies have shown that ced-9(lf) mutations enhance cell
survival in the pharynges of ced-3(weak lf) mutants
(Hengartner and Horvitz,
1994), indicating that ced-9 possesses death-promoting as
well as death-preventing activities. Surprisingly, we found that tail-spike
cell death proceeded normally in ced-9(n2812 lf);
ced-3(n2427 weak lf) double mutants
(Table 1B), and that the
tail-spike cell death defect in ced-9(n2812);
ced-3(n2436 weak lf) double mutants was identical to that of
ced-3(n2436 weak lf) single mutants
(Table 1C). These results
indicate that ced-9 lacks death-promoting activity in the tail-spike
cell. We also found that, unlike other somatic cells, tail-spike cell death
was only weakly affected by the ced-9(n1950) gainof-function
(gf) mutation, a glycine to glutamic acid substitution at a highly conserved
CED-9 residue (Hengartner and Horvitz,
1994). The ced-9(n1950gf) mutation alone had no
effect on tail-spike cell death (Table
1D), and it only partially enhanced the cell death defects
observed in ced-3(weak lf) and ced-5(lf) backgrounds
(Table 1D). Previous studies
argue that the ced-9(n1950gf) mutation prevents cell death
by disrupting a physical interaction between CED-9 and EGL-1
(Parrish et al., 2000;
Yan et al., 2004); however,
such a mechanism predicts that ced-9(n1950gf) and
egl-1(n1084n3082lf) mutants should exhibit identical
tail-spike cell death defects. The weaker defect observed in
ced-9(n1950gf) mutant animals suggests that the
n1950gf allele may only partially disrupt the interaction between
CED-9 and EGL-1 in the tail-spike cell (see Discussion).
Induction of ced-3 transcription determines the time of tail-spike cell death onset
Previous studies in C. elegans suggested that the regulation of
cell death initiation may be achieved by direct or indirect posttranslational
regulation of caspases by, for example, transcriptional regulation of the
egl-1 gene (Conradt and Horvitz,
1999; Metzstein and Horvitz,
1999; Thellmann et al.,
2003; Liu et al.,
2006). Similar mechanisms have been proposed in vertebrates and in
Drosophila (Meier et al.,
2000). However, the partial effects of egl-1 mutations on
tail-spike cell death and the unusual effects of ced-9 mutations in
this cell strongly suggested to us that a mechanism independent of
egl-1 must control the timing of the onset of tail-spike cell death.
Such a mechanism must function upstream of or in parallel to the
ced-3 caspase, because a ced-3(strong lf) allele completely
blocked tail-spike cell death (Table
1A). Unexpectedly, several lines of evidence implicated
transcription of ced-3 as a potential step in regulating the timing
of tail-spike cell death (see below).
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). Neither the transgene nor the smg-1(r861)
allele had any effect on programmed cell death in the pharynx (data not shown)
and, as expected, the transgene occasionally perturbed tail-spike cell death
(see above; data not shown). Three transgenic lines were scored.
Examination of transgenic embryos at different developmental stages
revealed that GFP was expressed in all or nearly all cells, except intestinal
cells and their precursors, starting at around 100-150 minutes
post-fertilization and continuing throughout the comma stage of embryogenesis
(Fig. 2A). Reporter expression
disappeared after the threefold embryonic stage, and only 2-3 cells expressed
GFP faintly in larva and adults (Fig.
2C). These cells, one of which was probably the head mesodermal
cell, were present in the head and did not reliably express GFP. Reporter
expression was similar between males and hermaphrodites, except that, in
males, intense staining was observed in the tails of L4 animals
(Fig. 2D). Interestingly, a
bout of cell death occurs in the late L4 in the male during tail morphogenesis
(Sulston and Horvitz,
1977).
Most cells expressing GFP in embryos or in the male tail were not cells
destined to die, consistent with previous functional studies
(Shaham and Horvitz, 1996a);
and, although we could detect GFP staining in dying cells, staining was
generally weaker than in other cells (Fig.
2B). GFP expression in dying cells could reflect de novo
transcription or residual GFP expressed in a precursor cell and perduring in
the dying cell. To distinguish between these possibilities, we observed the
GFP expression pattern in smg-1(r861);
ced-3(n717) animals carrying the same ced-3::GFP
transgene described above. GFP expression was nearly identical to that
observed in smg-1(r861) animals. Specifically, expression
was not detected in inappropriately surviving cells in larvae or adults
(Fig. 2E) (although see below
for tail-spike cell), as would be predicted if ced-3 transcription is
normally induced in these cells. These results are consistent with the
hypothesis that ced-3 is not actively transcribed in cells destined
to die, but is transcribed in their precursors. Supporting this notion, of the
131 somatic cells that die during hermaphrodite development, 18 cells die in
the L1 and L2 stages. No expression of the ced-3::GFP transgene was
ever detected in these cells in either smg-1(r861) or
smg-1(r861); ced-3(n717) animals, even
though the ced-3 genomic region we used to construct the GFP reporter
was sufficient to rescue the inappropriate survival of these cells in
ced-3(n717) mutants (S.S., unpublished). The 18 cells that
die postembryonically are descendents of the W, Pn, T and ABpl/rapapaa blast
cells that are born at the 200-cell stage, prior to the comma stage.
ABpl/rapapaa are the immediate precursors of the QL/R and V5L/R cells. As
mentioned above, these blast cells do express GFP in the embryo after they are
born. Thus, it is possible that ced-3 is normally expressed in the
blast cells of postembryonic cells fated to die, and that perduring
ced-3 RNA or protein eventually leads to their death.
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To test this hypothesis, we sought to follow the kinetics of ced-3 expression in the tail-spike cell in greater detail. Because a good antibody recognizing CED-3 has been difficult to generate (C.W.M. and S.S., unpublished), and because RNA in situ hybridization cannot be used reliably to detect ced-3 expression (S.S., unpublished), we could not directly follow the production of ced-3 mRNA or protein in the tail-spike cell. We decided, therefore, to follow ced-3 expression kinetics indirectly, in embryos carrying either the C. elegans or C. brigssae ced-3 promoter::GFP reporters (lines nsEx723 and nsIs25, respectively), as were described in the first subsection of the Results. Intriguingly, whereas expression of either reporter was detected in many cells, and throughout embryogenesis, in a pattern similar to that shown in Fig. 2 (data not shown), expression in the tail-spike cell was only observed during the threefold stage of embryogenesis (Fig. 1B,C), hours after the tail-spike cell is born. We followed individual embryos through this stage and found, surprisingly, that GFP fluorescence in the tail-spike cell first appeared 32.1±4.9 minutes (mean±s.d; n=6; C. elegans reporter) or 24.8±2.5 minutes (n=5; C. briggsae reporter) prior to visible signs of cell death (Fig. 1B-D). Similar findings were obtained by scoring ced-3 expression at specific time points in populations of synchronized embryos (n=10-15 embryos per time point, with ten equally spaced time points examined between 3-5 hours after the embryonic comma stage; data not shown). The finding that multiple lines carrying C. elegans and C. brigssae reporter transgenes, as well as multiple derivatives of the C. elegans transgenes (see below), exhibited identical kinetics of ced-3 expression in the tail-spike cell suggests that these reporters are likely to faithfully represent endogenous expression of ced-3. To further support this notion, we examined whether the same C. elegans ced-3 promoter used in our GFP reporter construct could promote tail-spike cell death in ced-3(n717) mutants when used to drive expression of a ced-3 cDNA. We found that this construct was sufficient to promote tail-spike cell death. For example, in three different lines, tail-spike cell death was rescued in 29/30, 26/30 and 28/30 animals examined, indicating that ced-3 expression at this late stage in the development of the tail-spike cell is sufficient to promote the demise of the cell, and supporting the hypothesis that ced-3 transcription may normally be the temporal trigger for cell death initiation in the tail-spike cell. Such a mechanism for cell death initiation has not been previously described.
To further investigate whether ced-3 transcription plays a role in
tail-spike cell death, we examined ced-3 promoter function in greater
detail. The C. elegans ced-3 promoter contains a 349 bp sequence that
is conserved in C. briggsae (Fig.
3A and see Fig. S1A in the supplementary material), and is
sufficient to drive GFP reporter expression in the tail-spike cell
(Fig. 3B). To identify regions
within the conserved promoter required for ced-3 expression in the
tail-spike cell, we deleted consecutive 14-16 bp sequences within this
promoter and assessed the effects of these deletions on reporter transgene
expression (Fig. 3B and see
Fig. S1B in the supplementary material). As shown in
Fig. 3B, three regions (A, B
and C) were required for ced-3 promoter::GFP expression in the
tail-spike cell. The sequences of regions B and C are highly conserved in
C. briggsae (Fig. 3A).
Although deleting regions A, B or C individually in the larger 1.5 kb
ced-3 promoter::GFP transgene did not fully block GFP expression
(although a significant partial effect was seen upon deletion of region B),
double deletions abolished GFP expression in the tail-spike cell
(Fig. 3C), suggesting that
these sequences function redundantly to control ced-3 expression in
this cell. To assess the functional relevance of these redundant promoter
sequences, we deleted them singly or in combination in the context of a 7.6 kb
rescuing C. elegans ced-3 genomic clone containing the same 1.5 kb of
5' promoter sequences (Yuan et al.,
1993). These clones were individually introduced into
ced-3 mutants, and their ability to rescue the cell death defect was
assessed. We found that transgenes lacking two or more of these sites did not
restore tail-spike cell death, but did rescue inappropriate cell survival in
the anterior pharynx (Fig. 3C),
indicating that the sites are required specifically for tail-spike cell death.
Taken together, the expression and rescue results strongly suggest that
induction of ced-3 transcription is a key step in controlling the
timing of tail-spike cell death initiation.
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ns114; ced-3(n717) animals exhibited weak (42%, n=88, three lines scored) or absent (58%) C. elegans ced-3 promoter::GFP expression in the tail-spike cell. Similar results were obtained using the C. briggsae reporter transgene nsIs25 (Fig. 4A,B), leading us to hypothesize that ns114 might also block tail-spike cell death. Two observations suggested that this was indeed the case. First, in an otherwise wild-type genetic background, 16 out of 75 (21%) ns114; nsIs25 animals had an inappropriately surviving tail-spike cell, as scored by weak GFP expression in this cell. By contrast, cell death in the anterior pharynx was unaffected (data not shown). This proportion is probably an underestimate of cell survival, because about 50% of cells did not express GFP at all and were not scored. Second, whereas 31 out of 33 (94%) of ced-5(n1812) animals exhibited a persistent tail-spike cell corpse, a persistent cell corpse was only seen in 20 out of 46 (43%) ns114; ced-5(n1812) animals (Fig. 4C,D), further indicating that this mutation blocked tail-spike cell death. Interestingly, the tail-spike cell death defect was greatly enhanced in ns114; egl-1(n1084n3082) double mutants compared with either single mutant alone (Fig. 4D), suggesting that ns114 affected a gene acting in parallel to egl-1 to promote tail-spike cell death. Consistent with this result, 10 out of 32 (31%) ns114; ced-9(n2812 lf); ced-3(n2427) animals had inappropriately surviving tail-spike cells, as scored in cells weakly expressing GFP, whereas ced-9(n2812); ced-3(n2427) mutants had no tail-spike cell survival (Table 1), indicating that ns114 must affect a gene that functions in parallel to ced-9.
In addition to defects in ced-3 expression and tail-spike cell death, some ns114 animals exhibited defects in tail-spike cell fusion (see Fig. S2A in the supplementary material) and some possessed a mild tail deformity (see Fig. S2B in the supplementary material). Tail-spike cell death defects were consistently observed in animals displaying neither cell fusion nor tail morphology abnormalities, suggesting that the defects are independent of one another. To avoid errors in cell identification, inappropriate tail-spike cell survival was only scored in animals with an otherwise wild-type tail morphology.
PAL-1 controls the expression of ced-3 in the tail-spike cell
We used single nucleotide polymorphism differences between the
ns114 and CB4856 C. elegans strains
(Wicks et al., 2001) to map
ns114 to a 4.5 map unit interval on chromosome III containing the
homeodomain transcription factor pal-1. Previously isolated
pal-1 mutants displayed tail morphogenesis defects
(Edgar et al., 2001),
suggesting that ns114 might be an allele of this gene. Indeed, we
found that both a 6 kb pal-1 genomic DNA fragment and W05E6, the
cosmid containing pal-1 (Edgar et
al., 2001), fully rescued the ns114 tail-spike cell GFP
expression defect (Fig. 5A).
Consistent with these results, we identified a C to T alteration at position
1944 of the pal-1 genomic sequence in ns114 animals
(Fig. 5B). This mutation
creates an ectopic consensus splice-donor site
(Fig. 5C) that, if used, would
result in a frame shift after codon 234, altering the C-terminal region of the
homeodomain, including the residues proposed to make up the DNA-binding domain
(Mlodzik et al., 1985). Unlike
ns114, pal-1-null alleles result in fully penetrant lethality
accompanied by severe defects in posterior patterning
(Edgar et al., 2001),
suggesting that the ectopic splice donor site in ns114 animals may be
used only occasionally. In pal-1(ok690)-null mutants, no
detected ced-3 expression in posterior regions was ever detected (0%,
n=30); however, we could not reliably identify the tail-spike cells
in these animals because of the patterning defects that they displayed.
|
PAL-1 can bind ced-3 promoter sequences
pal-1 is expressed in a number of posterior cells, including in
the tail-spike cell (Edgar et al.,
2001), and functions cellautonomously in the V6 cell
(Waring et al., 1992) and in
cells of the C and D lineages (Edgar et
al., 2001). These results suggested to us that the PAL-1 protein
may directly bind to the ced-3 promoter to allow ced-3
expression in the tail-spike cell. Although the consensus DNA-binding site of
pal-1 has not been defined, the similarities between the homeodomains
of PAL-1 and its closest Drosophila and vertebrate homologs, Caudal
and Cdx1/Cdx2, respectively, suggested to us that the proteins might share
similar DNA-binding affinities. Intriguingly, the caudal consensus
DNAbinding site, TTTAT(G) (Dearolf et al.,
1989), appears in two of the three ced-3 promoter sites
that we established as being crucial for tail-spike cell ced-3
expression and death (sites B and C; Fig.
3A). We therefore tested the ability of PAL-1 to bind these
promoter sites in an electrophoretic mobility-shift assay. As shown in
Fig. 6, a fusion protein
between glutathione Stransferase (GST) and the PAL-1 homeodomain (residues
203-270) bound 32P-labeled 22 bp oligonucleotides from the
ced-3 promoter containing either sites B or C
(Fig. 6A or 6B,
respectively). Binding was competed by cold wild-type oligonucleotides,
but was less efficiently competed by mutant B or C oligonucleotides in which
the consensus binding site was mutated to TGGAT. Similar results were obtained
using oligonucleotides in which all five consensus binding residues were
altered to ACGCC (data not shown). Furthermore, wild-type oligonucleotides
derived from site C competed efficiently with labeled site B oligonucleotides;
however, PAL-1-binding-site mutant C oligonucleotides competed less
efficiently (Fig. 6C). These
results demonstrate that PAL-1 is able to bind sites within regions B and C in
a sequence-specific manner. Consistent with this conclusion, the PAL-1 fusion
protein did not bind 32P-labeled mutant B or C oligonucleotides
(Fig. 6A,B). Additionally, when
identical mutations were created in the ced-3 promoter::GFP reporter,
expression in the tail-spike cell was greatly compromised. For example, in
four lines containing the B site mutation, 4/100 animals examined had weak GFP
expression and 96/100 had no GFP expression in the tail-spike cell. Similarly,
in four lines containing the C site mutation, 74/120 animals examined had weak
GFP expression and 28/120 had no expression in the tail-spike cell.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
this is not the case in the tail-spike cell. A number of observations support the model described here. For example, the model predicts that mutations in pal-1 should affect tail-spike cell death independently of mutations in ced-9. Indeed pal-1(lf); ced-9(lf); ced-3(lf) mutants have significantly more tail-spike cell survival than ced-9(lf); ced-3(lf) animals (see Results). Similarly, pal-1 functions independently of egl-1 (Fig. 4). In addition, we failed to detect expression in the tail-spike cell of two egl-1 promoter::GFP reporter transgenes, consistent with the idea that egl-1 plays a minor role in tail-spike cell death.
The time-estimates for ced-3 transcriptional induction described
in the Results section do not account for GFP folding rates; however, several
observations suggest that GFP folds rapidly. GFP folds in vitro within 2
minutes (Enoki et al., 2004;
Merkel and Regan, 2000) and
GFP expression is observed in cultured cells that have been microinjected with
a GFP reporter plasmid within 30 minutes of injection
(Schmoranzer et al., 2003). To
define an upper limit for GFP folding rates in C. elegans, we
engineered animals containing a heat-shock promoter::GFP transgene, subjected
them to a 20-minute heat shock at 34°C and observed robust GFP expression
within 30 minutes following heat shock (A. Valentine and S.S., unpublished).
Thus, it takes at most 50 minutes for animals to sense increased heat,
transcribe and translate GFP, and properly fold the protein. These
observations suggest that ced-3 transcription is induced at a maximum
of 80 minutes before the onset of tail-spike cell death. Given that we scored
the onset of ced-3 promoter::GFP expression when the signal is barely
detectable and that the signal increases several fold over the ensuing 30
minutes, we estimate that the true amount of time between ced-3
expression and the onset of cell death is significantly less than 80
minutes.
|
Our studies describe a role for the PAL-1 protein in controlling
ced-3 expression in the tail-spike cell. However, PAL-1 is unlikely
to act alone in this cell to promote ced-3 transcription. PAL-1 is
expressed in many cells in the animal that do not die
(Edgar et al., 2001).
Furthermore, within the tail-spike cell, PAL-1 is expressed several hours
before tail-spike cell death occurs. Thus, PAL-1 must either associate with
other factors that promote ced-3 transcription or must be
post-translationally activated to induce ced-3 transcription. It is
interesting to notice that we could not detect specific binding of PAL-1 to
the A site of the ced-3 promoter, suggesting that this site may
indeed be occupied by another protein that functions together with PAL-1 to
induce ced-3 transcription.
Our studies suggest that the control of caspase transcription may be an important mechanism for exercising temporal control of cell death initiation in other animals as well. In this context, it is worth noting that caspase expression during the development of any organism has not been extensively studied.
Control of cell death timing in other C. elegans cells
Although the egl-1 gene is required for the death of most somatic
cells destined to die in C. elegans, it is clear that additional
mechanisms must exist that control the onset of these deaths. Specifically, in
ced-9(lf); ced-3(weak lf) double mutants, some cells
destined to die do so appropriately
(Hengartner and Horvitz,
1994), suggesting that cells that normally die during C.
elegans somatic development can die in the absence of ced-9 and,
by extension, in the absence of egl-1. How might cell death timing be
controlled in these cells? Transcriptional control of ced-3 is
unlikely to be the main timing mechanism in these cells, because
ced-3 may not be transcribed within dying cells, but rather, may be
expressed in precursors of cells destined to die. Thus, asymmetric
segregation, or activation of CED-3 protein (or mRNA) during cell division may
be important for other cell deaths in C. elegans. Intriguingly, most
cells that die in the C. elegans soma do so within 30-60 minutes
after being born. This time interval is of the same order as the gap between
the onset of ced-3 transcription and the first signs of death in the
tail-spike cell. This observation suggests a model in which asymmetric
segregation/activation of CED-3 protein (or mRNA), which may occur in most
C. elegans cells fated to die, and transcriptional upregulation of
ced-3 in the tail-spike cell, are the key rate-limiting steps for
cell death initiation.
The ced-9 (n1950) gain-of-function allele does not only block the association of CED-9 and EGL-1
Our studies have demonstrated that egl-1 and ced-9 behave
differently in the tail-spike cell as compared with other cells destined to
die in two ways. First, we found that egl-1(lf) mutations blocked
tail-spike cell death in 30% of the animals examined, whereas
ced-9(1950gf) mutations did not block tail-spike cell death
at all. In all other somatic or germ cell deaths that have been examined,
ced-9(n1950gf) and egl-1(lf) mutants have exhibited
identical phenotypes (Conradt and Horvitz,
1998; Gumienny et al.,
1999; Gartner et al.,
2000). Second, in the tail-spike cell, ced-9 lacks its
death-promoting function. Specifically, ced-9 has been shown to have
both death-preventing, as well as death-promoting, functions in many somatic
cells (Hengartner and Horvitz,
1994). The nature of the death-promoting function of
ced-9 is not understood; however, genetic-interaction studies suggest
that ced-9 has the capacity to inhibit two alternatively spliced
ced-4 transcripts: ced-4S, which promotes cell death, and
ced-4L, which inhibits cell death
(Shaham and Horvitz, 1996b).
Inhibition of the former by ced-9 could explain the death-preventing
function of ced-9, and inhibition of the latter by ced-9
could explain the death-promoting function of ced-9
(Shaham and Horvitz, 1996b).
Alternatively, the death-promoting function of ced-9 may be mediated
by its role in mitochondrial fragmentation
(Jagasia et al., 2005).
How might the disparities between egl-1 and ced-9
functions in the tail-spike cell be resolved? Genetic and structural studies
suggest that the ced-9(n1950gf) mutation may block the
association of CED-9 with EGL-1 (Parrish
et al., 2000; Yan et al.,
2004). However, if this were the sole mechanism of n1950
function, then egl-1(lf) and ced-9(n1950gf) mutants
should exhibit identical phenotypes, which is not the case in the tail-spike
cell. One resolution of this apparent contradiction is to suggest that the
ced-9(n1950) mutation has two effects. First, this mutation
may only partially block association of CED-9 with EGL-1. Second,
n1950 may fully block the death-promoting function of CED-9; for
example, n1950 may block the ability of CED-9 to inhibit CED-4L
function. Thus, according to this hypothesis, n1950 should behave
like a weak egl-1 mutation in the tail-spike cell, only very mildly
preventing cell death, because the death-promoting function of ced-9
is not present in this cell. In other somatic cells, ced-9 does
possess a death-promoting function (perhaps because CED-4L is expressed in
these cells and not in the tail-spike cell, or due to a role in mitochondrial
fragmentation) and, thus, n1950 interferes both with that function
and with binding to EGL-1, resulting in extensive cell survival. Testing this
hypothesis will require a clearer understanding of the nature of the
ced-9 death-promoting function.
Caspase transcription and the control of tumorigenesis
Caspases have been demonstrated to play a role in tumorigenesis
(Stupack et al., 2006),
although the mechanism by which these proteases suppress tumor progression,
and the factors regulating their expression, remain poorly understood. Our
results suggest that mutations in transcriptional regulators of caspases may
promote tumorigenesis by blocking cell death. Intriguingly, mutations in the
vertebrate homolog of pal-1, Cdx2, promote digestive tract tumor
formation (Chawengsaksophak et al.,
1997; Aoki et al.,
2003; Bonhomme et al.,
2003), and tumor aggressiveness is inversely correlated with the
level of Cdx2 expression (Ee et al.,
1995; Hinoi et al.,
2001). Furthermore, in the intestinal epithelium, Cdx2 is
expressed at only low levels in less-differentiated cells near the intestinal
crypt, and at high levels in fully differentiated cells of the epithelium,
which continually undergo apoptosis
(Silberg et al., 2000). Taken
together, these observations raise the possibility that Cdx2 promotes
vertebrate caspase transcription to effect programmed cell death in the
intestinal epithelium in a manner similar to pal-1 regulation of
ced-3 expression in C. elegans.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/7/1357/DC1
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ACKNOWLEDGMENTS |
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