1 The University of Texas M.D. Anderson Cancer Center, Department of Biochemistry and Molecular Biology, 1515 Holcombe Boulevard Unit 117, Houston, TX 77030, USA
Author for correspondence (e-mail:
abergman{at}mdanderson.org)
Accepted 15 February 2005
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SUMMARY |
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Key words: Dronc (Nc), CARD, Caspase, Apoptosis, Cell death, Drosophila, Diap1
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Introduction |
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Molecular genetic studies performed in the last 10 to 15 years revealed
that the basic principles of regulation and execution of apoptosis are
conserved. Genetic studies in Caenorhabditis elegans have implicated
caspases as central to the apoptotic program
(Yuan et al., 1993). Caspases
are a highly specialized class of cysteine proteases that cleave target
proteins specifically after Asp residues. At least 11 human caspases are
known, while the Drosophila genome contains seven caspase genes
(Salvesen and Abrams, 2004
).
Caspases are synthesized as catalytically inactive zymogens, the activation of
which is tightly controlled and involves both positive and negative input (for
reviews, see Danial and Korsmeyer,
2004
; Salvesen and Abrams,
2004
). Activation occurs through proteolytic processing,
generating a large and small subunit that then form a tetramer containing two
large and two small subunits (Danial and
Korsmeyer, 2004
). Caspases are negatively regulated by inhibitor
of apoptosis proteins (IAPs), a highly conserved class of proteins with
members in all eukaryotic species (Miller,
1999
). IAPs directly bind to and inhibit caspases. Thus, IAPs
represent the last line of defense for a cell against apoptotic stimuli.
Two classes of caspases have been defined based on the length of the
prodomain. Initiator caspases such as Caspase 9 contain long prodomains that
harbor regulatory motifs such as the caspase activation and recruitment domain
(CARD). Through homotypic interactions of the CARD motif of Caspase 9 with the
CARD motif of Apaf-1, Caspase 9 is recruited into the apoptosome, a large
multi-subunit complex, where it undergoes autoprocessing and activation
(Danial and Korsmeyer, 2004).
Once activated, Caspase 9 cleaves and activates effector caspases (Caspase 3,
-6 and -7), which are characterized by the presence of short prodomains.
Effector caspases execute the cell death process by cleaving a large number of
cellular proteins (Danial and Korsmeyer,
2004
).
The Drosophila genome contains a total of seven caspase genes,
three of which encode putative initiator caspases [Dronc (Nedd2-like caspase
FlyBase), Dredd and Strica (Dream FlyBase)], whereas the
remaining four are putative effector caspases [DrICE (Ice FlyBase),
DCP-1, Decay and Damm] (reviewed by Kumar
and Doumanis, 2000; Salvesen
and Abrams, 2004
). Dronc is the only Drosophila caspase
that carries in its prodomain a CARD motif
(Dorstyn et al., 1999
), which
interacts with the CARD of Dark (Ark FlyBase), the Drosophila
Apaf-1 homolog, also known as D-Apaf-1 or Hac-1
(Rodriguez et al., 1999
;
Kanuka et al., 1999
;
Zhou et al., 1999b
). In this
respect, Dronc is functionally most similar to human Caspase 9. Consistent
with its function as an initiator caspase, Dronc can cleave and activate the
effector caspase DrICE in vitro (Hawkins
et al., 2000
). dronc is ubiquitously expressed throughout
development and is a target of the insect hormone ecdysone, which stimulates
increased dronc expression during metamorphosis
(Dorstyn et al., 1999
).
Several observations suggest that dronc is an important component of
the apoptotic machinery in Drosophila. Overexpression of
dronc in the developing fly eye induces cell death and tissue loss
(Meier et al., 2000
;
Quinn et al., 2000
). Dominant
negative constructs and RNA interference experiments support a role for
dronc in developmental cell death
(Meier et al., 2000
;
Quinn et al., 2000
). However,
without mutations in the endogenous gene, a definitive role of dronc
in developmental apoptosis cannot be defined.
Like Caspase 9, Dronc is subject to negative regulation by IAPs, in
particular Drosophila IAP1 [Diap1 (Thread FlyBase)
(Meier et al., 2000)]. Diap1
is characterized by two tandem repeats of approximately 70 amino acids each,
known as the Baculovirus IAP Repeat (BIR; for a review, see
Deveraux and Reed, 1999
), and
one C-terminally located RING domain. The BIR domains are required for binding
and inhibiting caspases (Zachariou et al.,
2003
). The RING domain has been shown to encode an E3 ubiquitin
ligase (Yang et al., 2000
).
Ubiquitin ligases mediate the transfer of ubiquitin from E2 conjugating
enzymes to target proteins that are subsequently degraded by the 26S
proteasome (Joazeiro and Weissman,
2000
). Target proteins of Diap1/RING-mediated ubiquitination
include Dronc in the absence of apoptotic signals
(Wilson et al., 2002
) and
Diap1 itself in the presence of apoptotic signals
(Ryoo et al., 2002
;
Yoo et al., 2002
;
Bergmann et al., 2003
).
Loss-of-function diap1 mutations cause a dramatic cell death
phenotype, in which nearly every cell in mutant embryos is apoptotic,
suggesting an essential genetic role for diap1 in cellular survival
(Wang et al., 1999
;
Goyal et al., 2000
;
Lisi et al., 2000
). This
phenotype is presumably caused by inappropriate activation of caspases
(Meier et al., 2000
;
Rodriguez et al., 2002
).
In Drosophila, the genes reaper, hid (wrinkled
FlyBase) and grim are both necessary and sufficient for the
induction of apoptosis (White et al.,
1994; Grether et al.,
1995
; Chen et al.,
1996
). Deletion of these genes, as seen in the H99
deficiency, results in a complete lack of developmental cell death in
Drosophila embryos (White et al.,
1994
). Overexpression of any of those genes in the fly eye using
the eye-specific enhancer GMR (for example, GMR-hid or
GMR-reaper) causes a severe eye ablation phenotype resulting from
inappropriate apoptosis (Grether et al.,
1995
; White et al.,
1996
) (see also Fig.
2A,E). Subsequent genetic and biochemical analyses have shown that
these genes induce apoptosis through direct inhibition of Diap1
(Wang et al., 1999
;
Goyal et al., 2000
). In
response to expression of reaper and hid, the RING domain of
Diap1 changes its substrate specificity, self-ubiquitinates and induces its
own degradation (Ryoo et al.,
2002
; Yoo et al.,
2002
; Bergmann et al.,
2003
). Caspases, most notably Dronc, are thus relieved from Diap1
inhibition and can induce apoptosis. In mammals, the factors Smac/DIABLO and
HtrA2 are known to relieve caspase inhibition by IAPs
(Du et al., 2000
;
Verhagen et al., 2000
;
Verhagen et al., 2002
;
Suzuki et al., 2001
;
Hegde et al., 2002
;
Martins et al., 2002
). These
factors share with Reaper, Hid, and Grim a conserved N-terminus that is
required for interaction with IAPs.
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Materials and methods |
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A recombinant chromosome containing en-Gal4 and UAS-GFP transgenes (referred to as en::GFP), located on the second chromosome, was crossed into a droncI24/droncI29 trans-heterozygous mutant background, and GFP expression in the wing was monitored.
Mosaic eye clones were obtained from ey-FLP; droncI24 FRT80/ubi-GFP FRT80 pupae and analyzed by anti-Discs large (Dlg) labeling.
For germline clone (GLC) analysis, droncI24 and
droncI29 were recombined onto the FRT2A chromosome. GLC
were induced by the DFS-FRT method as described
(Chou et al., 1993;
Chou and Perrimon, 1996
).
To visualize midline glia (MG) cells, males of the genotype P[sli-1.0-lacZ]; droncI24/TM6B, ubx-lacZ were crossed to GLC droncI24 or droncI29 females.
Immunohistochemistry
TUNEL and immunohistochemistry were done as described
(Goyal et al., 2000;
Patel, 1994
). CM1
(anti-cleaved Caspase 3) antibody was used at a dilution of 1:50, Elav
antibody at dilution of 1:20, anti-Krüppel antibody at 1:50, and anti-Dlg
antibody at 1:300. The MG was visualized by ß-gal immunohistochemistry.
Ovary dissections were done as described
(Rodriguez et al., 2002
).
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Results |
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We therefore developed a method that allows screening for recessive
suppressors of GMR-hid in a homozygous mutant condition. However, we
were uncertain as to whether dronc is an essential gene for
development. If it were, homozygous dronc mutants would die, which
would prevent us from screening modifications of the GMR-hid
eye phenotype. Instead, we screened for suppressors of
GMR-hid in homozygous mutant eye clones obtained by
FLP/FRT-mediated recombination in otherwise heterozygous animals.
Specifically, we used ey-FLP
(Newsome et al., 2000) to
express the FLP recombinase under eyeless (ey) enhancer
control in the developing eye to induce homozygous mutant clones. We termed
this approach the GheF method for GMR-hid ey-FLP. Because
the ey enhancer used to express FLP is active before the GMR
enhancer, the eye tissue is already mosaic for any induced mutation when
GMR begins to drive hid expression. If a gene required for
GMR-hid-induced apoptosis and eye ablation was mutagenized, the
homozygous mutant clone cells would be resistant to the effects of
GMR-hid. However, the twin-spot and heterozygous cells would contain
either two or one functional copies of the mutagenized gene, respectively, and
would be sensitive to GMR-hid-induced apoptosis. As a result, any
surviving eye tissue in the adult organism would be homozygous for the
mutagenized gene.
We conducted an EMS mutagenesis screen using the GheF method to isolate mutations in the dronc gene (Fig. 1). Because dronc maps to the left arm of the third chromosome (3L), we selected for mutations on this chromosome arm using FRT80, which is specific for 3L (Fig. 1). dronc is the only caspase known to map to 3L, so we expected to identify only dronc mutants in this screen. Among 45,000 F1 progeny screened, four mutations were isolated that suppressed the GMR-hid eye ablation phenotype. These mutations were subsequently confirmed to be mutant alleles of dronc (see next section). Three of them, droncI24, droncI29 and dronc2, rescued the GMR-hid eye ablation phenotype almost entirely in ey-FLP/FRT-induced clones (Fig. 2B). The fourth allele, droncL32, was weaker and suppressed the GMR-hid-eye phenotype partially to medium size (Fig. 2C). It is important to note that the dronc mutants suppressed GMR-hid only in homozygous mutant clones. In a heterozygous condition, even a null allele (droncI24) did not modify the GMR-hid phenotype (Fig. 2D). This finding is consistent with our assumption that a 50% reduction in the gene dose of dronc is not sufficient to visibly modify GMR-hid and provides an explanation for why dronc alleles were not recovered in the dominant modifier screen (see above). The dronc mutants also suppressed the GMR-reaper-induced small eye phenotype (Fig. 2E-G).
In summary, we isolated four dronc alleles as strong or medium-strength suppressors of GMR-hid in ey-FLP/FRT-induced clones. Our genetic analysis shows that dronc mutants recessively rescued the effect of GMR-hid and GMR-reaper expression in the eye, suggesting that dronc+ is genetically required for GMR-hid- and GMR-reaper-induced apoptosis.
Molecular analysis of the dronc alleles
Inter se complementation studies indicated that the four suppressor
mutations of GMR-hid all affected the same genetic function. These
mutations were semi-lethal when carried in trans to each other. Usually, they
died during pupal stages; however, at a low rate (less than 10% of the
expected progeny), homozygous mutant escaper flies of the strong alleles could
be recovered. These escapers are characterized by an abnormal wing phenotype
(see below, Fig. 4), a weak
rough eye phenotype (not shown) and a short life span. They died 2-3 days
after eclosion.
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droncL32 behaves genetically as a hypomorph
(Fig. 2C,G), changing Leu25 in
the CARD domain to Glu. Leu25 is a conserved residue in the CARDs of Caspase
9, Caspase 2, Apaf-1 and Ced-4. Interestingly, structural analyses of the
CARD/CARD interaction between human Caspase 9 and Apaf-1 showed that the
equivalent residue in human Caspase 9, Leu16, is not directly involved in the
interaction between the two CARDs (Qin et
al., 1999; Zhou et al.,
1999a
). Nevertheless, the fact that this residue is conserved in
various CARD motifs and that its mutation results in partial loss of function
suggests that it is important for appropriate CARD activity.
In summary, this analysis suggests that the isolated suppressor mutations of GMR-hid-induced eye phenotypes represent dronc alleles. With respect to droncI24, droncI29 and dronc2, our genetic and molecular analyses suggest that they are complete loss-of-function alleles, whereas droncL32 is a hypomorphic allele.
dronc mutants have an abnormal wing phenotype and extra cells in the eye
As mentioned above, most homozygous dronc mutant animals died
during pupal stages. However, at a low rate (<10%), homozygous escapers,
even of the null alleles (droncI24 and
droncI29), did eclose. These flies were characterized by a
short life span (they died within 3 days of eclosion), an abnormal wing
phenotype (Fig. 4A,B) and a
rough eye (data not shown). The mutant wing phenotype, although difficult to
illustrate in photographs, is readily scored under the dissecting microscope
using low magnification (8-10x). The mutant wing appeared opaque by
comparison to wild type, occasionally contained trapped fluid, and was curved
downward (Fig. 4A,B). This
phenotype is similar to the wing phenotype seen in dark and
hid mutant flies (Rodriguez et
al., 1999; Abbott and Lengyel,
1991
). Because these genes are involved in cell death, the wing
phenotype seems likely to be the result of decreased cell death.
It has been recently shown that a wave of cell death occurs in the wing
within the first hour after eclosion. Kimura et al.
(Kimura et al., 2004) used
en-Gal4 and UAS-GFP transgenes (referred to as
en::GFP) to drive expression of GFP as a marker in the posterior
compartment of the wing to analyze this cell death
(Fig. 4C). The majority of
en::GFP-positive cells are removed within one hour after eclosion in
wild-type wings (Kimura et al.,
2004
). Co-expression of the caspase inhibitor P35 blocks the
removal of en::GFP-expressing cells, suggesting that it is the result
of a Caspase-driven cell death process in wild-type wings
(Kimura et al., 2004
). We
examined whether this wave of cell death occurs in dronc mutants. By
contrast to wild type, en::GFP expression was still detectable in
wings of 24-hour-old dronc mutants
(Fig. 4D,E). These data suggest
that the persistence of en::GFP is the result of loss of
developmental apoptosis in dronc mutant wings. Thus, lack of
apoptosis probably contributes to the abnormal wing phenotype of
dronc mutants.
Homozygous dronc mutant flies also exhibit a mild rough eye
phenotype (data not shown). Using an antibody against the Discs large (Dlg)
protein to visualize cell outlines, we determined that mid-pupal (50 hours)
retinae of dronc mutants contained on average three additional
inter-ommatidial cells (Fig.
4G). These cells usually die in wild-type retinae
(Cagan and Ready, 1989;
Wolff and Ready, 1991
)
(Fig. 4F), suggesting that
dronc+ is genetically required for developmental cell
death in the retina. However, this result is contradictory to a recent study
by Chew et al. (Chew et al.,
2004
), which reported fine patterning defects in the developing
eye of dronc mutants rather than defects in cell death. However, the
authors came to this conclusion by using photoreceptor markers to analyze
dronc mutant eye discs (Chew et
al., 2004
). However, photoreceptors are not known to undergo
developmental cell death. Furthermore, the dronc mutant by Chew et
al. (Chew et al., 2004
) is
derived from an imprecise P-element excision, which also affects a neighboring
gene, CG6685. Thus, the reported fine patterning defect may be due to
inactivation of CG6685, and not of dronc.
In summary, this analysis provides strong evidence that dronc+ is genetically involved in cell death during imaginal disc development of the eye and the wing in the fly.
dronc is essential for apoptosis during embryogenesis
We analyzed the genetic requirement of dronc for apoptosis during
embryogenesis. Because of the large maternal contribution, dronc
mutant animals survived embryogenesis and most of them died during pupal
stages. The few homozygous escapers died within the first 3 days after
eclosion; homozygous females were sterile and could not provide embryos for
analysis. To analyze the genetic requirement of dronc for cell death
during embryogenesis, we removed the maternal contribution by inducing germ
line clones (GLC) in otherwise heterozygous females
(Chou et al., 1993;
Chou and Perrimon, 1996
) (see
Materials and methods). Embryos obtained from GLCs of the null mutants
droncI24 and droncI29 were embryonic
lethal if they were also zygotically mutant for dronc. These embryos
exhibited a head defect similar to that of hid mutants
(Abbott and Lengyel, 1991
)
(data not shown). Notably, the maternal loss of dronc was paternally
rescuable; that is, dronc+ provided by the father's sperm
was sufficient to rescue the embryonic phenotypes due to maternal loss of
dronc. Paternally rescued animals obtained from dronc GLC
gave rise to normal and fertile adult flies (data not shown).
We performed TUNEL assays to confirm the possibility that maternally and
zygotically mutant dronc embryos are embryonic lethal because they
lack apoptosis. TUNEL detects DNA fragmentation, a hallmark of apoptosis
(Wyllie, 1980;
Gavrieli et al., 1992
).
Compared with wild-type embryos, the number of TUNEL-positive cells was
substantially reduced during embryogenesis in both
droncI24 and droncI29 embryos
(Fig. 5A-C). We also analyzed
whether downstream caspases such as DrICE were activated in dronc
mutants. The CM1 antibody has been shown to detect only the activated form of
DrICE (Yu et al., 2002
).
Compared with wild-type embryos, CM1 labeling of both
droncI24 and droncI29 embryos was
substantially reduced (Fig.
5D-F). Thus, consistent with its identity as an initiator caspase,
Dronc+ is required for activation of downstream caspases including
DrICE. However, even though TUNEL-positive cell death is significantly reduced
in dronc embryos, it is not completely blocked.
This finding suggests that dronc+ is not required for all
embryonic cell death and that a dronc-independent cell death pathway
exists in the embryo (see Discussion).
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In addition, we used the Elav antibody to visualize neurons in both the CNS and the PNS, in particular the mechanosensory chordotonal organs. We found that dronc mutants contain on average about three additional neurons in each chordotonal cell cluster compared with wild type (Fig. 6E,F). We also found examples of unidentified neurons, which are increased in number in dronc mutants compared with wild type (data not shown).
Taken together, these data support the notion that dronc+ is essential for embryonic cell death. Thus, the dronc gene is genetically required for cell death in embryogenesis.
Analysis of dronc diap1 double mutants
Diap1 is an essential inhibitor of apoptosis during Drosophila
embryogenesis. diap1 mutant embryos die during early embryonic
development due to massive inappropriate apoptosis
(Wang et al., 1999;
Goyal et al., 2000
;
Lisi et al., 2000
). Because
diap1 and dronc mutants have opposite phenotypes, and
because their gene products directly interact with each other, it was proposed
that Diap1 acts as an inhibitor of Dronc
(Meier et al., 2000
;
Chai et al., 2003
). To
determine the genetic relationship between dronc and diap1,
we analyzed the phenotype of double mutants of these genes. One allelic
combination of weak diap1 alleles
(diap16B/diap18) generates viable, but
sterile, females due to ovarian atrophy
(Rodriguez et al., 2002
)
(Fig. 7A,C). We used this
phenotype to analyze the genetic relationship between diap1 and
dronc. In a double-mutant combination with dronc
(droncI24 diap18/droncL32
diap16B), the ovarian atrophy phenotype of
diap16B/diap18 females was partially
reversed. The size of the egg chamber was significantly enlarged compared with
that of diap16B/diap18 single mutants
(Fig. 7B,C). This finding
suggests that dronc mutations are able to rescue the diap1
phenotype, and places dronc genetically downstream of diap1
(see also Discussion). Despite this rescue, the double-mutant females still
did not produce functional embryos and were sterile (data not shown).
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Discussion |
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Using GheF screening, we isolated four EMS-induced point mutations
of the initiator caspase dronc in Drosophila, demonstrating
feasibility of the GheF screening method. Our genetic
characterization of these mutants in the wing, eye and embryo is consistent
with an essential role for dronc+ in developmental cell
death. The importance of caspases for programmed cell death was first revealed
in genetic studies in C. elegans
(Yuan et al., 1993), and later
confirmed by targeted gene disruptions in mice
(Kuida et al., 1996
;
Kuida et al., 1998
). In
Drosophila, the first report implicating caspases as important
mediators of programmed cell death took advantage of the universal caspase
inhibitor P35. In P35-overexpressing animals, cell death is significantly
reduced (Hay et al., 1994
).
More recently, dominant-negative constructs of cloned caspases and RNAi
experiments further supported the involvement of caspases in the cell death
response in Drosophila (Meier et
al., 2000
; Quinn et al.,
2000
).
The Drosophila genome encodes seven caspase genes
(Kumar and Doumanis, 2000;
Salvesen and Abrams, 2004
).
Despite considerable efforts in multiple mutagenesis screens, mutations in any
of the Drosophila caspases have not been reported. The only exception
is dredd (Chen et al.,
1998
); however, dredd does not appear to be an apoptotic
caspase, as dredd mutations do not affect the global cell death
pattern. Instead, genetic analysis has established that dredd has a
fundamental role in innate immunity
(Leulier et al., 2000
).
Mutations in the effector caspase dcp-1 have also been reported
(McCall and Steller, 1998
).
However, it was recently found that dcp-1 lies embedded in an intron
of another gene, called pita, and the reported phenotypes are due to
combined inactivation of both pita and dcp-1
(Laundrie et al., 2003
).
In this study we show that a 50% reduction in the gene dose of
dronc is not sufficient to modify the GMR-hid phenotype.
This result is in contrast to previous reports that interpreted the dominant
suppression of GMR-hid by the dronc deficiency
Df(3L)AC1 as evidence that dronc is the underlying cause of
this suppression (Meier et al.,
2000; Quinn et al.,
2000
). However, in addition to dronc, Df(3L)AC1 deletes a
number of other genes including gap1, which is a known suppressor of
GMR-hid (Bergmann et al.,
1998
). Because the dronc mutants we isolated failed to
dominantly modify GMR-hid (Fig.
2D), the authors of the aforementioned reports scored the
suppression by Df(3L)AC1 due to the absence of gap1 rather
than dronc.
Interestingly, the fact that a 50% reduction of dronc is
insufficient to dominantly modify GMR-hid suggests that
dronc is produced in excess over its genetic requirement. A similar
conclusion can be made about dark, mutations of which modify
GMR-hid and GMR-reaper only in homozygous mutants
(Rodriguez et al., 1999;
Kanuka et al., 1999
). This
conclusion is also consistent with the large maternal supply of dronc
provided by the mother to the oocyte (see below). Because caspases including
dronc are synthesized as inactive zymogens, which rely on association
with scaffolding proteins such as Apaf-1/Dark or proteolytic processing for
activation, the cell can afford to produce excessive amounts of these
potentially dangerous proteins without damaging consequences.
Most homozygous dronc mutant animals die at pupal stages. However, embryos obtained from dronc GLCs are embryonic lethal. This suggests that the maternal contribution compensates for the loss of zygotic dronc until pupal stages, at which time the maternal contribution is depleted and most animals die. However, a few homozygous animals survive and hatch as adults, presumably because the maternal stores lasted slightly longer in these flies than in others. These escaper flies are characterized by an abnormal wing phenotype. We determined that lack of cell death contributes to this phenotype. They also exhibit a rough eye phenotype due to additional inter-ommatidial cells. However, homozygous dronc escapers live for only 2 to 3 days after eclosion. It is not clear why they die, but the fact that they do suggests that dronc might also have important functions for adult survival.
Despite the fact that endogenous cell death was significantly reduced in
dronc mutants, it is not completely blocked, even for the putative
null alleles. Using TUNEL and CM1 antibody labeling as two independent cell
death assays, we consistently detected a few cells that underwent cell death
in dronc mutants. This observation suggests that dronc is
not required for all embryonic cell death. This is in contrast to the
H99 deficiency, in which reaper, hid and grim are
deleted. Homozygous H99 embryos completely lack developmental
apoptosis (White et al.,
1994). These observations suggest that the H99 genes can
induce at least a few apoptotic deaths independently of dronc. The
nature of this dronc-independent pathway is not known. However,
dredd, which encodes an initiator caspase most similar to human
Caspase 8 (Chen et al., 1998
),
is a good candidate to mediate dronc-independent cell death. Although
dredd mutants do not change the global cell death pattern visibly
(Leulier et al., 2000
), it is
still possible that a few cells are dependent on dredd+
function for apoptosis. In addition to Dronc and Dredd, a third potential
initiator caspase is encoded by the strica gene. Strica bears an
unusual N-terminal prodomain that does not contain any of the known
interaction motifs (Doumanis et al.,
2001
). Overexpression of strica causes cell death, but
mutants are not available for analysis of the role of strica in
developmental cell death. Finally, it is possible that an unknown mechanism
leads to dronc-independent cell death. Identification of the cells
that die in a dronc-independent manner and development of sensitive
cell death assays will be required to address this issue in the future. We are
also using the GheF screening method to identify genes involved in
the dronc-independent cell death pathway.
dronc is epistatic to diap1 in the ovary
Based on binding studies in vitro and overexpression studies in vivo, a
model has emerged that predicts Diap1 to be an important negative regulator of
Dronc (Meier et al., 2000;
Hawkins et al., 2000
;
Quinn et al., 2000
;
Muro et al., 2002
). However,
because of the lack of dronc mutants, the genetic relationship
between dronc and diap1 was unknown. We addressed the
genetic relationship between dronc and diap1 in the female
ovary.
There are at least two different phenotypes associated with the loss of
diap1 function in the ovary. The first is an ovary degeneration
phenotype generated by combination of two viable diap1 alleles in
trans to each other, diap16B and
diap18 (Rodriguez et
al., 2002). Removing dronc in
diap16B/diap18 mutant females strongly
suppresses the ovarian degeneration phenotype
(Fig. 7), demonstrating a
strong genetic interaction between dronc and diap1. The
second phenotype, described recently by Geisbrecht and Montell
(Geisbrecht and Montell,
2004
), involves border cell migration defects due to an
apoptosis-independent role of diap1. The two phenotypes in the ovary
are independent of each other, because the diap16B allele
that alters one key residue in the RING domain does not display border cell
migration defects, suggesting that the RING domain is not required for the
non-apoptotic function of Diap1
(Geisbrecht and Montell,
2004
). RING domain mutants of diap1 have been shown to
display a strong apoptotic phenotype in the embryo
(Lisi et al., 2000
), implying
that the degeneration phenotype of
diap16B/diap18 mutant ovaries is
likely to be the consequence of excessive apoptosis. Therefore, the rescue of
the ovary degeneration phenotype in the dronc diap1 double mutants
appears to result from suppression of apoptosis, as it is clearly not related
to border cell migration. Furthermore, the rescue strongly suggests that
dronc acts genetically downstream of diap1.
However, we also wanted to analyze the genetic relationship between
dronc and diap1 in a better-characterized apoptotic setting,
such as early in embryonic development. At this stage, strong diap1
mutants show extensive TUNEL-positive nuclei and inappropriate caspase
activation, resulting in developmental arrest and organismal death shortly
after gastrulation (Wang et al.,
1999; Goyal et al.,
2000
; Lisi et al.,
2000
). Ideally, we wished to analyze this embryonic cell death
phenotype of diap1 mutants in the absence of dronc function;
that is, in a dronc diap1 double mutant. Unfortunately, despite the
ovarian rescue of diap16B/diap18
mutants by removal of dronc function, the dronc diap1 double
mutant females were still sterile and did not produce embryos that would have
allowed us to analyze the embryonic cell death phenotype of dronc
diap1 mutants.
We therefore attempted to address this problem in GLCs. Both dronc
and diap1 map to the left arm of chromosome 3. Thus, we induced GLCs
that were double mutant for dronc and diap1. We used two
different diap1 alleles,
diap1109.07 and
diap15, which both behave genetically as null alleles
(Lisi et al., 2000).
Unfortunately, females with double mutant GLCs were sterile, and we did not
recover embryos for phenotypic analysis. We are currently designing
alternative methods to address this issue.
In summary, we have isolated and characterized four mutant alleles of dronc. At least two, but probably three, of them are complete loss-of-function alleles. These mutants lack most developmental cell death, suggesting that dronc is required for most cell death. However, a few cells die in a dronc-independent manner. Future studies will identify the nature of the dronc-independent pathway and clarify the genetic relationship between dronc and diap1.
Note added in proof
While this paper was in preparation, two additional studies appeared that
reported similar results about dronc mutants
(Chew et al., 2004;
Daish et al., 2004
).
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ACKNOWLEDGMENTS |
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Footnotes |
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