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First published online 15 December 2008
doi: 10.1242/dev.019042
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1 Department of Cell Biology, The University of Texas Southwestern Medical
Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9039, USA.
2 Department of Biochemistry and Cellular and Molecular Biology, University of
Tennessee, Knoxville, TN 37996, USA.
* Author for correspondence (e-mail: john.abrams{at}utsouthwestern.edu)
Accepted 12 November 2008
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Key words: Apoptosis, Bcl-2 genes, Cell death, Drosophila
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INTRODUCTION |
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SUMMARY
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MATERIALS AND METHODS
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DISCUSSION
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). Unlike worms, where a direct physical link to the
apoptosome is seen, the regulation of apoptosis by the mammalian Bcl-2 gene
family occurs indirectly through the regulation of mitochondrial properties.
For example, several pro- and anti-apoptotic Bcl-2 proteins impact the
mitochondrial outer membrane (MOM) permeability
(Kluck et al., 1997;
Zou et al., 1997), resulting
in the release of cytochrome c and subsequent formation of the apoptosome
(Green and Reed, 1998;
Gross et al., 1999).
Like worms and mammals, the Drosophila genome encodes at least two
Bcl-2 family members (Chen and Abrams,
2000). However, unlike mammals, fly cytochrome c is not required
for apoptosome formation (Yu et al.,
2005), and the roles of Drosophila Bcl-2 family members
as potential regulators of PCD remain unclear
(Arama et al., 2003;
Arama et al., 2005;
Dorstyn et al., 2004;
Dorstyn et al., 2002;
Mendes et al., 2006;
Zimmermann et al., 2002). To
date, Drosophila cytochrome c (cyt-c-d) has been linked to
caspase activation during spermatid differentiation
(Arama et al., 2003) and to the
proper timing of cell death in the pupal eye
(Mendes et al., 2006).
Previous studies, based on forced expression and RNAi, reported pro-apoptotic
functions for debcl (Brachmann et
al., 2000; Colussi et al.,
2000; Igaki et al.,
2000; Senoo-Matsuda et al.,
2005; Zhang et al.,
2000) and potentially anti-apoptotic functions for Buffy
(Brachmann et al., 2000;
Quinn et al., 2003). Recently,
genetic studies found limited roles for either gene in stress-induced
apoptosis, but, because partial debcl function is likely to occur in
these mutants, pivotal questions remain unresolved
(Sevrioukov et al., 2007).
To illuminate the functional role of Bcl-2 proteins in PCD, we generated definitive null alleles at debcl. We exclude a global role for this gene in developmental PCD, but do find selective roles for debcl in regulating cell death and cell numbers in the CNS. debcl genetically interacted with the ced-4/Apaf1 counterpart dark, but was not required for killing by RHG proteins. We found no overt role for debcl in regulating stress-induced cell death, cell-cycle checkpoint kinetics, genomic instability, or autophagy. In related studies, debclKO mutants were not significantly affected for mitochondrial density or volume. Surprisingly, in a model of caspase-independent cell death, we found that heterologous killing by murine Bax required debcl to exert its pro-apoptotic activity. Hence, although it regulates PCD in limited contexts during normal development, the action of debcl can be effectively recruited for killing by pro-apoptotic mammalian members of the Bcl-2 gene family.
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MATERIALS AND METHODS |
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MATERIALS AND METHODS
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DISCUSSION
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).
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Southern blotting
For verification of targeting, genomic DNA was prepared from flies carrying
the candidate targeted allele using the Wizard Genomic Purification Kit
(Promega). Genomic DNAs were digested as indicated
(Fig. 1A), separated by 0.8%
agarose gel electrophoresis and transferred to positively charged nylon
membranes. The membranes were probed with [32P]dCTP-labeled DNA
from PCR product using primers 5'-GCT ACA GTC GAG TGT GCT GGG TTG TTT
GCG-3' and 5'-GAC GGC GGA TTC CAG ACG CTT TCA GAA CGG-3' for
verification of the left recombination arm, and primers 5'-GCG TAC AAT
TAG ACC AGC CGT TGT GTT GGC-3' and 5'-AAG AGG ACA ACA GCG AGG TGG
AGG AGG ACG-3' for verification of the right recombination arm, and
hybridized using Express Hyb Hybridization Solution (BD Biosciences).
PCR and RT-PCR
A PCR strategy was used to recognize candidate targeted recombination
alleles. The primers 5'-GGT TAT CAT ACC ATT CCT GCT CTT TGG-3'
(Primer C) and 5'-GTC CTG AAG GAG ATC TGC GAA GAG GAC AAC AGC-3'
(Primer D) were used to amplify a PCR fragment unique to targeted
recombination. The same strategy using sequence specific primers was used to
verify the left recombination arm (data not shown). Additionally, primers
flanking the debcl ORF, 5'-AAC GAG AAC GGG AAC TCG AAA GAA CCT
AGA TCG-3' (Primer A) and 5'-AAG ACG AAT TGT CGT ACT CAA AAT ATT
GGC ACC-3' (Primer B), were used to distinguish native debcl or
replacement by the white+ gene. The genomic sequences from
the PCR reactions were fully sequenced, and the sites of recombination at the
right and left recombination arms matched endogenous sequence. To access
transcript levels, total RNA was prepared from debclKO and
wild-type (yw, WT) L3 larvae using the High Pure RNA Isolation Kit
(Roche). The Superscript One-step RT-PCR System with Platinum Taq (Invitrogen)
was used for RT-PCR reactions. The following primers were used to amplify
transcript in debcl and neighboring genes: rp49 (RT-PCR
control) 5'-ATG ACC ATC CGC CCA GCA TAC A-3' and 5'-ACA AAT
GTG TAT TCC GAC CAG G-3'; fmo-2 5'-ATC AAA ACT TCA GTG
GAC AAG CGT CGT GTT TGC-3' and 5'-ATC GTA TAC TTG TTG CTC CTG TAC
GTG TCC-3'; debcl 5'-CCA AGT TCA AGT CCT CGT CGC TGG
ACC-3' and 5'-GCG AAT CTA GAA CAG CAG CGA ATA CAG TTG
ACC-3'; CG30443 5'-GAG CTG GAC CAG TTC TAC TGC GAA ATA
TGC-3' and 5'-AGC CAA TCT GTA ATA ACT TCC TCG CTG TGG-3';
and geminin 5'-CCA GGG TCT ACA TCC AAG TCG AGA CAG AGG-3'
and 5'-TTG ACC TTG TCC TCG TCA CCC GTA GTG TCG-3'.
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), using
the ApopTag Fluorescein In Situ Apoptosis Detection Kit (Chemicon
International). Acridine Orange staining was performed as described by Sogame
et al. (Sogame et al.,
2003).
Fly strains
darkCD4 was meiotically recombined with
debclKO to generate double knock-out flies for genetic
interaction studies. The following stocks were also used for various analyses:
UAS-mito-GFP (Cox and Spradling,
2003), pGMR-Gal4, da-GAL4, pGMR-rpr,
-hid and -grim4 (Bloomington Stock Center). UAS-Bax
flies were kindly provided by B. Mignotte (Université de
Versailles/Sain-Quentin). Recognizing that these strains tended to accumulate
modifiers, we mobilized the UAS-Bax transgene by conventional
methods. Briefly, we crossed UAS-Bax flies to flies carrying
2-3 transposase, recovered candidate alleles for transgene
mobilization, and mapped the new insertion sites.
Immunohistochemistry
Embryos or L3 tissues were collected and treated as described by Chew et
al. (Chew et al., 2004).
Primary antibody was incubated overnight at 4°C [1:600 guinea pig
-Kr (Kosman et al.,
1998); 1:800 -βGal Ab (Promega); 1:500
-dHb9, 1:1 -LBe and 1:500 -Eg
(Rogulja-Ortmann et al.,
2007); 1:200 rabbit -phosphohistone H3 (Upstate); 1:50
-cleaved Caspase 3 (Cell Signaling)]. Secondary antibodies from
Molecular Probes were used at a 1:500 dilution.
Microscopy and imaging
All embryos and imaginal disc imaging was done on a Zeiss LSM 510 META
laser scanning confocal microscope, a Leica TCS SP5 spectral confocal
microscope, or a Zeiss Axioplan 2E digital light microscope. Images were
processed using ImageJ and Adobe Photoshop software. Photographs of fly eyes
were taken on a Zeiss SteREO Discovery.V12 microscope.
Notched wing assays
Wandering L3 larvae were treated with 0, 1500 or 2500 Rads of ionizing
radiation. Adults with notched wings were scored as a percentage of the total
number of adults counted.
Autophagy assays
Lysotracker dye was used as a marker for autophagy, and assays were
performed as described by Rusten et al.
(Rusten et al., 2004). The
following modifications were made: fat bodies were stained with LysoTracker
Red DND-99 (Molecular Probes) diluted 1:4000 in PBS with 1 µM Hoechst for
5-10 minutes. Fat bodies were washed several times in PBS and mounted in 70%
glycerol/PBS and visualized immediately. Images were captured using a
40x dry lens. For quantification analysis, three fat body lobes from
five independent animals of each genotype were obtained and imaged. The number
of lysotracker-positive structures was quantified from z-stacked
images taken on a Zeiss Axioplan 2E digital light microscope and processed
using ImageJ and Adobe Photoshop. A total area of 800 square pixels was
quantified for each image. These results are expressed as mean values
±s.d.
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RESULTS |
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MATERIALS AND METHODS
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) to
replace the endogenous debcl gene with the white+
marker gene. The debcl gene is located on the right arm of the second
chromosome 42C2, 4.0 kb downstream of fmo-2, and 2.7 kb upstream of
CG30443 (Fig. 1A). We
screened 56,000 flies for recombination events and obtained 10 candidate
alleles (Fig. 1B, top panel).
Seven of these were confirmed for the predicted event by PCR and Southern blot
analysis (Fig. 1B, middle
panel). RT-PCR was used to verify normal expression of the flanking genes
(fmo-2, CG30443 and geminin) and the absence of the
debcl transcript from these mutant alleles
(Fig. 1B, bottom panel, data
shown for debcl22). For most studies, a combination of
transheterozygous individuals were examined. debclKO
animals are viable, fertile and exhibit a normal life-span (data not shown).
Hence, debcl is not essential for survival or fertility. At variable
penetrance (25%-57%), extra scutellar bristles were found on the notum of
debclKO adults (Fig.
1C, white arrow). Notably, this extra bristle phenotype also
occurs in dark, dredd, dronc (Nc - FlyBase), Dcp-1,
cyto-c-d and mir-9a (microRNA-9a) mutant adults
(Chen et al., 1998;
Laundrie et al., 2003;
Li et al., 2006;
Rodriguez et al., 1999;
Xu et al., 2005). Other
phenotypes, observed at lower penetrance, include rotated male genitalia,
imperforate vaginas in females, melanotic-like cells in the maxillary palps,
and patterning abnormalities of tergites and sternites.
debcl regulates PCD and proper cell number in the developing central nervous system
debclKO embryos were examined for PCD and, when
compared with WT, a moderate decrease in the number of TUNEL-positive cells
was consistently observed (Fig.
2A,B). We also examined debclKO embryos using
well-established antibodies that mark cells which normally die in the nervous
system, but which fail to die in PCD mutants
(Rogulja-Ortmann et al.,
2007). For instance, the number of Kr+ cells
in the ventral nerve cord (VNC; Fig.
2C,D) was determined and quantification of these cells
(Fig. 2E) in abdominal segments
A2 and A3 showed no significant differences in cell number. However, extra
Kr+ cells were consistently present in segments A4 and A5,
and in Bolwig's organ of debclKO embryos
(Fig. 2E-H). We also examined
midline glia using two distinct markers for slit-expressing cells
(-βGal Ab and a slit-lacZ reporter),
and both detected supernumerary slit+ cells in
debclKO embryos (Fig.
2I-L). Quantification of these cells is shown
(Fig. 2M). Not all markers,
however, exhibited extra cells in debclKO animals. For
example, antibodies directed against the neuronal transcription factors
dHb9 (exex - FlyBase; see Fig. S1A,B in the supplementary
material), lbe and eg (not shown) showed wild-type cell
numbers in all segments and normal PCD of Crz+ neurons in
the pupal CNS (see Fig. S1C,D in the supplementary material)
(Choi et al., 2006). Likewise,
examination of pupal eyes by staining for discs large revealed normal
loss of interommatidial cells (see Fig. S1G,H in the supplementary
material).
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We also examined irradiated wing imaginal discs for damage-induced cell cycle checkpoint phenotypes using an anti-phosphohistone H3 antibody to label mitotic cells (Fig. 3H,I). After treatment with IR, proliferative arrest (see Fig. 3K,L) and resumption of proliferation (not shown) occurred with similar kinetics in WT and debclKO discs. Likewise, no genotype-specific differences in wing sizes before and after IR stress were seen and, using mwh as a loss of heterozygosity readout, we did not observe evidence that the debcl status affected levels of genomic instability (not shown).
Interaction with other pro-apoptotic death genes: Debcl is not required for killing by RHG proteins, but is required for heterologous killing by murine Bax
Adult eyes of debclKO flies show normal gross
patterning (Fig. 5B), and eye
ablation phenotypes caused by forced expression of the pro-apoptotic genes
grim, rpr and hid were unaffected by
debclKO genotypes (Fig.
5C-H). Hence, killing by forced expression of IAP antagonists does
not require debcl function. We also tested for possible genetic
interactions between debcl and other members of the apoptotic
pathway. For example, flies hypomorphic for the apoptosomal gene
dark, develop progressive melanized blemishes that can be quantified
as shown in Fig. 4G and, using
this phenotype as an indicator of defective PCD
(Link et al., 2007), positive
genetic interactions between dark and other cell death genes,
dp53 (Sogame et al.,
2003) and dronc (Chew
et al., 2004), have previously been established. Similarly, we
found that both the incidence and severity of melanized wing blemishing was
clearly exacerbated in debclKO,
darkCD4 double mutants
(Fig. 4E,F), compared with
single darkCD4 mutants
(Fig. 4D). This observation was
quantified in Fig. 4G.
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) and, as previously shown, Bax provoked eye ablation
phenotypes (Fig. 5I,K). We
therefore tested whether Bax-induced cell death might be affected by
debcl status. Surprisingly, these phenotypes were almost completely
suppressed in the debclKO background
(Fig. 5J,L). Together, these
data indicate that a debcl-dependent step is required for
heterologous killing by Bax.
debcl and autophagy
We examined the possibility that debcl, like other Bcl-2 family
members (Shimizu et al.,
2004), may play a role in autophagy. Using lysotracker as a marker
for starvation-induced autophagy (Munafo
and Colombo, 2001), we examined larval fat bodies during fed and
starved conditions. Autophagy was quantified in larval fat bodies, and in all
trials no significant differences between WT and debclKO
animals were observed (Fig.
6B). Examples of fat body staining imaged for quantification
analysis using lysotracker and Hoechst are shown
(Fig. 6C-F). Likewise, WT and
debclKO larvae exhibit similar survival curves when
transferred from fed to starved conditions
(Fig. 6A), with both genotypes
displaying an LD50 of approximately 6.5 days after challenge.
Mitochondrial properties are independent of Debcl status
We examined the possibility that debcl, like other Bcl-2 family
members (Karbowski et al.,
2006), may function to specify mitochondrial properties. We first
measured steady-state ATP levels in larval and adult tissues and found no
indication that respiration was perturbed in debclKO
mutants (not shown). Next, using the Gal4-UAS system together with a
GFP reporter (mito-GFP), we visualized mitochondria in imaginal wing
disc cells and in salivary gland cells before and after metamorphosis. In both
tissues, mitochondria in WT (Fig.
7A,C) and debclKO cells
(Fig. 7B,D) appeared to have
similar morphological distributions. Using Imaris software for unbiased
comparisons, we surveyed distributions of mitochondrial volume and found no
differences between WT and debclKO with respect to this
parameter (Fig. 7G,H). We also
determined that, on a per cell basis, mitochondrial densities were comparable
in WT and debclKO animals
(Fig. 7E,F). We also note here
that mitochondrial fragmentation was associated with PCD in pupal salivary
glands, but that this change was not evidently impacted on by debcl
genotypes (not shown).
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DISCUSSION |
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SUMMARY
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MATERIALS AND METHODS
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DISCUSSION
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),
or by indirectly promoting apoptosome formation in mammals, a step that
requires cytochrome c (Jurgensmeier et
al., 1997; Kluck et al.,
1999; Li et al.,
1998; Wei et al.,
2001). The fly genome encodes two well-conserved Bcl-2 family
members, yet, in this animal, cytochrome c is dispensable for apoptosome
assembly (Yu et al., 2005),
raising the possibility that insect Bcl-2 genes could mediate cytochrome c
independent activities that may or may not be related to cell death. To
clarify the functional roles of this gene family in the Drosophila
model, we genetically eliminated debcl, the fly ortholog of the
mammalian pro-apoptotic members Bax, Bak and Bok.
Using a targeted recombination strategy, we recovered seven allelic strains
that are definitively amorphic for debcl. Our studies exclude a
general requirement for debcl as a global apoptotic effector, which
had been suggested from gene silencing analyses
(Colussi et al., 2000;
Senoo-Matsuda et al., 2005).
Nevertheless, three compelling lines of evidence establish that debcl
does function to regulate a limited number of developmental cell deaths.
First, in every allelic combination tested, TUNEL labeling was consistently
and markedly reduced (see Fig.
2). Second, in every allele tested, debcl genetically
interacted with a hypomorphic allele of the apoptosomal gene dark.
Third, extra cells in debcl embryos were detected using markers that
visualize persisting or `undead' cells in canonical PCD mutants
(Chew et al., 2004;
Rodriguez et al., 2002;
Rogulja-Ortmann et al., 2007;
White et al., 1994). Although
the impact caused by eliminating debcl was modest, we note that
reproducible and consistent PCD phenotypes were observed for all alleles
tested. Furthermore, it is also worth noting that our data reflect counts of
marked cell populations, only a small fraction of which actually die
(Rogulja-Ortmann et al.,
2007). Hence, if we consider only the cells that are lost and
compare these against benchmarks seen in animals completely defective for PCD,
the effects caused by eliminating debcl are substantial. For example,
in H99 animals where no PCD occurs, a 37% excess of Kr+
cells is seen in Bolwig's organ (Link et
al., 2007) and, by comparison, an excess of up to 23% Kr+
cells was seen in debcl mutants
(Fig. 2). Graded effects along
the anteroposterior axis is another familiar phenotype seen here, and,
likewise, was previously reported for persisting motoneurons in H99
mutants (Rogulja-Ortmann et al.,
2007). Specifically, in cases where extra neuronal cells were
observed (e.g. -Kruppel, see
Fig. 2), these cells tended to
appear more commonly among the posterior segments. This trend of extra cells
in more posterior segments is consistent with previous studies reporting the
prominence of neuronal degeneration in the abdominal ganglion
(Kimura and Truman, 1990;
Robinow et al., 1993).
Segments with supernumerary cells averaged 17% additional cells in
debclKO animals (e.g.
Fig. 2) and, by comparison,
mutations in the apoptosomal genes dark and dronc produced a
range from 33-50% excess cells (Chew et
al., 2004; Rodriguez et al.,
2002). From these combined results, we speculate that perhaps
debcl augments apoptotic signaling in certain cell types. Not all
tissues were impacted in debclKO animals, however. Notable
examples of Drosophila PCD that were unaffected by debcl
status included PCD of interommatidial cells in the eye, PCD of the salivary
gland and elimination of Crz+ neurons during larval to
pupal transition (Choi et al.,
2006), as well as of markers specific for motoneurons during
embryogenesis (see Fig. S1 in the supplementary material). Hence, to the
extent represented by the markers studied here, the impact of debcl
status on Drosophila cell death appears to be limited to certain
embryonic neurons and glia, but not on the PCD of motoneurons. Taken together,
these studies exclude a universal requirement for debcl in PCD, yet
establish a limited role for this gene in the death of certain cell types.
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). Second, the markers used in the two studies were
quite different. In studies presented here, we applied established markers
proven to detect extra cells that persist in canonical PCD mutants (H99,
dronc and dark) by virtue of specific failures in
caspase-dependent cell death (Chew et al.,
2004; Rodriguez et al.,
2002; Rogulja-Ortmann et al.,
2007). By contrast, Sevrioukov et al. applied markers that
highlight axonal bundles (Mab+ cells) and a glial cell
marker (repo+ cells), neither of which previously detected
supernumerary cells in PCD defective H99 mutants
(Rogulja-Ortmann et al.,
2007).
We also carefully examined other cellular processes in addition to PCD.
debcl mutants showed normal levels of starvation-induced autophagy
and, likewise, were unperturbed for radiation stress responses, including cell
cycle arrest and loss-of-heterozygosity post-challenge. Interestingly,
however, we did find elevated levels of `notched' phenotypes in
debclKO wings after IR challenge. Although the cellular
basis for this observation is not clear, we suggest that debcl may be
recruited to promote effective compensation when development is perturbed
(de la Cova et al., 2004;
Ryoo et al., 2004).
In numerous models of apoptosis, including in flies, mitochondria are
remodeled through fusion and fission
(Abdelwahid et al., 2007;
Goyal et al., 2007;
Green and Reed, 1998;
Karbowski et al., 2006;
Youle and Karbowski, 2005). In
mammals, Bax and Bak are involved in this pathway of mitochondrial remodeling
(Gross et al., 1999;
Karbowski et al., 2006;
Wei et al., 2001;
Youle and Karbowski, 2005),
but whether this activity plays a role in transducing or amplifying the
apoptotic response is hotly debated, in part because it has been difficult to
separate these activities. Indications that these activities can, in fact, be
uncoupled was shown by Delivani et al., who found that expression of the worm
Bcl-2 proteins Egl-1 and Ced-9 in mammalian cells influenced mitochondrial
dynamics, but not the translocation of cytochrome c from mitochondria during
apoptosis (Delivani et al.,
2006). Recently, mitochondrial remodeling was also linked to PCD
in flies (Abdelwahid et al.,
2007; Goyal et al.,
2007). Therefore, we investigated the effect of debcl
genotypes upon constitutive mitochondrial dynamics in both larval and pupal
stages. Using automated imaging analyses, we showed that mitochondrial density
and mitochondrial volume in WT and debclKO cells were
similar in both L3 wing imaginal discs and salivary glands
(Fig. 7E-H). These results
suggest that the ortholog of Bax in flies does not play a crucial role in the
regulation of mitochondrial properties, as is seen in mice lacking Bax and Bak
(Karbowski et al., 2006). It
is also worth noting here that mitochondrial fragmentation was associated with
histolysis of the pupal salivary gland but that these changes occurred
irrespective of the debcl status (not shown).
Despite the absence of mitochondrial phenotypes, our findings highlight a
role for debcl in regulating a limited number of cell deaths. At the
same time, we can exclude a general requirement for this gene in PCD, but the
possibility remains that Buffy may play redundant roles in the
tissue-specific regulation of Drosophila PCD. Therefore, during
evolution, insects may have de-emphasized ancestral roles for the Bcl-2
proteins in PCD or, alternatively, mammals and worms may have evolved in ways
that emphasized roles for this gene family in apoptotic cell death
(Cory and Adams, 2002;
Danial and Korsmeyer, 2004;
Karbowski et al., 2006).
Remarkably, debcl function was also required for the heterologous
killing of fly cells by murine Bax. This observation rules out non-specific
toxicity as an explanation for Bax killing in this system. Instead, to kill
fly cells, Bax evidently recruits a native activity encoded by debcl
that is not otherwise essential for all apoptotic cell death in
Drosophila. Together with known actions of Bax
(Karbowski et al., 2006;
Wei et al., 2001;
Youle and Karbowski, 2005) and
with a previously described insensitivity to p35 (Gaumer et al., 2000b), these
findings suggest that, in order to kill fly cells, Bax instigates a
debcl-dependent, but caspase-independent, pathway of cell death.
Extending this rationale, it may be possible to use this system to identify
effectors of Bax without the potentially confounding effects that are linked
to coincident caspase activation in mammalian cells.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/2/275/DC1
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Footnotes |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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5.2 kb). Primer pair C and D were used
to verify the right arm of recombination during the screening process for
potential recombinants. Primer pair E and F were used to confirm targeted
recombination of the left arm of recombination (data not shown). WT refers to
the yw parental strain, wild type at the debcl locus on the
second chromosome; Donor refers to the debcl donor construct on the
third chromosome, wild-type at the debcl native locus on the second
chromosome; debcl6,22,23,27,59,61 represent different
debcl knock-out candidates disrupted at the native locus.
debcl6 was a potential debcl targeted deletion,
but failed the PCR screen and was therefore eliminated from further
characterization analysis. An additional debcl disruption allele,
debcl47/48, was also confirmed (not shown). Southern blot
analysis using genomic DNA from the indicated fly strains was used to confirm
both the right (SBP1) and left (SBP2) arm of recombination. For SBP1
(BglII digest) and SBP2 (SacI digest), the black arrowheads
indicate the expected 6.4 kb and 8.3 kb genomic fragments in the WT and donor
strains for SBP1 and SBP2, respectively. The white arrowheads indicate
aberrant genomic fragments of 9.6 kb and 5.8 kb, indicative of gene-targeted
replacement, for SBP1 and SBP2, respectively. RNA from L3 larvae was used as a
template to confirm abolishment of the debcl transcript in
debclKO flies, and to confirm that transcript levels are
unaffected in the neighboring genes fmo-2, CG30443 and geminin.
rp49 was used as a control for RT-PCR. The white asterisk indicates the
absence of the debcl transcript in mutant flies. (C)
Photograph of extra scutellar bristles in debclKO flies
(white arrow).
