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First published online 15 March 2006
doi: 10.1242/dev.02332
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2
1 Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX 75390,
USA.
2 Institute of Experimental Endocrinology, Slovak Academy of Sciences, Vlarska
3, 83306 Bratislava-Kramare, Slovakia.
3 Department of Genetics, Cell Biology and Development, University of Minnesota,
6-160 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA.
4 Department of Genetics, Faculty of Science, Comenius University, 84215
Bratislava, Slovakia.
5 Department of General Zoology, Lorand Eotvos University, Pazmany setany 1/C,
H-1117 Budapest, Hungary.
6 Department of Biochemistry, UT Southwestern Medical Center, Dallas, TX 75390,
USA.
7 Genome Sciences Centre, BC Cancer Research Centre, 675 West 10th Avenue,
Vancouver, BC V5Z 1L3, Canada.
8 Wellcome Trust, Sanger Institute, Genome Campus, Cambridge CB10 1SA, UK.
* Author for correspondence (e-mail: John.Abrams{at}utsouthwestern.edu)
Accepted 17 February 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Autophagy, Apoptosis, Drosophila, Histolysis, dark
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Upon interaction with adaptor molecules, apical caspases are stimulated to
activate effector caspases by proteolysis. dark, the
Drosophila homolog of nematode Ced-4 and mammalian Apaf1, is thought
to regulate the apical caspase Dronc (Nc - FlyBase), through
interactions involving respective caspase recruitment domains (CARD) (reviewed
by Mills et al., 2005). As in
mammalian systems, fly caspases are also subject to negative regulation by IAP
proteins (Danial and Korsmeyer,
2004), and, among Drosophila members of this family,
Diap1 (Thread - FlyBase) is known to exert important control over apoptosis
(Goyal, 2001;
Wang et al., 1999). This
protein binds Dronc and the effector caspase Drice
(Ice - FlyBase), inhibiting the activity of each via multiple
mechanisms (Ditzel et al.,
2003; Hays et al.,
2002; Martin,
2002; Meier et al.,
2000; Ryoo et al.,
2002; Wilson et al.,
2002; Wing et al.,
2002b). Diap1 itself is under tight regulation and is effectively
antagonized by proapoptotic proteins [reaper (rpr),
grim, hid (also known as Wrinkled) and skl] encoded
in the reaper region (Chai et al.,
2003; Christich et al.,
2002; Silke et al.,
2004; Wing et al.,
2002a; Wing et al.,
2002b; Wing et al.,
2001; Wu et al.,
2001; Yoo et al.,
2002; Zachariou et al.,
2003). Together, these linked genes specify virtually all
programmed cell death (PCD) in the fly embryo, as the combined deletion of
these eliminates PCD at this stage (Abrams,
1999).
Three broadly conserved protein families, represented by Ced-9/Bcl2,
Ced-4/Apaf1 and Ced-3/Caspase 9, define fundamental components in pathways of
caspase control. However, a unified mechanism for their action in cell death
remains elusive, as analogous physical interactions seen between nematode
Ced-9 and Ced-4 do not occur among orthologous mammalian counterparts
(Moriishi et al., 1999).
Instead, mammalian Bcl2 proteins indirectly engage Apaf1 by controlling the
mitochondrial release of cytochrome c, which promotes the formation of a
multimeric complex referred to as the apoptosome
(Danial and Korsmeyer, 2004;
Spierings et al., 2005).
Although the fly counterparts of these genes add provocative clues,
particularly with respect to the negative regulators of caspase activity
(Salvesen and Abrams, 2004),
they also complicate the picture, as cytochrome c appears dispensable for
Drosophila Apaf1 (Dark)-dependent cell death, despite the
conservation of a WD domain thought to be necessary for cytochrome c binding
and regulation (Adrain et al.,
1999; Dorstyn et al.,
2004; Hu et al.,
1998; Rodriguez et al.,
1999; Zimmermann et al.,
2002). Previous data from us, and from others, on viable
hypomorphic alleles (Kanuka et al.,
1999; Rodriguez et al.,
1999; Zhou et al.,
1999) have established that Dark shares functional
properties with its counterparts in C. elegans, where Ced-4 is
required for all PCD, and in the mouse, where context-specific apoptogenic
requirements for Apaf1 are seen. However, central questions, approachable only
with a null allele, remained open.
Here, we isolate a single-gene null mutation at dark and demonstrate a general requirement for this gene in PCD and stress-induced apoptosis. The role for dark in PCD was not absolute, however, as rare cell deaths were observed. We show that a required focus of dark- organismal lethality maps to the central nervous system and also describe the first hypermorphic allele within the Apaf1/Ced-4 gene family. In a model of tissue histolysis, dark was essential for cell death but dispensable for characteristic features of the autophagic program, indicating that the stimulation of autophagy per se is not the mechanism of cell killing but lies upstream, or parallel to dark. These data establish that common effector pathways, regulated by the apoptosome, specify apoptotic and histolytic forms of PCD.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was remobilized and candidates were tested in trans to
existing alleles and against lethal mutations in flanking genes. Promising
`hits' were screened by PCR. dark82 failed to complement
darkCD4, but complements adjacent lethal alleles in the
neighboring genes, RhoGEF and a new lethal P mutation in CG8963 that we
fortuitously obtained in our first round of mutagenesis. Genomic PCR across
the deletion junction and RT-PCR were used to validate the mutation and define
the dark82 lesion. yw was the parental wild-type
strain for molecular analysis and for ex vivo hemocyte studies. RNA extraction
and QRT-PCR were conducted as described by Gorski et al.
(Gorski et al., 2003). Ages at
25°C were normalized from 18°C
(Park et al., 1996). Genomic
PCR and RT-PCR were performed as described by Chew et al.
(Chew et al., 2004), with
relevant gene-specific primers.
Transgenic `rescue' and genetic manipulation
Full-length dark with 8xHis-tags at the N terminus and
3xMyc-tags at the C terminus was cloned into the
BamHI/XhoI sites of the pFastBac1 vector (Invitrogen). The
BamHI/XhoI insert was then subcloned into the pUAST vector
to produce pUAST-darkWT. pUAST-darkV was generated by
changing Aspartate 1292 to Alanine using a QuikChange Site-Directed
Mutagenesis Kit (Stratagene). The pUAST constructs were injected into fly
embryos following standard procedures to obtain transformants. Independent
transgenic lines were mapped and crossed to the dark82
background. For rescue experiments, dark82/CyO,
actin-GFP;UAS-darkWT or dark82/CyO,
actin-GFP;UAS-darkV flies were crossed to
dark82/CyO, actin-GFP; Tub-Gal4/TM3, Sb flies (or other
drivers). The number of homozygous dark82;
UAS-dark/Tub-Gal4 progeny and number of heterozygous
dark82/CyO, actin-GFP; UAS-dark/Tub-Gal4 progeny were
counted. The percent of rescue was calculated by dividing the number of
rescued dark82 homozygotes by the Mendelian value expected
if dark82 homozygotes were fully viable. Similar crosses
were used to obtain dark82; UAS-dark/Hml-Gal4 L3 larvae
for hemocyte isolation. c81-Gal4, c833-Gal4 and Hml-Gal4
strains (Drapeau et al., 2003;
Goto et al., 2003;
Hrdlicka et al., 2002;
Manseau et al., 1997) were
obtained from the Bloomington Stock Center.
Germline clones and AO staining
The dark82 allele was recombined onto the
FRT2R-G13 chromosome. To generate
dark82 maternal-null embryos, the Dominant
Female Sterile (DFS) technique was used, as described previously
(Chou and Perrimon, 1996).
hs-Flp/+; OvoD FRT2R-G13/dark82
FRT2R-G13 females were crossed with dark82/CyO,
actin-GFP males to generate maternal and zygotic dark-null
embryos. To detect cell death, Acridine Orange (AO) staining was carried out
(Abrams et al., 1993).
Ex vivo hemocyte analyses
Wandering L3 instar larvae were prepared as described by Chew et al.
(Chew et al., 2004), with the
following modifications. Hemolymph was collected from six larvae and agents
were added after media addition. Membrane blebbing, a characteristic feature
of apoptosis, was used to quantify apoptosis. At 
6 hours post-treatment,
cells were stained with a fluorescent membrane dye, 10 uM CellTracker
(Molecular Probes) in DMSO, to facilitate the visualization of apoptotic
membrane blebbing (without fluorescent labeling it was difficult to assess
membrane blebbing owing to the phagocytic nature of hemocytes).
Immunohistochemistry and western blotting
Immunohistochemistry on dissected salivary glands was conducted as
described by Farkas and Mechler (Farkas
and Mechler, 2000). For actin counterstaining,
fluorescein-conjugated phalloidin (1:200; Molecular Probes), or
AlexaFluor488-Phalloidin or AlexaFluor546-Phalloidin (Molecular Probes) was
used; for nuclear counterstaining, 0.5 µg/ml Hoechst 33258 (Calbiochem) or
1 µg/ml OliGreen (Molecular Probes); and for caspase activity, rabbit
anti-cleaved caspase-3 antibody (1:500; Cell Signaling Technology). Optical
sections (0.5 µm thick) were collected using a Zeiss LSM-510 Meta laser
confocal microscope equipped with a 40x planapochromat oil objective.
Recombinant Dark protein was prepared as described previously
(Yu et al., 2005). Transgenic
Dark protein was detected in extracts from adult heads, with an anti-Myc
antibody at a dilution of 1:2000. Anti-Dark polyclonal antibody was used at a
dilution of 1:3000.
Electron microscopy
Samples were prepared and processed for electron microscopy as described
previously (Juhasz and Sass,
2005).
Visualization of MDC and GFP-LC3
Transgenic flies containing UAS-GFP-LC3 were kindly provided by the Harald
Stenmark Laboratory (Rusten et al.,
2004) and crossed to flies containing the salivary gland driver
D59-Gal4 (Gustafson and Boulianne,
1996) (kindly provided by Carl Thummel). Salivary glands of the
progeny were dissected in Drosophila Schneider's medium (Invitrogen)
and transferred to the same medium with MDC (0.1 mM) for 30 minutes at room
temperature. Samples were then rinsed once and mounted (both in Schneider's
medium). Salivary glands were analyzed by fluorescence microscopy using a
Zeiss Axioplan 2 microscope.
|
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
dark82 is a 6.3-kb deletion spanning the entire
open-reading frame and nearly the entire transcription unit
(Fig. 1A-C). Animals homozygous
for this allele arrest as late pupae and often present a characteristic dark
blister located centrally along the midline. The mutation fails to complement
all existing hypomorphic dark alleles, but complements flanking genes
(see Materials and methods). Homozygous dark82 animals
were rescued to viability using a transgene containing a full-length
dark cDNA (see Table
1). Hence, dark82 is a lethal, single-gene
null mutation.
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|
;
White et al., 1994) and
dronc (Chew et al.,
2004). At the same time, gastrulation, segmental patterning and
extension of the germ band appeared grossly normal in the absence of
dark. Hence, to the extent that these events involve migration and/or
movement, we note that the proposed role for dark in cell motility
evidently does not generalize to these morphogenic processes
(Geisbrecht and Montell,
2004). We also tested larval hemocytes in ex vivo models of
stress-induced cell killing (Fig.
2E-G). In contrast to wild-type counterparts,
dark- hemocytes were completely resistant to a Smac
mimetic, which antagonizes inhibitor of apoptosis proteins (IAPs) and is
thought to simulate the action of reaper proteins
(Li et al., 2004;
Salvesen and Abrams, 2004).
Likewise, dark- cells were completely insensitive to the
apoptogenic effects of cycloheximide, a protein synthesis inhibitor. Together,
these data establish a central role for the action of dark in
programmed and unprogrammed apoptosis.
|
|
).
Together, these observations indicate that leaky expression of
darkV restores gene function to null animals, not to the wild-type
level but, instead, to levels comparable to those seen in
darkcd4. Hypermorphic properties related to
darkv were also noted in ex vivo hemocyte assays. Expression of the
wild-type cDNA in dark82 hemocytes only mildly restored
stimulus-dependent apoptosis after treatment with a Smac mimetic. However,
darkV almost completely restored this apoptotic response to
dark82 hemocytes (Fig.
3G). We considered the possibility that altered expression of
UAS-darkv might explain the hypermorphic properties conferred by
darkV but, as shown in Fig.
3F, the expression levels of wild-type and variant transgenes were
equal. Therefore, in studies of organismal viability and hemocyte apoptosis,
darkV conferred striking hypermorphic gene activity without
detectable effects upon steady-state expression. These results are consistent
with negative-feedback models whereby the action of the Dark protein may be
directly repressed by effector caspases, thereby setting an apoptotic
threshold in cells that are specified for death.
|
;
Thummel, 2001). In
dark mutants, these organs did not histolyse and, instead, persisted
intact, even in 36-hour APF animals (Fig.
4B). Wild-type and dark glands were also stained for
immunoreactivity with anti-cleaved caspase 3, an antibody that detects
effector caspase activity in Drosophila tissues
(Yu et al., 2002). In early
pupariation stages (4 and 8 hours APF), wild-type salivary glands show little
or no immunoreactivity (data not shown), but, four hours later (12 hours APF),
widespread staining with anti-cleaved caspase 3 can be observed in the
cytoplasm of these cells (Fig.
4C). By contrast, levels of anti-cleaved caspase 3 staining in
dark- glands were starkly attenuated for reactivity at
comparable stages and later (Fig.
4D).
These defects could reflect specific functional requirements for
dark in histolysis or, alternatively, could result from a more
generalized arrest in prepupal development. We can exclude the latter
possibility, as persisting glands were always sampled from animals that had
passed through the `head eversion stage' into pupation
(Ashburner, 1989) and numerous
associated landmarks also proceeded on schedule (see below). Like many changes
that occur during metamorphosis, salivary gland histolysis is tightly
controlled by ecdysone and, hence, failure to histolyse might formally derive
from a disruption of this hormonal axis
(Yin and Thummel, 2005). To
address this possibility, we examined ecdysone-dependent signaling events
known to occur during the period from 3 to 9 hours prior to histolysis (7-13
hours APF). For example, without dark function, ecdysone receptor
(EcR) and other regulatory factors, such as BFTZ-f1
(Fig. 4E), E74A
(Fig. 4F) and Kruppel
homolog (not shown), accumulated in the nucleus of salivary gland cells.
Likewise, in a survey of transcripts that anticipate salivary gland histolysis
(Gorski et al., 2003;
Lee et al., 2003), gene
expression profiles from wild-type and dark82 glands were
highly comparable (see Fig.
4G). As an indicator of developmental progression, equivalent
expression profiles (Fig. 4G)
offer considerable statistical power, as a bulk analysis of 20-30 pairs of
glands is represented at each time point. Therefore, by each criterion
examined, hormonal signaling and associated target responses were unperturbed
in dark animals. Together, these observations establish a specific
requirement for the action of dark in salivary gland cell death and
exclude generalized arrest or developmental delay as an explanation for
defective histolysis.
|
;
Jiang et al., 1997;
Juhasz and Sass, 2005). By
stark contrast, comparably aged dark glands show no signs of
vesicular saturation (Fig. 5B).
Similarly, pre-histolytic changes that otherwise occur in the nucleus are not
seen (Fig. 5C) and, instead,
features characteristic of earlier-staged nuclei are retained. Like wild-type
counterparts, however, numerous autolysosomes were evident in dark
glands (Fig. 5B,C), indicating
that dark function is not required for autophagy per se. To extend
this analysis, we confirmed that monodansylcadaverine (MDC), an acidic marker
that detects autolysosomes (Munafo and
Colombo, 2001), overlapped with the signal derived from GFP-LC3
(Rusten et al., 2004), a
transgenic marker of autophagy (Fig.
5F). Next, we established that, in wild-type salivary glands,
dramatic accumulation of MDC staining anticipates PCD several hours prior to
overt histolysis (Fig. 5D-G).
We applied this methodology in dark animals and, likewise, observed a
comparable abundance of MDC-stained structures in mutant glands
(Fig. 5H,I), indicating that
stimulation of autophagy, which normally anticipates histolysis, is not
dependent on dark activity. Therefore, in this tissue, the action of
dark in histolysis functions downstream of, or parallel to, an
autophagic program. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Together, these observations establish that, for a small
population of embryonic cells, apoptotic activators in the reaper
region can specify apoptosis without engaging the fly apoptosome. Similar
pathways might occur in post-embryonic stages, but we caution against deriving
firm conclusions in unaffected larval tissues, given the caveats relating to
perdurance of maternally derived product.
Unlike its counterparts in the worm or the mouse, genetic elimination of
dark produced a strictly lethal phenotype. Because ubiquitous and
`driver-specific' expression of a dark transgene complemented this
phenotype, it was possible to map the focus of genetic activity responsible
for restoring viability. We found that dark82 lethality
was reversed when expression was restored to cells of the post-embryonic CNS,
but that complementation failed if dark was restored to hemocytes or
imaginal discs. These results highlight essential functions for zygotic
dark in the post-embryonic CNS and suggest that the action of this
gene within other tissues may not be necessary for viability. Transgenic
complementation also proved to be an effective means for distinguishing the
wild-type gene action from that of derivatives with altered activities. By
this approach, we determined that darkv encodes striking
hypermorphic activity without affecting transgenic expression levels. As
darkv is mutated at a caspase cleavage site
(Fig. 3A-D), the data are
consistent with negative-feedback models whereby the action of Dark is
directly repressed by effector caspases, perhaps setting an apoptotic
threshold in cells that are specified to die. These findings describe the
first hypermorphic point mutation among all known alleles in the ced-4/Apaf1
gene family, and raise intriguing possibilities for investigating how life
histories and stress responses might be impacted in adults with excessive
apoptosomal activity. It is worth noting that, unlike cultured cell models,
where full-length dark exhibits mild killing activity
(Rodriguez et al., 1999), we
found no evidence of dominant phenotypes associated with the forced
overexpression of either the wild-type or the variant transgenes in tissues
presented here (Fig. 3), or in
other tissues, such as the eye (not shown). The different effects seen in
culture cells versus transgenic animals might reflect authentic
context-specific variance, or, alternatively, there may be a mild killing
activity that does not manifest as a gross phenotype in the animal.
Nevertheless, at least for most tissues and cells, it is unlikely that the
levels of Dark protein alone qualify as a determinant of apoptosome
activity. This inference, together with studies that exclude a fundamental
requirement for Drosophila cytochrome c in formation of the
apoptosome (Yu et al., 2005)
or in models of apoptosis (Dorstyn et al.,
2004; Zimmermann et al.,
2002), suggests that, to function properly, Dark must be
activated through an unknown mechanism.
Regression of Drosophila salivary glands in pupal development is a
classic model of histolytic cell death, and dying cells in this gland appear
morphologically distinct from cells undergoing apoptosis, indicating that
novel cell death pathways may control forms of histolytic cell death (reviewed
by Thummel, 2001). We assessed
morphological, ultrastructural and molecular indicators to establish that,
without dark, developmental progression was unperturbed, histolytic
regression of this organ failed and salivary gland cells remained
morphologically intact. Our results clearly establish a requisite function for
Dark in the histolysis of salivary gland cells, despite the fact that PCD of
these cells appears dissimilar from classical apoptosis. These observations
are consistent with effects produced by p35, a broad-spectrum caspase
inhibitor (Jiang et al., 1997;
Lee and Baehrecke, 2001;
Martin and Baehrecke, 2004),
and with animals mutated for the apical caspase dronc
(Daish et al., 2004). Because
apoptotic and histolytic forms of cell death are similarly impacted by the
same mutation, we conclude that common effector pathways, regulated by the
apoptosome, underlie morphologically distinct forms of PCD.
The induction of autophagy that anticipates salivary gland histolysis may
act as part of a novel killing mechanism in these cells
(Lee and Baehrecke, 2001;
Myohara, 2004;
Thummel, 2001), and in
mammalian cell death models as well
(Shimizu et al., 2004;
Yu et al., 2004). However, in
other circumstances, `self-digestion' clearly promotes survival when apoptosis
in prevented (Lum et al.,
2005), and, consequently, it is important to understand how links
between autophagy and cell death may instruct cell fates
(Levine and Klionsky, 2004).
As dark82 organs do not regress like their wild-type
counterparts, dark82 animals afford a unique opportunity
to dissect the relationship between histolysis and autophagy. Because the
stimulation of autophagy continued in glands that failed to histolyse, we
suggest that induced autophagy per se is not the `lethal event' mediating
histolysis of this organ. Instead, the epistasis experiments described here
demonstrate that the induction of autophagy lies upstream of, or parallel to,
the apoptosome in this model of histolytic cell death.
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ACKNOWLEDGMENTS |
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