Centro de Biología Molecular CSIC-UAM, Universidad Autónoma de Madrid, Madrid 28049, Spain
* Author for correspondence (e-mail: gmorata{at}cbm.uam.es)
Accepted 6 September 2004
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SUMMARY |
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Key words: Drosophila, Apoptosis, Caspase activity, Wing disc, wg, dpp
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Introduction |
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The molecular and subcellular events that characterize apoptosis are well
known and are conserved among nematodes, insects and vertebrates. A critical
step is the activation of cystein proteases (caspases) that degrade the
cellular substrates, causing cell death. During development, the activity of
caspases is kept in check by the Inhibitor of Apoptosis Proteins (IAP), which
allow survival of the cells. Initiation of apoptosis requires the inhibition
of the IAPs, which can be triggered by various inducing factors (reviewed in
Bergmann et al., 2003).
In Drosophila the apoptotic machinery is similar to that reported
in other organisms. The induction of apoptosis is mediated by the activity of
the genes reaper (rpr), head involution defective
(hid; Wrinkled, W FlyBase) and grim
(White et al., 1994;
Grether et al., 1995
)
(reviewed by Richardson and Kumar,
2002
). Their products inhibit the activity of the Drosophila
IAP1 (DIAP1; thread FlyBase) by promoting the
degradation of the DIAP1 protein (Goyal et
al., 2000
; Wang et al.,
1999
; Ryoo et al.,
2002
; Yoo et al.,
2002
), thus allowing caspase activity. In the absence of these
three genes (as in Df(3L)H99) there is very little apoptosis. One
additional pro-apoptotic factor is Dmp53, the homologue of the
mammalian p53 gene, which responds to radiation-induced DNA damage
(Ollman et al., 2000; Brodsky et al.,
2000
).
Some aspects of normal development in Drosophila require apoptotic
activity. A recent example (Lohman et al., 2002) is the formation of the cleft
that separates the embryonic mandibular and maxillary segments. In this case
the upstream factor is the Hox gene Deformed (Dfd), which
activates the pro-apoptotic gene rpr. However, for many other
developmental processes apoptosis is not a major factor, as most patterns are
formed normally in homozygous Df(3L)H99 embryos
(White et al., 1994;
White and Steller, 1995
).
Regarding the imaginal discs, the application of the TUNEL (TdT-mediated
dUTP-Nick-End Labelling) method, which detects the chromosomal fragmentation
associated with apoptosis (Chen et al.,
1996; Milan et al.,
1997
), reveals that there is apoptotic activity in the eye disc,
necessary to eliminate the interommatidial cells
(Wolff and Ready, 1991
), and
in the genital disc where the pro-apoptotic gene hid is involved in
the rotation of male genitalia (Grether et
al., 1995
). By contrast, there is very little apoptosis in the
wing disc (Milan et al., 1997
;
Ollmann et al., 2000
;
Brodsky et al., 2000
).
In addition to the developmentally regulated apoptosis that occurs
normally, such as rpr induction by Dfd, apoptosis can also
be induced by stress events. For example, although apoptotic levels are low in
the wing disc, X-rays cause a manifold increase in apoptosis (Ollman et al.,
2000; Brodsky et al., 2000).
This inducible apoptosis results from the activation of the Dmp53
gene, which in turn triggers rpr
(Brodsky et al., 2000
).
X-ray-induced apoptosis causes massive cell death; it has been estimated
(Haynie and Bryant, 1977
) that
a dose of 1000R eliminates between 40 and 60% of the cell population.
Interestingly, the adult flies emerging from these treatments present
cuticular patterns of normal size and shape, indicating the existence of
mechanisms that compensate for the loss of cells.
In this report we characterise some aspects of inducible apoptosis. We show that besides caspase activation, the expression of the apoptotic pathway includes the induction of the wg and dpp signalling genes, which may play a role in the mechanism of compensation for cell loss. Preventing caspase activity in apoptotic cells results in gross morphological alterations characterised by excess of proliferation and modifications of normal compartment boundaries. These anomalies are presumably caused by the persistent activity of wg and dpp in caspase-inhibited apoptotic cells.
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Materials and methods |
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To induce marked clones of p35-expressing cells we crossed yw FLP122; act<y+<Gal4 Sp UAS-GFP/SM5-TM6B Tb/TM2 to UAS-p35 flies. Imaginal discs from non-Tb larvae were fixed and stained. Clones of p53-expressing cells are marked by GFP fluorescence.
Stress treatments
Eggs were collected during a 24-hour laying period and different batches
were irradiated or heat shocked after 24, 48, 72 or 96 hours. Mature
third-instar larvae were collected for dissection as they left the medium. In
some experiments adults were allowed to lay eggs for several days in order to
obtain a population of larvae of all ages at the time of the treatment. Larvae
were collected as they matured at different times after the treatment.
Irradiations were carried out in a Philips X-ray machine at the standard dose
of 1500R and the heat shocks were given in incubators at 37°C.
Histochemistry
For antibody staining, imaginal discs were dissected in PBS and fixed with
4% paraformaldehyde in PBS for 25 minutes at room temperature. They were
blocked in PBS, 1% bovine serum albumin (BSA), 0.3% triton X-100 for 1 hour,
incubated with the primary antibody overnight at 4°C, washed four times in
blocking buffer, and incubated with the appropriate fluorescent secondary
antibody for 1 hour at room temperature in the dark. They were then washed and
mounted in Vectashield (Vector Laboratories). The TUNEL assay was performed
following the in-situ cell death detection kit as in Milan et al.
(Milan et al., 1997).
For BrdU staining, larvae were dissected in cold PBS, incubated in BrdU 0.01 mM for 15 minutes at 37°C, washed three times with PBS, fixed for 2 minutes with Carnoy (3 ethanol;1 acetic acid) and washed four times for 5 minutes in PBS. Then they were hydrolysed with HCl 2 N for 10 minutes, washed three times for 10 minutes with PBS and incubated overnight with the primary antibody at 4°C (BrdU labelling and detection Kit I-Roche). The remaining was performed as for standard antibody staining.
The rabbit antibody to cleaved human caspase 3 (Cell Signalling Technology)
has been shown to cross-react with activated Drosophila caspase 3
(Yu et al., 2002). The
monoclonal anti-Wg antibody was obtained from the Hybridoma Center, and the
ß-Gal antibody (rabbit) was purchased from Cappel. Images were taken in
confocal microscopes MicroRadiance (Bio-Rad) or LSM510 META (Zeiss), and
subsequently processed using Adobe Photoshop.
Measurement of compartment size
MetaMorph software, version 5.07, provided by Universal Imaging, was used
to measure compartment size. We obtained the P/A ratio by measuring areas of
posterior GFP-expressing cells versus anterior non-GFP ones in irradiated
(1500R) or non-irradiated (control) hh-Gal4>UAS-p35 UAS-GFP wing
discs.
Preparation of adult cuticles
Adult flies were dissected in water and cut into pieces. They were then
treated with 10% KOH at 95°C for 3-5 minutes to digest internal tissues,
washed with water, rinsed in ethanol and mounted in Euparal. The preparations
were studied and photographed using a Zeiss photomicroscope.
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Results |
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Blocking caspase activity during apoptosis causes developmental aberrations
We interfered with inducible apoptosis by using the baculovirus caspase
inhibitor P35 (Clem et al.,
1991), which inactivates downstream effector caspases (reviewed by
Goyal, 2001
), thus preventing
the death of cells that have entered the apoptotic pathway. It has been shown
to be an efficient caspase suppressor in Drosophila cells
(Hay et al., 1994
). As
expected, considering the low level of apoptosis in normal wing development,
the presence of the P35 protein did not affect wing pattern: using the
Gal4/UAS method (Brand and Perrimon,
1993
) we forced the presence of P35 in the entire wing blade
(nub-Gal4/UAS-p35, ap-Gal4/UAS-p35), or in the posterior (P)
compartment (hh-Gal4/UAS-p35, en-Gal4/UAS-p35). All these
combinations resulted in adult flies with virtually normal wings. The only
variation with respect to the wild-type pattern was the partial elimination of
a cross-vein and of the distal tip of vein 5.
Having established that caspase inhibition was inconsequential in normal wing development, we then studied the effects of inhibiting caspase function after apoptosis induction by X-rays or heat shock. Again, we made use of the Gal4/UAS method to force P35 in wing cells. We selected the combinations of UAS-p35 with the hh-Gal4, en-Gal4 and ap-Gal4 lines. In hh-Gal4>UAS-p35 and en-Gal4>UAS-p35 discs, caspase activity should be inhibited in the P compartments, whereas the anterior (A) compartments serve as a control. In ap-Gal4>UAS-p35 wing disc cell death was prevented in the dorsal, but not in the ventral, compartment. The UAS-GFP construct was added to label the cells containing P35. In all these combinations X-rays or heat shock could induce high apoptotic levels, but caspase activity was effectively suppressed in the compartments containing P35 (Fig. 2).
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The treated discs presented several other anomalies, which were probably caused by the abnormal Wg and Dpp signalling. To conduct a detailed study, we collected a large number of hh-Gal4>UAS-p35 UAS-GFP wing discs from larvae X-rayed in the first or the second instar. All the discs showed very abnormal morphology in the P compartments. A significant variation is that the P compartments were larger than normal (which can be seen in Fig. 3D,G,I). Using the GFP marker to delineate the borders, we compared the relative size of the A and P compartments in control and in treated discs. In a sample (n=10) of control discs (non-irradiated hh-Gal4>UAS-p35 UAS-GFP discs) that P/A size average ratio was 0.83. In an unselected sample (n=33) of irradiated discs of the same genotype, the P/A ratio was 1.19, reaching 1.8 in some cases.
The larger size of P compartments of irradiated hh-Gal4>UAS-p35 UAS-GFP discs suggested that caspase inhibition during apoptosis gives rise to an increase in cell proliferation rate. We tested this possibility by comparing the levels of BrdU incorporation and of phospho-histone 3 (PH3) activity in the A and P compartments. The results indicated that caspase inhibition in apoptotic cells causes an increase in cell division (Fig. 4): in discs fixed 4 hours after irradiation, only one out of seven showed greater BrdU incorporation in the P compartment, but in those fixed at +24 hours the ratio was 5/11; for the +48-hour series, the ratio was 5/7; and for +72 hours it was 15/16. None of the controls showed greater incorporation in the P compartment. One additional observation is that in the majority of these discs the increase of BrdU incorporation or of PH3 staining was not restricted to the P compartment but also affected A compartment cells close to the AP border (Fig. 4). This suggests that cells in the P compartment are producing proliferation signal(s) that not only diffuse in the P compartment but can also travel across the AP border.
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Discussion |
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The production and emission by the apoptotic cells of the secreted Wg and
Dpp signals is probably responsible for the non-autonomous effect on
proliferation. These two signals have been shown to control pattern and growth
in imaginal discs (Neuman and Cohen, 1996;
Burke and Basler, 1996;
Martin-Castellanos and Edgar,
2002
; Martín et al.,
2004
) and therefore may provide a proliferative signal. As
suggested independently by Ryoo et al.
(Ryoo et al., 2004
) and by Hu
et al. (Hu et al., 2004
), this
mitogenic effect may be responsible for the additional proliferation necessary
to compensate for the elimination of apoptotic cells. This provides an
explanation for the observation that high levels of induced apoptosis were
compatible with final structures of normal size. It might also have a role in
generating additional proliferation and signalling during regeneration
processes (Bryant, 1971
) in
which the apoptotic programme is likely to be involved. The finding that the
Hh pathway is activated during imaginal disc regeneration
(Gibson and Schubiger, 1999
)
is also consistent with this possibility.
The second set of findings concerns the overall response of compartments to
caspase inhibition during apoptosis. Our experiments permitted the
discrimination of two different aspects of the apoptotic programme: the
initiation and execution of apoptosis. By combining pro-apoptotic treatments
(X-rays or heat shock) with caspase inhibition we can uncouple the apoptotic
programme and cell death. A particularly interesting consequence of removing
death from the apoptotic programme is that it causes a permanent developmental
defect (Figs 2,
3). The perdurance of the
apoptotic cells generates an abnormal and self-maintained epigenetic
programme. We believe that the reason for this phenomenon lies in the finding
that these cells generate the secreted Wg and Dpp signals, which are primary
pattern determinants in imaginal discs (reviewed in
Lawrence and Struhl, 1996),
although it is conceivable that they may activate other signals as well. The
continuous production and emission of these signals by caspase-inhibited cells
is expected to produce developmental aberrations and growth defects,
especially if, as we show in Fig.
6, apoptotic cells can carry these signals into neighbouring
compartments.
We noted that some of the alterations observed after cell death inhibition
changes of cell size and shape, invasiveness and excess of
proliferation resembled those of tumorous cells of vertebrates. As
apoptosis inhibition is frequently associated with tumour formation
(Hanahan and Weinberg, 2000),
it could be speculated that some of the cellular transformations leading to
tumorogenesis might be provoked not by a series of individual somatic
mutations (Hanahan and Weinberg,
2000
) but by the acquisition of an abnormal epigenetic programme
triggered by stress events in conditions in which caspase activity is
compromised. They could also be caused by the normal developmentally regulated
apoptosis when caspase function is defective. It is known that many human
cancers are associated with inappropriate activity of the Hh or the Wnt
pathway (Bienz and Clevers,
2000
; Taipale and Beachy,
2001
). These two pathways are misexpressed in apoptotic
caspase-inhibited cells.
Besides, a number of animal viruses are known to promote oncogenic
transformations in host mammalian cells (reviewed by
Moore and Chang, 2003). As
some viruses encode caspase inhibitors to prevent death of the host cells
and the baculovirus P35 protein is a typical case it is
possible that some virus infections provoke a process similar to the one we
report here: the initiation of the apoptotic pathway in host cells coupled
with inhibition of cell death. This may produce abnormal signalling of growth
factors, which may result in the acquisition of a permanent and abnormal
epigenetic programme by groups of cells.
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ACKNOWLEDGMENTS |
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