|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 25 February 2009
doi: 10.1242/dev.034017
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Centro de Biología Molecular CSIC-UAM, Universidad Autónoma de Madrid, Madrid, Spain.
* Author for correspondence (e-mail: gmorata{at}cbm.uam.es)
Accepted 3 February 2009
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Apoptosis, Compensatory proliferation, Hyperplastic overgrowths, JNK, dpp, wg
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
) or the
adult leg articulations (Manjon et al.,
2007). This type of apoptosis has to be regulated during
development. (2) The apoptosis involved in the elimination of cells that have
been damaged or injured during development. This is a stress response
function, which is not developmentally regulated in a strict sense, as it does
not occur normally unless the organism is subjected to tissue damage or to
various forms of physiological stress
(Ollmann et al., 2000;
Brodsky et al., 2000).
The molecular/genetic mechanism implicated in Drosophila apoptosis
is well known: after an apoptotic stimulus, one or several of the
pro-apoptotic genes reaper, head involution defective (hid;
Wrinkled - FlyBase), grim or sickle (skl)
are activated and, in turn, their products inactivate that of the
diap1 gene (thread - FlyBase)
(Goyal et al., 2000;
Ryoo et al., 2002;
Wang et al., 1999;
Yoo et al., 2002), whose
function is essential for cell viability. The loss of DIAP1 activity allows
the catalytic activation of the caspases, which are responsible for
dismantling the cell substrates, causing the death of the cells.
The imaginal discs of Drosophila provide a convenient system in
which to study the properties of apoptotic cells. The wing disc shows very
little apoptosis during development (Milan
et al., 1997), but responds with elevated apoptotic levels after
irradiation or heat-shock treatments
(Perez-Garijo et al., 2004).
It has been estimated that the proportion of cell death after such treatments
is greater than 50% (Haynie and Bryant,
1977; Pérez-Garijo et
al., 2004). In spite of this massive cell elimination, the disc
recovers and eventually forms adult structures of normal size. The implication
is that surviving cells undergo additional proliferation to compensate for the
cell loss.
Several reports (Huh et al.,
2004; Perez-Garijo et al.,
2004; Ryoo et al.,
2004) described unexpected properties of apoptotic cells that
suggested a mechanism for compensatory proliferation. In those experiments
apoptosis was induced by various stimuli (forced activation of pro-apoptotic
genes or stress treatments such as X-rays or heat shock), but the death of
apoptotic cells was prevented by the presence of the baculovirus caspase
inhibitor P35 (Hay et al.,
1994). Under these conditions, these `undead' cells remain alive,
while retaining all the features of apoptosis (reviewed by
Martin et al., 2009).
Those experiments reported two crucial observations. The first was that
undead cells appear to stimulate the proliferation of non-apoptotic cells in
their vicinity. The second was that undead cells exhibit ectopic expression of
the dpp and wg signalling genes, which are known to act as
mitogens in the imaginal discs (Burke and
Basler, 1996; Giraldez and
Cohen, 2003). Moreover, Ryoo et al. and Pérez-Garijo et al.
provided evidence that induction of dpp/wg expression also
occurs in normal (e.g. not containing P35) apoptotic cells
(Ryoo et al., 2004;
Pérez-Garijo et al.,
2004).
These two observations suggested a mechanism for compensatory proliferation: before dying, the apoptotic cells secrete Wg and Dpp, which stimulate the proliferation of non-apoptotic cells located nearby that would restore the normal size of the disc. It is clear from this definition that the additional proliferation needed for size compensation would be caused by the adventitious activation of the Dpp and Wg pathways in the proximity of the apoptotic cells, and not by the normal Dpp and Wg activities of the disc. These are of course required for the normal growth of the disc, but would not be involved in compensatory proliferation.
It was also observed that keeping apoptotic cells alive with P35 results in
abnormal development of the affected compartments. This was especially clear
in discs in which the posterior compartment contained P35 but the anterior one
did not; the anterior compartment recovered after massive apoptosis to form a
structure of normal size and pattern, but the posterior compartment grew in
excess and showed morphological aberrations
(Ryoo et al., 2004;
Perez-Garijo et al., 2004).
Thus, although there is normal compensatory proliferation in the anterior
compartment, the presence of undead cells in the posterior compartment gives
rise to hyperplastic overgrowths. Measurements of cell division levels
(Pérez-Garijo et al.,
2004) indicated abnormally high proliferation rates in the
posterior compartments, which would account for the excess of growth.
The discovery that apoptotic cells emit Dpp and Wg signals also suggested
an explanation for these overgrowths. As undead cells retain dpp and
wg expression indefinitely after the initiation of apoptosis
(Perez-Garijo et al., 2004;
Martin et al., 2009), it
appears likely that the excess of growth is due to the continuous supply of
these signals. In support of this, Ryoo et al. showed that the activity of the
Wg pathway contributes to the developmental anomalies induced by undead cells
(Ryoo et al., 2004).
In the experiments reported here, we aimed to test the role of the Dpp and Wg signals emitted by apoptotic cells in the following two processes: (1) the compensatory proliferation that occurs after massive apoptosis in response to irradiation; and (2) the formation of hyperplastic overgrowths when apoptotic (undead) cells are kept alive with P35.
We wish to point out that we addressed the second issue in a previous paper
(Pérez-Garijo et al.,
2005). We reported that undead cells lacking wg but
possessing dpp activity give rise to neoplastic tumours in the wing
disc. Those clones showed higher proliferation rates than the surrounding
cells and produced massive overgrowths. We interpreted the overgrowths as
being caused by unbalanced signalling from undead cells: the mitogenic
influence of Dpp was not counteracted by the growth repressing function of the
Wg signal. However, subsequent work showed that the wg mutant
chromosome used in those experiments inadvertently contained a mutation at the
lethal giant larvae (lgl) gene. lgl mutations are
known to produce neoplastic tumours (reviewed by
Hariharan and Bilder, 2006),
suggesting that the lgl mutation may be the cause of the appearance
of tumours in our experiments. It also indicated that our previous
interpretation of the appearance of tumours was incorrect. The role of the
lgl mutation in the formation of those tumours is presently being
studied.
The results that we present in this report indicate that the ectopic Dpp and Wg signals do not mediate compensatory proliferation, because it can occur in compartments in which they cannot be produced. However, ectopic Dpp and Wg are major factors involved in the appearance of the hyperplastic overgrowths caused by keeping apoptotic cells alive. We also present evidence indicating that the activation of dpp and wg in apoptotic cells, and hence the formation of overgrowths, is caused by a non-apoptotic role of the JNK pathway, which is itself activated by the irradiation.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
), UAS-FLP (Bloomington Stock Center), UAS-GFP,
UAS-hepAct (Bloomington Stock Center) and UAS-shmi
Dpp2 (Haley at el.,
2008). Mutations: the allele dppd12 eliminates
adult dpp disc activity (St.
Johnston et al., 1990); the wgRF is a null
wg mutation (gift of Gary Struhl, Columbia University, New York, NY,
USA). Mutant wgRF embryos exhibit a very strong phenotype
identical to that reported for null wg alleles. The
droncl29 mutation effectively inhibits apoptosis
(Xu et al., 2005). Other
mutations, dpp-lacZ (P{PZ}dpp10638),
arm-lacZ (Bloomington Stock Center), puc-lacZ, crinkled
(ck), were used as markers. The Minute allele used in the experiments
was M(2L)24F (FlyBase). Flies with posterior compartment mutant for dppd12 were of the genotype dppd12 ck FRT40A/M(2L)24F ubi-GFP FRT40A; hh-Gal4/UAS-Flp. Similarly flies with posterior compartment mutant for wg were wgRF FRT40A/M(2L)24F ubi-GFP FRT40A; hh-Gal4 UAS-Flp. To generate clones mutant for both dpp and wg we built a recombinant dppd12 wgRF FRT40A chromosome.
Apoptosis induction by irradiation
Larvae arising in the crosses described above were irradiated with 1500
rads at 48-72 hours after egg laying, which corresponds to the second larval
period. They were allowed to grow, then were dissected and wing discs
extracted when they reached the wandering or prepupal stage, usually 72-96
hours after the irradiation.
Histochemistry
Fixation and immunohistochemistry of imaginal discs were carried out as
described previously (Aldaz et al.,
2003). The following antibodies were used: anti-casp3 (Cell
Signaling), anti-Wg (Hybridoma Bank), anti-Dronc, anti-Hid (gifts of Hermann
Steller, Rockefeller University, New York, NY, USA), anti-PH3 (Upstate),
rabbit anti-β-Gal (Cappel). Secondary antibodies used were purchased from
Jackson ImmunoResearch.
For the double in situ hybridisation/antibody staining, we followed the
protocol of Goto and Hayashi (Goto and
Hayashi, 1997), with some modifications. After fixation, larvae
were washed three times and stained with primary antibody overnight at 4°C
in PBTH (DEPC-treated PBS, 0.1% Tween 20, 50 µg/ml heparin, 10 µg/ml
salmon sperm) with 0.26 U/ml RNAse inhibitor (Roche). Incubation with
secondary antibody was carried in PBTH for 4 hours at 4°C before fixation
for 20 minutes in 4% paraformaldehyde. After fixation, larvae were washed for
15 minutes with PBT (1xPBS, 0.1% Tween) + HSS (0.02 M Tris HCl pH 8.2,
0.25 mM EDTA, 0.3 M NaCl, 1xDenhardts, 50% formamide) 1:1, prehybridized
for 60 minutes in HSS at 55°C and incubated with the probe overnight at
55°C. After incubation the discs were washed three times with HSS at
55°C and three times with PBT at room temperature before incubation with
anti-DIG (Roche Diagnostics; diluted 1:2000) overnight at 4°C. Staining
was done with FastRed (Boehringer Mannheim).
Measurement of the P:A size ratio
To measure the size of anterior and posterior compartments, we used the
WCIF ImageJ Software. As the posterior compartments were labelled with GFP, we
measured the size of the P compartment (in pixels) and also that of the entire
disc. The P:A ratio was calculated with Microsoft Excel. To measure the
relative size of the spalt domain in the experiments involving JNK
activation, we used the same method, measuring the size of the spalt
domain in comparison with that of the entire disc.
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
In the compensatory proliferation experiments the rationale was to induce apoptosis by X-rays in cells that were defective in either dpp and/or wg function, so that they could not bring about ectopic Dpp/Wg signalling. The region covered by the dpp or wg defective cells needed to be sufficiently large, so that the possible effect on size could easily be noticed; we therefore performed our experiments in the posterior (P) compartment.
|
;
Giraldez and Cohen, 2003), it
was important to ensure that the normal dpp and wg
expression domains were not altered, or that the alterations introduced were
compatible with the growth of the disc. The elimination of dpp activity in the P compartment was not expected to have a developmental effect, as the Dpp signal is synthesised in the A compartment, from where it diffuses to A and P compartment cells. As illustrated in Fig. 1J, this is the case: the P compartment:A compartment size ratio is not affected by the lack of dpp. However, we observed that the lack of wg causes a diminution of the P compartment size (Fig. 1D-J).
Using mitotic recombination methods, we have generated wing discs in which
the P compartment is entirely mutant for dppd12, which
eliminates adult dpp function (St
Johnston et al., 1990). To abolish dpp activity in the P
compartment, we have also used a transgenic strain carrying the
UAS-shmiR-dpp2 construct, which has been shown to degrade the mRNA of
dpp (Haley et al.,
2008).
To remove wg function in the P compartment by the same system, we used the wgRF mutation, which behaves as a null allele: wgRF cells show no trace of anti-Wg staining (see Fig. 1F,I) and wgRF embryos display a strong wg phenotype (not shown). We also tested the possibility of redundant roles of dpp and wg by generating discs in which the P compartment was deficient for both gene activities.
The experiments to study the role of dpp and wg in the hyperplastic overgrowths were very similar to those above, but adding the UAS-p35 transgene to the P compartment cells. The elimination of dpp or wg activity in the undead cells allowed the contribution of either signal to the overgrowths to be tested.
The wing disc exhibits compensatory proliferation in the absence of Dpp and Wg signalling
To test the compensatory response in dpp- compartments,
we used a combination of the FRT/FLP and Minute methods
(Martin and Morata, 2006;
Foronda et al., 2008) to generate discs in which virtually all of the cells in
the P compartment were homozygous for dppd12. In discs of
genotype dppd12 ck FRT40A/M(2)24F ubi-GFP FRT40A; hh-Gal4
UAS-Flp, the high levels of Flipase generated by the hh-Gal4
driver would induce FRT-mediated mitotic recombination in many cells in the
posterior compartment. The dpp- M+ clones will
have a proliferation advantage (Morata and
Ripoll, 1975) and will eventually fill the posterior compartment
(see Fig. S1 in the supplementary material). The conversion of the affected
compartment from M/+ to M+ occurs early in the
development of the disc (Martín and
Morata, 2006). The dpp- M+ cells
are identified in the disc by loss of the ubi-GFP transgene, and in
the adult wing by homozygosity for the cuticular marker crinkled
(ck). One advantage of using the hh-Gal4 line is that
dpp is eliminated only in the P compartment, thus the A compartment
serves as a control. We also used this line in the experiments to suppress
dpp activity by RNA interference with the UAS-shmiR-dpp2
construct (Haley et al.,
2008).
|
). Irradiated second instar larvae were allowed to develop
until the wandering/prepupal stage before extracting imaginal discs for fixing
and staining. Then, the size ratio of P and A compartments was measured and
compared with that of non-irradiated controls.
If compensatory proliferation after apoptosis is inhibited in the
dpp- P compartments, the P:A ratio should decrease
significantly. Inspection by compound microscopy of discs and adult wings from
irradiated and from non-irradiated larvae did not indicate any significant
difference in the P:A size ratio of dpp+ and
dpp- compartments (Fig.
1A,C). Nevertheless, we carried out a careful measurement of the
sizes of irradiated compartments and compared the P:A ratio with that of
non-irradiated controls. The results are summarised in
Fig. 1J and Table S1 in the
supplementary material, and show that the P:A ratio is similar in control and
irradiated discs. These results clearly indicate that the P compartment is
able to compensate for growth in the absence of dpp function. This is
also supported by the results obtained with discs of the genotype
hh-Gal4>UAS-shmiR-dpp2 in which dpp transcripts are
degraded, resulting in at least a 75% reduction of dpp activity
(Haley et al., 2008). In these
discs, the P:A ratio is also unaffected by the irradiation
(Fig. 1J; see also Table S1 in
the supplementary material).
Next, we studied the compensatory response to X-rays of compartments in which wg activity was eliminated. The protocol was the same as that used in the dppd12 experiment but in this case the M+ clones were homozygous for the wgRF mutation. These posterior compartments show no sign of wg activity (Fig. 1D,F) and are also smaller than normal P compartments (Fig. 1J). The results are illustrated in Fig. 1D,F,J and in Table S1 in the supplementary material. Although the size of P compartments that are entirely wgRF is smaller than that of normal ones, the irradiation does not affect the P:A ratio, indicating that there is compensatory proliferation.
As there was the possibility of redundant functions of dpp and wg, we also studied the apoptotic response of P compartments defective in both dpp and wg, by generating P compartments doubly mutant for dppd12 and wgRF. As illustrated in Fig. 1G,I,J and in Table S1 in the supplementary material, the P:A ratio remains unaltered after irradiation, indicating that there is size compensation.
The overall conclusion from all of the experiments described above is that the elimination of dpp and/or wg activity in the P compartment does not prevent compensatory proliferation after radiation-induced apoptosis. The implication of this result is that the ectopic activation of wg and dpp observed in apoptotic cells does not play a significant role in the compensatory proliferation process.
The Dpp and Wg signals are necessary for the hyperplastic overgrowths caused by undead cells
It is known (Perez-Garijo et al.,
2004; Kondo et al.,
2006; Wells et al.,
2006) that preventing the death of apoptotic cells by inhibiting
caspase function causes overgrowths and pattern abnormalities. After
stress-induced apoptosis (Perez-Garijo et
al., 2004) in hh>p35 discs, the A compartment exhibits
high apoptotic levels for about 24 hours, but eventually recovers and forms
structures of normal size and pattern. By contrast, the P compartment
(Fig. 2A, see Table S2 in the
supplementary material) becomes larger than normal and shows aberrant
morphology.
The principal difference between the A and the P compartments in these
experiments is that apoptotic cells are kept alive in the P compartment. These
cells can be identified because they express the pro-apoptotic gene
hid, as well as other apoptotic markers, such as Dronc and Drice
(Martin et al., 2009). In
addition, they frequently show ectopic dpp and wg
expression, which persist during the rest of the development. Dpp and Wg
function are pattern organizers as well as mitogenic signals in the wing disc
(reviewed by Lawrence and Struhl,
1996), suggesting that their inappropriate activities might be
responsible for the hyperplastic overgrowths.
To test the role of dpp, we made P compartments containing P35 that were also homozygous for the dppd12 mutation. The actual genotype of the discs was dppd12 ck FRT40A/M(2)24F ubi-GFP FRT40A; hh-Gal4 UAS-Flp/UAS-p35.
|
), in hh>p35 dppd12 discs these
alterations were much less abundant, and the size and pattern of the P
compartments were normalized (Fig.
2B). The P:A size ratio (Fig.
2D; Table S2 in the supplementary material) was similar to that of
normal discs. This shows that the Dpp signal is a major factor in the
production of the hyperplastic overgrowths in hh>p35 discs. It is
worth noting that we use the dppd12 allele, and that it
results in the abolishment of the overgrowths caused by apoptotic
dpp+ undead cells, indicating that the
dppd12 allele eliminates the growth-inducing capacity of
apoptotic cells. The requirement for wg was tested by irradiating discs of genotype wgRFFRT40A/M(2)24F ubi-GFP FRT40A; hh-Gal4 UAS FLP/UAS-p35, in which the P compartment was defective in wg activity. The result was that these discs are of normal aspect; the P compartments showed very few morphological alterations and the P:A ratio was similar to that of discs in which the P compartment was wgRF (compare Fig. 1J with Fig. 2D, see also Table S2 in the supplementary material). This result indicates that wg is also required for the appearance of the overgrowths.
The finding that the elimination of either dpp or wg results in almost complete abolishment of the hyperplastic overgrowths was an unanticipated result. It suggested that both genes are necessary and that there is a mutual requirement. Therefore, we examined wg expression in compartments containing undead cells that are mutant for dpp, and conversely dpp expression in compartments with undead cells mutant for wg. The results are shown in Fig. 3 and indicate clearly that the ectopic expression patterns of wg and dpp are mutually dependent: the function of either gene is required for the activation of the other. These results explain why the elimination of either gene prevents hyperplastic overgrowths, but at the same time it poses the problem of their mutual requirement at the transcriptional level.
Role of the JNK pathway in the activation of dpp and wg
The activation of dpp and wg is one of the features of
apoptotic cells but the mechanism of activation is not known. A crucial factor
in the establishment of apoptosis in Drosophila is the JNK pathway.
Its activity leads to apoptosis, whereas in absence of JNK function apoptosis
is much reduced (Adachi-Yamada et al.,
1999; McEwen and Peifer,
2005). In addition, there is no ectopic dpp or
wg activation in undead cells generated by rpr induction
(Ryoo et al., 2004).
The JNK pathway also has other roles in development that are not connected
with apoptosis, such as conferring epithelial cells with the ability to
migrate during dorsal closure and disc fusion
(Glise et al., 1995;
Martin-Blanco et al., 2000),
or its involvement with dpp activation in the border cells during
embryonic dorsal closure (Glise and
Noselli, 1997; Hou et al.,
1997; Riesgo-Escovar and
Hafen, 1997).
We have studied some aspects of JNK activity after X-ray-induced apoptosis,
in particular its possible role in the activation of dpp and
wg. First, we checked whether the normal dose of X-rays used in our
experiments induced JNK activity. Using the puc-lacZ insert to
monitor JNK function (Martin-Blanco et
al., 1998), we examined puc levels 8 hours after
irradiation. The results are illustrated in
Fig. 4A-C. In non-irradiated
discs there was no expression of puc, except in a band of cells in
the proximal-thoracic region (Fig.
4A), as was previously known
(Martin-Blanco et al., 2000).
In irradiated discs there was an overall increase of JNK activity in the rest
of the disc (Fig. 4B), which
was especially clear in the wing pouch and was associated with high apoptotic
levels (Fig. 4C). This
activation was not unexpected as JNK mediates most or all stress-induced
apoptosis in Drosophila (McEwen
and Peifer, 2005; Luo et al.,
2007). We also observed that undead cells in irradiated
hh>UAS-p35 discs co-express JNK and wg even 72 hours
after irradiation (Fig.
4D-F).
Because JNK becomes active during apoptosis but also has other
non-apoptotic functions, there was the possibility that the activation of
dpp and wg in apoptotic cells might be independent of
apoptosis. To test this possibility, we made use of the
UAS-hepact construct
(Adachi-Yamada et al., 1999) to
force JNK activity in dronc mutant discs in which apoptosis is
greatly reduced (Daish et al.,
2004; Chew et al.,
2004; Xu et al.,
2005). We have confirmed that droncI29 mutant
discs show a very low apoptotic response to X-rays. The line
spalt-Gal4 drives expression in the wing pouch
(Fig. 5A) and, when directing
hepact in dronc+ discs, induces high
levels of caspase and wg activity in the spalt domain.
Moreover, in spalt>hepact droncI29 discs the
amount of apoptosis is much reduced (see Fig. S2 in the supplementary
material).
The significant result is shown in Fig. 5C-E: wing discs of genotype spalt>hepact; droncI29 exhibit ectopic activation of both wg and dpp in the region of the wing pouch corresponding to the spalt domain. The expression domains of wg and dpp appear to be co-extensive in most cells of the spalt domain. This experiment strongly suggests that the expression of dpp and wg in apoptotic cells is not a consequence of apoptosis, but of the activation of JNK. The mechanism by which the JNK pathway induces wg and dpp is not known.
|
|
It follows from these two observations that ectopic JNK activity should be able to cause excess growth in the absence of apoptosis. We tested this by examining the size and morphology of discs of genotype spalt>hepact GFP dronc-. The results are illustrated in Fig. 6 and see Table S3 in the supplementary material. In these discs there is a clear overgrowth in the spalt domain in comparison with the control spalt>GFP droncI29 discs. These overgrowths are associated with folding and pattern abnormalities.
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Perez-Garijo et al., 2004;
Huh et al., 2004), led to the
model that compensatory proliferation is caused by the mitogenic activity of
the ectopic Dpp and Wg signals emitted by apoptotic cells.
In irradiated discs, the ectopic Dpp/Wg signalling generated by the
apoptotic cells is superimposed on the normal Dpp/Wg signalling. The latter is
essential for the normal growth of the wing compartments; in
dppd12 homozygous discs the wings are reduced to a
rudiment (St Johnston et al.,
1990), and as we show here
(Fig. 1F,I) the lack of
wg activity results in smaller compartments. Our experiments have
tested the role of the ectopic Dpp/Wg signalling in size restoration of
irradiated discs, that is, the contribution of the apoptotic cells to the
process.
We have examined the ability of P compartments to compensate growth in conditions in which apoptotic cells can produce neither the Dpp nor the Wg signal, or are defective in both signals. The results indicate that the model of compensatory proliferation mentioned above is incorrect. The elimination of ectopic dpp and wg functions in wing discs subjected to massive apoptosis does not impede the restoration of normal size and pattern; in other words, there is compensatory growth without contribution of the Dpp and Wg signals emitted by the apoptotic cells.
Having studied compensatory growth only in P compartments, it is just
conceivable that Dpp and Wg originated by apoptotic cells in the A compartment
might diffuse to the P compartment where they could induce the additional
growth necessary to compensate size. In our view this is very unlikely for two
reasons. (1) The undead apoptotic cells induce additional proliferation only
in their own vicinity (see Ryoo et al.,
2004; Perez-Garijo et al.,
2004). Thus, it is hard to imagine that Dpp/Wg of anterior origin
could have an affect on proliferation extending to the entire posterior
compartment. Moreover, in our experiments the cells are not protected by P35;
they are not undead cells but regular apoptotic cells that die soon after
initiating apoptosis. Therefore the proliferation stimulus they provide would
be very short lived. (2) If the Dpp and Wg of apoptotic origin were able to
travel a long way across compartment borders, it would be expected that the
overgrowths produced by undead cells were not restricted to compartments. For
example, in irradiated hh>p35 discs or in en>hid + p35
(and other similar genotypes), in which undead cells belong to the P
compartment, the A compartment should also overgrow, stimulated by the Dpp and
Wg of posterior origin. In all cases reported
(Ryoo et al., 2004;
Perez-Garijo et al., 2004;
Kondo et al., 2006)
(Fig. 2A;
Fig. 3A), the effect is
essentially restricted to the posterior compartment.
|
; Burke and Basler,
1996) and an excess of Dpp activity causes additional growth
(Martín-Castellanos and Edgar,
2002; Martín et al.,
2004). Moreover, in the experiments in which apoptotic cells are
protected with P35, we find that the absence of Dpp and Wg prevents the
appearance of overgrowths, strongly suggesting that these signals are
responsible for the additional growth associated with apoptotic cells.
We outline our ideas for compensatory growth in
Fig. 7A. We believe that it
does not require any special mechanism involving the participation of
apoptotic cells. It is the normal process that regulates compartment size that
is responsible for restoring normal size after massive apoptosis. It has been
shown recently (Martín and Morata,
2006) that A and P compartments are autonomous units of size
control in the wing disc, i.e. A and P compartments grow autonomously until
they reach the final correct size. It has also been shown that the size
control mechanism is highly homeostatic. It can adjust to changes in cell size
and number (Neufeld et al.,
1998; Johnston et al.,
1999), and to differential cell division rates
(de la Cova et al., 2004;
Moreno and Basler, 2004) -
alterations in any of these parameters do not produce changes in the final
compartment size. As stated above, only the overproduction of Dpp results in
breakdown of the size control mechanism.
In our view, the compensatory growth after the loss of cells because of
irradiation (or any other stress event) is another example of the versatility
of the size control mechanism. As illustrated in
Fig. 7A, we propose that the
massive cell death caused by the irradiation would be equivalent to making the
compartment smaller. The irradiated compartment would then restore the correct
size simply by performing some additional division. It would be, in effect, an
overall regeneration process of the entire blastema, which would be achieved
by lengthening the proliferation period, an idea that is supported by
observations such that damage to growing discs results in a prolonged growth
period (Wells et al., 2006).
Even a loss of 50% of the cells can be restored if all of the surviving cells
divided once. In the wing disc, the length of the division period is about
8-12 hours (Garcia-Bellido and Merriam,
1971; Johnston and Sanders,
2003; Neufeld et al.,
1998) and therefore only a short delay may be sufficient to allow
time for recovery. Thus, irradiated discs would, after some delay caused by
the stress, resume growth and the normal control mechanism would stop growth
once compartments have reached the final size
(Martin and Morata, 2006).
Although the ectopic Dpp and Wg signals do not have a role in compensatory proliferation, they are required for the appearance of overgrowths caused by undead cells (Fig. 2). A key difference between undead cells and normal apoptotic cells is that the former persistently express Dpp and Wg (probably as a result of JNK activity, see below). In irradiated posterior compartments that comprise undead and non-apoptotic cells, such as, for example, in irradiated hh>p35 discs, the undead cells keep producing the Dpp and Wg signals from shortly after the irradiation and until the end of the proliferation period of the disc (illustrated in Fig. 7B). Thus, the non-apoptotic cells receive a continuous supply of the Dpp and Wg mitogens from the undead ones. The result is an overgrowth, which is also associated with abnormal cell differentiation. Both additional growth and abnormal differentiation would be expected in these circumstances, as Dpp and Wg are growth inducers as well as morphogens determining cell pattern and differentiation.
The JNK pathway is responsible for the ectopic Dpp/Wg signalling and the hyperplastic overgrowths caused by undead cells
The overall conclusion from the above is that the ectopic Dpp and Wg
signals generated by apoptotic cells are irrelevant for compensatory
proliferation, but are prime factors in the generation of hyperplastic
overgrowths caused by undead cells. The question then is why are dpp
and wg activated in normal apoptotic cells. In our view, their
activity is a collateral effect of the activation of the JNK pathway after an
apoptotic stimulus: 
-irradiation induces JNK activity in the wing disc
and radiation-induced apoptosis depends on JNK activity
(McEwen and Peifer, 2005). As
expected, in our experiments X-irradiation also induced JNK activity
(Fig. 4B).
|
;
McEwen and Peifer, 2005). In
experiments in which cell death is blocked with P35 after apoptosis induction,
the JNK pathway becomes continuously activated in undead cells and appears to
be associated with ectopic wg expression
(Fig. 4E)
(Ryoo et al., 2004;
McEwen and Peifer, 2005). It
is therefore possible that the ectopic activation of dpp and
wg in the apoptotic cells could be a consequence of JNK function,
rather than a consequence of the apoptotic program. Our results strongly
support this view: direct activation of JNK via the
UAS-hepact construct in dronc mutant discs, in
which apoptosis is much reduced, induces wg and dpp
expression (Fig. 5D-F).
Furthermore, these mutant discs show hyperplastic overgrowths in the
spalt domain, where JNK is active
(Fig. 6B,C).
It has been shown that JNK activity induces several cellular functions: the
initiation of the apoptotic program, and also other non-apoptotic functions,
such as the capacity for cell migration
(Glise et al., 1995;
Martín-Blanco et al.,
2000) and the ability to induce dpp
(Glise and Noselli, 1997;
Hou et al., 1997). It is
probable that normal apoptotic cells acquire these other JNK-dependent
properties, but that they die very quickly and so these other functions have
minimal effects. This is different in undead cells because the JNK activity
becomes persistent (Fig. 4E)
and, therefore, they can manifest some or all of the JNK non-apoptotic
functions: these cells can move and invade neighbouring compartments
(Pérez-Garijo et al.,
2004) (Fig. 2A),
and express dpp and wg continuously
(Fig. 3C,
Fig. 4C). In our opinion, it is
the persistent manifestation of these two non-apoptotic JNK-mediated
properties, dpp/wg activation and the induction of cell
migration that causes the hyperplastic overgrowth
(Fig. 7B).
The implication of the Dpp and Wg signals in hyperplastic overgrowths in
Drosophila might have some general significance as their vertebrate
homologues, BMP/TGFβ and Wnt, are known to be involved in the generation
of tumours in mammals (Katoh,
2007; Polakis,
2007). Moreover, inappropriate function of the JNK pathway is also
connected with tumour formation in vertebrates
(Heasley and Han, 2006;
Kennedy and Davis, 2003). We
speculate that situations similar to those described here might also occur in
mammalian cells in which caspase activity is blocked, by virus infections or
other causes. This could result in continuous activation of the JNK pathway
and, subsequently, of BMP/TGFβ and Wnt, and could eventually produce a
tumour.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/7/1169/DC1
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Adachi-Yamada, T., Fujimura-Kamada, K., Nishida, Y. and
Matsumoto, K. (1999). Distorsion of proximodistal information
causes JNK-dependent apoptosis in Drosophila wing.
Nature 400,166
-169.[CrossRef][Medline]
Aldaz, S., Morata, G. and Azpiazu, N. (2003).
The Pax-homeobox gene eyegone is involved in the subdivision of the thorax of
Drosophila. Development
130,4473
-4482.
Brodsky, M. H., Nordstrom, W., Tsang, G., Kwan, E., Rubin, G. M.
and Abrams. J. M. (2000). Drosophila p53 binds a damage
response element at the reaper locus. Cell
101,103
-113.[CrossRef][Medline]
Burke, R. and Basler, K. (1996). Dpp receptors
are autonomously required for cell proliferation in the entire developing
Drosophila wing. Development
122,2261
-2269.[Abstract]
Chew, S. K., Akdemir, F., Chen, P., Lu, W. J., Mills, K., Daish,
T., Kumar, S., Rodriguez, A. and Abrams, J. M. (2004). The
apical caspase dronc governs programmed and unprogrammed cell death in
Drosophila. Dev. Cell 7,897
-907.[CrossRef][Medline]
Daish, T. J., Mills, K. and Kumar, S. (2004).
Drosophila caspase DRONC is required for specific developmental cell
death pathways and stress-induced apoptosis. Dev. Cell
7, 909-915.[CrossRef][Medline]
de la Cova, C., Abril, M., Bellosta, P., Gallant, P. and
Johnston, L. (2004). Drosophila myc regulates organ size by
inducing cell competition. Cell
117,107
-116.[CrossRef][Medline]
Foronda, D., Perez-Garijo, A. and Martin, F. A.
(2009). Dpp of posterior origin patterns the proximal region of
the wing. Mech. Dev. (in press).
Garcia-Bellido, A. and Merriam, J. (1971).
Parameters of the wing imaginal disc development of Drosophila melanogaster.
Dev. Biol. 2461
-87.[CrossRef][Medline]
Giraldez, A. J. and Cohen, S. M. (2003).
Wingless and Notch signaling provide cell survival cues and control cell
proliferation during wing development. Development
130,6533
-6543.
Glise, B. and Noselli, S. (1997). Coupling of
Jun amino-terminal kinase and Decapentaplegic signaling pathways in Drosophila
morphogenesis. Genes Dev.
11,1738
-1747.
Glise, B., Bourbon, H. and Noselli, S. (1995).
hemipterous encodes a novel Drosophila MAP kinase kinase, required for
epithelial cell sheet movement. Cell
83,451
-461.[CrossRef][Medline]
Goto, S. and Hayashi, S. (1997). Specification
of the embryonic limb primordium by graded activity of Decapentaplegic.
Development 124,125
-132.[Abstract]
Goyal, L., McCall, K., Agapite, J., Hartwieg, E. and Steller,
H. (2000). Induction of apoptosis by Drosophila reaper, hid
and grim through inhibition of IAP function. EMBO J.
19,589
-597.[CrossRef][Medline]
Haley, B., Hendrix, D., Trang, V. and Levine. M.
(2008). A simplified miRNA-based gene silencing method for
Drosophila melanogaster. Dev. Biol.
15,482
-490.
Hariharan, I. K. and Bilder, D. (2006).
Regulation of imaginal disc growth by tumor-suppressor genes in Drosophila.
Annu. Rev. Genet. 40,335
-361.[CrossRef][Medline]
Hay, B. A., Wolff, T. and Rubin, G. M. (1994).
Expression of baculovirus P35 prevents cell death in Drosophila.
Development 120,2121
-2129.[Abstract]
Haynie, J. L. and Bryant, P. J. (1977). The
effects of X-rays on the proliferation dynamics of Cells in the imagina wing
disc of Drosophila melanogaster. Roux's Arch.
183,85
-100.[CrossRef]
Heasley, L. E. and Han, S. Y. (2006). JNK
regulation of oncogenesis. Mol. Cells
21,167
-173.[Medline]
Hou, X. S., Goldstein, E. S. and Perrimon, N.
(1997). Drosophila Jun relays the Jun amino-terminal kinase
signal transduction pathway to the Decapentaplegic signal transduction pathway
in regulating epithelial cell sheet movement. Genes
Dev. 11,1728
-1737.
Huh, J. R., Guo, M. and Hay, B. A. (2004).
Compensatory proliferation induced by cell death in the Drosophila wing disc
requires activity of the apical cell death caspase Dronc in a nonapoptotic
role. Curr. Biol. 14,1262
-1266.[CrossRef][Medline]
Johnston, L. A. and Sanders, A. L. (2003).
Wingless promotes cell survival but constrains growth during Drosophila wing
development. Nat. Cell Biol.
5, 827-833.[CrossRef][Medline]
Johnston, L. A., Prober, D. A., Edgar, B. A., Eisenmann, R. N.
and Gallant, P. (1999). Drosophila myc regulates
growth during development. Cell
98 779-790.[CrossRef][Medline]
Katoh, M. (2007). Networking of WNT, FGF,
Notch, BMP, and Hedgehog signaling pathways during carcinogenesis.
Stem Cell Rev. 3,30
-38.[CrossRef][Medline]
Kennedy, N. J. and Davis, R. J. (2003). Role of
JNK in tumor development. Cell Cycle
2, 199-201.[Medline]
Kondo, S., Senoo-Matsuda, N., Hiromi, Y. and Miura, M.
(2006). DRONC coordinates cell death and compensatory
proliferation. Mol. Cell. Biol.
19,7258
-7268.
Lawrence, P. A. and Struhl, G. (1996).
Morphogens, compartments and pattern: lessons from Drosophila.
Cell 85,951
-961.[CrossRef][Medline]
Lohmann, I., McGinnis, N., Bodmer, M. and McGinnis, W.
(2002). The Drosophila Hox gene deformed sculpts head
morphology via direct regulation of the apoptosis activator reaper.
Cell 110,457
-466.[CrossRef][Medline]
Luo, X., Puig, O., Hyun, J., Bohmann, D. and Jasper, H.
(2007). Foxo and Fos regulate the decision between cell death and
survival in response to UV irradiation. EMBO J.
26,380
-390.[CrossRef][Medline]
Manjon, C., Sanchez-Herrero, E. and Suzanne, M.
(2007). Sharp boundaries of Dpp signalling trigger local cell
death required for Drosophila leg morphogenesis. Nat. Cell
Biol. 9,57
-63.[CrossRef][Medline]
Martin, F. A. and Morata, G. (2006).
Compartments and the control of growth in the Drosophila wing
imaginal disc. Development
133,4421
-4426.
Martín, F. A., Pérez-Garijo, A., Moreno, E. and
Morata, G. (2004). The brinker gradient controls wing growth
in Drosophila. Development
131,4921
-4930.
Martin, F. A., Pérez-Garijo, A. and Morata, G.
(2009). Apoptosis in Drosophila: compensatory
proliferation and undead cells. Int. J. Dev. Biol. (in
press).
Martín-Blanco, E., Gampel, A., Ring, J., Virdee, K.,
Kirov, N., Tolkovsky, A. M. and Martinez-Arias, A. (1998).
puckered encodes a phosphatase that mediates a feedback loop regulating JNK
activity during dorsal closure in Drosophila. Genes
Dev. 12,557
-570.
Martin-Blanco, E., Pastor-Pareja, J. C. and Garcia-Bellido,
A. (2000). JNK and decapentaplegic signaling control
adhesiveness and cytoskeleton dynamics during thorax closure in
Drosophila. Proc. Natl. Acad. Sci. USA
97,7888
-7893.
Martín-Castellanos, C. and Edgar, B. A.
(2002). A characterization of the effects of Dpp signaling on
cell growth and proliferation in the Drosophila wing.
Development 129,1003
-1013.[Medline]
McEwen, D. G. and Peifer, M. (2005). Puckered,
a Drosophila MAPK phosphatase, ensures cell viability by antagonizing
JNK-induced apoptosis. Development
132,3935
-3946.
Milan, M., Campuzano, S. and Garcia-Bellido, A.
(1997). Developmental parameters of cell death in the wing disc
of Drosophila. Proc. Natl. Acad. Sci. USA
94,5691
-5696.
Morata, G. and Ripoll, P. (1975). Minutes:
mutants of Drosophila autonomously affecting cell division rate.
Dev. Biol. 42,211
-221.[CrossRef][Medline]
Moreno, E. and Basler, K. (2004). dMyc
transforms cells into super-competitors. Cell
117,117
-129.[CrossRef][Medline]
Neufeld, T. P., de la Cruz, A. F., Johnston, L. A. and Edgar, B.
A. (1998). Coordination of growth and cell division in the
Drosophila wing. Cell
93,1183
-1193.[CrossRef][Medline]
Ollmann, M., Young, L. M., Di Como, C. J., Karim, F., Belvin,
M., Robertson, S., Whittaker, K., Demsky, M., Fisher, W. W., Buchman, A. et
al. (2000). Drosophila p53 is a structural and
functional homolog of the tumor suppressor p53. Cell
101,91
-101.[CrossRef][Medline]
Perez-Garijo, A., Martin, F. A. and Morata, G.
(2004). Caspase inhibition during apoptosis causes abnormal
signalling and developmental aberrations in Drosophila.
Development 131,5591
-5598.
Perez-Garijo, A., Martin, F. A., Struhl, G. and Morata, G.
(2005). Dpp signaling and the induction of neoplastic tumors by
caspase-inhibited apoptotic cells in Drosophila. Proc.
Natl. Acad. Sci. USA 102,17664
-17669.
Polakis, P. (2007). The many ways of Wnt in
cancer. Curr. Opin. Genet. Dev.
17, 45-51.[CrossRef][Medline]
Riesgo-Escovar, J. R. and Hafen, E. (1997).
Drosophila Jun kinase regulates expression of decapentaplegic via the
ETS-domain protein Aop and the AP-1 transcription factor DJun during dorsal
closure. Genes Dev. 11,1717
-1727.
Ryoo, H. D., Bergmann, A., Gonen, H., Ciechanover, A. and
Steller, H. (2002). Regulation of Drosophila IAP1 degradation
and apoptosis by reaper and ubcD1. Nat. Cell Biol.
4, 432-438.[CrossRef][Medline]
Ryoo, H. D., Gorenc, T. and Steller, H. (2004).
Apoptotic cells can induce compensatory cell proliferation through the JNK and
the Wingless signaling pathways. Dev. Cell
7, 491-501.[CrossRef][Medline]
St Johnston, R., Hoffmann, F., Blackman, R., Segal, D.,
Grimaila, R., Padgett, R., Irick, H. and Gelbart, W. (1990).
Molecular organization of the decapentaplegic gene in Drosophila
melanogaster. Genes Dev.
4,1114
-1127.
Wang, S. L., Hawkins, C. J., Yoo, S. J., Muller, H. A. and Hay,
B. A. (1999). The Drosophila caspase inhibitor DIAP1
is essential for cell survival and is negatively regulated by HID.
Cell 98,453
-463.[CrossRef][Medline]
Wells, B. S., Yoshida, E. and Johnston, L.
(2006). Compensatory proliferation in Drosophila
imaginal discs requires requires Dronc-dependent p53 activity.
Curr. Biol. 16,1606
-1615.[CrossRef][Medline]
Xu, D., Li, Y., Arcaro, M., Lackey, M. and Bergmann, A.
(2005). The CARD-carrying caspase Dronc is essential for most,
but not all, developmental cell death in Drosophila.
Development 132,2125
-2134.
Yoo, S. J., Huh, J. R., Muro, I., Yu, H., Wang, L., Wang, S. L.,
Feldman, R. M., Clem, R. J., Muller, H. A. and Hay, B. A.
(2002). Hid, Rpr and Grim negatively regulate DIAP1 levels
through distinct mechanisms. Nat. Cell Biol.
4, 416-424.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  L. A. Baena-Lopez, X. Franch-Marro, and J.-P. Vincent Wingless Promotes Proliferative Growth in a Gradient-Independent Manner Sci. Signal., October 6, 2009; 2(91): ra60 - ra60. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
