European Molecular Biology Laboratory, Meyerhofstraße 1, 69117 Heidelberg, Germany
* Author for correspondence (e-mail: cohen{at}embl.de)
Accepted 1 October 2003
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SUMMARY |
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Key words: Cell proliferation, Apoptosis, Pattern formation
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Introduction |
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Secreted signaling proteins of the Hedgehog, Wingless (Wg) and Dpp families
organize spatial pattern in the imaginal discs of Drosophila. These
signaling proteins have also been implicated in control of imaginal disc
growth. Hedgehog and Dpp activate cell proliferation in a region specific
manner in the imaginal discs (Duman-Scheel
et al., 2002;
Martin-Castellanos and Edgar,
2002
). Dpp signaling also supports cell survival (Moreno et al.,
2002a). Notch and Wg have been implicated in both growth and cell survival
signaling. Reduction of Notch or Wg activity leads to a reduction in the size
of the wing (Couso et al.,
1994
; de Celis and Garcia
Bellido, 1994
; Neumann and
Cohen, 1996a
; Neumann and
Cohen, 1997
; Go et al.,
1998
; Klein and
Martinez-Arias, 1998
; Baonza
and Garcia-Bellido, 1999
; Chen
and Struhl, 1999
; Thompson et
al., 2002
). Ectopic activation of Notch induces high levels of Wg
expression and causes tissue overgrowth during wing development
(Diaz-Benjumea and Cohen,
1995
; Neumann and Cohen,
1996b
; Go et al.,
1998
). Toward the end of the third larval instar these signals
cause cells near the dorsoventral (DV) boundary to exit proliferation before
cells in other regions of the disc do so
(Phillips and Whittle, 1993
;
Johnston and Edgar, 1998
).
Notch and Wg regulate expression of each other at the DV boundary of the
wing disc (Rulifson and Blair,
1995; Diaz-Benjumea and Cohen,
1995
; de Celis et al.,
1996
; Neumann and Cohen,
1996b
; Micchelli et al.,
1997
; de Celis and Bray,
1997
). Previous reports have not adequately distinguished the
individual contributions of the two pathways to control of cell proliferation
and survival. In this report, we examine the effects of reducing Wg activity
in ways that do not compromise the signaling centers. We compare the effects
of ectopically expressing Wg with those of ectopic activation of the Notch
pathway under conditions where Wg cannot be activated. We present evidence
that Wg serves as both a proliferation signal and a cell survival signal, and
that it does so to different degrees in different regions of the wing pouch.
These effects can be distinguished from the effects of Wg on cell fate
specification outside the wing pouch. Notch acts through a signal relay
mechanism to stimulate cell proliferation non-autonomously. Some of the
effects of Notch can be attributed to the induction of Wg expression; however,
we present evidence that Notch also acts through another relay signal. We
suggest that Wg and Notch act synergistically to control tissue growth and
cell survival.
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Materials and methods |
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Genotypes used to generate mosaic analysis
hsFLP122/+; FRT42D arm-lacZ/FRT42D Ubi-GFP
(Fig. 1C);
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hsFLP122/+; FRT42D arrow2/FRT42D Ubi-GFP; pucE69/+ (Fig. 3);
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hsFLP122/+; FRT42D arm-lacZ/FRT42D hsP(myc)
M(2)531 (Fig.
5D);
hsFLP122/+; FRT42D arrow2/FRT42D
hsP(myc) M(2)531
(Fig. 5E);
hsFLP122/+; FRT42D arrow2/FRT42D Ubi-GFP; FRT82B pygoS28/FRT82B arm-lacZ (Fig. 5H-K);
hsFLP122 UAS-CD8-GFP/+; FRT42D arm-lacZ/FRT42D Gal80; UAS-p35/tubGal4 (Fig. 4A, Fig. 7F);
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hsFLP122 UAS-CD8-GFP/+; FRT42D arm-lacZ/FRT42D Gal80; UAS-puc/tubGal4 (Fig. 4F);
hsFLP122 UAS-CD8-GFP/+; FRT42D arrow2/FRT42D Gal80; UAS-puc/tubGal4 (Fig. 4H-J);
hsFLP122 UAS-CD8-GFP/+; FRT40A arm-lacZ/FRT40A Gal80; UAS-NotchIntra/tubGal4 (Fig. 7B,E; Fig. 8B; Fig. 9A,E);
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hsFLP122 UAS-CD8-GFP/+; FRT40A arm-lacZ/FRT40D Gal80; +/tubGal4 (Fig. 7A,E; Fig. 8A); and
hsFLP122 UAS-CD8-GFP/+; tubGal4/+; FRT82B AxinS044230 /FRT arm-lacZ (Fig. 7D,E; Fig. 8C).
Measurement of clone and compartment size
For area measurements on compartment size, larvae expressing the transgenes
were grown in the same vial with the control larvae expressing only the Gal4
driver. An average of 10 female larvae of each genotype were analyzed 120
hours after egg laying (AEL). Larvae were genotyped by antibody staining.
Using Wg and Nubbin expression as landmarks, the area of the dorsal (D) versus
ventral (V) wing blade was measured using NIH image software. En-Gal4 UASGFP
was used to distinguish anterior (A) and posterior (P) compartments. Crosses
with UAS-NotumGT and UAS-DFz2-GPI were kept at 22°C.
For control, arrow and pygopus mutant clones, embryos were collected for 12 hours. Larvae were heat-shocked at 60±6 hours AEL, or 66±6 hours, for 20 minutes at 37°C, and dissected at 116±6 hours AEL. For Minute+ (M+) clones, embryos were collected for 6-8 hours. The wild-type and the mutant larvae were combined in the same vial 24 hours AEL to provide identical growth conditions. Larvae were heat-shocked at 76±4 hours AEL for 20 minutes at 37°C, and dissected at 160±4 hours AEL. Mutant and wild-type clone areas were measured from confocal images using NIH image. The size of wild-type clones was quantified in the apical surface, whereas in the arrow clones it was quantified in the basal region, were most of the arrow mutant cells were found; the section used for the quantification was that of maximal clone area. In M+ experiments, some clones were obviously fused; however, there was usually a clear border between them, which allowed an approximation of the contribution of the separate clones. The presence of a few heterozygous cells in the middle of a clone was indicative of two fused clones, and thus these were quantified as two separate clones.
To measure tissue overgrowth due to expression of NotchIntra in
wild-type, wgcx4 or vg83b27R
mutant cells, we generated positively marked clones using the Gal80 system
(Lee and Luo, 1999). Embryos
were collected for 8 hours. Larvae were heat-shocked at 48±4 hours AEL
for 20 minutes at 37°C, and dissected at 118±4 hours AEL. Clones
were scored for obvious tissue overgrowth, including alteration of shape in
the surrounding tissue (bulging), compared with clones that did not produce
obvious overgrowth. Three different areas of the wing disc were considered
separately: (1) distal, adjacent to the DV boundary defined by the Wg staining
(3-5 cell diameters); (2) medial, between the proximal and the distal region;
and (3) proximal, adjacent to the fold that delimitates the wing pouch (7-10
cell diameters). All three areas of the wing pouch express Nubbin and
Vestigial protein. The `hinge and pleura' was defined as the area outside the
fold that defines the wing pouch, coincident with Wg expression in the two
rings and Nubbin expression. These cells do not normally express Vg
protein.
BrdU incorporation
Larvae were dissected in Grace's insect medium (Sigma), incubated for 1
hour in medium containing 0.2 mg/ml of BrdU (Sigma), washed and fixed for
labeling with rabbit anti-GFP and anti-BrdU antibodies
(Usui and Kimura, 1992). To
compare BrdU incorporation in clones expressing NotchIntra in the
presence or the absence of Wg, we generated positively marked clones in larvae
of genotype hsFLP122, UAS-CD8-GFP; FRT40A arm-lacZ (or FRT40A,
wgcx4)/FRT40A Gal80; UAS-NotchIntra/tubGal4.
Embryos were collected for 8 hours. Larvae were heat-shocked at 48±4
hours AEL for 20 minutes at 37°C, and dissected at 118±4 hours AEL.
Larvae were labeled with anti-GFP, anti-BrdU and DAPI. Figs
6 and
7 show projections of four
single sections taken at 4 µm intervals for the BrdU channel. The GFP and
the DAPI channel are single sections.
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Antibodies
Mouse anti-BrdU was used at a dilution of 1:50 (PharMingen). Rabbit
anti-activated caspase-3 was used at 1:20 (Cell Signaling Technology). Rabbit
anti c-Myc (A-14) was used at 1:200 (Santa Cruz Biotechnology). Guinea pig
anti-Notum were used at 1:100 (Giraldez et
al., 2002). Mouse anti-Cut was used at 1:100
(Micchelli et al., 1997
). Rat
anti-Dll was used at 1:200 (Wu and Cohen,
1999
). Rat anti ß-gal was used at 1:500
(Strigini and Cohen, 1997
).
Rabbit anti-Vg was used at 1:200 (Williams
et al., 1993
). Monoclonal mouse anti-Wg was used at 1:10
(Brook et al., 1996
). Mouse
anti-Nubbin was used at 1:100 (Ng et al.,
1996
). Mouse anti-Cyclin D was used at 1:5; and rabbit anti-Cyclin
E (#3433) was used at 1:25 (Duman-Scheel,
2002
).
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Results |
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Cell autonomous requirement for Wg activity
To determine whether these phenotypes reflected a property of the
compartment as a whole, or whether they reflected a cell-autonomous
requirement for Wg activity, FLP-induced mitotic recombination was used to
produce clones of homozygous mutant cells and sister clones that were
homozygous wild type. Each pair of clones derives from a single cell division.
Consequently, relative growth can be compared by measuring the areas of the
individual pairs of mutant and wild-type twin clones after a period of time.
Mutations in two components of the Wg signaling pathway were examined.
arrow encodes the Wg co-receptor
(Wehrli et al., 2000). The
arrow2 allele truncates the protein before the
transmembrane domain and should be a null allele. pygopus encodes a
nuclear protein required for Wg signaling
(Kramps et al., 2002
;
Thompson et al., 2002
). The
pygoS28B allele truncates the protein before the
PHD domain and appears to be a strong hypomorphic mutant.
Clones generated before 48 hours after egg laying (AEL) were poorly
recovered at 120 hours (Thompson et al.,
2002; Wehrli et al.,
2000
), therefore we induced recombination at 60±2 hours AEL
and examined the clones at 120 hours. For wild-type control clones, there was
no difference in average clone and twin-spot areas in wing pouch or in the
notum (Fig. 1C,F; pouch,
P=0.9; notum, P=0.94, using T-test). By contrast,
arrow2 and pygoS28
clones were on average considerably smaller than their wild-type twins in the
pouch (Fig. 1D,E,G,H). These
differences were statistically significant (arrow,
P=1.6x10-7; pygo, P<0.001). In the notum,
the arrow2 and
pygoS28 mutant clones were not significantly
smaller than their twins (arrow, P=0.07; pygo, P=0.41).
These results indicate that Wg signaling is required cell-autonomously for
clonal growth in the wing pouch. Reduced clone size could reflect either a
decrease in the rate of cell proliferation (cell growth and division) or an
increase in cell death.
Wg signaling provides a survival cue
To determine whether the reduced area of
arrow2 and pygoS28
clones could be attributed to increased apoptosis, we examined mutant clones
for activation of caspase-3 (Yu et al.,
2002). High levels of activated caspase-3 were found toward the
basal surfaces of arrow2 mutant clones,
indicating apoptosis (Fig.
2A,B). A smaller increase in the number of apoptotic cells was
found in pygoS28 mutant clones
(Fig. 2C,D). The difference in
the proportion of apoptotic cells seen in arrow2
and pygoS28 mutant clones was consistent with the
differences in their average size (Fig.
1G,H), suggesting that at least part of the growth defect an be
attributed to reduced cell survival.
The Jun kinase (JNK) pathway is activated during apoptotic cell death in
wing disc (Adachi-Yamada et al.,
1999; Adachi-Yamada and
O'Connor, 2002
; Moreno et al.,
2002
). puckered (puc) encodes a dual-specificity
phosphatase that is transcriptionally upregulated by JNK pathway activation
(Martin-Blanco et al., 1998
).
A puc-lacZ reporter gene is induced in cells undergoing
developmentally triggered apoptosis; for example, due to reduced Dpp signaling
(Adachi-Yamada et al., 1999
;
Adachi-Yamada and O'Connor,
2002
; Moreno et al.,
2002
). To test whether the JNK pathway was activated in cells with
reduced Wg signaling, we examined puc-lacZ expression in
arrow2 mutant clones. puc-lacZ was
induced in 17/28 clones examined (Fig.
3A). puc-lacZ was observed in apical sections. In more
basally located optical sections, activated caspase-3 was observed in the same
clones (Fig. 3B). In addition,
arrow2 mutant clones showed a genetic interaction
with puckered. Recovery of arrow2 mutant
clones was reduced in the puc-lacZ/+ genetic background compared with
an otherwise wild-type background (10/25 versus 21/29 clones/twins examined).
These results indicate a contribution of JNK-mediated amplification to
apoptosis in cells deprived of Wg signaling.
Clone growth defect is only partly due to increased cell death
To assess the contribution of cell death to the reduced size of arrow
mutant clones, we made use of the FRT/Gal80 MARCM system
(Lee and Luo, 1999) to direct
Gal4-dependent expression of UAS-p35 transgene in FLP/FRT generated mutant
clones. The viral caspase inhibitor protein p35
(Hay et al., 1994
) was
expressed and clone areas were measured
(Fig. 4A-D). Expression of p35
allowed the recovery of arrow2 mutant cells that
accumulated activated caspase-3. Many of these cells sorted out below the disc
epithelium, but did not undergo apoptosis
(Fig. 4D,E). Suppression of
cell death only partially rescued clonal growth.
arrow2 clones expressing p35 were on average 56%
of the size of wild-type clones expressing p35 in the wing pouch
(Fig. 4B; P<0.001),
compared with 17% wild-type clone size for arrow2
mutant clones. As a second way of reducing apoptosis in the mutant clones, we
expressed puckered to reduce JNK pathway activity. This led to
reduced activation of Caspase-3 and fewer cells with pyknotic nuclei
(Fig. 4H-J). Reducing
JNK-mediated apoptosis partially rescued of the growth defect of
arrow2 clones compared with comparable wild-type
clones (Fig. 4G).
arrow2 clones expressing puckered were
32% the size of control clones expressing puckered
(P=3x10-6), compared with 17% of the wild-type clone
size for arrow2 alone. As blocking apoptosis only
partially suppressed the arrow2 mutant clonal
growth deficit, we conclude that reduced Wg signaling also reduces cell
proliferation. Thus Wg signaling appears to be required autonomously to
support cell proliferation in the wing disc.
Cell competition
Slower-growing cells can be eliminated by competition with faster-growing
cells in the developing wing disc (Morata
and Ripoll, 1975; Simpson,
1979
; Simpson et al.,
1981
). If cell survival in a clone is reduced as a result of cell
competition, providing the mutant cells with a relative growth advantage by
impairing the growth of the other cells can reduce cell death in the mutant
clone (e.g. Moreno et al.,
2002
). To assess the contribution of cell competition, we produced
clones of arrow2 M+ and
pygoS28 M+ at
76±4 hours AEL in discs heterozygous for both mutations. Because
homozygous M mutant cells die, we generated wild-type
M+/+ clones in a M+/- background as a
control. The experimental and control larvae were grown in the same vial at
low density to ensure comparable growth conditions. Recovery of
pygoS28 M+ mutant
clones was comparable to control M+ clones
(Fig. 5A,B), but average clone
area was 59% of the controls (Fig.
5C; P=4x10-10). We found similar levels
of activated caspase-3 in control M+ and
pygopusS28 M+ mutant
cells (Fig. 5F,G). Thus, the
reduced clonal growth observed under these conditions cannot be attributed to
elevated cell death.
Even when provided with a growth advantage, recovery of
arrow2 M+ clones was
low compared with control M+ clones
(Fig. 5D,E). Caspase activation
was high in arrow2
M+ and we observed many pyknotic nuclei
(Fig. 5E, inset). The fact that
arrow mutant cells died despite having been given a growth advantage
suggests that the reduction of Wg signaling might be stronger in the
arrow2 mutant than in the
pygoS28 mutant clones. To test this, we generated
genetic mosaics for both mutants in the same disc, and assessed the level of
expression of Distal-less (Dll), a Wg target gene that is sensitive to the
level of Wg activity (Neumann and Cohen,
1997; Zecca et al.,
1996
). Dll protein levels were reduced to a greater extent in
arrow2 mutant cells than in
pygoS28 mutant cells, indicating that
arrow2 compromises Wg signaling more severely
that pygoS28. Thus we can conclude that the lower
levels of Wg signaling in arrow2 mutants resulted
in apoptosis, even when clones were given a growth advantage. The level of Wg
signaling activity in pygoS28 mutants reduced
clonal growth but not primarily as a result of cell death. These findings
suggest that Wg serves both as a survival factor and to stimulate cell
proliferation during wing development. Thus Wg activity appears to be
comparable to that of Dpp with respect to proliferation and cell survival
(Martin-Castellanos and Edgar,
2002
; Moreno et al.,
2002
).
Wg promotes cell proliferation
In view of the foregoing results, we examined the ability of Wg to promote
cell proliferation in the wing disc. We used the FRT/Gal80 MARCM system
(Lee and Luo, 1999) and
Dpp-Gal4 to ectopically activate Wingless signaling, and used BrdU
incorporation to label cells that had undergone DNA replication during a
one-hour-labeling period. Cells in wing discs from control dpp-Gal4 UAS-GFP
larvae incorporated BrdU in a uniformly random pattern
(Fig. 6A; except for the normal
zone of reduced proliferation near the DV boundary known as the ZNC)
(Phillips and Whittle, 1993
;
Johnston and Edgar, 1998
).
Discs expressing Wg showed considerable expansion of the proximal wing pouch,
as well as hinge and notum regions (Fig.
6C). BrdU incorporation was elevated in the proximal part of the
wing pouch, indicating an increased rate of cell division. This was
accompanied by an increase in the relative area of the proximal wing pouch, so
we infer that Wg stimulated a net increase in cell proliferation (cell growth
and division) in the proximal part of the wing. We noted a lower level of BrdU
incorporation in the center of the wing pouch compared with control discs,
which may reflect an expansion of the normal ZNC [which depends on Wg
signaling from the DV boundary (Johnston
and Edgar, 1998
)]. The reduced proliferation observed in the
center of the wing pouch presumably reflects an expansion of the endogenous
ZNC, which contrasts with the increased cell proliferation observed toward the
proximal edge of the wing pouch.
To further examine the contribution of Wg signaling to proliferation,
clones of cells were generated in which the Wg pathway was autonomously
activated using mutants for the repressor protein axin
(Hamada et al., 1999;
Willert et al., 1999
).
axin mutant clones incorporated more BrdU than nearby cells in the
pulse label, indicating that their proliferation rate was increased. This
effect was cell autonomous, and stronger in the portions of the clones located
at the proximal part of the wing pouch and in the hinge region
(Fig. 7D, red arrow). Flow
cytometry analysis showed fewer axin mutant cells in G1 compared with
control cells in the same discs (at 115±4 hours AEL;
Fig. 7E), and we observed a
reduction in the levels of cyclin E in axin mutant cells in the wing
pouch (Fig. 8C). As cyclin E
regulates G1/S during imaginal disc development
(Knoblich et al., 1994
;
Duman-Scheel et al., 2002
),
the effect of Wg in the proximal wing pouch may be to accelerate G1/S
transition leading to an increase in S phase (increased BrdU incorporation)
and to an accumulation of cells in G2. We also noted that axin mutant
clones in the center of the wing pouch did not show elevated BrdU
incorporation (similar to dpp-Gal4 UAS-Wg discs).
To examine cell cycle profiles for cells mutant for components of the Wg signaling pathway, we compared cell cycle profiles of control cells expressing GFP and the apoptosis inhibitor p35 with those of arrow mutant cells expressing GFP and p35. Expression of p35 increased the fraction of cells in G2 at expense of G1, compared with control cells in the same disc. Taking this into account, arrow mutant p35 expressing cells showed an increase in the percentage of cells in G1 at expense of G2 (Fig. 7F).
These observations suggest that Wg signaling induces net cell proliferation
in the proximal wing pouch, perhaps by accelerating the G1 phase of the cell
cycle, whereas blocking Wg signaling leads to an increase of cells in G1.
Similar observations on the effects of Wg signaling on cell cycle phasing were
recently reported by Johnston and Sanders, who also noted that the cells with
elevated Wg signaling resemble cells from younger discs
(Johnston and Sanders, 2003).
In our view, this reflects Wg induced proliferation during the rapid phase of
disc growth. As illustrated in Figs
6 and
7, Wg signaling continues to be
able to induce net proliferation in the proximal part of the wing pouch,
whereas the high levels of Wg activity near the DV boundary directs cells to
pause in the G2 phase of the cell cycle
(Johnston and Edgar, 1998
).
The basis for the proximodistal difference in the cellular response to Wg is
not known, but parallels the normal formation of the ZNC in the distal wing
pouch.
Notch promotes cell proliferation via Wg-dependent and Wg independent
signals
Notch activity has also been implicated in the control of growth in the
wing disc. Notch activation induces Wg expression in the wing pouch
(Rulifson and Blair, 1995;
Diaz-Benjumea and Cohen,
1995
), so we expected that part of the effect of Notch could be
mediated by induction of Wg expression. We have therefore assessed the effects
of Notch activation on cell proliferation and tissue growth in the wing disc
under conditions that allow Wg expression, and under conditions where Wg
cannot be produced. Expression of the constitutively active form of Notch
(NotchIntra), under dpp-Gal4 control, resulted in overgrowth of the
wing pouch, and an increase in BrdU incorporation in the proximal pouch and
hinge regions (Fig. 6B),
indicating increased cell proliferation. Activated Notch differs from Wg in
that it also causes more proliferation throughout the notum. Clones of cells
expressing NotchIntra produced comparable results, causing extra
cell proliferation in the proximal part of the wing pouch, as well as in hinge
and notum (Fig. 7A,B). Within
the wing pouch proximal cells proliferated more, whereas cells closer to the
endogenous DV boundary proliferated less. NotchIntra-expressing
clones induce Wg expression and, when induced early enough, caused
considerable overgrowth in the proximal wing pouch, as well as in the hinge
and the notum (Fig. 9A,E; see
Fig. S2 at
http://dev.biologists.org/supplemental/).
We noted that cells adjacent to NotchIntra-expressing clones in the
proximal wing pouch also over-proliferated
(Fig. 7B;
Fig. 9A), perhaps due to their
ability to induce Wg, which can induce proliferation of proximal wing pouch
cells (Fig. 6). Interestingly,
NIntra expression in wgcx4 mutant
cells was still able to cause a non-autonomous increase in cell proliferation,
measured by BrdU incorporation (Fig.
7C). However, we noted that in the absence of Wg expression,
Notch-induced proliferation was mostly restricted to the wing hinge and
pleura, with little proliferation observed in the proximal part of the wing
pouch (Fig. 7C and
Fig. 9B,C). In the proximal
wing pouch the induction of ectopic growth was considerably reduced. Although
NIntra-expressing cells (wild type for the wg gene)
induced overgrowth in the proximal pouch in 75% of the cases, only 20% of
NIntra-expressing clones (mutant for wg) caused overgrowth
(Fig. 9E).
FACS analysis showed no difference in the cell cycle parameters of control cells and cells expressing NIntra in the same disc, whether or not they can produce Wg (Fig. 7E). We observed reduced levels of Cyclin E close to the DV boundary in NIntra-expressing cells (Fig. 8B). These observations suggest three conclusions. (1) Wg expression contributes significantly to the ability of Notch to induce tissue growth in the wing pouch. (2) Notch appears to be able to induce another signaling protein that contributes to non-autonomous induction of tissue growth in the proximal parts of the wing pouch, and in the hinge and pleura regions. (3) As Notch activity drives balanced cell proliferation without causing cells to accumulate in G2, the second Notch dependent signal may promote G2/M transition, so that, when expressed with Wg, cells do not accumulate in G2 (as they do when Wg is expressed alone).
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Discussion |
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Wingless
Our findings suggest that Wg signaling is required to support cell survival
and to promote cell proliferation during the rapid phase of wing disc growth.
Cells deprived of Wg signaling die as a result of cell competition and
apoptosis. In the absence of cell competition, loss of Wg signaling still
results in cell death, thus supporting the idea that Wg functions as a
survival factor to the cells in the wing. In addition, two types of evidence
suggest that Wg is required to support a normal rate of cell proliferation and
tissue growth. (1) Clones of cells deprived of Wg signaling grow and divide
more slowly than normal cells, even when cell death is prevented. In our
hands, arrow mutant clones expressing p35 or puckered, to
block cell death, were always smaller than control clones
(Fig. 4). In this respect, our
findings differ from those of Johnston and Sanders, who reported that clones
expressing a dominant-negative form of TCF and p35 to prevent cell death were
larger than control clones (Johnston and
Sanders, 2003). (2) We have also presented evidence that Wg
expression or activation of Wg signaling in axin mutant clones
increased cell proliferation in the wing, as measured by BrdU incorporation.
In mature third instar discs, Wg increased cell proliferation in the proximal
part of the wing, while at the same time inhibiting proliferation distally,
near the endogenous ZNC. This was also true of axin mutant clones,
which were very small distally, but proliferated more rapidly than control
cells proximally. Thus, we suggest that Wg-induced proliferation reflects the
effects of Wg during the rapid growth phase of the disc. Indeed, reduced Wg
function during second and early third instar can reduce the size of the
entire wing pouch (Couso et al.,
1994
; Neumann and Cohen,
1996b
; Neumann and Cohen,
1997
).
At later stages the role of Wg becomes more complex. High levels of Wg
signaling during the last 30 hours of the larval period promote a band of
distal cells to exit proliferation and pause in G2 to form the ZNC
(Phillips and Whittle, 1993;
Johnston and Edgar, 1998
). In
this context, Wnt signaling has been shown to contribute to regulation of the
bantam miRNA, which controls the rate of cell proliferation in the
ZNC (Brennecke et al., 2003
).
Our findings suggest that proximal and distal cells continue to respond
differently to Wg signaling even at this stage of wing disc development. The
basis for this proximal-distal difference in the cellular response to Wg is
not known. We suggest that Wg contributes to controlling survival and
proliferation during the phase of rapid disc growth, and that the opposing
effects seen in distal cells in the late third instar reflects a distinct
process. Dpp signaling has a similar profile of effects on cell growth and
survival. Ectopic activation of the Dpp pathway autonomously induces cell
proliferation in the wing
(Martin-Castellanos and Edgar,
2002
). As for Wg, cells responded differently to according to
their position. Cells far from the normal source of Dpp respond more strongly
than cells close to the source. Dpp signaling also provides survival cues
(Moreno et al., 2002
).
Notch
Activation of Notch has similar effects on wing disc growth to Wg
expression. In particular, we note that Notch activity stimulated cell
proliferation proximally, and not evenly, in all cells of the clones
expressing activated Notch. This observation is difficult to reconcile with
the idea that Notch activity per se drives proliferation. We provide evidence
that the non-autonomous effects of Notch are mediated in part through Wg and
in part through at least one other signaling protein. We observed two
different responses to Notch activation. (1) NotchIntra induced
non-autonomous overproliferation that was dependent on the ectopic expression
of Wg in the wing pouch. (2) NotchIntra induced non-autonomous
overgrowth in the wing hinge and the notum independently of Wg production.
These cells still required a Wnt signal as arrow2
mutant clones expressing NotchIntra did not overgrow (not shown).
These observations support a model in which the Notch and Wg pathways act
synergistically to induce cell proliferation and tissue growth during early
larval stages. We propose that Wg activity may stimulate cells to enter S
phase, and thus may contribute to Notch induced proliferation. The other
Notch-induced signal may promote mitosis.
Wnt signaling proliferation in development and cancer
Comparable roles for the Wnt pathway in controlling tissue growth have been
reported in vertebrate development. Wnt1-mediated signaling stimulates cell
proliferation in the developing CNS
(Dickinson et al., 1994) and
in the pituitary gland (Treier et al.,
1998
). The Wnt pathway also controls cell proliferation and
self-renewal of the intestinal epithelia through ß-catenin/Tcf4-mediated
transcription of the oncogene Myc (van de
Wetering et al., 2002
). The Wnt pathway has been shown to act
through the transcription factor Pitx2 to promote cell proliferation in the
developing pituitary and heart (Kioussi et
al., 2002
). These few examples illustrate that Wnt signaling plays
a role in developmentally controlled cell proliferation in vertebrates.
Mutations that activate the Wnt pathway have been associated with
uncontrolled proliferation in humans and mice leading to cancer (reviewed by
Polakis, 2000;
Taipale and Beachy, 2001
).
Mutations seem to be preferentially associated with specific types of tumors.
Notably, APC mutations occur frequently in colorectal cancer. Humans with
germ-line mutations in APC develop familial adenomatous polyposis coli
referred to as Gardner Syndrome. These patients mainly develop colorectal
cancer and hepatoblastoma, as well as jaw and sebaceous cysts. Other tissues
are more weakly affected (see
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=OMIM).
This might reflect cell-type specific growth responses to altered Wnt
signaling, perhaps analogous to the region-specific effects of Wg that we
observed in the wing disc.
The dependence of cells on extrinsic survival factors provides a powerful
mechanism to regulate cell number during development (reviewed by
Conlon and Raff, 1999).
Although cell death does not seem to play a major role in regulating tissue
size during wing development, cell number is controlled by apoptosis in other
contexts, such as the Drosophila eye and nervous system. In a subset
of midline glial cells, activation of the EGFR pathway controls cell number by
suppression of the pro-apoptotic protein Hid
(Bergmann et al., 2002
).
Interestingly, cells in the intestinal epithelia far away from the domain of
Wnt activation undergo apoptosis and/or extrusion into the lumen
(van de Wetering et al.,
2002
). Given our observation that Wg provides survival cues in the
wing disc, it seems worth considering whether APC/ß-catenin mutations
might contribute to the development of colorectal carcinoma by making
intestinal cells refractory to apoptotic signals.
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ACKNOWLEDGMENTS |
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Footnotes |
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