1 Department of Biological Sciences, Columbia University, 701 West 168th Street,
HHSC 1104, New York, NY 10032, USA
2 Department of Biochemistry and Molecular Biophysics, Columbia University, 701
West 168th Street, HHSC 1104, New York, NY 10032, USA
* Author for correspondence (e-mail: rsm10{at}columbia.edu)
Accepted 14 September 2004
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SUMMARY |
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Key words: Drosophila, Wing, Teashirt, Homothorax, Wingless, Decapentaplegic, Polycomb
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Introduction |
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A number of observations support the idea that both wg and
dpp act together to induce distal fates in the wing disc. wg
mutant discs fail to repress tsh distally, whereas ectopic activation
of the wg pathway represses tsh proximally
(Azpiazu and Morata, 2000;
Wu and Cohen, 2002
). Wg and
Dpp signal transduction also appear to be necessary for complete hth
repression in the pouch (Azpiazu and
Morata, 2000
). However, the relationship between Dpp signaling and
tsh regulation remains unclear. Ectopic activation of the Dpp pathway
in early-induced clones only represses tsh in the most lateral
regions of the disc (Wu and Cohen,
2002
), and hypomorphic dpp mutant larvae still show some
tsh repression in the distal wing disc
(Cavodeassi et al., 2002
). The
combinatorial model of Wg and Dpp function in the wing is also weakened by the
observation that wg activity is only required for hth
repression in a subset of the wing pouch
(Azpiazu and Morata, 2000
).
Differences in the timing of tsh and hth repression also
indicate that the two genes are regulated by distinct mechanisms in the wing
disc. tsh is repressed in distal wing disc cells in the early second
larval instar, coincident with the ventro-anterior wedge of wg
expression (Wu and Cohen,
2002). By contrast, hth repression begins shortly after
tsh repression. Furthermore, Notch-dependent activation of
wg at the DV compartment boundary
(Diaz-Benjumea and Cohen,
1995
; Neumann and Cohen,
1996
) is not required for tsh repression
(Wu and Cohen, 2002
), raising
the question of what maintains tsh repression as wing development
progresses. Notch also activates the gene vestigial (vg), an
essential factor for wing blade growth and patterning
(Halder et al., 1998
;
Kim et al., 1996
;
Simmonds et al., 1998
;
Williams et al., 1991
;
Williams et al., 1994
).
However, vg is neither necessary nor sufficient for tsh
repression in the wing disc (Wu and Cohen,
2002
). By contrast, the loss of vg activity causes
hth de-repression in a subset of distal clones. Because vg
is activated by Dpp and Wg (Kim et al.,
1997
; Kim et al.,
1996
; Neumann and Cohen,
1997
; Zecca et al.,
1996
), these results have been interpreted to suggest that
vg may be responsible for Dpp- and Wg-mediated repression of
hth (Azpiazu and Morata,
2000
; Wu and Cohen,
2002
). However, the endogenous vg and hth
expression domains overlap considerably outside of the wing pouch, suggesting
a more complex relationship between these two factors.
To better understand how PD domains in the wing disc are established and maintained, we executed a genetic screen to identify genes involved in these processes. Two of the genes identified by this screen, which we focus on in this report, were Medea (Med), a downstream component of the Dpp pathway, and Su(z)12, a member of the Polycomb group (PcG) of genes. The identification of these genes prompted us to examine the role of the Dpp, Wg and PcG pathways in the formation of the PD axis of the wing disc. We demonstrate that Dpp signal transduction is necessary for hth, but not tsh, repression in the distal cells of the wing disc throughout larval development. Wg signal transduction is also dispensable for the maintenance of tsh repression. We also elucidate the distinct temporal and spatial requirements of vg as a mediator of hth repression. Finally, we show that PcG-mediated gene silencing maintains the separation between wing and body through tsh, but not hth, repression, accounting for the inability of distal cells to express tsh, even when both the Dpp and Wg signaling pathways are compromised.
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Materials and methods |
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Medadro is homozygous larval lethal, and lethal in
trans to the strong hypomorph Med13
(Hudson et al., 1998) and to
Df(3R) tll-e. The transheterozygous phenotype is slightly more severe
than the homozygous phenotype, indicating that Medadro is
not a null allele. In clones, Medadro and
Med13 phenotypes are indistinguishable.
Su(z)12daed was mapped by its failure to complement
Df(3L)kto2 and the strong Su(z)123 allele
(Kehle et al., 1998). Hox gene
de-repression in Su(z)12daed clones closely resembles the
effect reported for strong Su(z)12 hypomorphic alleles
(Birve et al., 2001
).
Fly stocks
Mutant alleles: arr2
(Tearle and Nusslein-Volhard,
1987) (see FlyBase); brkXH
(Campbell and Tomlinson, 1999
);
MadB1 (Wiersdorff et
al., 1996
); PcXT109
(Franke et al., 1995
);
vg83b27R (Williams et
al., 1991
); tkv8
(Tearle and Nusslein-Volhard,
1987
) (see FlyBase).
Gal4 drivers: vgBE-Gal4
(Simmonds et al., 1995);
act>hs-CD2>Gal4 (Pignoni
and Zipursky, 1997
).
UAS lines: UAS-GFP; UAS-brk
(Lammel et al., 2000);
UAS-dTCFDN (van de
Wetering et al., 1997
).
Mutant clones
vgBE-Gal4, UAS-flp (vgBE::flp) clones:
Heat shock clones:
Larvae were heat shocked for 45 minutes to an hour at 37°C. The developmental stage of clone induction for each experiment is indicated in the results section.
Misexpression
yw hs-flp; act>hs-CD2>Gal4, UAS-GFP was used to drive
expression of UAS-dTCFDN in clones. Prior to heat shock,
larvae were grown at 22°C. Second instar larvae were heat shocked for 30
minutes at 35°C, then transferred to 25°C until dissection.
yw hs-flp; act>hs-CD2>Gal4, UAS-GFP was used to drive expression of UAS-brk in clones. Prior to heat shock, larvae were grown at 22°C. Late first instar larvae were heat shocked for 30 minutes at 37°C, then transferred to 25°C until dissection.
Larvae of the genotype yw hs-flp tub>Gal4, UAS-GFP; UAS-dTCFDN; FRT82B Med13/FRT82B tub>Gal80 hs-CD2 y+ M(3)w124 were heat shocked for 45 minutes to an hour at 37°C during the second instar. Perdurance of Gal80 prevented strong expression of UAS-GFP until 12 to 24 hours after clone induction.
Larvae of the genotype yw hs-flp tub>Gal4, UAS-GFP; UAS-Nrt-wg; PcXT109 FRT2A tub>Gal80 hs-CD2 FRT2A were heat shocked for one hour at 37°C during the early second instar.
Immunostaining
Antibodies: mouse anti-ß-galactosidase (Promega); mouse anti-rat CD2
(Serotec); mouse anti-Dll (Cohen et al.,
1993); guinea pig anti-Hth
(Casares and Mann, 1998
);
mouse anti-Nub (Ng et al.,
1995
) (from Michalis Averof); rabbit anti-Tsh (from SK Chan);
rabbit anti-Vg (Williams et al.,
1991
); mouse anti-Wg (4D4;
(Neumann and Cohen, 1997
)
(Iowa University Hybridoma bank). Fluorescent secondary antibodies (FITC, Cy3,
Texas Red and Cy5) were from Jackson Laboratories. All imaginal discs were
analyzed with a Bio-Rad 1024 confocal system.
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Results |
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To identify factors necessary for the establishment and maintenance of
these PD domains, we performed an F1 genetic screen (see Materials and
methods). This screen used a wing-specific source of Gal4 to drive expression
of the FLP recombinase, generating clones by the FRT-FLP system
(Golic, 1991;
Xu and Rubin, 1993
). We
selected the vestigial boundary enhancer-Gal4, UAS-flp
(vgBE::flp) combination for its robust wing expression and its
ability to drive clones along the entire PD axis. Although primarily active at
the dorsoventral (DV) margin under the control of the Notch pathway
(Kim et al., 1996
), the
vgBE is transiently active throughout the wing disc
(Vegh and Basler, 2003
). This
property allowed us to screen adult wings for PD defects caused by large
mutant clones.
One mutation identified by this screen, dubbed aerodrome
(adro), profoundly affected the growth and patterning of the wing,
and mapped to the Medea (Med) gene (see Materials and
methods). Medea, a homolog of vertebrate Smad4, functions downstream of Dpp as
a DNA-binding partner for Mothers against Dpp (Mad)
(Hudson et al., 1998;
Wisotzkey et al., 1998
).
Medadro clones driven by vgBE::flp caused a
non-autonomous reduction of wing size and growth along the PD axis
(Fig. 2A), evident in both the
blade and the hinge. Wing blade clones tended to sort out from the surrounding
wild-type cells, forming vesicles (Fig.
2B). Clones located close to the DV boundary sometimes induced
non-autonomous duplications of margin structures
(Fig. 2C). We also observed
cell autonomous differentiation of lateral hinge elements in
Medadro clones near the AP boundary of the hinge
(Fig. 2D).
|
To discriminate between a requirement for Med in the initiation
versus the maintenance of hth repression, we examined
HS:flp-induced clones generated in the mid-third instar, after
hth repression in the pouch has occurred
(Casares and Mann, 2000;
Wu and Cohen, 2002
).
Medadro clones induced during the mid-third instar also
de-repressed hth throughout the wing pouch
(Fig. 2I-K). Like
Medadro clones, clones of two other Dpp pathway
components, thickveins (tkv), which encodes a type I Dpp
receptor (Brummel et al., 1994
;
Nellen et al., 1994
;
Penton et al., 1994
;
Ruberte et al., 1995
), and
Mad, also de-repressed hth, but not tsh (data not
shown). Thus, Dpp signaling is necessary to maintain hth, but not
tsh, repression, at least until mid-third instar stage.
Although the above results demonstrate that, by the late second instar, Dpp
signaling is not required to repress tsh in the pouch, early-induced
clones expressing a constitutively active form of Tkv (Tkv*) have
been reported to repress tsh in the lateral region of the wing disc,
suggesting that Dpp has the potential to repress tsh in some contexts
(Wu and Cohen, 2002). It was
therefore possible that Dpp might play an early role during the establishment
of tsh repression in the second instar. However, according to another
report, a strong dpp hypomorphic combination is not sufficient to
de-repress tsh in the distal wing disc
(Cavodeassi et al., 2002
). We
addressed the requirement for Dpp signal transduction during the establishment
of tsh repression by compromising the Dpp pathway in the early wing
disc. First, we induced flip-out clones expressing brinker
(brk) in late first instar larvae. brk encodes a repressor
of Dpp target genes and is normally expressed in the lateral regions of the
wing imaginal disc, as its transcription is itself negatively regulated by Dpp
(Campbell and Tomlinson, 1999
;
Jazwinska et al., 1999a
;
Jazwinska et al., 1999b
;
Minami et al., 1999
). Most
ectopic Brk-expressing cells in the pouch are eliminated from the epithelium
soon after clone induction. However, we obtained a small number of
Brk-expressing clones in the distal regions of early and mid-third instar wing
discs. Such clones de-repressed hth, but had no effect on the
expression of tsh or the distal marker, nubbin
(nub) (Ng et al.,
1995
) (Fig. 2L,M).
In addition, ectopic expression of brk using the Dpp-Gal4 driver line, which
is active before tsh is initially repressed, also results in
hth, but not tsh, de-repression (data not shown).
As a second test for an early role of Dpp in tsh repression, we
induced Medadro clones by heat shock during the first
larval instar in a Minute background. Even in these early-induced clones,
tsh expression was absent from the distal-most portion of these
almost entirely mutant discs (Fig.
2P-W). By contrast, hth was expressed in the mutant cells
(Fig. 2P-R). As expected, these
discs are much smaller than wild type, and resemble those obtained from
dppd12/dppd14 larvae, which also maintain some
tsh repression in distal cells
(Cavodeassi et al., 2002).
Together, these experiments strongly suggest that the Dpp pathway is not required for the establishment or maintenance of tsh repression. By contrast, Dpp signaling is required to both establish and maintain hth repression throughout wing disc development.
Temporal and spatial requirements for vg repression of hth
As a downstream target of Dpp, vg is a good candidate to mediate
the Dpp-dependent repression of hth. Two previous results suggest
that vg plays a role in hth repression in the pouch: (1)
some vg loss-of-function clones in the pouch ectopically express
hth; and (2) ectopic vg expression in the hinge represses
hth (Azpiazu and Morata,
2000). However, the latter observation is complicated by the fact
that vg and hth are normally co-expressed in the wild-type
lateral hinge (Fig. 3A-C). To
better assess the requirement for vg in hth repression, we
used the Minute technique to recover multiple vg mutant clones, which
tend to survive poorly. Unlike Med mutant clones, which de-repress
hth at all positions along the AP axis of the pouch, vg
mutant clones, induced in early third instar larvae, only de-repressed
hth in pouch cells far from the AP compartment boundary
(Fig. 3D-F). The failure of
vg mutant clones to de-repress hth near the AP boundary
the source of secreted Dpp suggests that vg is not
necessary for hth repression in cells that receive high levels of
this signal.
|
The absence of Dpp signaling permits co-expression of vg and hth
To further characterize the relationship between Dpp, vg and
hth, we examined their expression in Medadro
clones. In Medadro clones near the DV boundary of the
pouch, we observed strong hth de-repression but no effect on
vg expression, leading to the co-expression of these two
transcription factors (Fig.
4A-D). The inability of Vg to repress hth in the absence
of Dpp signaling might provide an explanation for the co-expression of these
two factors in the wild-type lateral hinge. In this region, low Dpp signaling
leads to high levels of brk, which represses Dpp target genes. We
tested whether brk expression is required for hth expression
in the lateral hinge by inducing brk loss-of-function clones in
second instar larvae. Lateral distal hinge (DH) brk
clones expressed vg but not hth, and tended to grow larger
than their wild-type twin spots (Fig.
4E-H). This result complements our observation of vg and
hth co-expression in Medadro clones
(Fig. 4A-D), and suggests that
Brk represses an as yet unidentified repressor of hth (see
Discussion).
|
Wg is not required for the maintenance of tsh repression
Previous results suggested that Wg signaling is necessary for both
hth repression and the early establishment of tsh repression
in the wing pouch. Clones mutant for the Wg signal transducer
dishevelled (dsh) ectopically express hth when
located far from the AP compartment boundary
(Azpiazu and Morata, 2000), and
transheterozygotes of a disc-specific regulatory allele and a null allele of
wg fail to repress tsh in the wing pouch
(Wu and Cohen, 2002
). The DV
stripe of wg expression, however, is dispensable for tsh
repression (Wu and Cohen,
2002
). To demonstrate directly that Wg signal transduction is not
necessary for the maintenance of tsh repression in the pouch, we
eliminated the ability of pouch cells to respond to Wg, by generating clones
of cells mutant for the Wg co-receptor arrow (arr)
(Wehrli et al., 2000
).
arr mutant clones located in the pouch far from the AP boundary,
expressed hth, but never expressed tsh
(Fig. 5A-C). Similarly, pouch
clones expressing dTCFDN, a dominant-negative form of the
Wg pathway transcription factor (van de
Wetering et al., 1997
), also expressed hth, but not
tsh (Fig. 5D-F). Thus,
although Wg signal transduction is required for the initiation of tsh
repression, it is not required to mantain tsh repression in the wing
pouch. We also note that the subset of pouch cells that requires wg
signaling to maintain hth repression is the same subset that requires
vg for hth repression. Because vg is a target of
wg, these observations suggest that the requirement for wg
to maintain hth repression in the lateral wing pouch may be mediated
through vg.
|
Dpp and Wg are not redundantly required to repress tsh
Based on the Dpp and Wg pathway loss-of-function experiments described
above, as well as the previous analysis of wg mutant discs
(Wu and Cohen, 2002), we
suggest that Wg signaling establishes tsh repression in the second
instar wing pouch, but that neither signaling pathway is necessary to maintain
this repression. One possibility not addressed by our previous experiments is
that Dpp and Wg are able to redundantly repress tsh expression. We
tested this question by making clones that are compromised for both signaling
pathways. We used the MARCM system (Lee
and Luo, 2001
) to ectopically express dTCFDN
in Med13 clones. The experiment was performed in a Minute
background to ameliorate the severe growth disadvantage of these clones. We
included a UAS-GFP transgene to mark these clones and also monitored
Dll (a target of Wg signaling) expression to confirm that Wg
signaling was compromised (Neumann and
Cohen, 1996
; Zecca et al.,
1996
). By the late third instar, tub>Gal4;
UAS-dTCFDN; Med13 clones strongly expressed
GFP and hth, but showed no tsh expression (Fig.
G-I). We also observed the loss of Dll expression in these clones
(Fig. 5J). Because the
morphology of the disc is severely disrupted by these doubly mutant clones, we
were concerned that the mutant cells could be derived from the peripodial
membrane. To rule this out, we examined hth and tsh in
mutant peripodial cells, and found no change in the endogenous expression
levels of both genes (Fig. 5K). Thus, tsh remains repressed in the main epithelium even when the
activities of both long-range signaling pathways of the wing pouch are
simultaneously compromised.
PcG genes are required to maintain repression of tsh in the wing pouch
If neither Dpp nor Wg are required for the maintenance of tsh
repression, what, then, performs this essential function? In the course of our
F1 screen, we identified a mutation, dubbed daedalian
(daed), that caused non-autonomous reduction of wing blade and hinge
size along the PD axis (Fig.
6A), and mapped to the Suppressor of zeste 12
(Su(z)12) gene. Su(z)12 is a PcG member, required to
maintain the heritable silencing of Hox genes throughout development
(Birve et al., 2001).
Su(z)12daed clones tended to sort out from the surrounding
wild-type tissue, frequently forming vesicles
(Fig. 6B). Some wing blade
clones cell autonomously differentiated bristles characteristic of proximal
hinge and notum (Fig. 6C). When
examined in the wing imaginal disc, we found that
Su(z)12daed clones induced by vgBE::flp weakly
expressed hth in some of the mutant pouch cells
(Fig. 6D,E). However, the same
clones ectopically expressed high levels of tsh throughout the mutant
tissue (Fig. 6D,F). Thus, in
contrast to Wg and Dpp pathway mutations, Su(z)12daed
clones de-repressed tsh more readily than hth.
|
Temporal and spatial regulation of tsh in PcG mutants
The extent and timing of loss of PcG-mediated silencing depends on both the
PcG member and the particular target gene being examined
(Beuchle et al., 2001). To
confirm that PcG mutant clones are defective in the maintenance of
tsh repression, and to better assess the kinetics of tsh
de-repression, we induced Pc clones by heat shock
at different time-points during larval development
(Fig. 7A).
|
The spatial pattern of tsh de-repression in PcG mutant clones
differs significantly from the pattern for other reported PcG targets.
Ubx, and other Hox genes, are de-repressed first in the prospective
wing pouch, and later in the hinge and notum
(Beuchle et al., 2001). By
contrast, late induced Pc clones only expressed
tsh along the periphery of the wing pouch
(Fig. 7B,C). Earlier induced
clones expressed tsh in more distal regions
(Fig. 7D-I). However, even in
large clones induced during the first or second instar, tsh
expression was not observed close to the DV boundary or in most of the hinge
(Fig. 7H,I). As both of these
regions of the disc express Wg, this pattern points to the possibility that
tsh expression remains sensitive to repression by Wg signaling even
in the absence of PcG function. To test this, we used the MARCM system to
express the membrane tethered, non-diffusable, Nrt-Wg
(Zecca et al., 1996
) in
Pc cells. We found that Nrt-Wg strongly repressed
the ectopic tsh resulting from loss of Pc function
(Fig. 7J-M), indicating that Wg
retains the ability to repress tsh in the absence of PcG-mediated
silencing.
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Discussion |
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Dpp signaling during PD axis formation in the wing
In the course of a screen for mutations affecting the PD axis of the wing,
we isolated an allele of the Drosophila Smad4 homolog Med.
Like other Dpp pathway mutations, Medadro clones located
in the wing pouch cell autonomously de-repress hth. This is evident
even in late-induced clones, demonstrating the continuous role of Dpp
signaling in shaping the wing blade/hinge subdivision during larval
development. By contrast, we did not detect any de-repression of tsh
resulting from any of our manipulations of the Dpp pathway.
The ability of ectopic Dpp activity to repress tsh in
early-induced proximal clones was interpreted to suggest that Wg and Dpp
cooperate to repress tsh in the early pouch
(Wu and Cohen, 2002). However,
because Dpp is dispensable for tsh repression, this model must be an
over-simplification. We conclude that Wg, not Dpp, must be considered the
primary repressor of tsh in the wing. The lack of synergy between the
two pathways is reminiscent of the regulation of Dll, which is
activated in the leg by the combined activities of Wg and Dpp, but requires
only Wg for its expression in the wing pouch
(Neumann and Cohen, 1996
;
Zecca et al., 1996
).
In the absence of Dpp signaling, wing pouch cells co-express hth,
nub and Dll, but not tsh. This combination of factors
is normally only found in the distal hinge (DH), suggesting a transformation
from pouch to DH when the Dpp pathway is compromised
(Azpiazu and Morata, 2000;
Casares and Mann, 2000
). The
expression of the Iro-C genes, normally restricted to the notum, extends to
the distal limit of the tsh domain in dpp mutant discs,
leading to the hypothesis that Dpp signaling is essential for the separation
of wing and body wall (Cavodeassi et al.,
2002
). However, because loss of Dpp signaling transforms wing
pouch to DH, we propose an alternative view, in which Dpp further divides an
already extant appendage/trunk subdivision by repression of hth in
the pouch and Iro-C in the proximal hinge (PH). According to this proposal,
the distal limit of tsh expression, initiated by early Wg expression
and maintained by PcG silencing, denotes the boundary between the appendage
and the body.
hth repression through multiple mechanisms
Our results suggest that repression of hth in the wing disc only
occurs in cells with a history of vg expression and continuous Dpp
input. Consistent with this, ectopic vg expression in the medial DH
(Azpiazu and Morata, 2000) and
loss of brk in the lateral DH both result in hth repression.
The requirement for vg can be separated into two distinct stages. The
first stage occurs in the second instar, when vg expressed at the DV
compartment boundary determines which cells are competent to repress
hth in response to Dpp signaling
(Fig. 8B). Thus, both
vg or Dpp-pathway mutant clones induced at this early stage fail to
repress hth.
|
A model that encompasses these observations is that Vg and Dpp activate
another factor that directly represses hth
(Fig. 8C). This factor would be
activated in Vg-positive cells by Dpp signaling beginning in the late second
instar. By the third instar, high levels of Dpp signaling would be sufficient
to maintain its activation, with additional input by Vg and Wg required only
at the lateral regions of the pouch. Even further from the source of Dpp, in
the lateral hinge, high levels of Brk would prevent expresssion of this
factor, thus allowing hth expression despite the presence of Vg. This
model is consistent with the idea that Brk is a transcriptional repressor
(Hasson et al., 2001;
Kirkpatric et al., 2001; Rushlow et al.,
2001
; Sivasankaran et al.,
2000
; Zhang et al.,
2001
) and Vg is a transcriptional activator
(Halder and Carroll, 2001
;
Halder et al., 1998
). There is
also precedent for the idea that early vg expression predisposes
cells to a particular Dpp response, which was also proposed for the activation
of the vgQE (Klein and Arias,
1999
).
We note that the above model does not apply to PH cells, which have a
distinct response to Dpp signaling. For example, we found that
Medadro clones located near the AP boundary of the PH
ectopically expressed vg (data not shown). tsh is an
attractive candidate for mediating this switch in response to Dpp signaling,
as it is expressed in the PH but not the DH, and is reported to bind Brk in
vitro (Saller et al., 2002).
However, the absence of reagents to readily examine tsh
loss-of-function clones prevents us from testing this idea at this time.
PcG genes and the maintenance of tsh repression
If tsh repression marks a fundamental subdivision along the PD
axis, then the maintenance of tsh repression is crucial for the
maintenance of this subdivision. Although Wg signaling is clearly required for
the initiation of tsh repression, it is dispensable by the time the
DV margin is established (Wu and Cohen,
2002). The elbow-no ocelli (el-noc) gene complex
has been identified as a target of both Dpp and Wg that is necessary for
tsh repression in the wing (Weihe
et al., 2004
). However, tsh de-repression is only
observed in el-noc loss-of-function clones induced in first or early
second instar larvae. tsh repression must therefore be maintained by
a wg- and el-noc-independent mechanism. We ruled out the
possibility of redundant Wg- and Dpp-mediated tsh repression by
making clones doubly mutant for both signaling pathways. Such clones
upregulated hth, and lost Dll expression, but showed no
ectopic tsh expression. Thus, neither of the two major long-range
signaling systems of the wing pouch is involved in the maintenance of
tsh repression.
Instead, our analysis of Su(z)12daed and Pc
mutant clones indicates that the maintenance of tsh repression is
mediated by a heritable silencing mechanism. By inducing Pc mutant
clones in third instar discs, we demonstrated that this ectopic tsh
expression represents a failure to maintain rather than a failure to establish
repression. The weak hth levels observed in some PcG mutant clones
may be due to the previously noted ability of tsh to upregulate
hth (Azpiazu and Morata,
2000; Casares and Mann,
2000
). This interpretation is supported by the fact that
hth expression is only seen in large Pc mutant clones, and
only in cells expressing the highest levels of tsh. The general
absence of hth expression in PcG mutant clones, together with the
ectopic hth expression resulting from late Dpp pathway disruption,
points to the need for continuous signaling input to maintain hth
repression. By contrast, tsh requires PcG gene activity, but not
continuous Wg or Dpp input, to maintain its repression during the third
instar.
We cannot at this stage rule out the possibility that the affects of PcG
mutant clones on tsh repression described here are indirectly due to
the de-repression of another factor. We suggest that this is unlikely,
however, in part because the spatial distribution of tsh
de-repression in PcG mutant clones differs significantly from reports of Hox
gene de-repression (Beuchle et al.,
2001). Additionally, the ectopic tsh expression in
Pc mutant clones is repressible by Nrt-Wg, indicating that
tsh is still subject to regulation by Wg signaling.
In the embryo, Hox genes are repressed in some segments by the transient
presence of the gap genes (reviewed by
Bienz and Muller, 1995). This
initial repression is then maintained by the PcG proteins through a heritable
silencing mechanism. Our model of tsh repression follows this general
outline, whereby Wg signaling is required transiently to establish the limits
of the tsh expression domain (Fig.
8A). PcG proteins subsequently maintain the tsh silenced
state, while the appendage is further subdivided along the PD axis
(Fig. 8A-C). Similar mechanisms
may be important for tsh regulation in other tissues, as was
suggested by a recent report showing tsh de-repression in PcG mutant
clones in the eye disc (Janody et al.,
2004
).
tsh and hth repression are distinct events during the development of the wing imaginal disc. The requirement for PcG activity in tsh, but not hth, repression points to the primacy of tsh repression in determining appendage versus trunk fate. PcG regulation ensures a strict and inflexible pattern of gene expression, ideal for defining the fundamental divisions of the disc. Within the specified appendage domain, Wg and Dpp signaling can then modify the shape and size of the hinge and wing blade through continuous input into transcription factors that control patterning and growth. In the absence of Tsh, Hth is an essential mediator of this process, as it promotes hinge development at the expense of wing pouch growth. The complexity of hth relative to tsh regulation may, therefore, reflect the greater need for plasticity in the response of hth to the Wg and Dpp morphogen gradients.
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ACKNOWLEDGMENTS |
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