Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
Authors for correspondence (e-mail:
shashi{at}ccmb.res.in)
Accepted 12 November 2003
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SUMMARY |
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Key words: Drosophila, Arm, Wg
![]() |
Introduction |
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The ability of Axin to bind Protein Phosphatase 2A (PP2A)
(Willert et al, 1999;
Hsu et al., 1999
) suggests
that this phosphatase can interact with the Axin-APC-GSK3ß-catenin
complex. It might modulate the effect of the kinase on one or more substrates,
thereby affecting Wnt signaling. However, studies involving the inhibition of
PP2A by okadaic acid in mammalian cell lines are conflicting. Seeling et al.
(Seeling et al., 1999
) and Li
et al. (Li et al., 2001
)
report that PP2A is a negative regulator of Wnt signaling, while studies by
Willert et al. (Willert et al.,
1999
) suggest that PP2A is a positive regulator. The predominant
form of PP2A in cells has a heterotrimeric-subunit structure, consisting of a
core dimer of
36 kDa catalytic (C) and
65 kDa scaffold (A) subunits
complexed to a third variable regulatory (B) subunit. Knockout mouse embryos
lacking the catalytic subunit of PP2A show decreased levels of ß-catenin,
suggesting its positive role in Wnt signaling upstream of ß-catenin
(Gotz et al., 2000
).
Relatively large number of B or B-related polypeptides are known, many of
which are present in multiple isoforms (reviewed by
Janssens and Goris, 2001).
They express in tissue- and temporal-specific manner during development.
Reports also indicate that they may target the PP2A catalytic complex to
intracellular sites such as microtubules
(Sontag et al., 1995
) or the
nucleus (McCright et al.,
1996
). These features of the PP2A regulatory subunits suggest that
it is the B subunit that defines the substrate specificity of a given PP2A
heterotrimeric complex and its physiological role. Thus, identification of the
B subunit(s) that interacts with components of Wnt pathway will elucidate the
mechanism by which PP2A regulates Wnt signaling. Indeed, evidence is available
for the involvement of a regulatory subunit in the Wnt pathway. For example,
overexpression of B56 in mammalian cells or in Xenopus
embryos/explants results in the reduction of ß-catenin levels suggesting
a negative role upstream of ß-catenin
(Seeling et al., 1999
;
Li et al., 2001
).
Overexpression of B'/PR61 inhibits ß-catenin-induced axis
duplication in Xenopus embryos, suggesting that this subunit is also
a negative regulator of Wnt signaling
(Ratcliffe et al., 2000
).
These reports do not reconcile with the findings from mouse knockout
experiments that the catalytic subunit positively regulates Wnt signaling
(Gotz et al., 2000
) as well as
to a recent report that shows loss of Wnt signaling in embryos depleted with
PR/B56
transcripts (Yang et al.,
2003
). However, none of the above studies combine both loss- and
gain-of-function approaches to examine the role of a given subunit, which may
help in better understanding the role of PP2A in Wnt signaling.
We have earlier reported that overexpression of human APC (hAPC) in
Drosophila downregulates Wingless (Wg; Drosophila homologue
of Wnt) signaling (Bhandari and
Shashidhara, 2001). We had observed that deficiency mutation
Df(3R)by62 enhanced hAPC-induced eye phenotypes, but not its
overlapping deficiency Df(3R)by10. Thus, the locus/loci that enhances
hAPC-induced phenotypes was mapped to 85E11-85F16, the cytological location of
tws, which encodes Drosophila homologue of B (of type PR55)
regulatory subunit of PP2A. We have examined a possible requirement of Tws in
Wg signal transduction. We have employed both loss- and gain-of-function
genetic studies to determine the precise role of Tws in Wg signaling. Results
outlined in this report suggest that tws is a positive regulator of
Wg signaling. Tws is required for the stabilization of Armadillo (Arm;
Drosophila homologue of ß-catenin) in response to Wg signaling.
Its primary role in Wg pathway appears to be inhibition of Shaggy (Sgg;
Drosophila homologue of GSK3ß) activity.
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Materials and methods |
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Full-length (2.5 kb) tws cDNA clone (LD12394;
http://www.fruitfly.org)
was subcloned into the P-element vector, pCaSpeR-UAS
(Brand and Perrimon, 1993). The
construct was first sequenced to ensure that no mutations have been introduced
during cloning. All the transgenic flies (29 independent lines) generated from
this construct were crossed to various GAL4 drivers to express Tws in
different tissues. All transgenic lines showed similar phenotypes, although
they differed in the severity of the phenotypes. UAS-Tws23 gave the strongest
effect as a single insertion, which was used in all the experiments reported
here.
Other UAS lines used were, UAS-armS2 and UAS-armS10
(Pai et al., 1997),
UAS-Flu-
Arm (Zecca et al.,
1996
), UAS-Dsh (Neumann and
Cohen, 1996
), UAS-DN-TCF/pan (van der Wetering et al., 1997),
UAS-APC/FL (expressing full-length human APC) and UAS-hAPC/CBD (expressing the
ß-catenin binding domain of human APC)
(Bhandari and Shashidhara,
2001
), UAS-Sgg (Steitz et al.,
1998
), UAS-DNGSK-3ß (dominant negative form of
Xenopus Sgg/GSK-3ß) (Jia et
al., 2002
) and UAS-Cadi5 (intracellular domain of
Drosophila E-cadherin) (Sanson et
al., 1996
). GAL4 strains used were en-GAL4
(Brand and Perrimon, 1993
),
vg-GAL4 (Simmonds et al.,
1995
), hs-GAL4 (Bloomington stock list; originally developed by
Andrea Brand) and 405-GAL4 (expressed only in the differentiating neurons of
wing and eye imaginal discs) (Bhandari and
Shashidhara, 2001
).
Histology
X-gal staining and immunohistochemical staining were essentially as
described (Ghysen and O'Kane,
1989; Patel et al.,
1989
). The lacZ reporter gene constructs used were vg-DV
enhancer-lacZ and vg-quadrant enhancer-lacZ
(Kim et al., 1996
).
FITC-labeled actin-Phalloidin was purchased from Molecular Probes. The primary
antibodies used were, monoclonal anti-Ac
(Skeath and Carroll, 1991
),
anti-Arm (Riggleman et al.,
1990
), anti ß-galactosidase (Sigma, St Louis, MO), anti-Ci
(Motzny and Holmgren, 1995
),
anti-Cut (Blochlinger et al.,
1993
), anti-Dll (Vachon et
al., 1992
), anti-Fas3 (Patel
et al., 1987
), anti-Myc-tag (purchased from Sigma, St. Louis, MO),
anti-Sca (Mlodzik et al.,
1990
) and anti-Wg (Brook and
Cohen, 1996
) and polyclonal anti-Arm
(Ruel et al., 1999
),
ß-galactosidase (raised by A. Khar, CCMB, India), anti-Tws
(Gomes et al., 1993
), anti-Vg
(Williams et al., 1993
).
Anti-Ac, Anti-Arm, anti-Fas3, anti-Sca and anti-Wg were obtained from the
Development Studies Hybridoma Bank, University of Iowa, USA. Fluorescence
images were obtained either on a Zeiss Axiocam digital camera, on Meridian
Ultima confocal microscope or on Zeiss LSM/Meta. Control and experimental
images were digitized always at identical fluorescence microscope and camera
settings. The adult appendages were processed for microscopy as described
before (Shashidhara et al.,
1999
).
![]() |
Results |
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|
Growth and patterning during fly wing development are mediated by signaling
from its dorsoventral (DV) organizer. Interactions between dorsal and ventral
cells of the wing pouch set up the organizer by activating Notch (N) in the DV
boundary (Diaz-Benjumea and Cohen,
1993; Diaz-Benjumea and Cohen,
1995
; Williams et al.,
1994
; Irvine and Wieschaus,
1994
; Kim et al.,
1996
; de Celis et al.,
1996
). N, in turn, activates Wingless (Wg), Cut (Ct) and Vestigial
(Vg) in the DV boundary (Couso et al.,
1995
; Kim et al.,
1995
; Rulifson and Blair,
1995
; Kim et al.,
1996
; Neumann and Cohen,
1996
). Wg is known to diffuse to non-DV cells from the DV boundary
and acts as a morphogen (Zecca et al.,
1996
; Neumann and Cohen,
1997
). First, it collaborates with N to activate Cut (Ct) in a
cell-autonomous manner (de Celis and Bray,
1997
). Highest levels of secreted Wg are required to activate
Achaete (Ac) and Scabrous (Sca) expression in the sensory mother cells (SMC)
along the DV boundary (Zecca et al.,
1996
; Neumann and Cohen,
1997
), whereas moderate levels are enough to activate Distal-less
(Dll) and low-levels to activate Vg
(Neumann and Cohen, 1997
).
Thus, Vg is expressed in both DV and non-DV cells. Two different enhancers
regulate Vg expression in DV and non-DV cells
(Kim et al., 1996
). They are
vg-boundary enhancer (vg-BE) and vg-quadrant
enhancer (vg-QE).
Anti-Wg antibody staining of twsP mutant discs revealed that the levels of Wg are marginally reduced, but the pattern of expression is unchanged (Fig. 1G,I). However, vg-BE expression remained robust (Fig. 1K), suggesting that N pathway is not affected in tws mutant discs. We then examined a number of targets of Wg pathway. Ct expression, which is dependent on both N and Wg signaling, is normally seen as a continuous narrow line along the entire DV boundary (Fig. 1L). In tws mutant wing discs, its expression is irregular and discontinuous (Fig. 1M). We observed partial to complete loss of Ac (Fig. 2B) and Sca (Fig. 2D) expression, particularly in the presumptive margin SMCs, and moderate to severe reduction in Dll and vg-QE expression (Fig. 2F,H). Downregulation of Ac, Sca, Dll and vg-QE suggests that Wg signaling is down regulated in twsP wing discs. Observed downregulation of Wg expression could be due to interference in its autoregulation.
|
Clonal analysis of twsP allele
Serrated wing margin phenotypes are characteristics of loss of Wg
(hypomorphic wg mutants)
(Phillips and Whittle, 1993)
or loss of its upstream regulators such as N and Ser
(Neumann and Cohen, 1996
).
Downregulation of targets of Wg such as Ac and Sca in twsP
wing discs predict serrated anterior margin in adult wings. As
twsP mutants die in late larval/early pupal stages, we
employed a clonal analysis approach to examine the requirement of tws
gene product in adult wing margin formation. We observed higher frequency of
mitotic clones when FLP expression was induced at 48-72 hours AEL. Consistent
with our prediction on downregulation of Wg signaling in
twsP discs, a large number of adult flies with
twsP/twsP clones showed serrated wing
margin phenotype (n=27; Fig.
3A,B). Interestingly, the frequency of the clones observed in
imaginal discs was far higher than the number of clones observed in pharate
adult/adult flies. Consistent with this observation, imaginal discs showed
large clusters of tws+/tws+ cells with
small or no associated twsP/twsP spots
(Fig. 3C). Because we also
observed larger twsP/twsP clones
(Fig. 3D), it is likely that
they grow slowly and subsequently eliminated by neighboring cells.
|
Arm is not stabilized in tws mutant wing discs
We then examined the levels of Arm, the key regulator of Wg signaling. In
addition to being an effector of Wg signaling, Arm binds to intracellular
domain of E-cadherin and participates in modulating the cell-adhesion
properties of the cell. Stabilization of cytoplasmic Arm is a key step in the
transduction of Wg signaling. Thus, although Arm is present in all the cells,
cytoplasmic levels of Arm, which transduces Wg signaling, are higher only in
cells wherein Wg signaling is active
(Peifer et al., 1994). In wing
discs, cells immediately adjacent to the DV boundary show higher levels of Arm
than non-DV cells (Jiang and Struhl,
1998
; Collins and Treisman,
2000
; Bhandari and Shashidhara,
2001
; Mohit et al.,
2003
) (Fig. 4A).
Unlike in wild-type discs, Arm levels are lower in the DV boundary of all
tws homozygous mutant discs examined (n>40;
Fig. 4B). In
twsP/twsP wing discs, peripodial and
disc proper cells are often in the same focal plane, suggesting that these
discs are flatter than the wild-type discs
(Fig. 4B). We therefore
examined if loss of Arm in tws- background is due to
changed disc morphology. Noncytoplasmic Arm (Cadherin-bound form) is normally
localized to the apical/subapical surface of wing epithelial cells. However,
there was no difference between wild-type and
twsP/twsP discs in the intensities of
Arm in adherens junctions of cells around the DV boundary
(Fig. 4B). In addition to Arm,
we used actin-phalloidin to mark the apical surface and Fas3 to mark the
basolateral surface. Confocal examination of tws homozygous mutant
discs stained with these markers suggested normal apicobasal polarity of disc
cells (data not shown). In addition, normally observed intense staining of
actin-phalloidin in the cells adjacent to the DV boundary
(Fig. 4D) was not affected in
tws- wing discs (Fig.
4F), indicating similar cell densities around the DV boundary of
wild-type and mutant wing discs. Thus, observed reduction in Arm levels in
tws- discs is not due to loss of apicobasal polarity, or
to changes in cell morphology or to a decrease in the density of cells in and
around the DV boundary. Finally, clonal removal of twsP
from the cells abutting the DV boundary caused cell-autonomous downregulation
of Arm to the levels normally seen in distant non-DV cells
(Fig. 4G). Therefore, reduction
in Arm in tws- discs is probably due to lowering of the
levels of cytoplasmic Arm.
|
Tws functions downstream of Dsh and upstream of Sgg
We further examined if twsP modifies phenotypes induced
by the overexpression of known positive and negative effectors of Wg
signaling. Positive regulators such as Dsh and activated Arm and negative
regulators such as Sgg, dominant-negative TCF/pan (DN-TCF/pan), intracellular
domain of Cadherin (Cadintra) were overexpressed in wild-type and
in twsP heterozygous backgrounds
(Table 1). Severity of wing
margin phenotypes (supernumerary bristles;
Fig. 5A) induced by ectopic Dsh
was significantly reduced when it was expressed in twsP
heterozygous background (Fig.
5B). Conversely, wing-to-notum transformations induced by ectopic
Sgg (Fig. 5C) were
significantly enhanced when expressed in twsP heterozygous
background (Fig. 5D).
Interestingly, phenotypes generated by the ectopic expression of
degradation-resistant forms of Arm (UAS-Arm or UAS-armS10
crossed to vg-GAL4) were not even marginally affected by
twsP (data not shown).
|
|
In both Drosophila and in mammalian cells, APC binds to
Arm/ß-catenin even when Wg/Wnt is active
(Papkoff et al., 1996;
Bhandari and Shashidhara,
2001
). In those cells, wherein Sgg is inactivated, APC sequesters
Arm/ß-catenin rather than recruiting it to degradation machinery. For
example, overexpression of human colon cancer gene APC in wing discs
sequesters Arm only in DV cells (Bhandari
and Shashidhara, 2001
; Mohit
et al., 2003
) (Fig.
5G). In other cells, overexpressed APC participates in the
Arm-degradation machinery and hence no change in Arm expression is observed.
Because only unphosphorylated Arm/ß-catenin is sequestered and not the
phosphorylated form (Munemitsu et al.,
1996
), the amount of Arm sequestered by overexpressed APC would
provide a relative estimate of Sgg activity. Overexpressed APC in
twsP/twsP background did not sequester
Arm (Fig. 5H), indicating that
in this genetic background Sgg is active in DV cells. Thus, the normal
function of tws gene appears to be inactivation of Sgg in response to
Wg signal.
Ectopic Tws induces gain-of-Wg-function phenotypes
Results described above show that Tws functions as a positive regulator of
Wg signaling, which is consistent with the phenotypes observed in mice lacking
the catalytic subunit of PP2A. Most of the observations against this, wherein
PP2A is shown as a negative regulator of Wnt/Wg signaling comes from studies
involving overexpression of the regulatory subunit. We therefore analyzed the
effect of ectopic expression of Tws in the context of Wg signaling.
We generated transgenic flies that enable overexpression of Tws using GAL4-UAS system. We first tested if the transgene expresses normal protein. We expressed Tws using hs-GAL4 driver in homozygous twsP and tws60 backgrounds and examined if UAS-Tws could rescue the mutations. Temperature-shift experiments, wherein the developing animals were kept at temperatures between 25°C and 28°C for varying lengths of time, did not show any rescue of twsP mutant phenotypes. We also tried a number of heat-shock regimes (1 hour pulse at 37°C), but with no success. However, low levels of expression of UAS-Tws driven by the basal activity of the heat-shock promoter at 25°C was sufficient to rescue late larval/early pupal lethal phenotype of tws60. Rescued pharate adults showed distinctive adult structures (Fig. 6B,C, n=20), but were unable to eclose. Significantly, wing blades of rescued pharate adults showed normal wing margin, suggesting recovery of Wingless signaling. No wing disc showed pattern duplication (n=28), suggesting rescue of both Wingless-dependent and independent developmental events. Inability to rescue twsP, the stronger of the two alleles, suggests that survival of homozygous twsP larvae may require higher levels of tws gene product. Indeed, expression of UAS-Tws with the help of a stronger driver (en-GAL4) resulted in the rescue of twsP mutant flies at the levels of both pattern duplication in discs (data not shown) and adult wing blade (Fig. 6D). Close observation of the cuticle in the rescued pharate adults indicated that the rescue was limited to posterior compartment (data not shown), which reflects the fact that Tws was expressed only in that compartment.
|
Overexpression of Tws in the wild-type background functions as dominant negative
Results described in the previous section clearly indicate that UAS-Tws
construct we employed in our study functions like wild-type Tws. It rescued
tws mutation and also showed gain-of-Wg signaling. However, when Tws
was overexpressed in wild-type background, we observed downregulation of Wg
signaling. Overexpression of Tws specifically in the DV boundary using
vg-GAL4 was enough to cause downregulation of Ct expression in the DV
boundary (Fig. 7A), Sca
expression in the presumptive margin SMCs
(Fig. 7B), and Dll
(Fig. 7C) and Vg
(Fig. 7E) expression in non-DV
cells. Arm levels along the DV boundary were also downregulated in wing discs
overexpressing Tws using vg-GAL4
(Fig. 7G). Adult flies
displayed characteristic serrated wing margin
(Fig. 7H), which is similar to
the phenotype observed in wing blades carrying twsP
mitotic clones. In addition, we observed enhanced notching of wing margin when
Tws was overexpressed in wgP/+ background (data not
shown). These results suggest that ectopic Tws in wild-type background
functions as dominant-negative that recreates the tws mutant
phenotypes both at the molecular levels in discs and adult phenotypic levels.
This is further supported by the observation that co-expression of degradation
resistant form of Arm (armS10) with Tws was able to suppress wing
margin phenotype (Fig. 7I).
However, degradation sensitive form of Arm (armS2) did not show any
effect on Tws-induced phenotypes (data not shown). This suggests that, similar
to loss of tws, overexpressed Tws causes downregulation of Wg
signaling by interfering with an event downstream of Dsh and upstream of
Arm.
|
![]() |
Discussion |
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Similar to many other signal transduction pathways, changes in phosphorylation-dephosphorylation status of Wg/Wnt pathway components are key to its regulation. Although the role of PP2A as a dephosphorylating agent is established by studies on Pp2a- mice, there has not been a consensus on (1) whether PP2A positively or negatively regulates Wg/Wnt signaling, (2) does PP2A function downstream or upstream (or both) of ß-catenin and (3) which regulatory subunit of PP2A functions to modify Wg/Wnt signaling.
tws is a positive regulator of Wg/Wnt signaling
Results of our studies described in this report show that
Drosophila homologue of B/PR55 subunit of PP2A is involved in
modifying Wg signaling. We have observed partial to complete downregulation of
short- (Ct and Sca) and long-range (Dll and Vg) targets of Wg pathway in
tws- background. The downregulation of Wg signaling in
wing discs was reflected in adult phenotypes, such as serrated wing margin in
mitotic clones of tws. We have also observed that loss-of-Wg
phenotypes (induced by the overexpression of DN-TCF/pan or Sgg or
Cadintra) are enhanced in tws heterozygous mutant
background. In addition, mutation in tws suppressed the phenotypes
induced by Dsh, a positive component of Wg signaling. Finally, some of the
phenotypes induced by the overexpression of Tws are characteristic of
gain-of-Wg phenotypes. These results suggest that Tws functions as a positive
regulator of Wg signaling.
We have further shown that overexpression of, otherwise normal, Tws protein induce dominant-negative phenotypes. The dominant-negative phenotype is unlikely to be neomorphic or antimorphic, as UAS-Tws rescued tws alleles (at the levels of both Wingless-dependent and independent developmental events) and also induced gain-of-Wg phenotypes. The dominant-negative phenotype is probably due to imbalance in the relative amounts of the three subunits in the heterotrimeric complex, proper formation of which is obligatory for PP2A function. Thus, the conflicting reports on the role of PP2A in Wnt signaling could be due to the dominant negative effect caused by the overexpression of one of the subunits.
Mechanism of Tws function
In tws mutant background, cytoplasmic Arm levels are
downregulated. Even the overexpressed Arm was degraded in
tws- background. Furthermore, loss of tws had no
effect on the degradation-resistant form of Arm, which suggests that Tws
functions upstream of Arm to mediate Wg signaling. We could not confirm these
results directly by western blotting, as only a very small fraction (such as
DV cells) of wing disc shows changes in Arm levels in response to Wg
signaling. Nevertheless, results presented in this report suggest that
stabilization of the cytoplasmic form of Arm by Wg signaling is dependent on
Tws function.
A dominant-negative form of Sgg/GSK-3ß was able to rescue
tws- phenotype at the level of Dll expression. However,
overexpression of Dsh failed to rescue Dll expression in
tws- discs, suggesting that Tws functions downstream of
Dsh and upstream of Sgg to stabilize cytoplasmic Arm in response to Wg
signaling. Preliminary results presented here suggest that function of Tws in
Wg pathway is inactivation of Sgg. Normally, overexpressed APC sequesters Arm
only in those cells in which Sgg activity is downregulated
(Bhandari and Shashidhara,
2001). In other cells, APC participates in Arm-degradation
machinery. In tws- wing discs, overexpressed APC failed to
sequester Arm in DV cells, suggesting that loss of tws results in
upregulation of Sgg activity. However, recently, Yang et al.
(Yang et al., 2003
) have
reported that PR/B56
functions upstream of Dsh to regulate Wnt signaling
in Xenopus embryos. The PR/B56
homologue in Drosophila
is widerborst (with 80% identity at the protein level), which is
involved in the determination of planar cell polarity
(Hannus et al., 2002
).
widerborst is also known to be functional upstream of Dsh, but not in
the canonical Wg/Wnt pathway. Although Tws homologues in other organisms have
not been well characterized, our studies are consistent with a role for PP2A
in dephosphorylation of Axin (Willert et
al., 1999
).
The next question is on the substrate of PP2A function in Wg pathway. In
mammalian cells, Axin is dephosphorylated in response to Wnt signaling
(Willert et al., 1999).
Furthermore, dephosphorylated Axin binds ß-catenin less efficiently than
the phosphorylated form. Thus, dephosphorylation of Axin would free
ß-catenin from the degradation machinery
(Willert et al., 1999
). Thus,
Tws may function by inhibiting the activity of Axin, which acts a scaffold
protein to bring Sgg and Arm to close proximity. Further biochemical work is
in progress to determine phosphorylated status of Arm in
tws- background and to determine if Tws directly binds to
Sgg or Axin or both.
Role of tws in other signal transduction pathways
The current study was mainly concerned with the role of Tws in Wg
signaling. tws alleles were initially isolated based on two different
kinds of phenotypes, both of which are not related to Wg signaling. The
catalytic subunit of PP2A in Drosophila is encoded by microtubule
star (mts). Loss-of-function of either mts causes
overcondensation of chromatin and blocks the cell division at mitosis, between
prophase and anaphase (Snaith et al.,
1996). Similar phenotypes are reported for the
aar1 allele of tws
(Gomes et al., 1993
). These
phenotypes reflect the interaction between Tws and cyclinB/cdc2, which are
required for G2-M transition (Minshull et
al., 1996
).
The pattern duplication in twsP is always limited to
posterior compartment and is mainly due to loss of En, which in turn causes
ectopic Dpp expression (Uemura et al.,
1993). Vn/EGFR signaling is known to specify notum by antagonizing
wing development and by activating notum-specifying genes
(Baonza et al., 2000
;
Wang et al., 2000
;
Pallavi and Shashidhara,
2003
). Thus, loss of EGFR signaling would lead to notum-to-wing
transformation. Furthermore, Vn/EGFR is required for the maintenance of En in
the posterior compartment, defect in which would lead to pattern duplications
of the kind seen in twsP background. Thus, it is likely
that Tws is also functional in EGFR signal transduction pathway. Consistent
with this, tws alleles are known to interact with Ras
alleles and enhance eye phenotypes of the latter (Wassarman, 1996).
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
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