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First published online March 6, 2009
doi: 10.1242/10.1242/dev.021428
1 Centro de Biología Molecular `Severo Ochoa' (C.S.I.C.-UAM), Universidad
Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain.
2 Department of Physiology, Development and Neuroscience, Anatomy Building,
University of Cambridge, Downing Street, Cambridge CB2 3DY, UK.
* Author for correspondence (e-mail: iguerrero{at}cbm.uam.es)
Accepted 3 February 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Lines, Drumstick, Bowl, Wingless, Hedgehog, Notch, Groucho, Hairless, Drosophila wing development
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The Drosophila wing is a discrete organ that has been used to
study the coordination of signaling pathways during development. The
developing wing disc is a sac-like structure composed of the columnar
epithelium or disc proper cells (DP), the cuboidal marginal cells (MC) and the
overlying squamous cells (SC); MC and SC constitute the peripodial epithelium
(PE) (Fig. 1A-D). During larval
development, imaginal cells proliferate extensively and are patterned. After
metamorphosis, the DP cells differentiate into the cuticle that forms the
adult wing and notum, whereas PE cells contribute little to these structures
(Milner et al., 1984
).
Here, we summarize key signaling events that take place in the DP during
development. At very early stages, localized Wingless (Wg) signaling restricts
the activity of the EGFR pathway to the proximal region to subdivide the wing
imaginal disc into wing and body wall (notum) precursors, where it appears to
be required continuously to allocate notal cell fates from neighboring wing
fates (Wang et al., 2000;
Zecca and Struhl, 2002b). This
subdivision is the primary manifestation of the proximodistal (PD)
patterning.
The growth and patterning of the wing disc depends on the establishment of
two organizing/signaling centers. One is established at the boundary between
anterior (A) and posterior (P) cells through the activity of Hh, produced in
the P compartment. Hh induces expression of the secreted signaling molecule
Decapentaplegic (Dpp; a member of the TGF-beta family) in a thin stripe of A
cells that acts as a long-range morphogen to coordinate patterning and growth
along the AP boundary (reviewed by Blair,
2007). A second organizer is established during the second larval
instar to subdivide the wing disc into dorsal (D) and ventral (V) compartments
(Diaz-Benjumea and Cohen,
1995), resulting in a stripe of cells with elevated N activation
at the interface of DV cells (de Celis et
al., 1996; Diaz-Benjumea and
Cohen, 1995; Doherty et al.,
1996). N in turn activates the expression of Wg in cells along the
DV boundary (Diaz-Benjumea and Cohen,
1995; Rulifson et al.,
1996), and further refinement involves a series of positive- and
negative-feedback loops between both pathways. In addition to these primary
signaling events, several other pathways are also important for coordination
of cell proliferation in developing tissues, including the JAK/STAT pathway
(Mukherjee et al., 2005).
One key question is how can different pathways be integrated and
coordinated during the wing development? In the nucleus, several of these
pathways use to share common regulators that act simultaneously on their
transcriptional control. One of these components is the co-repressor
Groucho/TLE (Gro). Gro is recruited to target promoters by association with
DNA-binding proteins through conserved eh1 or WRPW domains
(Buscarlet and Stifani, 2007).
Transcriptional regulators of the N, Wnt, Dpp, EGFR and Hh pathways all
interact with Gro (Hasson et al.,
2005). A second possible integrator is the regulatory cassette
formed by Lines (Lin) and the Odd-skipped gene family of zinc finger proteins
[bowl, drumstick (drm), odd-skipped (odd)
and sister of odd and bowl (sob)]
(Bras-Pereira et al., 2006;
de Celis Ibeas and Bray, 2003;
Hao et al., 2003;
Hatini et al., 2005;
Iwaki et al., 2001). The
cassette Drm/Lin/Bowl controls the morphogenesis at several stages: in the
embryo, they coordinate epidermal cell differentiation through regulating Hh
and Wg signaling inputs (Bokor and DiNardo,
1996; Hatini et al.,
2000; Hatini et al.,
2005); and, in the gut, they regulate morphogenesis by controlling
the JAK-STAT proliferative pathway (Green
et al., 2002; Iwaki et al.,
2001). During imaginal disc development, they are regulated by the
N signaling pathway in the leg disc (de
Celis Ibeas and Bray, 2003;
Hao et al., 2003); in the eye
disc, the Odd-skipped family regulates the activation of Hh during
retinogenesis (Bras-Pereira et al.,
2006).
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
bowl2 (Wang and
Coulter, 1996), gro1
(Preiss et al., 1988),
STAT92E-lacZ
(http://flystocks.bio.indiana.edu/),
Hh-lacZ (Lee et al.,
1992), brk-lacZ
(Minami et al., 1999),
kekkon-lacZ (Musacchio and
Perrimon, 1996) and puckered-lacZ (pucE69)
(Martin-Blanco et al., 1998).
The following Gal4 drivers were used: ptc-Gal4
(Hinz et al., 1994),
ubx-Gal4 (Pallavi and
Shashidhara, 2003), pnr-Gal4, ap-Gal4
(Calleja et al., 1996) and
sd-Gal4 (Mullor et al.,
1997).
Transgenic fly lines previously described are: UAS-bowl
(de Celis Ibeas and Bray,
2003), UAS-drm (gift from Judith Lengyel),
UAS-armS10 (Pai et
al., 1997), UAS-dTcf
(van de Wetering et al.,
1997), UAS-H (Klein
et al., 2000), UAS-gro
(Apidianakis et al., 2001),
UAS-mtv (Funakoshi et al.,
2001), UAS-dicer
(Dietzl et al., 2007) and
UAS-linRNAi, UAS-bowlRNAi (VDRC,
http://stockcenter.vdrc.at).
To generate the Bowleh1- construct, a fragment of the bowl
cDNA lacking the last 13 codons, which encodes the eh-1 domain (RTGFFSIEDI),
was amplified. To generate the Bowleh1-VP16 construct, this
bowl cDNA was ligated in frame with herpes virus protein VP16
(pHK3NVP16).
Overexpression experiments and generation of clones
Mitotic recombination clones Random clones were generated by FLP-mediated
recombination. Flies of the genotype FRT42D
linG1/CyO were crossed to flies FLP; FRT42D
arm-lacZ/CyO or FLP; FRT42D Ubi-GFP/CyO, and mosaic
clones were induced by incubating larvae at 37°C for 30 minutes at 24-48,
48-72 and 72-96 hours after egg laying (AEL).
Flip-out clones
The transgene abx/ubx<FRT, stop, f+, FRT<
Gal4-UAS-lacZ (de Celis et al.,
1998) and the transgene Act>CD2>Gal4
(Pignoni and Zipursky, 1997)
were used to generate ectopic expression clones by incubating larvae at
37°C for 15 minutes at 48-72 hours AEL.
MARCM clones
To generate linG1 clones that ectopically express
ArmS10 or Gro males UAS-armS10;
FRT42DlinG1/CyO or
FRT42DlinG1/CyO; UAS-Gro were crossed to
females: y,w,FLP,Tub Gal4,UAS-GFP; FRT42D Gal80/CyO. To generate
bowl2 clones marked by UAS-GFP, males
bowl2 FRT40A were crossed to females y,w,FLP,Tub
Gal4,UAS-GFP; Gal80 FRT40A/CyO. In all cases, larvae were incubated at
37°C for 30 minutes at 48-72 hours AEL.
Transient expression of transgenes
Transient expression of UAS transgenes was induced using different Gal4
drivers and maintaining crosses at 18°C and inactivating the
Gal80ts for 7 to 36 hours at the restrictive temperature
(29°C).
In situ hybridization
Dioxigenin (Roche) probes were used to detect lin, bowl and
drm mRNA in imaginal discs. To prepare the antisense riboprobes,
fragments from lin (clone LD 43682), drm (clone LD 26791)
and bowl (clone LD 15350) cDNAs were cloned into pGEMT, pOT2 or pBS
SK vectors.
Generation of the anti-Lin antibody
For generating the anti-Lin antibody, a region of 1.6 kb of the
lin cDNA was amplified and subcloned in the
BamHI/KpnI site of the expression vector pT7-7. The induced
Lin protein was purified by electrophoresis in acrylamide-SDS gels and
extracted and injected in guinea pigs.
Antibodies and immunohistochemistry
Immunostaining was performed according to standard protocols. Antibodies
were used at the following dilutions: rabbit anti-β-gal 1/1000 (Jackson
Laboratories); rabbit anti-β-gal 1/100 (Promega); anti-Dl 1/5, anti-Wg
1/50 and anti-Ubx antibody 1/10 from the Hybridoma Bank; rabbit anti-Bowl
1/500 (de Celis Ibeas and Bray,
2003); rabbit anti-Hth 1/200
(Aldaz et al., 2005); rat
anti-Iro 1/200 (Diez del Corral et al.,
1999); mouse anti-MAPK-P 1/1000 (Sigma); mouse anti-Nub 1/50
(Yeo et al., 1995); guinea-pig
anti-Sens 1/1000 (Nolo et al.,
2000); rat anti-STAT92E 1/20 (gift from Aurel Betz); rabbit
anti-STAT-p 1/1000 (Cell Signaling Technology); rabbit anti-Tsh 1/1000
(Gallet et al., 1998); rabbit
anti-Zfh2 1/250 (Whitworth and Russell,
2003); rabbit anti-Caspase3 1/50 (Hybridoma bank); mouse anti-Ptc
1/50 (Capdevila and Guerrero,
1994); rabbit anti Hh antibody 1/800
(Takei et al., 2004); and
rabbit anti-phospho-Histone-3 1/400 (Cell Signaling Technology).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Hatini et al., 2000;
Hatini et al., 2005). To
investigate whether similar mechanisms apply in the wing imaginal disc, we
monitored lin mRNA and protein distribution. Both, the lin
transcript and Lin protein, were ubiquitously expressed
(Fig. 1E,H); however, the
subcellular localization of the protein was clearly modulated. Whereas Lin was
detected in both nucleus and cytoplasm in most of DP cells
(Fig. 1H,J), it was restricted
to the cytoplasm in the cubiodal MC, a narrow stripe of cells along the
anterior border of the notum and pleura
(Fig. 1I), and in SC
(Fig. 1K). If the Lin/Drm/Bowl
cassette functions as in the embryo, the cytoplasmic localization should
indicate where Lin is inactive. Conversely, the presence of nuclear Lin in
most of the wing imaginal cells would be suggestive of a functional role.
To analyze the role of lin during wing development, we induced
mitotic recombinant clones of the null allele linG1
(lin-) (Bokor and
DiNardo, 1996). Clones induced mid-way through larval development
(48-72 hours AEL) gave rise to dramatic overgrowths that segregated from
wild-type tissue, forming smooth borders. The increased in division rate in
lin- clones is monitored by higher expression of
phospho-Histone 3 (PH3), a marker of cell division. Only few of such clones
persisted into adult structures, most probably as a consequence of cell death
observed using the apoptotic markers puckered-lacZ, and activated
caspase 3. Notably, clones induced earlier (24-48 hours AEL) fail to survive
even to larval stages, as only wild-type twin clones were detected under these
conditions. Strikingly, some late induced clones (72 hours AEL) were able to
regenerate a complete wing or notum when they were located near the wing hinge
region (see Fig. S1 in the supplementary material).
In many developmental contexts, Lin is regulated by Drm, which prevents the
interaction between Lin and Bowl, allowing nuclear accumulation of Bowl
(Bras-Pereira et al., 2006;
de Celis Ibeas and Bray, 2003;
Hao et al., 2003;
Hatini et al., 2005;
Iwaki et al., 2001). In the
wing imaginal disc, drm transcript was detected in both the MC along
the anterior notum border and in the SC, where Lin is cytoplasmic
(Fig. 1F). However, although
bowl transcript was present uniformly throughout the disc
(Fig. 1G), Bowl protein was
found only in the nucleus of drm-expressing cells
(Fig. 1L,M). As predicted, in
lin- clones Bowl protein accumulated dramatically,
independently of the position of the clone in the DP cells
(Fig. 2A). Therefore, we
conclude that in the wing disc Lin regulates Bowl protein stability, probably
through similar mechanisms as during embryonic development
(Hatini et al., 2005).
Furthermore, ectopic expression of Drm in the DP cells results in a relocalization of Lin to the cytoplasm (Fig. 2B) and a corresponding stabilization of Bowl (Fig. 2C) supporting our model. Therefore, Drm overexpression in DP cells should cause the same phenotypes as lin- clones. Indeed, ectopic Drm expression gave overgrowths and cell identity changes similar to lin- clones (see below) (see Fig. S2 in the supplementary material). Therefore, in subsequent experiments, we use both Drm gain-of-function (GOF) and lin- clones for analyzing the lin requirement in the wing imaginal disc.
To determine whether the phenotypes observed in lin-
(or Drm GOF) clones were a consequence of Bowl stabilization, we knocked out
both lin and bowl functions by co-expressing RNAi against
both genes. We first tested the functionality of the corresponding RNAi.
Expression of Lin-RNAi in D cells of the wing disc severely compromised Lin
protein expression and led to Bowl protein stabilization
(Fig. 2D). Expression of
Bowl-RNAi in P cells in the leg disc, where Bowl has a characteristic rings
pattern corresponding to the joint primordia
(de Celis Ibeas and Bray,
2003; Hao et al.,
2003; Hatini et al.,
2005), is able to ablate Bowl expression in these cells,
demonstrating efficient knock down (Fig.
2E).
To confirm that the effects of removing Lin depend on Bowl, we examined the expression of either Lin-RNAi or Bowl-RNAi, or both, in DP cells of the wing pouch. Expression of Bowl-RNAi did not cause any wing alteration (Fig. 2F). However, Lin-RNAi expression resulted in a drastic reduction of wing size, indicating that the development was severely compromised (Fig. 2G). This phenotype was completely suppressed when Lin-RNAi and Bowl-RNAi were co-expressed (Fig. 2H). Hence, we can conclude that Bowl is the primary effector of the changes observed in lin- (and Drm GOF) clones in the wing disc.
|
; Ng et al.,
1995; Whitworth and Russell,
2003) was absent in lin- clones in the wing
pouch (Fig. 3A). Conversely,
genes normally expressed in the hinge and notal regions of the disc, such as
the zinc finger homeodomain-2 (zfh-2), teashirt
(tsh) and homothorax (hth)
(Fig. 3F,H,J)
(Azpiazu and Morata, 2000;
Casares and Mann, 2000;
Terriente et al., 2008;
Whitworth and Russell, 2003;
Wu and Cohen, 2002;
Zirin and Mann, 2007), were
now expressed at high levels in lin- cells, indicating a
change in their PD identity (Fig.
3G-I).
As the PD specification involves an antagonistic interaction between Wg and
EGFR (Zecca and Struhl, 2002a;
Zecca and Struhl, 2002b), the
phenotypes might be caused by a requirement of lin for the activation
of Wg target genes. To test whether this was the case, we generated
lin- clones that simultaneously expressed a constitutively
active form of Arm (ArmS10) or dTcf (Pangolin), transcriptional
effectors of the Wg pathway (reviewed by
Tolwinski and Wieschaus,
2004). Neither rescue of over-proliferation nor aberrant gene
expression was detected in either combination
(Fig. 3D,E), suggesting that
Lin acts in parallel to or downstream of Arm and dTcf/Pangolin to regulate Wg
pathway, as it was observed in the embryo
(Hatini et al., 2000;
Hatini et al., 2005).
Lin/Bowl regulate Wg signaling at the D/V compartment border of the wing disc
Wg is required at multiple stages during wing development. To investigate
whether Lin could regulate the later Wg function at the DV compartment
boundary, we analyzed Senseless (Sens) expression, a specific Wg target, and
found that its expression was absent in both lin- and Drm
GOF clones (Fig. 4B). These
results are consistent with the role of Lin regulating Wg signaling, as at
earlier stages. Moreover, the initial DV border specification involves an
antagonistic interaction between N and Wg pathway; thus, an alternate
possibility could be that N signaling is ectopically activated in the
lin- cells. In support of this hypothesis, we observed
that lin- clones induced close to the DV boundary
ectopically expressed two N targets, as Wg and Cut (Ct)
(Fig. 4D,F). This result
suggests that the effect on Sens expression, a Wg target gene, could be an
indirect consequence of N pathway activation.
To distinguish whether the effects on the Wg activity is caused directly or
through N activation, we used the Gal4/Gal80ts technique to induce
Drm expression (representing lin- function) at different
developmental times. Using this approach, we examined the effect of Drm on
targets of both Wg and N pathways after the DV boundary is established. The
earliest effect caused by ectopic Drm expression was Bowl stabilization, 7
hours after induction (Fig.
4G). Next, we detected total repression of Sens before 18 hours
(Fig. 4H); at that time, the
ectopic expression of N targets Wg and Ct was also observed in and adjacent to
the Drm-expressing clones (Fig.
4I,J). Thus, upregulation of N and Wg targets appear at similar
times, suggesting that effects on these pathways are independent. We note that
the strongest induction of Ct occurs at the boundary of the Drm-expressing
clone, suggesting that it might be augmented due to the induction of N ligand
expression within these clones (see Fig. S2B in the supplementary material), a
characteristic of ectopic Notch pathway activity in the late stages of wing
margin development (de Celis and Bray,
1997).
|
). We
therefore analyzed Vg expression in Drm-expressing clones induced for 25
hours. We observed Vg expression in clones at the DV border, but in the clones
located at a distance from the DV border Vg is repressed. These spatially
distinct phenotypes indicate that Drm-expressing cells can both upregulate N
pathway activity (to maintain Vg at DV) and downregulate Wg pathway activity
(to repress Vg at distant positions; see Fig. S2C in the supplementary
material). As Bowl appears to be the primary effector in lin- or Drm GOF clones in the wing disc, we examined N and Wg targets in clones where Bowl and Lin were simultaneously eliminated by co-expression of UASLin-RNAi and UASBowl-RNAi. Under these conditions, neither the repression of Sens nor the activation of the N targets was detected (Fig. 4K). These data suggest that the inhibition of Bowl by Lin is essential for normal Wg and N functions.
Lin/Drm/Bowl regulates Hh expression
In the dorsal embryonic epidermis lin plays an essential role
regulating the antagonistic interaction between the Wg and Hh pathways
(Hatini et al., 2005). We
therefore analyzed whether the Hh pathway was also affected in
lin- or Drm GOF cells in the wing disc. We transiently
overexpressed Drm in a stripe in the A compartment and monitored the
expression of Hh and its target, Patched (Ptc). Both were ectopically
expressed in the A compartment cells (Fig.
5B, compare with Fig.
5A). Using the hh-lacZ reporter, we confirmed that this
regulation occurs at transcriptional level. The Hh derepression was more
pronounced in V cells, as we also observed for N pathway targets
(Fig. 5C, see also
Fig. 4I,J), although the reason
for this is unclear. Next, inducing UAS-linRNAi clones randomly we
observed that hh was only activated in the A compartment clones close
to the AP border (Fig.
5D,D'). However, as discussed earlier, repression of the Wg
target Sens occurred in all ectopic UAS-linRNAi clones that touch the
DV border (Fig. 5D,D'').
The spatially restricted hh induction (in A clones close to the AP
compartment border) is similar to that seen in gro
(Apidianakis et al., 2001) and
mtv (Apidianakis et al.,
2001; Bejarano et al.,
2007) mutant clones. Mtv is a target of Hh at the AP compartment
border, which, together with Gro, helps to maintain hh repression in
the responding cells. Taken together, these results suggest that Lin/Bowl
plays a similar role in the wing pouch to that observed in the dorsal
embryonic epidermis, regulating the antagonistic interaction between the Wg
and Hh pathways in both contexts (Hatini
et al., 2000; Hatini et al.,
2005).
|
;
Barolo et al., 2002;
Bejarano et al., 2007;
Cavallo et al., 1998;
de Celis and Ruiz-Gomez, 1995;
Lawrence et al., 2000;
Morel et al., 2001;
Nagel et al., 2005). As Bowl
contains an eh-1 motif that recruits Gro, one way that Lin could exert its
effects is by regulating Bowl/Gro interactions
(Goldstein et al., 2005).
Thus, nuclear Bowl in lin- or Drm GOF cells could interact
with Gro and sequester it from the N and Hh repressor complex. To investigate whether the effects of Lin/Bowl could be mediated through sequestration of Gro, we tested whether the phenotypes caused by ectopic Bowl could be suppressed by co-expressing Gro. On its own, ectopic Bowl induces expression of Ct (or Wg) and Hh (Fig. 6A,E; and data not shown) in a similar manner to lin- or Drm GOF clones (albeit to a much weaker extent because Lin is still competent to destabilize the ectopically expressed Bowl protein). Co-expression of Gro and Bowl was sufficient to prevent the activation of these targets (Fig. 6B). However, when the eh-1 motif was eliminated in Bowleh1- (Fig. 6C,F) or substituted by the VP16 activation domain in Bowleh1-VP16 (Fig. 6D), Bowl is unable to activate ectopic expression of Ct, Wg or Hh. These results indicate that Bowl needs to interact with Gro to activate N targets and Hh expression. Therefore, we propose that Lin prevents the Bowl/Gro interaction. As Gro is required for repression in the N pathway and Hh expression, in lin- or Drm GOF cells, Bowl sequesters Gro from the repressor complexes, triggering ectopic expression of the target genes.
Conversely, the effect on Sens expression suggests that Bowl might act through a different mechanism to regulate the Wg pathway. First, co-expression of Bowl and Gro yields the same effects on Wg targets (Fig. 6H) as when either Gro (see Fig. S4A in the supplementary material) or Bowl (Fig. 6G) are expressed alone, arguing against the sequestration model. Second, expression of Bowleh1-, still repressed Sens (Fig. 6I), indicating that Bowl may acts as a transcriptional repressor independently of its interaction with Gro. Third, expression of Bowleh1-VP16 can activate Sens expression (Fig. 6J), although activation was variable and primarily detected in clones close to the endogenous source of Wg. Nevertheless these results suggest that the repression of Sens by Bowl can be reversed by the presence of an activation domain (VP16) and it is independent of its interaction with Gro. Therefore, Bowl represses Wg targets via a Gro-independent mechanism.
Our model implies a functional relationship for lin/bowl and gro, which might be detected by genetic interactions between alleles of lin and gro genes. gro1 individuals display tufts of bristles in the dorsal head and in the scutelum. Removing one dose of lin in this background (linG1/+; gro1/+) results in a high incidence of lethality and the few escapers showed enhanced phenotypes, such as ectopic eyes, leg truncations and duplications, loss of proboscis, duplication of antenna segments and nicks in the wing margin (see Fig. S3A-H in the supplementary material). The interaction between lin and gro was also evident from the rescue of ectopic Wg expression when Gro was overexpressed in lin- clones (see Fig. S3I,I' in the supplementary material).
Bowl recruits Gro from the N and Hh repressor complexes
Our results suggest that the effects of lin on the N and Hh
pathways are a consequence of the ability of Bowl to bind Gro, a crucial
component for repression of both pathways. Thus, the sequestration of Gro by
Bowl can explain both the ectopic activation of N targets and the Hh
expression.
Repression of N target genes by Gro is mediated by Suppressor of Hairless
[Su(H)], and Hairless (H), the adaptor that binds directly to Gro
(Barolo et al., 2002;
Furriols and Bray, 2000;
Morel et al., 2001). If the
activation of N targets in lin- or Drm GOF clones is
caused by the sequestration of Gro, it might be possible to overcome this by
increasing the availability of H. We therefore tested whether co-expression of
H and Drm was sufficient to suppress the lin- phenotypes.
In agreement, the overproliferation and the deregulation of the N pathway,
caused by Drm overexpression (Fig.
7A), were largely normalized by H
(Fig. 7B-C'). However,
Sens expression was not recovered (Fig.
7C). Moreover, the normal Hh activity in the A cells was also
restored by the ectopic H (Fig.
7B). Similarly, we tested whether overexpression of Mtv was able
to recover the ectopic Hh expression induced in Drm GOF, as predicted if this
is also caused by Bowl sequestering Gro from the Mtv/Gro repressor complex.
Co-expression of Mtv with Drm prevents the ectopic activation of Hh
(Fig. 7D). However, like H, Mtv
was unable to normalize the expression of Wg
(Fig. 7D) or Sens
(Fig. 7E). These results
indicate that Bowl can unbalance the Mtv/Gro and H/Su (H)/Gro repressor
complexes by sequestering Gro.
|
). In distal
lin- clones (48-60 hours AEL), we observed expression of
several EGFR targets (Kekkon, Iroquois and phosphorylated MAPK), suggesting
aberrant EGFR signaling and changes in PD identity. Expression of other
markers, such as STAT92E-lacZ, phospho-STAT92E and the Dpp targets
genes brinker (brk) and spalt (sal), was
also altered (see Fig. S5 in the supplementary material). The activation of
proximal markers can be explained as a long-term consequence of ectopic
repression of the early Wg by ectopic stabilized Bowl in
lin- cells, because a pulse (24 hours) of Drm expression
at the end of the second instar was sufficient to induce changes in the PD
identity, even though Drm was not present continuously (see Fig. S2D in the
supplementary material).
|
).
Bowl function in the peripodial epithelium
The regulatory interaction between Lin, Drm and Bowl restricts Bowl protein
to the SC and MC within the PE. To determine the function of Bowl in these
domains, we induced early bowl- clones, marked by the
expression of GFP (MARCM clones). Although the frequency of recovered clones
in PE (Fig. 8A) is usually
lower than in the DP (Fig. 8B),
we could visualize large clones in the PE and observed that they still
expressed the peripodial markers Ubx (Fig.
8A,A') and Hth (Fig.
8A,A''). We also expressed Bowl-RNAi in the PE and in the MC
using ubx-Gal4 (Pallavi and
Shashidhara, 2003). We found that some
Ubx-Gal>UAS-bowlRNAi wing discs were smaller than wild-type discs
(Fig. 8C) and showed altered
expression of Ubx (Fig.
8D,D') and Hth (Fig.
8D,D''). ubx-Gal4>UAS-bowlRNAi adult wings
display (30%) reduction of the proximal wing and occasionally the whole wing
was missing (Fig. 8F,F'').
Likewise, expressing UAS-bowlRNAi with pnr-Gal4 (expressed
in the notum primordium, including the MC expressing Bowl) results in a cleft
in the thorax and absence of dorso/central bristles
(Fig. 8I). These results
suggest that Bowl is required for normal wing and notum development, possibly
differentiating the signaling response between the SC/MC and DP cells.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Hao et al.,
2003; Hatini et al.,
2005; Nusinow et al.,
2008). In all cases, the steady-state accumulation of Bowl is
regulated by the relative levels of Drm and Lin proteins. High levels of Drm
impede binding of Lin to Bowl and, thus, this transcriptional repressor
becomes stabilized in the nucleus. Here, we have found that regulatory
interaction Lin/Drm/Bowl also functions during wing development. In
lin- or Drm GOF cause ectopic expression of Bowl and
dramatic overgrowths within the wing disc. These overgrowths frequently showed
altered cell identity, resembling more proximal disc margin cells. Some of the
effects can be explained by the ability of Bowl to interact with Gro
co-repressor through the eh-1 motif, forming a complex that sequesters Gro
from other repressors complexes such as Su(H)/H/Gro and Mtv/Gro.
Lin/Drm/Bowl regulative interaction
Although Bowl is ubiquitously transcribed in the wing disc, Bowl protein is
present only in the SC and MC, being normally absent from the DP cells. The
spatial distribution of nuclear Bowl is dependent on Drm, which causes Lin to
relocalize to the cytoplasm. Drm is absent from most of the DP cells and,
therefore, Lin turns down the steady-state accumulation of Bowl protein in
these cells. In the absence of Lin, Bowl accumulates in the DP cell nuclei and
elicits the dramatic alterations observed in lin- mutant
cells. Therefore, the main function of Lin is to prevent Bowl accumulation in
the DP cells, restricting Bowl protein to MC and SC of the PE.
The main alterations in lin-, Drm GOF or Bowl GOF
clones can be classified according to the signaling pathways temporally
affected. The earliest defect observed is the repression of Wg pathway
responses and the evidence suggests that Bowl functions as a repressor of the
Wg pathway. However, activated forms of nuclear Wg pathway components, such as
ArmS10 or dTcf, cannot restore the expression of the
proximal-distal markers owing to repression of the Wg targets in
lin-, indicating that Bowl must act in parallel to or
downstream of Arm and dTcf, as was previously suggested
(Green et al., 2002;
Hatini et al., 2005).
|
). Our results indicate that this mechanism is also
important under conditions where Bowl accumulates in the wing disc. Most of
the alterations observed in lin- or Drm GOF clones can be
explained by Bowl sequestering Gro from other repression complexes (causing
activation of N targets and Hh). Several results support this model. First,
the strong genetic interaction between lin and gro alleles,
where trans-heterozygous combinations between lin and gro alleles result in
dramatic phenotypes, argue that Gro is a limiting factor. Second, removal of
eh-1 motif that recruits Gro, eliminates the effects of Bowl on the Hh and N
pathways. Third, ectopic expression of Gro, H or Mtv partially suppress the
phenotypes of ectopic Drm or Bowl. These observations imply a `tug of war'
between Bowl, H and Mtv for Gro. Increased H or Mtv would shift the balance
back in favor of N target repression and Hh repression. By contrast, the repression of Wg pathway observed in lin- cells appears to involve a different mechanism. Although the effect is Bowl dependent, repression of Wg targets also occurs with Bowleh1-, indicating that Gro sequestration is not required. Similarly, co-expression of Bowl with H or Mtv cannot re-establish the repression of the Wg targets. These results show that Bowl is able to repress Wg targets independently of Gro and the observation that Bowleh1- VP16 can cause some ectopic expression of Sens suggests that this may involve a direct effect of Bowl on Wg targets.
Wnt/Wg, N and Hh signaling represent major conserved signaling channels to
control cell identity and behavior during development. An antagonistic
interaction between the Wg and Hh has also been described in the embryo
(Hatini et al., 2005) and at
the intersection of the D/V and A/P compartment borders of the wing disc
(Glise et al., 2002).
Similarly, Wnt/Wg and N activities are closely entangled in many different
systems. Mutual dependent interactions between N and Wnt signaling have been
observed in vertebrate skin precursors
(Estrach et al., 2006), in
rhombomere patterning (Cheng et al.,
2004) and in somitogenesis
(Aulehla et al., 2003;
Dale et al., 2003;
Hofmann et al., 2004). It has
also been reported that orthologues of the Odd-skipped family, Osr1
and Osr2, function as transcriptional repressors during kidney
formation (Tena et al., 2007).
It is possible therefore that Lin/Bowl/Gro interaction is evolutionary
conserved and it will be interesting to discover whether lin is an
important regulatory factor in other systems.
Bowl function in wing development
By analyzing lin- clones in the wing primordium, we
have uncovered the consequences of stabilizing Bowl in the DP cells. There
are, however, two regions where Bowl accumulates normally, in the MC and SC
within the PE. Removal of Bowl in the PE might lead to ectopic Wg protein and
thus to ectopic activity of the Wg signaling to transform PE from squamous to
columnar cells (Baena-Lopez et al.,
2003). In this context, recently, it has shown that Bowl
inhibition by ectopic expression of Lin results in the replacement of the PE
by a mirror image duplication of the DP cells
(Nusinow et al., 2008).
However, we did not observe much alteration in cell morphology nor in the
expression of markers such as Ubx or Hth when Bowl was depleted in PE cells
(bowl- clones and UAS-BowlRNAi). It could be that
the recovered bowl- clones were not induced early enough
or that the levels of Bowl-RNAi were not sufficient to completely eliminate
the Bowl function in these cells. Nevertheless, our manipulations revealed
that bowl- phenotypes in the proximal wing and notum were
consistent with a functional role in MC. Therefore, we conclude that Lin has
an important role in restricting Bowl to the MC (and PE), delimiting a
Bowl-free territory that forms the DP cells and enables their responsiveness
to key signaling pathways such as Wg.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/7/1211/DC1
|
Footnotes |
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REFERENCES |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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