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First published online 28 February 2007
doi: 10.1242/dev.02823
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Centro de Biología Molecular Severo Ochoa, CSIC and UAM, Cantoblanco, 28049 Madrid, Spain.
Author for correspondence (e-mail:
scampuzano{at}cbm.uam.es)
Accepted 31 January 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Iroquois complex, Imaginal wing disc, Drosophila, Notum development, Dpp signalling, EGFR signalling
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
genes that actually carry out the specification of the different body regions
and fates, for instance the specification of body wall versus appendage or the
implementation of neural versus epidermal fate, decode this positional
information. Their cis-regulatory elements (REs) act as integrating devices
receiving inputs present in the cells, which consist of combinations of
transcription factors, and eliciting responses in the form of the restricted
patterns of expression of the genes they are controlling
(Modolell and Campuzano,
1998).
The araucan (ara), caupolican (caup)
and mirror (mirr) genes of the Iro-C
(Gomez-Skarmeta et al., 1996;
McNeill et al., 1997) belong
to the class of genes whose expression specifies territories. Thus, the
homeoproteins they encode are essential for, among other functions (reviewed
by Cavodeassi et al., 2001),
the specification of the notum territory and its subsequent patterning
(Diez del Corral et al., 1999;
Gomez-Skarmeta et al., 1996).
These diverse functions rely on their spatially and temporally restricted
expression patterns. Thus, at the second larval instar, these genes are
expressed in the most proximal region of the wing disc. There, they confer on
cells the ability to form notum since, in their absence, these cells give rise
to structures of the dorsal wing hinge
(Diez del Corral et al.,
1999). Furthermore, the confrontation of Iro-C-expressing and
nonexpressing cells generates an organizing border similar to those found
between compartments (Diez del Corral et
al., 1999; Villa-Cuesta and
Modolell, 2005). Afterwards, expression of Iro-C is restricted to
the prospective lateral notum where the Iro proteins function as components of
the prepattern that governs the expression of the achaete-scute complex (AS-C)
genes and, accordingly, help define the pattern of adult sensory organs
(Gomez-Skarmeta et al.,
1996).
To understand the development of the notum it is thus necessary to clarify
how expression of the Iro-C is controlled. Previous reports have shown that,
in the prospective notum region of the wing disc, Iro-C is under the control
of the Epidermal growth factor receptor (EGFR) and Decapentaplegic (Dpp)
signalling pathways. During the second larval instar, EGFR signalling is
necessary for notum development and expression of Iro-C
(Wang et al., 2000;
Zecca and Struhl, 2002a;
Zecca and Struhl, 2002b),
whereas the Dpp pathway contributes to the confinement of the expression of
Iro-C to the notum region (Cavodeassi et
al., 2002). In the early third larval instar, this pathway further
restricts expression of the Iro-C genes to the prospective lateral notum
(Cavodeassi et al., 2002). How
both pathways converge on the regulation of Iro-C genes is still unknown. To
address this point, we have characterized the regulatory sequences of the
Iro-C, a gene complex that spans approximately 150 kb of DNA
(Gomez-Skarmeta et al., 1996;
McNeill et al., 1997).
Expression of Iro-C genes is thought to be controlled by enhancer regulatory
sequences that would act jointly on, at least, ara and caup
(Gomez-Skarmeta et al., 1996).
We have identified five partially redundant wing disc-specific cis-regulatory
elements within the Iro-C (IroREs) and demonstrated the ability of some of
them to mediate regulation by the EGFR and Dpp signalling pathways. We show
that the transcription factor Pointed (Pnt) mediates the activation of Iro-C
by the EGFR pathway and the involvement of Pannier (Pnr), U-shaped (Ush) and
Mothers against Dpp (Mad) transcription factors in Dpp-dependent repression.
In addition, we propose a mechanism for the coexistence of Iro-C expression
and the activity of the Dpp pathway at the prospective lateral notum near the
anterior-posterior (AP) compartment boundary. Our results help clarify how the
territorial specification and patterning of the Drosophila thorax is
effected by the antagonistic/cooperative action of EGFR and Dpp pathways on
Iro-C expression.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Lee and Luo,
1999) in larvae of the following genotypes: y w hsFLP1.22; ubi-nlsGFP FRT40A/ushVX22 FRT40A (or tkva12 FRT40A, or yan443 FRT 40A or yan884 FRT 40A); IroRE-lacZ/+
y w hsFLP1.22; IroRE-lacZ/+; FRT82B ubi-nlsGFP/FRT82B
grn7L12 (or FRT82B
pnt
88 or FRT82B
pnrVX6)
y w hsFLP1.22; IroRE-lacZ/+; FRT82B M ubi-nlsGFP/FRT82B ttk1e11
yw hsFLP tub
1-Gal4, UAS-GFP; tub
1-Gal80 FRT40A/ushVX22 FRT40A;
UAS-tkvQD/IroRE-lacZ
Second instar (48-72 hours after egg laying; AEL) or early third instar (72-96 hours AEL) larvae were heat shocked for 1 hour at 37°C.
Misexpression experiments
Clones of cells overexpressing different genes were obtained as described
by Ito et al. (Ito et al.,
1997). Actin5C>yellow+>Gal4, UAS-GFP;
IroRE-lacZ males were mated with females carrying hsFLP1.22 and
different UAS transgenes (UAS-tkvQD, UAS-Mad, UAS-pnr, UAS-ush,
UAS-vein, UASRasV12, UAS-RafDN, UAS-RafAct,
UAS-pntP1). Larvae were treated 24-48 hours AEL or 72-96 hours AEL, for
10 minutes at 37°C. The clones were revealed by GFP expression.
Iro-C reporter constructs
EcoRI or HindIII fragments of genomic Iro-C DNA from the
lambda phage and P1 walk described by Gomez-Skarmeta et al.
(Gomez-Skarmeta et al., 1996),
encompassing 110 kb of the DNA proximal to the iroDFM2
breakpoint, were subcloned in the C4PLZ enhancer tester plasmid that contains
a weak P-element promoter (Wharton, Jr and
Crews, 1993). Fragments encompassing IroRE1 and
IroRE2 were ligated and subcloned into C4PLZ to obtain the
transgenic flies IroRE1-IroRE2-lacZ.
IroRE2-B1 to IroRE2-B5 fragments were obtained by PCR
amplification using 1.6 kb IroRE2-B as a template and appropriate
primer sets. (Primers sequences are available upon request.) PCR-amplified
fragments cloned into pGEM Teasy vector (Promega), were excised by
EcoRI digestion and subcloned into C4PLZ. The lacZ reporter
plasmids were introduced into y w1118 embryos by standard
P-element transformation (Ashburner,
1989). Three to six independent transgenic lines were established
and examined for each construct.
Immunohistochemistry
Imaginal discs were dissected and stained as described previously
(Gomez-Skarmeta et al., 1996).
Primary antibodies were: rabbit and mouse anti-ß-galactosidase (Cappel
and Promega), rat anti-Caup, an antibody that recognizes both Ara and Caup
(Diez del Corral et al., 1999)
and mouse anti-Wingless (DSHB). Secondary antibodies were from Jackson
Immunoresearch Laboratories and Molecular Probes. Confocal images were
acquired using Bio-Rad Microradiance and Zeiss LSM510 Meta confocal
microscopes. Images were imported and assembled using Adobe Photoshop 7.0
software.
Sequence analysis
Flyenhancer
(http://flyenhancer.org/Main)
(Markstein and Levine, 2002)
and cis-analyst
(http://rana.lbl.gov/cis-analyst/)
(Berman et al., 2002) programs
were used in the search of putative transcription factor binding sites.
Cross-species sequence conservation was monitored using the VISTA program
(http://genome.lbl.gov/vista/index.shtml)
(Couronne et al., 2003).
Mutagenesis of GATA and ETS putative binding sites
Mutagenesis was performed by the site directed QuikChange system
(Stratagene). To obtain the IroRE2B-2-GATA-mut,
IroRE2B-2 DNA subcloned into pGEM Teasy, was used as template to
mutate the putative GATA binding site GATAAG into CTGAAG
with the following primers: forward
5'-GATGGCGATGGCAGCctgAAGCCCCATGATTTTG-3' and reverse
5'-CAAAATCATGGGGCTTcagGCTGCCATCGCCATC-3'. Iro-RE2B-2
DNA was similarly used to obtain the Iro-RE2B-2-ETS-mut, with the
putative conserved ETS binding site CGGGATG changed into
CGCATTG, using the following primers: forward
5'-CCGGGGATTGGGAAATGGGTTCGcatTGGCCAGTTTAGTCG-3' and reverse
5'-CGACTAAACTGGCCAatgCGAACCCATTTCCCAATCCCCGG-3'. Altered bases are
shown in lowercase. Mutations were confirmed by sequencing.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Gomez-Skarmeta et al., 1996).
Later, in the third instar, their expression is restricted to the lateral
notum (Fig. 1A). In addition,
at this developmental stage, novel domains of expression appear in the
prospective regions of the L1, L3 and L5 veins, tegula, dorsal radius, dorsal
and ventral pleura and alula (Diez del
Corral et al., 1999;
Gomez-Skarmeta et al., 1996).
The expression of mirr is slightly different, being absent from the
L3, L5 and tegula domains but present at the other domains
(Kehl et al., 1998). The Iro-C
harbours two additional transcription units, lincoyan
(linc), whose pattern of expression at the notum is identical to that
of ara and/or caup and quilapan (quil),
which is ubiquitously expressed (R. Diez del Corral, PhD thesis, Universidad
Autónoma de Madrid, Spain, 1998). Previous genetic analysis suggested
the existence of enhancer-like REs that would drive the coincident expression
of ara and caup in the wing disc
(Gomez-Skarmeta et al., 1996).
Thus, In(3L)iroDFM2, associated with a breakpoint within
the ara transcription unit (Fig.
1B), removes ara expression in the wing disc except in
the L3 vein domain, in contrast to caup expression which is only lost
from that domain (Gomez-Skarmeta et al.,
1996). This suggests the existence of vein L3-specific RE(s)
distal to the In(3L)iroDFM2 breakpoint and other RE(s),
specific for the remaining domains of Iro-C expression, located proximal to
such breakpoint. To identify notum-specific REs, we analyzed the regulatory
potential of 31 different genomic fragments, spanning approximately 110 kb of
genomic Iro-C DNA (Fig.
1B). Only five of those fragments drove lacZ expression at specific regions of the imaginal wing disc (Fig. 1B). One of them, 3.3 kb in length and named Iro regulatory element2 (IroRE2), reproduced most of the expression pattern of Iro-C in the prospective notum (Fig. 1D,D',H,H'). Thus, IroRE2-lacZ was expressed in the proximal region of early third instar wing discs (the presumptive notum region; Fig. 1H,H') and at the presumptive lateral notum in third instar wing discs (Fig. 1D,D'). Note, however, that the pattern of IroRE2-mediated lacZ expression did not exactly coincide with that of ara/caup. Thus, ß-gal was not detected in a triangular area, located near the notum/hinge border and centred around the AP compartment boundary, where expression of ara/caup is enhanced (Fig. 1D, arrowhead, compare with D''). This is precisely the region where expression of lacZ was driven by another Iro-C genomic fragment of 3.9 kb, IroRE1 (Fig. 1C,C'). Accordingly, an IroRE1-IroRE2 composite RE was found to drive lacZ expression in a pattern very similar, albeit not identical, to that of the endogenous ara/caup genes (see Fig. S1 in the supplementary material).
Two other genomic fragments, IroRE3 and IroRE4 (3.4 and 3.7 kb), adjacent to each other (Fig. 1B), drove lacZ expression in a stripe of cells located at the proximal region of the presumptive lateral notum, which partially overlapped with the caup expression domain (Fig. 1E-F'). Finally, IroRE5 (2.8 kb) drove expression mainly in the prospective alula and peripodial membrane (Fig. 1G).
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EGFR and Dpp signalling regulate transcription mediated by IroRE2
In the proximal region of the wing disc, activation of EGFR by its ligand
vein leads to the expression of the Iro-C genes
(Wang et al., 2000;
Zecca and Struhl, 2002b). We
thus examined whether EGFR signalling similarly regulates expression driven by
IroRE2, by monitoring ß-gal accumulation in clones of cells
with altered EGFR signalling. Excess of signalling conditions
(UAS-RasV12 or UAS-RafAct
overexpression clones), promoted upregulation of lacZ expression,
similar to that observed with the endogenous ara/caup genes and with
similar topological restrictions (Fig.
2A-B') (Zecca and
Struhl, 2002b). Namely, in clones induced at the second instar,
lacZ activation occurred at the hinge and pleura domains
(Fig. 2A,A', arrows) but
not at the central wing pouch or proximal notum
(Fig. 2A,A', arrowheads
and not shown). In later induced clones, lacZ expression occurred
within the wing pouch, albeit at lower levels than at the hinge
(Fig. 2B,B', insets), and
in the anterior region of the medial notum
(Fig. 2B,B', arrow).
Conversely, lacZ expression was eliminated in
UAS-RafDN overexpressing clones
(Fig. 2C,C'). As
described for the endogenous ara/caup genes, overexpression of
vein had no effect (Zecca and
Struhl, 2002a). We conclude that EGFR signalling positively
controls IroRE2.
Dpp signalling represses Iro-C expression
(Cavodeassi et al., 2002).
Thus, we next investigated the ability of this pathway to modulate the
expression of IroRE2-lacZ. Inactivation of the pathway
(tkva12 clones) caused strong ectopic expression of
lacZ in the proximal notum (Fig.
3A,A') and a weaker one at the hinge
(Fig. 3C,C').
Furthermore, lacZ expression was upregulated in the extant domain of
IroRE2 (Fig.
3B,B'). Conversely, IroRE2-lacZ was
repressed when the Dpp pathway was overactivated in
UAS-tkvQD (a constitutively activated form of Tkv)
(Nellen et al., 1996) or
UASMad overexpressing clones (Fig.
3D,D' and not shown). Thus, Dpp signalling negatively
regulated IroRE2. Taken together the above results indicate that
the EGFR and Dpp pathways regulate ara/caup expression in the notum
mainly through IroRE2.
IroRE2 contains putative binding sites for effectors of the EGFR and Dpp signalling pathways
We next whished to identify the sites of IroRE2 that mediate its
response to the EGFR and Dpp pathways. We reduced the IroRE2 to a
1.6 kb subfragment (IroRE2-B;
Fig. 4B), which maintained
enhancer activity in the notum (Fig.
4C) and was activated by EGFR and repressed by Dpp signalling (not
shown). Next, we examined its sequence for putative binding sites of effectors
of these pathways using Flyenhancer
(Markstein and Levine, 2002)
and cis-analyst (Berman et al.,
2002) programs.
|
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). As shown in
Fig. 4B (and see Fig. S4 in the
supplementary material), IroRE2-B contains nine putative ETS
binding sites (Xu et al.,
2000) and five putative Ttk binding sites
(Brown and Wu, 1993;
Read et al., 1990). With
respect to the Dpp pathway, heteroliogomers of Mad and Medea act as
intracellular signal transducers (Affolter
et al., 2001). In accordance with its Dpp-dependent regulation, we
identified a putative binding site for Mad
(Kim et al., 1997) within
IroRE2-B (Fig. 4B;
see Fig. S4 in the supplementary material).
Previous genetic evidence has shown repression of ara/caup
expression in the prospective medial notum by the GATA transcription factor
Pannier (Pnr) (Calleja et al.,
2000), which is a target of Dpp signalling in the wing disc
(Sato and Saigo, 2000;
Tomoyasu et al., 2000).
Consistent with a direct regulation, in IroRE2-B we also found nine
putative GATA binding sites (Haenlin et
al., 1997; Martin and Orkin,
1990) (Fig. 4B; see
Fig. S4 in the supplementary material).
A high proportion of the putative binding sites found in our analysis were
located within conserved regions between D. melanogaster, D.
pseudoobscura, D. virilis and D. mojavensis as shown by
comparison with the VISTA program
(Couronne et al., 2003)
(Fig. 4A; see Fig. S4 in the
supplementary material).
Pnt is required for Iro-C expression
We identified putative binding sites for Pnt, Yan and Ttk in the
IroRE2-B sequence. Accordingly, we investigated the function of
these transcription factors in the regulation of
IroRE2-lacZ and ara/caup genes.
We first examined the function of pnt. Gain-of-function
pnt clones (induced at 72-96 hours AEL) caused ectopic accumulation
of ß-gal in the wing pouch, wing hinge and proximal notum and enhanced
accumulation (above the extant levels) at the lateral notum
(Fig. 5A,A', arrowheads).
(The central hinge and wing pouch were somehow refractory to such activation
when clones were induced at 48-72 hours AEL; not shown.) Consistently, loss of
function of pnt (pnt88
clones) reduced or totally removed lacZ expression driven by
IroRE2, especially in the central and posterior regions of the
lateral notum (Fig.
5B,B'). Moreover, expression of ara/caup was
similarly abolished in pnt clones
(Fig. 5C,C',
arrowhead).
The expression of IroRE2-lacZ and ara/caup genes was unaffected either in cells devoid of Yan (yan443 or yan884 clones) or Ttk (ttk1e11clones) (not shown). This suggested that Yan and Ttk do not regulate IroRE2-lacZ or ara/caup expression. Thus, Pnt appears to be the main effector of the EGFR pathway in the regulation of Iro-C through IroRE2.
|
; Haenlin et al.,
1997; Ramain et al.,
1993). Since Pnr represses ara/caup in the proximal
region of third instar wing discs (Calleja
et al., 2000), we assayed the effect of pnr or
ush loss-of-function on the transcription of
IroRE2-lacZ. pnrVX6 or
ushVX22 mutant clones, located at the most proximal region
of the disc, exhibited ectopic lacZ expression
(Fig. 6A,A' and
Fig. 7A,A', arrows),
indicating a repressor function of these proteins on transcription mediated by
IroRE2. Similarly, lack of pnr or ush caused
ectopic ara/caup expression (Fig.
7A,A'', arrows) (Calleja
et al., 2000). Consistent with a repressor role for Pnr/Ush,
UAS-ush was able to repress expression of
IroRE2-lacZ and ara/caup in the proximal most
domain of the lateral notum (Fig.
7B-C', arrows), most likely through the formation of Pnr/Ush
heterodimers.
Surprisingly, the effect of lack of pnr on transcription mediated
by IroRE2 differed along the proximodistal axis of the disc. Thus,
ß-gal was absent from pnrVX6 clones situated in a
region marked by wg expression
(Fig. 6B,B', arrows).
Since Pnr activates expression of wg whereas the heterodimer Pnr/Ush
represses it (Calleja et al.,
1996; Sato and Saigo,
2000; Tomoyasu et al.,
2000), expression of wg reveals cells containing Pnr but
devoid of Ush. These data suggested that Pnr activates expression of
IroRE2-lacZ in the absence of Ush and, indeed, activation
of this reporter gene was observed in pnr overexpression clones
(Fig. 6C,C'). On the
contrary, overexpression of pnr inhibited ara/caup
expression in the notum and did not ectopically activate it (not shown). Thus,
regulation of ara/caup and IroRE2-lacZ by Pnr
appears to be slightly different, most likely due to the fact that additional
regulatory elements, other than the IroRE2, cooperate to establish
the pattern of the endogenous ara/caup genes (see Discussion).
Considering that the expression of pnr and ush in
partially overlapping domains in the medial region of the wing disc depends on
Dpp activity (Sato and Saigo,
2000; Tomoyasu et al.,
2000), the above results suggest that Pnr/Ush dimers may mediate
Iro-C repression by the Dpp pathway at the medial notum through
IroRE2. However, these observations do not rule out a possible
direct repression of IroRE2-lacZ by Mad binding to its
putative binding sites present in this RE. To address this point, we made use
of the MARCM technique (Lee and Luo,
1999) to overexpress UAS-tkvQD in
ushVX22 clones. We reasoned that if the repressor effect
of UAS-tkvQD was solely due to activation of pnr
and ush (Sato and Saigo,
2000) and formation of the Pnr/Ush repressor heterodimer,
repression should not occur in the absence of Ush. However, absence of
ush did not relieve lacZ repression by the Dpp pathway
(Fig. 7D). Reduced expression
of IroRE2-lacZ was also found in UAS-Mad,
ushVX22 clones (not shown). These results suggest a role
for Mad, in addition to Pnr/Ush, in Dpp-dependent repression through
IroRE2 in the medial notum (see below).
|
|
; Haenlin et al.,
1997). This opened up the possibility that an Ush/Grn dimer might
mediate the Dpp-dependent Iro-C repression at the hinge. However, there was no
upregulation of ara/caup or IroRE2-lacZ
expression in ush or grn mutant clones at the hinge region
(Fig. 7A-A'', arrowheads,
and not shown), suggesting that this was not the case. Accordingly,
elimination of Mad-dependent repression could account for the enhanced
expression of lacZ found in tkva12 clones in the
wing hinge. A similar situation may apply to the lateral notum
(Fig. 3B,B'), a region
devoid of Pnr and Ush, where Mad could be the Dpp pathway effector.
Definition of a minimal notum enhancer
IroRE2-B contains putative binding sites for effectors of the
EGFR and Dpp pathways. To determine the in vivo functionality of
these binding sites, we first set to define the minimal functional length of
this RE by assaying the enhancer potential of five overlapping 500 bp
subfragments. Three of them (IroRE2-B1, IroRE2-B2 and
IroRE2-B4) drove lacZ expression in the wing disc in
overlapping regions contained within the Iro-expressing domain but also
outside of it (Fig. 4D-F).
Interestingly, putative ETS and GATA binding sites are not scattered within
the IroRE2-B sequence but appear clustered within these
subfragments (Fig. 4B).
Clustering of binding sites might contribute to synergistic regulation by the
transcription factors (Arnosti et al.,
1996). Removal of the 200 initial bp of the IroRE2-B,
which harbours several putative binding sites, did not affect its regulatory
potential (not shown), indicating the dispensability of these sites.
Furthermore, a lacZ construct containing exclusively the 200 initial
bp of the IroRE2-B was not expressed at the wing disc (not shown).
In sum, these data pointed to the 240 bp sequence shared by
IroRE2-B1 and IroRE2-B2 and the region covered by
IroRE2-B4 as the most relevant for the control of lacZ
expression. Note that the former region harbours a highly conserved sequence
among the Drosophila species analyzed
(Fig. 4A, asterisk) and
putative ETS and GATA binding sites (Fig.
4B).
Neither the IroRE2-B2 fragment, which contains a Mad binding site, nor the IroRE2-B1 and IroRE2-B4 fragment, devoid of such a site, drove expression in the prospective medial notum (Fig. 4D-F). Since the three fragments, on the contrary, contain GATA binding sites, this suggests that the GATA protein Pnr should be the main repressor of Iro-C at the medial notum through the IroRE2-B. A mutation was created in the unique GATA site of IroRE2-B2 to assess the relative contribution of Pnr to the establishment of the proximal border of Iro-C expression. The resulting IroRE2-B2GATAm-lacZ construct showed expansion of lacZ expression into the medial notum (Fig. 4G,G'), thus stressing the main contribution of GATA proteins. The potential contribution of Mad to IroRE2-B2-lacZ regulation was assayed in tkva12 clones generated in flies harbouring the IroRE2-B2GATAm-lacZ construct. Interestingly, further upregulation of lacZ expression was found in the clones (Fig. 4I-I''). These results, in agreement with those of Fig. 7D-D'', indicate that Mad, in addition to Pnr/Ush, should contribute to Dpp-dependent repression in the medial notum.
|
EGFR and Dpp signalling pathways activate transcription mediated by IroRE1
Although the Dpp pathway represses Iro-C expression
(Cavodeassi et al., 2002) and,
that mediated by IroRE2, this does not prevent the spatial
coincidence, in the lateral notum of third instar discs, of Iro-C proteins and
elevated levels of Dpp activity (Fig.
9C). Moreover, the overexpression of UAS-tkvQD
is unable to repress Iro-C expression in a domain of the lateral notum near
the Dpp source (Cavodeassi et al.,
2002). Interestingly, IroRE1 drives lacZ
expression in that domain (Fig.
1C,C'). Thus, we examined the effect of the Dpp pathway on
IroRE1-lacZ regulation. Overexpression of
UAS-tkvQD did not repress lacZ expression but
instead activated it, although exclusively in a domain restricted to the
lateral notum (Fig.
8A,A', arrows). Accordingly, loss of Dpp signalling
(tkva12 clones) significantly reduced ß-gal
accumulation (Fig. 8B,B',
arrows). IroRE1-mediated transcription was also activated by the
EGFR pathway, as shown by ectopic lacZ expression in
UAS-RafAct clones (Fig.
8C,C', arrows) and its converse reduction in
UAS-RafDN clones (Fig.
8D,D', arrow). Thus, Iro-RE1 may be instrumental
in allowing expression of Iro-C genes in the posterior lateral notum despite
the repressor effect of the Dpp pathway through other regulatory elements.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A
similar situation applies to the specification of prospective body regions
within the wing imaginal disc. Here, during the early second instar, EGFR and
Dpp pathways act antagonistically on the regulation of the Iro-C
(Cavodeassi et al., 2002;
Wang et al., 2000;
Zecca and Struhl, 2002b)
restricting its expression to the prospective notum region where it specifies
notum development rather than hinge (Diez
del Corral et al., 1999). Later, at the early third instar, again
the concomitant activity of EGFR and Dpp signals (the latter now also
emanating form the most proximal region of the wing disc) partition the
prospective notum into two different subdomains, the medial and the lateral
notum, the latter being specified by ara/caup expression
(Cavodeassi et al., 2002;
Diez del Corral et al., 1999).
Thus, to understand how regionalization of the adult fly body is achieved it
is important to elucidate the mechanisms responsible for the joint
interpretation of both signalling pathways.
The opposing effects of the EGFR and Dpp pathways on Iro-C expression may
have resulted from direct cross talk between these pathways. For instance,
both in vertebrates and invertebrates the ability of EGFR to antagonize BMP
signalling by phosphorylation and inhibition of Smad proteins has been
reported (Kretzschmar et al.,
1997; Kubota et al.,
2000). Although not previously described, Dpp signalling may
similarly interfere with the activity of the EGFR pathway. However, this
appears not to be the case in the wing disc since expression of
argos, a readout of the EGFR pathway
(Rebay, 2002) can be detected
in the proximal-most notum of the wing disc (not shown), a region with high
levels of Dpp signal. Furthermore, overexpression of activated tkv
does not repress expression of kekkon, another EGFR downstream gene
(Rebay, 2002) (our unpublished
results).
Here we show that the opposing effects of the EGFR and Dpp pathways on Iro-C expression result from the convergence of both pathways on at least two distinct Iro-C regulatory elements, IroRE1 and IroRE2. These two REs drive gene expression in two complementary domains of the prospective notum region of the wing disc, and appear to mediate most of the regulation of the Iro-C genes by the Dpp and EGFR pathways in this region of the wing disc. Furthermore, IroRE1 provides a regulatory mechanism for the coexistence at the prospective lateral notum of Iro-C expression and Dpp pathway activity, notwithstanding the negative regulation of Iro-C by such pathway (Fig. 9).
|
),
maintains some lacZ expression in part of the central notum (not
shown).
The identified REs might act simultaneously on ara and
caup expression to give rise to their almost coincident patterns of
expression. As previously proposed (R. Diez del Corral, PhD thesis,
Universidad Autónoma de Madrid, Spain, 1998), such coincidence cannot
be attributed to cross-regulation between ara and caup since
in irorF209 mutant discs (irorF209 is
an ara null allele, Fig.
1B) expression of caup is unmodified. Regulation of
ara/caup would be, accordingly, similar to that of the
achaete-scute genes of the AS-C, which show identical patterns of
expression due to the use of shared enhancers (reviewed in
Modolell and Campuzano, 1998).
Expression of the vertebrate Iroquois (Irx) genes appears to be
similarly regulated. Thus, the analysis of the regulatory potential of highly
and ultra conserved non-coding regions present in the intergenic regions of
the Irx clusters suggests these genes to be regulated by partially redundant
enhancers shared by the components of each cluster
(de la Calle-Mustienes et al.,
2005).
Expression of mirr in the notum region of the wing disc largely
coincides with that of ara/caup and most likely is under the control
of the same REs. Thus, activity of the IroRE2 may account for the
unmodified expression of mirr in iro1 imaginal
discs (associated with an inversion breakpoint located within the
caup transcription unit; Fig.
1B) (R. Diez del Corral, PhD thesis, Universidad Autónoma
de Madrid, Spain, 1998). In addition, differences in the expression of
ara/caup and mirr might be due to the presence of repressor
RE(s) or insulator sequences (Gerasimova
and Corces, 1996) that would prevent the action of the RE(s)
controlling ara/caup on the mirr promoter. This is
consistent with the previous observation of ectopic expression of
mirr in Mob1 mutants, a regulatory mutation mapped within
the Iro-C (Kehl et al.,
1998).
The Iro-C regulatory elements and the integration of developmental signals
Our identification of REs present in the Iro-C has allowed us to the unveil
some of the molecular mechanisms of its transcriptional regulation at the
level of DNA-protein interaction and to analyse the interplay of positive and
negative inputs from convergent signalling pathways.
EGFR activation in the proximal region of the wing disc leads to expression
of Iro-C (Wang et al., 2000;
Zecca and Struhl, 2002a;
Zecca and Struhl, 2002b). Here
we demonstrate that both IroRE1 and IroRE2 mediate
positive regulation by the EGFR pathway
(Fig. 9A,B). We show that Pnt
mediates activation of IroRE2-lacZ by the EGFR pathway.
Furthermore, as previously shown for the Iro-C genes
(Zecca and Struhl, 2002a;
Zecca and Struhl, 2002b),
EGFR-dependent activation is cell context dependent. This suggests the
existence, in the cells receiving EGFR signalling, of presently unknown
factors that would contribute to ara/caup activation and/or the
presence of counteracting repressing mechanisms, which should prevent their
activation. Clearly, the Dpp pathway is so far the best candidate, since it
has been shown that it can repress Iro-C
(Cavodeassi et al., 2002) and
the IroRE2-lacZ transgene (this work).
The molecular mechanism of Dpp-dependent regulation of Iro-C expression
appears to be more complex. The Dpp pathway can repress or activate Iro-C
through different REs and different effector proteins. IroRE2
appears to mediate Dpp-dependent repression at the medial notum (most probably
through direct binding of the heterodimer Pnr/Ush and Mad) and at the hinge
and lateral notum (independently of Pnr, Ush and the GATA factor Grn in these
domains). Dpp-dependent repression of Iro-C may be mediated, in addition,
through a different RE, namely, through a brk silencer element
(brkSE), shown to mediate Dpp-dependent repression of brk by binding
of a Medea/Mad/Schnurri repressor complex
(Pyrowolakis et al., 2004),
which is present at the Iro-C within IroRE5 (A.L. and S.C.,
unpublished observations).
|
). It
is noteworthy that, IroRE1 mediates lacZ expression
exclusively in that region of the wing disc and it appears to provide a
regulatory mechanism for the co-existence of Iro-C expression and Dpp pathway
activity, since the Dpp pathway does not repress but, on the contrary,
activates IroRE1-mediated lacZ expression
(Fig. 9B). Activation is
restricted to the lateral notum, most likely because of the presence, in the
hinge and medial notum territories, of repressors [Muscle segment homeobox,
Msh; also known as Drop (Villa-Cuesta and
Modolell, 2005) and Pnr/Ush, respectively] that would counteract
activation. Putative binding sites for both Msh [consensus sequence G/C
TTAATTG (Kuzuoka et al.,
1994)] and GATA proteins are indeed present in IroRE1
(not shown). Thus, IroRE1 and IroRE2 represent two
different REs in the same gene that respond in opposite ways to the same
positional information, i.e. Dpp signalling. A similar complex level of
regulation has been found in the case of segmentation genes such as even
skipped (eve) where the same transcription factor (Hunchback) is
able to activate or repress transcription mediated through different
eve position-specific enhancers
(Small et al., 1992;
Small et al., 1996).
In addition a Dpp-independent mechanism based in the mutual repression
between Iro-C and the homeoprotein Msh helps to maintain the distal border of
Iro-C expression (Villa-Cuesta and
Modolell, 2005). This repression could be mediated by direct
binding of Msh to one putative Msh binding site present in the
Iro-RE2-B sequence.
Further analysis of IroRE3, IroRE5, other putative RE(s) located outside of the tested region and putative repressor RE(s) that could not have been identified in our study, would help to establish the final pattern of Iro-C expression, which is essential for the specification of the notum and its subsequent patterning.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/7/1337/DC1
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ACKNOWLEDGMENTS |
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|
Footnotes |
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Present address: Functional Genomics, CIC bioGUNE, Bizkaia Technology Park,
Building 801A, 48160 Derio, Spain
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