Patterning fields of cells is of central importance for the
development of multicellular organisms. A few evolutionarily
conserved molecules, including Hox selector genes and
signaling molecules, play fundamental roles in these processes,
which raises the question of how a restricted number of
molecules instruct the cell fate complexity of developing
animals. Hox selector genes encode homeodomain (HD)-
containing transcription factors, whose functions distinguish
the identities of homologous groups of cells along the
anteroposterior axis (Lewis, 1978; McGinnis and Krumlauf,
1992). Previous experiments have established that Hox
proteins (about 40 in vertebrates and only eight in Drosophila)
play fundamental roles in coordinating the development of
groups of cells during morphogenetic processes. A Hox
combinatorial code has been proposed to instruct cellular and
pattern diversity (Lewis, 1978; Hunt et al., 1991a; Hunt et al.,
1991b), with cell fate determined by which combination of
Hox transcription factors is active within it. Although the
importance of the Hox code in patterning fields of cells is
established, strong experimental support demonstrating that
the combination of Hox proteins within a single nucleus
instructs cell-type diversity is lacking.
Signaling and Hox protein functions have been extensively
studied separately. However, how they act together to define
higher levels of control is a poorly understood emerging theme.
The Drosophila embryonic midgut provides an ideal model
system for studying the coordinated action of Hox genes and
signaling pathways. First, transcription of Hox genes in the
visceral mesoderm (VM) occurs in adjacent non-overlapping
expression domains (Tremml and Bienz, 1989), which allows
a simple assessment of Hox protein function without any
complication resulting from a potential Hox combinatorial
code. Second, differential transcription of Hox genes
directs localized production of two signaling molecules:
Decapentaplegic/Tgfβ (Dpp/Tgfβ) in parasegment 7 (PS7)
under Ultrabithorax (Ubx) control, and Wingless/Wnt
(Wg/Wnt) in PS8 under Abdominal A (AbdA) control (Reuter
et al., 1990; Bienz, 1994). The parasegmental boundary
between PS7 and PS8 thus constitutes a signaling center from
which the Dpp and Wg pathways organize morphogenetic
processes: positioning the central midgut constriction
(Staehling-Hampton and Hoffman, 1994) and establishing cell
fate diversification (Hoppler and Bienz, 1994; Hoppler and
Bienz, 1995). Third, the Drosophila midgut is the only tissue
where multiple Hox target genes have been identified; these
provide appropriate markers for investigating the mechanisms
of Hox transcriptional activity at the molecular level (Graba et
We explored the genetic and molecular mechanisms that
endow a single Hox protein with distinct transcriptional
properties by studying the function of AbdA during midgut
morphogenesis. AbdA is expressed and is active in the third
and fourth compartments of the midgut (PS8-PS12), and yet it
Hox proteins play fundamental roles in generating pattern
diversity during development and evolution, acting in
broad domains but controlling localized cell diversification
and pattern. Much remains to be learned about how Hox
selector proteins generate cell-type diversity. In this study,
regulatory specificity was investigated by dissecting the
genetic and molecular requirements that allow the Hox
protein Abdominal A to activate wingless in only a few cells
of its broad expression domain in the Drosophila visceral
mesoderm. We show that the Dpp/Tgfβ signal controls
Abdominal A function, and that Hox protein and signal-
activated regulators converge on a wingless enhancer. The
signal, acting through Mad and Creb, provides spatial
information that subdivides the domain of Abdominal A
function through direct combinatorial action, conferring
specificity and diversity upon Abdominal A activity.
Key words: Hox, Signaling, Drosophila, AbdA, Dpp/Tgfβ
Tgfβ signaling acts on a Hox response element to confer specificity
and diversity to Hox protein function
Aurélie Grienenberger1, Samir Merabet1,*, John Manak2,3, Isabelle Iltis1, Aurélie Fabre1, Hélène Bérenger1,
Matthew P. Scott2,3, Jacques Pradel1and Yacine Graba1,†
1Laboratoire de Génétique et Biologie du Développement, IBDM, CNRS, Université de la méditerranée, Parc Scientifique de
Luminy, Case 907, 13288 Marseille Cedex 9, France
2Department of Developmental Biology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA
3Department of Genetics, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305-5427,
*Present address: Biozentrum, University of Basel, Department of Cell Biology, Klingelbergstrasse 70, 4056 Basel, Switzerland
†Author for correspondence (e-mail: email@example.com)
Accepted 29 July 2003
Development 130, 5445-5455
© 2003 The Company of Biologists Ltd
activates the wg target gene only in PS8 (Immerglück et al.,
1990). Here, we report that the Dpp signal secreted from PS7
provides the spatial information required for PS8-localized wg
activation and that, acting through a newly identified 546 bp
enhancer, AbdA and Mad, a transcriptional effector of the Dpp
pathway, directly control wg transcription. The convergence of
Hox function and Dpp signaling therefore occurs at the levels
of DNA and transcription, and endows AbdA with PS8-specific
Materials and methods
Identification, mutation of the XC enhancer and
establishment of transgenic reporter lines
Restriction fragments from a 9 kb wg upstream regulatory region were
cloned into pBS(SK+) (Stratagene) and then transferred to the P-
element transformation vector pC4PLZ using standard cloning
procedures. Mutated versions of the XC enhancer were generated
using either the Sculptor mutagenesis kit (Pharmacia) or the Splicing
by Overlap Extension (SOE) method (Horton et al., 1989). Details on
the procedure and sequences of oligonucleotides used to generate XC
variants are available upon request. The point mutations (underlined)
introduced are as follows:
Creb1, TGGCGCCA→TGGTTGAG; and
XC(∆[Hox/Pbx2-3-4]) was generated by using an RsaI restriction
site to delete the promoter proximal region of the XC enhancer.
Mutated enhancers and an oligomer containing three copies of Box2
were transferred into the pC4PLZ reporter vector, and introduced into
the fly genome by P-mediated germline transformation (Rubin and
Spradling, 1982). At least four lines were established and analyzed
for each construct. In all experiments where lacZ expression levels
were compared, embryos were processed in the same conditions and
were stained for the same length of time.
wg midgut regulatory region from D. virilis and
A D. virilis EMBL3 phage genomic library (provided by J. Tamkun)
was screened with a 3.5 kb EcoRI/SphI genomic fragment of the D.
melanogaster wg upstream regulatory region. Hybridization was
carried out, at moderate stringency, in 4×SSPE, 1% SDS, 0.5% non-
fat dried milk. Washes were in 2×SSPE, 0.2% SDS, 0.05% sodium
phosphate at the same temperature. From a phage clone containing a
7 kb SalI fragment, a 1.4 kb BamHI/HindIII restriction fragment that
hybridizes to D. melanogaster XhoI/BamHI DNA was subcloned in
pUC19 and sequenced. The D. virilis sequence was PCR amplified,
its sequence was verified, and it was then cloned into the pC4PLZ
vector for P-mediated germline transformation. D. pseudoobsura
sequences were from the Drosophila Genome Project.
Fly stocks and in situ hybridization
Fly stocks were obtained as follows: wgIL114and wgCX4from A.
Martinez-Arias; dpps4, dpps6and dpps13 from W. Gelbart; mad12 from
S. Newfeld; UAS-abdA from M. Akam; UAS-Creb(DN), also termed
UAS-Cbz, UAS-dpp and UAS-Tcf(DN) from M. Bienz; hthP2from R.
Mann; UAS-hth-en from A. Salzberg; and HS-abdA from G. Morata.
The exdXP11allele and the 24B-Gal4 mesodermal driver were used.
Mutant embryos were identified by the absence of lacZ balancers. In
situ hybridization on wholemount embryos was performed as
described by Tautz and Pfeifle (Tautz and Pfeifle, 1989), using
antisense riboprobes produced by standard methods (Boehringer-
Mannheim Genius kit). Immunostaining was performed according to
Alexandre et al. (Alexandre et al., 1996), using the rabbit anti-β-
galactosidase (Cappel). Embryos were mounted in 80% glycerol and
photographed using Nomarski optics.
Protein production and gel shift assays
Full-length AbdA, Hth and Exd proteins for EMSAs were produced
using the TNT-coupled in vitro transcription/translation system
(Promega). The Drosophila
recombinant protein (Usui et al., 1993) was synthesized in E. coli and
purified using Ni2+chromatography (Qiagen). A GST-Mad fusion
protein was produced and purified according to standard procedures
(Pharmacia). It contained the first 159 amino acids of Mad, and thus
included the MH1 DNA-binding domain (Waltzer and Bienz, 1999).
The DIIRcon double-stranded oligonucleotides (Gebelein et al.,
2002), and the following oligonucleotides and their respective
complementary oligonucleotides, were used:
DRS3, 5′-TTTGACCCAATGCCGCCGACCCACGTAC-3′; and
Box2m, DRS1m, DRS2m, DRS3m, Creb1-2m oligonucleotides
and their complementary oligonucleotides are identical to the above
oligonucleotides except that they carry the mutations indicated in
the first section of Materials and methods. Oligonucleotides were
end-labelled with [γ32P]ATP, annealed with their respective
complementary oligonucleotides, and gel purified. EMSAs with in
vitro produced AbdA, Exd and Hth were performed in a volume of
20 µl as described by Pöpperl et al. (Pöpperl et al., 1995). Binding
experiments were also performed with AbdA and Exd proteins
produced in bacteria. In that case, His-tagged AbdA (from amino acid
79 to its carboxy terminus) and Exd (from amino acid 1 to 323) (Ryoo
and Mann, 1999) were purified using Ni2+chromatography (Qiagen).
Binding experiments using Mad and Drosophila CrebB proteins were
performed in similar conditions with 30,000 cpm radiolabelled
probes. Binding buffers for Mad and Drosophila CrebB gel shifts
were, respectively: 4% Ficoll, 20 mM Hepes (pH 7.9), 40 mM KCl,
1 mM EDTA and 4 mM DTT, with 2.5 µg BSA and 0.5 µg dAdT/10
µl of binding reaction; and 20 mM Hepes (pH 7.9), 20% glycerol, 100
mM KCl, 0.1% NP4O, 20 mM MgCl2 and 0.5 mM DTT, with 3 µg
BSA/10 µl of binding reaction. DNA-protein complexes were
analyzedby non-denaturing 6% PAGE in0.5×TBE and were detected
by autoradiography. The rabbit anti-AbdA antibody, raised against the
full-length protein, was provided by M. Cappovila.
Identification of a midgut enhancer that
recapitulates wg expression and regulation
To identify the enhancer responsible for wg expression in the
VM, subfragments of a 9kb genomic region known to drive wg
embryonic expression (A. Martinez-Arias and L. Owen,
personal communication) were analyzed in transgenic lines
transformed with lacZ reporter constructs (Fig. 1A). The
smallest fragment that drives accurate expression in the VM is
a 546 bp XhoI/ClaI (XC) restriction fragment. Its activity is
first detected during germ-band retraction (Fig. 1C), when wg
transcripts are visualized in the VM by in situ hybridization
(Fig. 1B), and only in PS8 VM cells. During subsequent
development, XC enhancer activity still mimics wg expression
(Fig. 1D,E), and is associated with the site of central midgut
Development 130 (22)Research article
5447 Integration of Hox and signaling activities
constriction formation (Fig. 1F,G). Thus, from
early on to the end of embryogenesis, the
XC enhancer exclusively and accurately
recapitulates wg spatiotemporal expression in
To establish that the XC enhancer obeys the
same regulatory inputs as wg (Immerglück
et al., 1990; Rauskolb and Wieschaus, 1994;
Rieckhof et al., 1997), its activity in embryos
homothorax (hth) and dpp was examined. Loss
of abdA (Fig. 1H), exd or hth (data not shown)
function results in the absence of lacZ
transcription factors are essential for XC enhancer activation,
as they are for wg transcription. In dpps4(Fig. 1I) or dpps6
mutants (not shown), the activity of the XC enhancer is
diminished, mimicing the decreased transcription of wg in
We analyzed in further detail the contribution of Hth to wg
expression and XC enhancer control. Hth fulfils two separable
functions in the regulation of Hox downstream target genes.
It is responsible for Exd nuclear import (Rieckhof et al., 1997)
and it can be a component of a tripartite Hox/Exd/Hth DNA-
binding complex (Ryoo et al., 1999). To discriminate between
these two functions, we used a fusion protein of Hth and the
repression domain of Engrailed (En), which behaves as a
dominant negative form of Hth but does not impair Exd
nuclear translocation (Inbal et al., 2001). Expression of the
Hth-En fusion protein in the mesoderm leads to the complete
loss of wg transcription (Fig. 1J) and XC enhancer activity
(Fig. 1K). This effect of Hth on wg is not a secondary
consequence of a primary effect on dpp, as dpp expression in
hth mutants is not abolished but is expanded anteriorly (data
not shown), as it is in exd-mutant embryos (Rauskolb and
that the three
Wieschaus, 1994). This suggests that Hth participates in a
Hox/Exd DNA-binding complex that is required for wg
Dpp signaling is essential for wg expression and XC
dpps4and dpps6regulatory mutations do not completely
abolish Dpp activity in the VM (Bilder et al., 1998): their
effect on odd paired (opa) in the VM is weaker than is the
effect of dpps13, a shortvein allele whose 3′ breakpoint is
closer to the dpp transcription unit (Hursh et al., 1993). We
found that wg transcription and XC enhancer activity are
totally abolished in dpps13embryos (Fig. 2B,D). Dpp therefore
is essential for wg transcription. A previous study reported that
Dpp affects the level and maintenance of wg transcription
(Immerglück et al., 1990), but we can see now, by using the
stronger dpps13allele, that Dpp has an essential off/on
influence. This is an important difference, as only an essential
requirement for dpp is compatible with the Dpp signal
providing the information responsible for PS8-restricted
activation of wg by AbdA.
Fig. 1. Identification and regulation of
a wg midgut enhancer. (A) wg
upstream regulatory elements that
drive (red bars) or do not drive (black
bars) expression in the midgut when
fused to a lacZ reporter.
(B-G) Comparison of wg transcription
and XC enhancer activity. wg
trancripts (B,D,F) and lacZ (C,E,G)
transcripts were detected by in situ
hybridization. Arrows indicate PS8, the site of wg
midgut expression. At all stages examined, shown
here as lateral views of stage 11 (B,C) and stage 13
(D,E) embryos, XC enhancer activity exclusively
mimics wg expression in the embryonic midgut.
(F,G) Magnified ventral views of stage 16 embryos.
The arrowheads indicate the central midgut
constriction. (H-K) XC enhancer regulation
recapitulates wg regulation. The activity of the XC
enhancer visualized by lacZ transcripts is lost in
abdAJX2homozygous-mutant embryos (H) and
diminished in dpps4homozygous mutants (I). wg
transcripts (J) and XC enhancer activity (K) are no
longer detected in the central midgut following
mesodermal expression of the Hth-En fusion protein
in 24B-Gal4/UAS-hth-en embryos.
Dpp signaling provides positional cues for local wg
expression and XC enhancer activity
To determine whether locally produced Dpp is responsible for
the restricted wg activation by AbdA, we analyzed the changes
in wg and XC enhancer expression patterns that result from
expression of abdA and dpp at ectopic positions in the VM.
Because the same conclusions were obtained for wg and the
XC enhancer, we will describe the behavior of the enhancer
only. We first provided the Dpp signal ubiquitously in the VM
and observed additional patches of β-galactosidase staining
(Fig. 2E). The sites of ectopic expression are posterior to PS8
within the AbdA expression domain. Most embryos exhibit
two additional patches, whereas in a few cases a third patch is
observed more posteriorly. This suggests that XC enhancer
activation requires a high level of Dpp signaling, which is best
achieved close to endogenous sources of Dpp, where
endogenous and 24B-driven Dpp signal are combined. At later
stages, ectopic Dpp and, consequently, ectopic wg expression,
here visualized by posterior ectopic XC enhancer activity (Fig.
2F), results in abnormal midgut morphogenesis, with ectopic
constrictions forming just posterior to the central one.
We next analyzed XC activation in response to ubiquitous
expression of AbdA in the mesoderm and could occasionally
detect a faint ectopic β-galactosidase staining anterior to the
normal site of wg expression, close to PS7 (Fig. 2G). This
experiment deserves two comments. First, the low frequency
and reduced levels at which ectopic staining occurs is a
consequence of two opposite functions of AbdA in the VM.
Besides activating wg, AbdA represses dpp (Reuter et al.,
1990), which indirectly impairs wg transcription. Thus, the
embryos in which ectopic lacZ expression is seen likely
correspond to embryos where AbdA has not completely
abolished dpp transcription. Second, the fact that ectopic
staining is only seen close to PS7, where the Dpp signal
originates, is further consistent with the requirement of both
AbdA and Dpp for XC enhancer activation.
Development 130 (22)Research article
Fig. 2. wg expression and XC enhancer activity depends on Dpp and
Wg signaling. (A,B) wg transcripts revealed by in situ hybridization.
Arrowheads indicate PS8, the site of wg midgut expression in wild-
type embryos (A). wg expression is completely abolished in the
midgut of dpps13homozygous-mutant embryos (B).
(C-L) Regulation of XC or XC(∆[Hox/Pbx2-3-4]) enhancer activity
by AbdA, Dpp and Wg visualized by in situ hybridization to lacZ
transcripts. All panels show magnifications of lateral views of the
midgut of stage 14 embryos, except panel F, which is from a stage 15
embryo. Embryos in C,J,K and L have been processed in the same
conditions and the length of staining time was identical. Arrows
indicate sites of ectopic enhancer activity. The embryo in C bears a
wild-type copy of the XC enhancer and serves as a reference for the
activity of XC variants. With respect to wg expression, XC enhancer
activity in the wild type (C) is lost in dpps13homozygous-mutant
embryos (D). The embryo in D has been overstained to ensure the
absence of lacZ staining. Mesodermal ubiquitous expression of Dpp
in 24B-Gal4/UAS-dpp embryos induces ectopic activity of the XC
enhancer posterior to PS8 (E) in cells where AbdA is present. At
stage 15, ectopic sites of XC activity coincide with the sites of extra
constrictions that form in this genotype (F). Mesodermal ubiquitous
expression of AbdA in 24B-Gal4/UAS-abdA embryos weakly
induces ectopic activity of the XC enhancer anterior to PS8 (G), in
close proximity to the PS7 Dpp source. In 24B-Gal4/UAS-abdA
embryos, ectopic XC(∆[Hox/Pbx2-3-4]) enhancer activity is stronger
than XC enhancer activity, and is also detected more anteriorly, close
the source of Dpp in VM cells close to PS3-4 (H). Simultaneous
expression of AbdA and Dpp in 24B-Gal4/UAS-dpp/UAS-abdA
embryos induces ectopic XC enhancer activity anterior and posterior
to PS8 (I). XC activity is diminished in wgIL114homozygous
embryos shifted to restrictive temperature at 7 hours of development
at 25°C (J) or in 24B-Gal4/UAS-Tcf(DN) embryos expressing the
dominant-negative form of the Wg transcriptional effector
Drosophila Tcf (K). In wgILL114homozygous mutants grown at
29°C, ectopic Dpp signaling provided by 24B-Gal4/UAS-dpp still
induces, although at lower levels, ectopic XC enhancer activity (L).
Table 1. In vivo activities of XC variants
XC enhancer variants
PS8 VM activity
NS, not shown.
5449 Integration of Hox and signaling activities
However, we never detected XC enhancer activity close to
PS3-4 of the VM, where Dpp is also produced. To examine this
point further, we used a XC enhancer version lacking the most
proximal sequence, XC(∆[Hox/Pbx2-3-4]), which has stronger
activity than does the full-length enhancer (see Table 1 and Fig.
4D). We first checked that ectopic Dpp, as with the XC
enhancer, induces posterior ectopic XC(∆[Hox/Pbx2-3-4])
activity (not shown). The improved activity of this enhancer
allowed a better visualization of the effect of ubiquitously
provided AbdA (Fig. 2H): as for XC, two sites anterior to the
normal site of wg expression were observed. In addition,
ectopic staining then also occurred more anteriorly, at the
site of Dpp production in PS3-4. These experiments clearly
emphasize the simultaneous requirement of Dpp and AbdA for
XC enhancer and wg transcriptional activation. Thus, the local
source of Dpp, secreted from cells of PS7, just anterior to the
large AbdA expression domain (PS8-12), allows PS8-restricted
activation of wg by AbdA.
To determine whether all VM cells are competent to express
wg in response to AbdA and Dpp, we looked at XC
andXC(∆[Hox/Pbx2-3-4]) enhancer activity following
simultaneous expression of AbdA and Dpp in the entire VM.
In this context, ectopic lacZ expression occurs both anterior
and posterior to PS8 (Fig. 2I). However, we did not observe
ectopic expression in all VM cells. Thus, although many VM
cells are competent to activate the enhancers when exposed to
Dpp and AbdA, some cells do not respond in our experimental
conditions. We suggest that the threshold of 24B-driven Dpp
is limiting. This is supported by the two following
observations. First, when providing 24B-driven Dpp, wg
expression or XC enhancer activity always occurs close to the
source of endogenous Dpp, but rarely at positions where
the ubiquitous source of Dpp is not implemented by the
endogenous signal. Second, when strong dpp expression is
induced in the anterior midgut by the ectopic expression of an
AbdA protein mutated in the hexapeptide motif, strong and
ubiquitous expression of wg in all VM cells of the anterior
midgut is achieved (Merabet et al., 2003).
Wg signaling implements the AbdA and Dpp
responsiveness of the XC enhancer
As Dpp and Wg act together in the regulation of a Ubx VM
enhancer (Eresh et al., 1997; Riese et al., 1997), we examined
whether XC activity in PS8 depends on Wg signaling. XC
activity is severely reduced in the absence of wg function
(Fig. 2J), or in the presence of a dominant-negative form of
Drosophila Tcf (Pan – FlyBase), a transcriptional effector of
Wg signaling (Brunner et al., 1997) (Fig. 2K). Consistent with
its dependency on Wg signaling, ectopic activation of the XC
enhancer by ubiquitous dpp expression in the VM occurs at
Fig. 3. Evolutionary
conservation of the wg
activities visualized by
in situ hybridization to
Embryos are shown in
a lateral view. The D.
virilis counterpart of
the XC enhancer drives
expression in the
central part of the
midgut (B), as does the
D. melanogaster XC
enhancer (A), although
at a reduced level.
(C) Alignment of the D.
melanogaster (mel), D.
and D. virilis (vir)
in red emphasize
sequence identity. Two
long stretches of
are boxed in yellow.
Consensus binding sites
are indicated in blue for
Hox and Hox/Pbx
complexes, and in
green for Mad/Medea
(referred to as DRS)
and Creb proteins.
Sequences after the
arrow are deleted in
high levels only when wg is also present (compare Fig. 2L with
Fig. 2E). In summary, these observations show that both Dpp
and Wg control wg transcription, each providing a distinct
contribution: Dpp is essential and instructive, allowing local
activation of wg by AbdA, whereas Wg is permissive,
necessary for XC enhancer activity but not controlling spatial
pattern. The conclusion reached here, from loss-of-function
experiments, that Wg maintains its own expression through an
auto-regulatory loop, is distinct from the conclusion obtained
by others, from gain-of-function experiments (Yu et al., 1998),
that high level Wg signaling represses its own expression.
Potential binding sites for AbdA and transcriptional
effectors of the Dpp signaling pathway are
evolutionarily conserved in the XC enhancer
To address whether AbdA and Dpp signaling could directly
regulate wg, we first examined the sequence of the XC enhancer
for the presence of putative binding sites for AbdA and for
Mad/Medea (referred to as DRS, for Dpp response sequence),
the canonical transcriptional effectors of the Dpp/Tgfβ
signaling pathway known to recognize identical target
sequences (Affolter et al., 2001). As genetic and molecular data
led to the proposal that, in Drosophila, the CRE sequences to
which Creb proteins bind are required to respond to Dpp in
addition to DRSs (Andrew et al., 1997; Eresh et al., 1997), we
also looked for potential Creb binding sites. Six TAAT core
sequences and four sequences resembling the consensual
Hox/Pbx binding sites (TGATNNATG/TG/A) were identified
as potentially mediating AbdA function (Fig. 3C). The Hox/Pbx
3 and 2 sequences strongly match the consensus, with seven or
six of the eight consensus nucleotides conserved, respectively.
Hox/Pbx sequences 1 and 4 only have five of the eight
consensus nucleotides conserved. The XC fragment contains
three sequences matching DRSs and two potential CRE sites.
To assess the evolutionary conservation of the XC enhancer,
an homologous fragment from Drosophila virilis was isolated
and analyzed for its in vivo activity by transgenesis in
Drosophila melanogaster. The D. virilis fragment drives
expression in a pattern very similar to that of the XC enhancer
(Fig. 3B), suggesting that sequences conserved between these
two enhancers may be important for wg regulation in the
midgut. Sequence comparison, including sequences from D.
pseudoobscura, revealed that a majority of the TAAT core
motifs, the DRSs and the putative Creb-binding sequences are
evolutionarily conserved, whereas sequences that match
heterodimeric Hox/Pbx consensus binding sites are not (Fig.
3C). We also noted the existence of two large conserved
sequences, Box 1 and 2. As Box1 lies in a fragment that does
not drive reporter gene expression in transgenic flies (XS in
Fig. 1A), particular attention was paid to Box2 (see below).
AbdA directly regulates wg and mediates its effect
through multiple binding sites
To test whether wg is a direct target of AbdA, and to identify
the cis-regulatory sequences responsible for this regulation, we
generated variants of the XC enhancer disrupted in one or
several of the potential Hox-binding sites and analyzed their
activities in vivo. We first looked at Hox6/7 motifs found in the
evolutionarily conserved Box2 and obtained evidence that they
are important for the wg response to AbdA. A variant deleted
of Box2 showed a severely reduced in vivo activity (Fig. 4B).
A similar loss of enhancer activity was obtained by mutating
the two Hox TAAT core motifs (Fig. 4C), suggesting that the
diminished activity observed following the deletion of Box2
results from impairing the AbdA-regulatory function.
Because the deletion of Box2 does not cause a complete loss
of lacZ gene expression, as was observed upon abdA mutation,
we investigated whether the four putative sites for Hox/Pbx
lying outside of Box2 play a role in AbdA-mediated activation
of the XC enhancer. Enhancer variants were generated and
tested in transgenic flies. Point mutations that alter Hox/Pbx
site 1, which lies between two Creb-binding sites, or Hox/Pbx
site 3, which closely matches the Hox/Pbx consensus, lead only
to a weak inactivation of the XC enhancer (data not shown;
summarized in Table 1). More drastically mutated variants,
XC(∆[Hox/Pbx2-3-4]), where the promoter-proximal region
containing Hox/Pbx sites 2, 3 and 4 is deleted, and
XC(Hox/Pbx1;∆[Hox/Pbx2-3-4]), which no longer contains
any potential Hox/Pbx binding sites, do not reduce enhancer
activity but, surprisingly, improve it (Fig. 4D and Table 1,
respectively). This suggests that the deleted region contains
sites used to downregulate the XC enhancer. In summary, these
data show that AbdA directly regulates wg, and that it does so
through multiple binding sites.
To establish more firmly the importance of Box2 in
mediating the response to AbdA, two additional experiments
were performed. First, we used the XC(∆[Hox/Pbx2-3-4]) that
displays a stronger enhancer activity than the full-length
enhancer version, and found that the two TAAT core sequences
Development 130 (22) Research article
Fig. 4. Two Hox binding
sites within Box2 are
required for XC enhancer
requirement of Hox6/7 sites
for XC or XC(∆[Hox/Pbx2-
3-4]) enhancer activity
visualized by in situ
hybridization to lacZ
transcripts. All embryos
have been processed under
the same conditions, with
identical staining times. All
panels show magnifications
of lateral views of the
midgut of stage 14 embryos.
The embryo in A bears a
wild-type copy of the XC
enhancer and serves as a
reference for the activity of
XC variants. The deletion of
Box2 (B), or the mutation of
the two Hox binding sites
(Hox6/7) found in Box2 (C),
results in a strong
diminution of XC enhancer
activity. The activity of the
enhancer (D) is stronger
than that of the full-length
XC (A), and is also
upon mutation of the Hox6/7
5451 Integration of Hox and signaling activities
of Box2 play an essential role, as their mutation
results in decreased enhancer activity (Fig. 4E).
Second, we assayed the ability of Box2 to drive, on
its own, reporter gene expression in transgenic flies.
Box2 initially promotes expression in a group of cells
within the prospective third midgut chamber (Fig.
5A), posterior to wg-expressing cells. Later in
development (stage 15), enhancer activity is detected
in the entire third midgut chamber and part of the
fourth gut chamber (Fig. 5B). Box2 thus promotes
expression in a posteriorly extended domain with
regards to the wg/XC domain. However, it is limited
to VM cells that express AbdA, suggesting a strict
dependence on AbdA. The lack of any β-
galactosidase staining in abdA mutants (Fig. 5C), and
the induction of lacZ expression in the whole VM of
embryos producing AbdA throughout this germ layer
(Fig. 5D), clearly demonstrates that Box2 activity is
controlled by AbdA.
As Box2 is sufficient to generate an AbdA-
contributes to XC enhancer activity, we assayed
for in vitro molecular interactions. Band-shift
experiments established that in vitro produced AbdA
protein specifically binds to Box2. This binding (Fig.
5E; lane 6) depends on the integrity of the two TAAT
core sequences (Fig. 5E; lane 11) and is abolished
when anti-AbdA antibodies, which impair AbdA
DNA binding (Fig. 5E; lane 23), are added to the
binding reaction (Fig. 5E; lane 10). Together with the
in vivo activity of Box2, these results indicate that
AbdA binding to Box2 directly regulates wg
Although Box2 does not contain any consensus
sequences for Hox/Pbx, EMSA experiments in the
presence of Exd were conducted. AbdA and Exd
produced in vitro do not form a dimeric complex on
Box2 (Fig. 5E; lane 7), contrasting with the ability of
the two proteins (same batches) to assemble on
DllRcon, an enhancer element of Distalless (Gebelein
et al., 2002) that recruits an AbdA/Exd complex (Fig.
5E; lane 21) (Merabet et al., 2003). EMSA performed
using AbdA (from amino acid 79 to the carboxy
terminus) and Exd (from amino acid 1 to 323) variant
proteins produced in E. coli led to the same
conclusion: that AbdA and Exd do not form a dimeric
complex on Box2 (data not shown). During these
experiments, we noticed that proteins produced in
vitro and in E. coli behaved differently with respect to
the effect of Exd on the DNA-binding activity of
AbdA: whereas DNA-binding was slightly decreased
using in vitro produced proteins (Fig. 5E; lane7), it
was significantly improved using proteins produced in
E. coli (not shown). This suggests either that the folding of the
in vitro and bacterially produced proteins are not equivalent,
or that domains absent from the proteins produced in E. coli
inhibit the improvement of AbdA DNA binding by Exd. A
similar improvement of Hox DNA binding activity by Exd in
the absence of Hox/Exd complex formation (Pinsonneault et
al., 1997; Ryoo and Mann, 1999; White et al., 2000) has
already been reported, suggesting that Exd/Pbx cofactors use
pattern and crucially
multiple molecular mechanisms for assisting Hox protein
In addition, we asked whether the presence of Hth allowed
the formation of an AbdA/Exd/Hth complex on Box2.
Consistent with the absence of a sequence matching a Hth
binding site, no AbdA/Exd/Hth complex was observed on
Box2 (Fig. 5E; lane 9), although the same preparations of
proteins do form a tripartite complex on DllRcon (Fig. 5E; lane
Fig. 5. Box2 binds in vitro to AbdA and is sufficient to drive an AbdA-
dependent expression pattern in the embryonic midgut. (A-D) Box2 responds
in vivo to AbdA. Enhancer activity is visualized by immunohistochemistry
using an anti-β-galactosidase antibody. All panels show magnifications of
lateral views of the midgut of stage 14 embryos. Arrowheads indicate PS8, the
site of wg midgut expression. Arrows indicate ectopic enhancer activity. An
oligomer consisting of three copies of Box2 drives lacZ expression in the
posterior midgut in the AbdA expression domain. Expression is first detected
posterior to the normal site of wg expression (A) and later in a domain that
includes the third, and part of the fourth, midgut chambers (B). Box2 enhancer
activity no longer occurs in abdAJX2homozygous mutants (C), and is induced
ectopically in the entire midgut VM when AbdA is ubiquitously provided by a
heat shock construct (D). (E) Gelshift experiments with AbdA, Exd and Hth
proteins produced in vitro were performed on wild-type (lanes 1-10) and
mutated (Box2m; lanes 11-14) forms of Box2. Box2m carries the same point
mutations as those introduced in XC(Hox6/7). 3 µl of the programmed lysate
were used for each protein and for the mock lysate (lanes 2 and 16). For the
binding experiments combining Exd and Hth, the two proteins were
simultaneously produced and 6 µl of the lysates were used. The anti-AbdA
serum was used at a 1/20 dilution. The activity of the proteins were assayed on
a DllR sequence (lanes 15-23), known to assemble an AbdA/Exd/Hth
complex. The asterisk and the dot mark the position of the AbdA/Exd and
AbdA/Exd/Hth complexes, respectively.
22). In summary, these observations do not favor a model
whereby AbdA, Exd and Hth act as a ternary protein complex
binding Box2 in the regulation of wg, as has been demonstrated
in the regulation of labial (Mann and Affolter, 1998). However,
they do not exclude that aided by additional proteins and cis-
regulatory sequences, such a ternary complex may form in
The Dpp transcriptional effector Mad and the
Drosophila CrebB protein directly regulate wg
First, we addressed whether Mad and Creb are involved in XC
enhancer activation. In embryos transformed with the XC-lacZ
construct and mutant for mad, no β-galactosidase staining
could be detected (Fig. 6B), indicating that Mad is essential for
XC enhancer activity. As no mutant for Drosophila CrebB, the
gene encoding the Creb isoform expressed in the VM, is
available, we used a dominant-negative form of Creb. Its
expression in the mesoderm strongly reduces β-galactosidase
staining (Fig. 6C), indicating that a Creb protein, most likely
Drosophila CrebB, is required for XC enhancer activity.
Next, we determined whether the evolutionarily conserved
consensus sequences for Mad/Medea (DRS1, 2, 3) and Creb
are used in vivo. The mutation of DRS 1 and 3 does not result
in a significant inhibition of the reporter gene (data not shown;
Table 1). When all three DRSs are simultaneously mutated, the
XC enhancer is inactive in the VM (Fig. 6D), clearly
demonstrating the essential role of Mad/Medea consensus
sequences for wg VM expression. This result indicates, in
addition, that the three DRSs individually contribute to the
control by Dpp transcriptional effectors, or ,alternatively, that
DRS2 is of special functional importance. Of note, the variant
mutated for the three DRSs gains a novel activity, as revealed
by ectopic lacZ expression near the foregut/midgut boundary
and in the midgut endoderm close to sources of Dpp signal.
Interaction of Mad/Medea with the DRSs therefore appears to
be distinctly used in PS8 of the VM for wg activation, in
the midgut endoderm and more anteriorly to prevent wg
expression. These observations suggest that the function
ultimately depends on locally specified, tissue-specific,
combinatorial interactions. Mutation of the two Creb-binding
sites reduces XC enhancer activity, indicating that, although
important, they are not essential (Fig. 6E). The complete loss
of XC enhancer activity observed when the three DRSs and the
two Creb consensus sequences are mutated (Fig. 6F) indicates
that the ectopic endoderm expression seen with XC(DRS1-2-
3) requires Creb binding.
In addition, we tested whether Mad and Drosophila CrebB
proteins directly bind their putative sites on the XC enhancer
in vitro. Band-shift experiments performed with purified
proteins show that DRS1, 2 and 3 bind to Mad with distinct
affinities (Fig. 7A-B; data for DRS3 not shown). The strongest
binding is to DRS2, which might be functionally significant as
XC(DRS1-3), a variant mutated in sites 1 and 3 only, possesses
an in vivo activity comparable to the wild-type version. The in
vitro association of Mad to each of the three sequences appears
specific, as shown by the impaired binding when each DRS is
mutated, as well as by the competition experiments. Similar
band-shift experiments conducted with Drosophila CrebB
purified proteins also led to the conclusion that Drosophila
CrebB specifically binds to Creb1 and 2 consensus sequences
(Fig. 7C). In vertebrates, Smads and the Creb-like proteins Fos
and Jun have been shown to co-activate artificial promoters
(Zhang et al., 1998). It therefore appears that Creb proteins
may play a rather general role in implementing the response to
Dpp and possibly other Tgfβ signaling molecules.
In summary, these experiments show that Mad, and most
likely Medea that is known to function in a complex with Mad,
as well as Creb proteins bind in vitro sites that are specifically
required for the activation of the wg XC enhancer in vivo. This
provides strong evidence that the Dpp signaling pathway
directly regulates wg.
Hox/signaling integration: interactions for reciprocal
Considerable interest has recently emerged about how selector
gene products and signaling molecules cooperate in organ
patterning (Curtiss et al., 2002). It was proposed that
combinatorial use of Scalloped (Sd), a transcription factor that
works together with Vestigial (Vg) to specify the wing field
(Bray, 1999), and transcriptional effectors of the Notch
[Suppressor of Hairless, Su(H)] (Lecourtois et al., 1995) and
Development 130 (22)Research article
Fig. 6. Requirement of Mad and Creb proteins for XC enhancer
activity. Enhancer activity is visualized by in situ hybridization to
lacZ transcripts. All panels show magnifications of lateral views of
the midgut of stage 14 embryos. Arrowheads indicate the site of wg
expression in PS8. Arrows indicate ectopic enhancer activity. All
embryos have been processed under the same conditions and the
staining times were identical. (A-C) Requirements in trans. (A) Wild-
type embryo carrying the XC enhancer. The activity of the XC
enhancer is lost in mad12-homozygous mutants (B) and is strongly
reduced upon expression of a dominant-negative form of Drosophila
CrebB in the mesoderm of 24B-Gal4/UAS-Creb(DN) embryos (C).
(D-F) Requirements in cis. Mutation of the three DRS in XC(DRS1-
2-3) results in the complete loss of enhancer activity in VM PS8 (D).
It also induces ectopic activity (arrows) of the enhancer in
endodermal cells from the central part of the midgut, and in a more
anterior region close to the foregut/midgut boundary (D). XC(Creb1-
2) that bears mutations in the two Creb binding sites shows a
severely reduced enhancer activity (E). The mutation of the three
DRSs and the two Creb binding sites results in a complete loss of
enhancer activity (F), including in the territories where the enhancer
is ectopically induced by XC(DRS1-2-3).
5453Integration of Hox and signaling activities
Dpp (Mad) signaling pathways regulate cut (ct) and the
vestigial quadrant enhancers (vgQ) in specific portions of the
wing disc (Guss et al., 2001). vgQ and ct are direct targets of
Sd, and the association of binding sites for Sd to those of Mad
or Su(H), creates synthetic enhancers that mimic vgQ or ct
expression. The absolute requirement for Sd-binding sites in
the synthetic enhancers provided an explanation for the
activation of ct and vgQ by the Notch and Dpp pathways in the
wing disc only. Two additional studies showed that the tissue
specific transcription factors Twist and Tinman also locally
specify the activity of signaling pathways (Halfon et al., 2000;
Marty et al., 2001; Xu et al., 1998). Thus, selector proteins
provide tissue-specificity for the action of signaling molecules,
allowing a few signals to be reiteratively used and yet achieve
distinct functions in different tissues. This conclusion also
holds for the Hox selector protein Lab, which is involved in a
positive autoregulatory loop in the endoderm. Although Dpp
signals in the central midgut both in the VM and in the
endoderm, lab expression and the activity of a lab Dpp-
responsive enhancer only occurs in the endoderm (Grieder et
al., 1997). It was further shown that the enhancer contains a
single Lab/Exd/Hth composite binding site responsible for the
endoderm-restricted activity (Marty et al., 2001).
Like signaling molecules, Hox proteins are also widely
expressed and reiteratively used during development. Although
the Lab/Dpp synergy provides the best documented example
of Hox/signaling combined action, it does not constitute a
suitable model to address whether signaling pathways
modulate and specify Hox protein activity, because synergy
between Lab and Dpp apparently occurs in all Lab-expressing
cells. In this study, Hox/signaling integration was examined to
determine whether signaling pathways contribute towards
specifying how a widely expressed Hox selector protein
controls the development of distinct pattern elements at
different locations. We show that the Dpp signal secreted from
PS7 provides the positional cue responsible for localized
activation of wg by AbdA. Biochemical and reverse genetics
experiments established that AbdA and Mad directly regulate
wg transcription through the XC enhancer, which thus serves
as an integrator of Hox and Tgfβ input. AbdA is impotent with
respect to this enhancer in the absence of the Dpp signal,
though it can function perfectly well on other genes without
Dpp (Bilder et al., 1998). Therefore, functional interactions
between selector proteins and signaling pathways confer
specificity to signaling pathways (Curtiss et al., 2002; Guss et
al., 2001), and reciprocally confer functional diversity to
selector proteins (this study).
Cis-regulatory read out of a Hox/signaling
combinatorial code: a mechanism to diversify Hox
Our study provides a conceptual framework for understanding
the molecular basis of regional Hox protein transcriptional
activity. We previously reported that Dpp/Tgfβ and Wg/Wnt
signaling subdivide the AbdA Hox domain (Bilder et al.,
1998), allowing activation of pointed (pnt) and opa target genes
in the third and fourth midgut chambers, respectively. Based
upon the data presented here, we suspect that the localized
activation of pnt and opa by AbdA also relies on direct
enhancer integration of Hox and signaling inputs (Fig. 8).
Accordingly, a Hox/signaling combinatorial code functionally
Fig. 7. Mad and Drosophila CrebB proteins bind in vitro to the XC
enhancer. (A) Gelshift experiments with Mad protein [50 ng (+) or
200 ng (++)] were performed on double-stranded oligonucleotides
corresponding to wild-type or mutated (DRS2m) versions of DRS2,
in the presence or absence of a 500-fold excess of cold DRS2
competitor. Lanes 1-3 show a dose-dependent binding of Mad to
DRS2. Lanes 4-7 indicate that binding is competed by cold DRS2
(lanes 4 and 5), and that the integrity of the DRS2 site is required for
Mad binding (lanes 6-7). (B) Similar experiments on double-stranded
oligonucleotides corresponding to the wild-type or mutated
(DRS1m) version of DRS1. Compared with the experiments in A,
this gelshift shows that Mad protein binds DRS2 with a stronger
affinity than DRS1. (C) Gelshift experiments with Drosophila CrebB
protein [20 ng (+) or 100 ng (++)] were performed on double-
stranded oligonucleotides corresponding to wild-type (Creb1-2) or
mutated (Creb1-2m) versions of Creb-binding sites, in the presence
or absence of a 500-fold excess of cold Creb1-2 competitor. Lanes 1-
3 show a dose-dependent binding of Drosophila CrebB. Lanes 4-7
indicate that the binding of Drosophila CrebB is competed by the
competitor, and that the integrity of the two Creb binding sites is
required for binding to occur.
subdivides the domain where a single Hox protein is made,
giving rise to discrete patterns of target gene activation. The
structures of relevant cis-regulatory regions of AbdA target
genes are instrumental for determining which signal is required
to allow activation by AbdA. The pnt midgut enhancer would
contain AbdA and Wg response elements and would be
activated by AbdA specifically in the third midgut chamber
through the combinatorial action of AbdA and the Drosophila
Tcf/Arm transcriptional effector of Wg signaling. Similarly,
the opa midgut enhancer would contain AbdA and Dpp
response elements and would be activated only in the fourth
gut chamber by AbdA, in this case because of an inhibitory
effect of the Dpp-regulated transcription factor on AbdA
Further studies are required to understand how Hox selector
proteins functionally interact with nuclear effectors of
signaling pathways to generate specific transcriptional
patterns. In the control of wg by AbdA, several scenarios can
be envisioned. In one, the effect of the Dpp transcriptional
effector Mad on AbdA activity would be indirect, by
antagonizing the function of a repressor that would otherwise
act on the XC enhancer to prevent wg expression. The absence
of a binding site for this hypothetical repressor in Box2 could
explain how Box2 drives AbdA-dependent transcription even
without Dpp transcriptional effector binding sites. In a second
scenario, Dpp transcriptional effectors would more directly
control the activity of AbdA by influencing its DNA binding
or transregulatory properties. A direct interaction of HoxC8
and Smad1 has been reported to induce osteoblast
differentiation (Shi et al., 1999; Yang et al., 2000), suggesting
that the coordinate action of AbdA and Dpp signaling might
rely on direct AbdA-Mad interaction. In wg regulation, the
situation may be different, as additional regulatory inputs are
involved. bin and hth are essential, and Wg signaling is
required for accurate levels of wg expression. The contribution
of Creb might indicate that the Ras/Mapk signaling pathway is
involved as well. Ras signaling has been proposed to play a
permissive role by acting on CRE sequences of the Ubx and
lab enhancers (Szuts et al., 1998). These observations suggest
that AbdA and Hox proteins in general attain specificity and
diversity by participating in a variety of protein interactions in
We are greatly indebted to A. Martinez-Arias and L. Owen for
sharing reagents and unpublished observations about the wg upstream
regulatory region, and to L. Mathies for her contribution to an early
part of this study. We also thank W. Gelbart, M. Bienz, A. Martinez-
Arias, M. Akam, R. Mann, M. Capovilla, S. Neufeld, A. Salzberg, S.
Smolik, G. Morata and J. Tamkun for providing fly stocks, expression
plasmids, anti-AbdA antibodies and genomic libraries. M.P.S. is an
Investigator of the Howard Hughes Medical Institute. The work was
supported by grants from the CNRS, La Ligue Contre le Cancer
(‘Equipe labéllisée’), the HHMI and the MENRT, and by fellowships
from MENRT, l’ARC, Boehringer and LNCC to A.G., A.F. and S.M.
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Fig. 8. A model for the regionalization of AbdA activity: cis-
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