1 Laboratoire de Génétique et Biologie du Développement,
IBDM, CNRS, Université de la méditerranée, Parc
Scientifique de Luminy, Case 907, 13288 Marseille Cedex 9, France
2 Department of Developmental Biology, Howard Hughes Medical Institute, Stanford
University School of Medicine, Stanford, CA 94305-5427, USA
3 Department of Genetics, Howard Hughes Medical Institute, Stanford University
School of Medicine, Stanford, CA 94305-5427, USA
Author for correspondence (e-mail:
graba{at}lgpd.univ-mrs.fr)
Accepted 29 July 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Hox, Signaling, Drosophila, AbdA, Dpp/Tgfß
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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/Tgfß (Dpp/Tgfß) 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 al.,
1997
).
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 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 regulatory properties.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
D. pseudoobscura
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 4xSSPE, 1% SDS, 0.5% nonfat
dried milk. Washes were in 2xSSPE, 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: wgIL114 and
wgCX4 from A. Martinez-Arias; dpps4,
dpps6 and 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; hthP2 from R. Mann;
UAS-hth-en from A. Salzberg; and HS-abdA from G. Morata. The
exdXP11 allele 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-ß-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 CrebB (CrebB-17A FlyBase) 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:
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
[32P]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
analyzed by non-denaturing 6% PAGE in 0.5xTBE and were detected by
autoradiography. The rabbit anti-AbdA antibody, raised against the full-length
protein, was provided by M. Cappovila.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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
Wieschaus, 1994
). This suggests that Hth participates in a Hox/Exd
DNA-binding complex that is required for wg control.
Dpp signaling is essential for wg expression and XC enhancer
activity
dpps4 and dpps6 regulatory
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 dpps13 embryos (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
dpps13 allele, 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.
|
We next analyzed XC activation in response to ubiquitous expression of AbdA
in the mesoderm and could occasionally detect a faint ectopic
ß-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.
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.
|
|
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 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/Tgfß 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.
|
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 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 ß-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.
|
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 multiple molecular mechanisms for assisting Hox protein function.
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 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 vivo.
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 ß-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 ß-galactosidase staining
(Fig. 6C), indicating that a
Creb protein, most likely Drosophila CrebB, is required for XC
enhancer activity.
|
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
Tgfß signaling molecules.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 Tgfß 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 protein function?
Our study provides a conceptual framework for understanding the molecular
basis of regional Hox protein transcriptional activity. We previously reported
that Dpp/Tgfß 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 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 activity.
|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Affolter, M., Marty, T., Vigano, M. A. and Jazwinska, A.
(2001). Nuclear interpretation of Dpp signaling in Drosophila.
EMBO J. 20,3298
-3305.
Alexandre, E., Graba, Y., Fasano, L., Gallet, A., Perrin, L., De Zulueta, P., Pradel, J., Kerridge, S. and Jacq, B. (1996). The Drosophila teashirt homeotic protein is a DNA-binding protein and modulo, a HOM-C regulated modifier of variegation, is a likely candidate for being a direct target gene. Mech. Dev. 59,191 -204.[CrossRef][Medline]
Andrew, D. J., Baig, A., Bhanot, P., Smolik, S. M. and
Henderson, K. D. (1997). The Drosophila dCREB-A gene
is required for dorsal/ventral patterning of the larval cuticle.
Development 124,181
-193.
Bienz, M. (1994). Homeotic genes and positional signalling in the Drosophila viscera. Trends Genet. 10, 22-26.[CrossRef][Medline]
Bilder, D., Graba, Y. and Scott, M. P. (1998).
Wnt and TGFß signals subdivide the AbdA Hox domain during
Drosophila mesoderm patterning. Development
125,1781
-1790.
Bray, S. (1999). Drosophila development: Scalloped and Vestigial take wing. Curr. Biol. 9,R245 -R247.[CrossRef][Medline]
Brunner, E., Peter, O., Schweizer, L. and Basler, K. (1997). pangolin encodes a Lef-1 homologue that acts downstream of Armadillo to transduce the Wingless signal in Drosophila.Nature 385,829 -833.[CrossRef][Medline]
Curtiss, J., Halder, G. and Mlodzik, M. (2002). Selector and signalling molecules cooperate in organ patterning. Nat. Cell Biol. 4,E48 -E51.[CrossRef][Medline]
Eresh, S., Riese, J., Jackson, D. B., Bohmann, D. and Bienz,
M. (1997). A CREB-binding site as a target for
decapentaplegic signalling during Drosophila endoderm
induction. EMBO J. 16,2014
-2022.
Gebelein, B., Culi, J., Ryoo, H. D., Zhang, W. and Mann, R. S. (2002). Specificity of distalless repression and limb primordia development by abdominal hox proteins. Dev. Cell 3,487 -498.[Medline]
Graba, Y., Aragnol, D. and Pradel, J. (1997). Drosophila Hox complex downstream targets and the function of homeotic genes. BioEssays 19,379 -388.[Medline]
Grieder, N. C., Marty, T., Ryoo, H. D., Mann, R. S. and
Affolter, M. (1997). Synergistic activation of a Drosophila
enhancer by HOM/EXD and DPP signaling. EMBO J.
16,7402
-7410.
Guss, K. A., Nelson, C. E., Hudson, A., Kraus, M. E. and
Carroll, S. B. (2001). Control of a genetic regulatory
network by a selector gene. Science
292,1164
-1167.
Halfon, M. S., Carmena, A., Gisselbrecht, S., Sackerson, C. M., Jimenez, F., Baylies, M. K. and Michelson, A. M. (2000). Ras pathway specificity is determined by the integration of multiple signal-activated and tissue-restricted transcription factors. Cell 103,63 -74.[Medline]
Hoppler, S. and Bienz, M. (1994). Specification of a single cell type by a Drosophila homeotic gene. Cell 76,689 -702.[Medline]
Hoppler, S. and Bienz, M. (1995). Two different thresholds of wingless signalling with distinct developmental consequences in the Drosophila midgut. EMBO J. 14,5016 -5026.[Abstract]
Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. and Pease, L. R. (1989). Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77,61 -68.[CrossRef][Medline]
Hunt, P., Gulisano, M., Cook, M., Sham, M. H., Faiella, A., Wilkinson, D., Boncinelli, E. and Krumlauf, R. (1991a). A distinct Hox code for the branchial region of the vertebrate head. Nature 353,861 -864.[CrossRef][Medline]
Hunt, P., Whiting, J., Nonchev, S., Sham, M. H., Marshall, H., Graham, A., Cook, M., Allemann, R., Rigby, P. W., Gulisano, M. et al. (1991b). The branchial Hox code and its implications for gene regulation, patterning of the nervous system and head evolution. Development Suppl. 1,63 -77.
Hursh, D. A., Padgett, R. W. and Gelbart, W. M.
(1993). Cross regulation of decapentaplegic and
Ultrabithorax transcription in the embryonic visceral mesoderm of
Drosophila. Development
117,1211
-1222.
Immerglück, K., Lawrence, P. A. and Bienz, M. (1990). Induction across germ layers in Drosophila mediated by a genetic cascade. Cell 62,261 -268.[Medline]
Inbal, A., Halachmi, N., Dibner, C., Frank, D. and Salzberg, A. (2001). Genetic evidence for the transcriptional-activating function of Homothorax during adult fly development. Development 128,3405 -3413.[Medline]
Lecourtois, M. and Schweisguth, F. (1995). The neurogenic suppressor of hairless DNA-binding protein mediates the transcriptional activation of the enhancer of split complex genes triggered by Notch signaling. Genes Dev. 9,2598 -2608.[Abstract]
Lewis, E. B. (1978). A gene complex controlling segmentation in Drosophila. Nature 276,565 -570.[Medline]
Mann, R. S. and Affolter, M. (1998). Hox proteins meet more partners. Curr. Opin. Genet. Dev. 8, 423-429.[CrossRef][Medline]
Marty, T., Vigano, M. A., Ribeiro, C., Nussbaumer, U., Grieder, N. C. and Affolter, M. (2001). A Hox complex, a repressor element and a 50 bp sequence confer regional specificity to a DPP-responsive enhancer. Development 128,2833 -2845.[Medline]
McGinnis, W. and Krumlauf, R. (1992). Homeobox genes and axial patterning. Cell 68,283 -302.[Medline]
Merabet, S., Kambris, Z., Capovilla, M., Berenger, H., Pradel, J. and Graba, Y. (2003). The hexapeptide and linker regions of the AbdA Hox protein regulate its activating and repressive functions. Dev. Cell 4,761 -768.[Medline]
Pinsonneault, J., Florence, B., Vaessin, H. and McGinnis, W.
(1997). A model for extradenticle function as a switch
that changes Hox proteins from repressors to activators. EMBO
J. 16,2032
-2042.
Pöpperl, H., Bienz, M., Studer, M., Chan, S.-K., Aparicio, S., Brenner, S., Mann, R. S. and Krumlauf, R. (1995). Segmental expression of Hoxb-1 is controlled by a highly conserved autoregulatory loop dependent upon exd/pbx. Cell 81,1031 -1042.[Medline]
Rauskolb, C. and Wieschaus, E. (1994). Coordinate regulation of downstream genes by extradenticle and the homeotic selector proteins. EMBO J. 13,3561 -3569.[Abstract]
Reuter, R., Panganiban, G. E., Hoffmann, F. M. and Scott, M. P. (1990). Homeotic genes regulate the spatial expression of putative growth factors in the visceral mesoderm of Drosophila embryos. Development 110,1031 -1040.[Abstract]
Rieckhof, G. E., Casares, F., Ryoo, H. D., Abu-Shaar, M. and Mann, R. S. (1997). Nuclear translocation of extradenticle requires homothorax, which encodes an extradenticle-related homeodomain protein. Cell 91,171 -183.[Medline]
Riese, J., Yu, X. N., Munnerlyn, A., Eresh, S., Hsu, S. C., Grosschedl, R. and Bienz, M. (1997). LEF-1, a nuclear factor coordinating signaling inputs from wingless and decapentaplegic. Cell 88,777 -787.[Medline]
Rubin, G. M. and Spradling, A. C. (1982). Genetic transformation of Drosophila with transposable element vectors. Science 218,348 -353.[Medline]
Ryoo, H. D. and Mann, R. S. (1999). The control
of trunk Hox specificity and activity by Extradenticle. Genes
Dev. 13,1704
-1716.
Ryoo, H. D., Marty, T., Casares, F., Affolter, M. and Mann, R.
S. (1999). Regulation of Hox target genes by a DNA bound
Homothorax/Hox/Extradenticle complex. Development
126,5137
-5148.
Shi, X., Yang, X., Chen, D., Chang, Z. and Cao, X.
(1999). Smad1 interacts with homeobox DNA-binding proteins in
bone morphogenetic protein signaling. J. Biol. Chem.
274,13711
-13717.
Staehling-Hampton, K. and Hoffman, F. M. (1994). Ectopic decapentaplegic in the Drosophila midgut alters the expression of five homeotic genes, dpp, and wingless, causing specific morphological defects. Dev. Biol. 164,502 -512.[CrossRef][Medline]
Szuts, D., Eresh, S. and Bienz, M. (1998).
Functional intertwining of Dpp and EGFR signaling during Drosophila endoderm
induction. Genes Dev.
12,2022
-2035.
Tautz, D. and Pfeiffle, C. (1989). A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81-85.[Medline]
Tremml, G. and Bienz, M. (1989). Homeotic gene expression in the visceral mesoderm of Drosophila embryos. EMBO J. 8,2677 -2685.[Abstract]
Usui, T., Smolik, S. M. and Goodman, R. H. (1993). Isolation of Drosophila CREB-B: a novel CRE-binding protein. DNA Cell Biol. 12,589 -595.[Medline]
Waltzer, L. and Bienz, M. (1999). A function of
CBP as a transcriptional coactivator during Dpp signalling. EMBO
J. 18,1630
-1641.
White, R. A., Aspland, S. E., Brookman, J. J., Clayton, L. and Sproat, G. (2000). The design and analysis of a homeotic response element. Mech. Dev. 91,217 -226.[CrossRef][Medline]
Xu, X., Yin, Z., Hudson, J. B., Ferguson, E. L. and Frasch,
M. (1998). Smad proteins act in combination with synergistic
and antagonistic regulators to target Dpp responses to the Drosophila
mesoderm. Genes Dev. 12,2354
-2370.
Yang, X., Ji, X., Shi, X. and Cao, X. (2000).
Smad1 domains interacting with Hoxc-8 induce osteoblast differentiation.
J. Biol. Chem. 275,1065
-1072.
Yu, X., Riese, J., Eresh, S. and Bienz, M.
(1998). Transcriptional repression due to high levels of Wingless
signalling. EMBO J. 17,7021
-7032.
Zhang, Y., Feng, X. H. and Derynck, R. (1998). Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-beta-induced transcription. Nature 394,909 -913.[CrossRef][Medline]
Related articles in Development: