1 Department of Biochemistry and Molecular Biology, Norris Cancer Hospital, USC
Keck School of Medicine, 1441 Eastlake Avenue, Los Angeles, CA 90033,
USA
2 Department of Developmental and Cell Biology, Developmental Biology Center, UC
Irvine, Irvine, CA 92697-2275, USA
3 Departments of Orthopedic Surgery and Biological Chemistry, David Geffen
School of Medicine, UCLA, Los Angeles, CA 90095-1737, USA
4 Department of Biology, California State University at Chico, Chico, CA 95929,
USA
* Author for correspondence (e-mail: maxson{at}hsc.usc.edu)
Accepted 5 August 2004
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SUMMARY |
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Key words: BMP, Msx2, Homeodomain, Smad, Transcription, Transgenic mouse, Evolution
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Introduction |
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BMPs are a large class of TGF-ß related ligands with diverse
activities in embryonic development. They can function as classical
morphogens, eliciting changes in cell fate in a concentration-dependent manner
(Gurdon and Bourillot, 2001).
Interaction of BMPs with their cognate receptors leads to the phosphorylation
of members of the receptor-regulated Smad family (R-Smads)
(Heldin et al., 1997
;
Massague, 1998
). Upon
phosphorylation, R-Smads associate with Smad4 and translocate to the nucleus,
where they serve as effectors of transcription
(Kretzschmar et al., 1997
;
Lagna et al., 1996
;
Liu et al., 1997
). Smads 1, 5
and 8 function in the BMP pathway, Smads 2 and 3 in the activin and TGF-ß
pathways, and Smad4 in all three branches of the TGF-ß superfamily
(Hoodless et al., 1996
;
Liu et al., 1996
;
Massague and Chen, 2000
;
Shi and Massague, 2003
;
Suzuki et al., 1997
;
Wiersdorff et al., 1996
).
Although Smads are crucial for the transcriptional activation of BMP target
genes, they bind DNA weakly and are expressed broadly in embryonic tissues
(Shi et al., 1998;
Wrana, 2000
). Therefore, the
tissue-specific activation of BMP target genes probably involves
sequence-specific transcription factors that cooperate with Smads. Despite the
importance of such factors, few have been identified and little is known about
how they interact with Smads to regulate transcription
(Hata et al., 2000
;
Henningfeld et al., 2002
). The
Msx2 promoter provides an ideal starting point in the search for such
factors.
Msx2 is one of three closely related genes in mammals
(Bell et al., 1993;
Davidson, 1995
;
Maxson et al., 2003
;
Pollard and Holland, 2000
). A
well-documented BMP target, Msx2 is expressed in a subset of known
domains of BMP function (Furuta and Hogan,
1998
; Graham et al.,
1994
; Hollnagel et al.,
1999
; Vainio et al.,
1993
). Targeted mutations in Msx2 have verified that it
acts downstream of BMPs and is required for a subset of BMP-mediated cellular
responses (Satokata and Maas,
1994
; Satokata et al.,
2000
). The regulatory elements controlling Msx2
expression thus provide a useful model for investigating how BMP signals are
processed at the level of the promoter to produce different BMP responses in
specific developmental settings.
Here, we report the identification and fine-structure analysis of a BMP-responsive enhancer (BMPRE) located upstream of the murine Msx2 gene. We demonstrate that consensus Smad elements and a consensus homeodomain element control the temporal and spatial pattern of BMP-dependent transcription directed by this enhancer. We show that the nucleotide sequence of this enhancer is highly conserved within mammals, but not in other vertebrate classes or nonvertebrates. Despite this lack of conservation outside mammals, this Msx2 BMPRE accurately interprets Dpp signals in Drosophila embryos and imaginal discs. Furthermore, as in mouse embryos, it exhibits a dependence on the consensus homeodomain as well as consensus Smad sites. These results demonstrate that the Msx2 BMPRE can respond to BMP/Dpp signals in two widely divergent animal groups, and suggest the functional design represented by this enhancer originated early in the evolution of the bilateria.
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Materials and methods |
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For the production of transgenic mice, Msx2 promoter fragments
were fused with the hsp68 minimal promoter and a lacZ
reporter (Kothary et al.,
1989). The 1.8 kb
3Msx2-hsplacZ and 560 bp
4Msx2-hsplacZ transgenic mice have been described
(Kwang et al., 2002
). The
5Msx2-hsplacZ construct contained a 220 bp PCR fragment
located between 3521 and 3310 bp upstream of the Msx2
translation start site. The 52 bp
6Msx2-hsplacZ construct
contained a tetramer repeat of the sequence between 3523 and
3471. This construct, and those bearing mutations in the Smad and
homeodomain sites, were generated using dimer repeat oligos that were ligated
together immediately upstream of the hsp68-lacZ-SV40
cassette. The 560
52bpMsx2-hsplacZ and
560bpMsx2-hdm-hsplacZ transgenes were generated as described
previously.
Msx2 promoter fragments used for producing transgenic flies were cloned into the CPLZN P-element vector. For the 560, 480 and 220 bp promoter fragments, inserts were generated by PCR. The 52 bp transgene consisted of a tetramer repeat.
Cell culture and transfection
10T1/2 cells were propagated in DMEM with 10% FBS. The cells were
transfected with the reporter constructs using Superfect (Qiagen) and 24 hours
later induced with BMP4 (60ng/ml final) (R&D). After another 24 hours in
culture, the cells were harvested and luciferase assays performed using the
dual luciferase system (Promega).
Bead implantations
Affigel agarose beads (BioRad) were washed in PBS prior to incubating in
0.1% BSA with 100 ng/µl BMP4 (R&D), 5 ng/µl TGFß1 (R&D) or
BSA (0.1%) for 30 minutes at 37°C. Beads were placed on tissues in
transwell membranes, immersed in DMEM. For limb-bud implantations, BMP4 beads
were placed on the dorsal side of left forelimbs prior to overnight incubation
in 6% CO2. Tissues were then fixed for 10 minutes in 4%
paraformaldehyde and stained for lacZ activity.
Chromatin immunoprecipitation (ChIP) assays
MLB13MYC-clone-14 (C-14) cells (Rosen
et al., 1994) were cultured in DMEM (10% FBS) and induced with 60
ng/ml BMP4 (R&D) for 30 minutes prior to fixation with formaldehyde (1.0%
final concentration) at room temperature for 10 minutes. ChIP assays were
performed as described (Ma et al.,
2003
) using an anti Phospho-Smad1 antibody (Upstate). PCR
amplification was performed using primers that span the 52 bp BMP-responsive
region of the Msx2 promoter (32 cycles). Primer sequences were: BMPRE
(forward) 5'-TCT GCC CAG TTG GAG GTT TGA-3' and (reverse)
5'-GCC GCG TTA ATT GCT CTC G-3'; upstream control (forward)
5'-GCA ACA AAC ATC CCT GAG A-3' and (reverse) 5'-CTG CCT CCT
AAC CTT CAT AG-3'.
Electophoretic mobility shift assays
GST-Smad4 (MH-1 domain) fusion proteins were expressed in BL21 bacteria and
purified by chromatography on glutathione beads (Pharmacia). Prx1b protein was
expressed from a mouse cDNA (Norris and
Kern, 2001) by coupled in vitro transcription-translation (TNT
kit, Invitrogen). All probes used were labeled with
-32P-dCTP. EMSA was performed as previously described
(Kwang et al., 2002
).
ß-galactosidase staining, in situ hybridization and immunohistochemistry
Mouse embryos were fixed in 4% paraformaldehyde at 4°C prior to
staining for lacZ (Liu et al.,
1994). Whole-mount in situ hybridization on mouse embryos was
performed as described previously (Hogan
et al., 1994
; Kwang et al.,
2002
). In situ hybridization and detection of ß-galactosidase
expression in Drosophila embryos was carried out as described in
Arora et al. (Arora et al.,
1994
). To detect phosphorylated R-Smads, tissue was fixed in 4%
paraformaldehyde and equilibrated in 30% sucrose prior to freezing in Histo
Prep (Fisher). Frozen sections were cut (8 µm) and fixed in cold acetone
for 10 minutes. Primary anti phospho-Smad1, 5, 8 antibody (Cell Signaling
Technology) was diluted 1/50 in PBST/1.0% BSA and incubated at 4°C
overnight. The secondary antibody, anti-Rabbit Rhodamine (Calbiochem), diluted
1/200 in PBST/1.0% BSA, was incubated for 1 hour at room temperature. Sections
were counterstained with DAPI and cover slipped using Vectashield (Vector
Labs). lacZ staining of frozen sections was performed as described in
Ishii et al. (Ishii et al.,
2003
).
Production of transgenic mice and flies
Transgenic mouse embryos and lines were generated by pronuclear injections
as described by Liu et al. (Liu et al.,
1994). Transgenic flies were produced by germline transformation
as described by Arora et al. (Arora et al.,
1994
).
Genotyping
DNA was prepared from mouse tails or embryo yolk sacs as described by Hogan
(Hogan, 1994).
Msx2-lacZ transgene genotypes were determined by PCR using primers to
lacZ as described by Kwang et al.
(Kwang et al., 2002
).
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Results |
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Alignments of Msx2 loci from mouse, rat, rabbit, human and
chimpanzee revealed a 480 bp block of homology positioned between 3 and 4 kb
upstream of the translation start sites
(Fig. 2). Located within the
560 bp BMP-responsive region, this block exhibited an average identity between
different orders of mammals of 90%. By contrast, the 3.3 kb promoter
region located between the conserved block and the translation start site had
an average identity of 56%, suggesting that selection has maintained the
sequence of the 480 bp block. No significant matches with this 480 bp region
were evident in non-mammals. Nor did mammalian sequences outside the
Msx2 locus match the 480 bp sequence. A survey (MatInspector 7.0,
2003) of consensus cis-regulatory elements within the conserved region
revealed several consensus binding sites with potential roles in BMP
signaling. These included Smad1 and Smad4 elements, as well as sites for OAZ,
Creb, Tcf/Lef1 and Hnf/Fox (Fig.
2) (Ionescu et al.,
2004
; Ishida et al.,
2000
; Johnson et al.,
1999
; Rice et al.,
2003
; Shim et al.,
2002
; Zawel et al.,
1998
).
|
|
|
We next asked whether the 52 bp sequence was sufficient to respond to a BMP signal in vivo. To minimize position effect, we produced a tetramer of this sequence, which we inserted upstream of the hsp68-lacZ reporter. In transgenic embryos, this construct was expressed in a subset of the sites where the parental 560bpMsx2-hsplacZ transgene and the endogenous Msx2 gene are expressed (Fig. 4A-I; Table 1) (10 independent lines). These included the cardiac outflow tract, pharyngeal arches, genital region, allantois, limb buds, mandible, nasal process, midbrain, eye, otic vesicle and hair follicles. An extended staining time was also required to achieve signal intensities comparable with those of larger Msx2 constructs, indicating reduced transgene activity.
|
|
Phosphorylated BMP R-Smads colocalize with 52 bp transgene expression in embryos and are recruited to the Msx2 BMP-responsive region in native chromatin
That the 52 bp transgene is expressed at sites of active BMP signaling was
confirmed by staining with an antibody against the phosphorylated (active)
form of the BMP restricted Smads 1, 5, 8 (R-Smads). Limbs exhibited nuclear
staining in a halo surrounding implanted BMP4-soaked beads
(Fig. 5D,E), coincident with
lacZ expression (Fig.
5B,C). Nuclear staining was also evident in the anterior limb,
overlapping with 52bpMsx2 transgene expression
(Fig. 5A,E, arrows). Similarly,
in the cardiac region, nuclear staining was apparent in pharyngeal arch
mesenchyme and the outflow tract (Fig.
5H,J). Thus, in the pharyngeal region the 52 bp fragment was
expressed in a subset of R-Smad positive cells
(Fig. 5G,I).
|
GCCG and TAAT sequences are necessary for BMP responsiveness of Msx2 transgenes
The 52 bp fragment contained a GC-rich partial inverted repeat flanking an
AT-rich region (Fig. 2,
Fig. 6A). Each repeat contained
two GCCG sequences, which resembled the GCCGNCGC consensus sequence to which
the Drosophila Mad protein binds
(Kim et al., 1997;
Raftery and Sutherland, 1999
;
Szuts et al., 1998
;
Xu et al., 1998
). The GCCG
sites flanked the sequence AGAGCAATTAACG, which matched closely the
consensus site for several Antennapedia superclass homeodomain proteins, as
well as paired class homeodomain proteins
(de Jong et al., 1993
;
Florence et al., 1991
;
Gehring et al., 1994
;
Laughon, 1991
) (MatInspector
7.0, 2003). As homeodomain proteins are candidates for tissue- and
stage-specific modulators of the BMP response
(Marty et al., 2001
), the
AATTAA site was of particular interest.
To test the function of the GCCG and AATTAA sequences in vivo, we produced
two mutant 52bpMsx2-hsplacZ transgenes. One contained mutations in
the four GCCG sequences, the other a mutation in the AATTAA site
(Fig. 6A). Gel shift
experiments verified that mutations in the GC-rich inverted repeats
significantly reduced binding by the Smad4 MH1 domain, a DNA-binding motif
conserved among several Smad proteins
(Massague, 1998). Low protein
inputs produced a single complex, higher inputs an additional, more slowly
migrating complex (Fig. 6B).
The latter complex probably resulted from the binding of additional Smad4
molecules. Mutation of the four GCCG sequences abolished the slower migrating
(2°) complex and reduced substantially (60-90%) the faster migrating
(1°) complex (Fig. 6B,C).
That the primary complex was not abolished may have been due to an additional
GCCG sequence located 3' of the inverted repeat, or to several GNCT
motifs, both of which might bind Smad4
(Fig. 6A)
(Ishida et al., 2000
;
Johnson et al., 1999
;
Kusanagi et al., 2000
;
Zawel et al., 1998
). Despite
this residual binding of Smad4 in vitro, the transgene bearing mutations in
the four GCCG sites (7 independent lines) was expressed at much lower levels
and in fewer regions than its non-mutated counterpart
(Fig. 6E,F;
Table 1). In addition, the
mutant transgene did not respond to BMP4-soaked beads implanted on the limb
bud (Fig. 6I) or mandible (data
not shown).
Protein titrations showed that oligonucleotides containing the TTAATT site
and flanking sequence were capable of binding the homeodomain proteins Prx1b
(Fig. 6D) and Msx2 (data not
shown). A mutation in the TTAATT site (Fig.
6A) reduced the binding of Prx1b to an undetectable level
(Fig. 6D), prompting us to test
this same mutation in vivo. Analysis of 14 independent lines revealed a
dramatic reduction in transgene expression in the limb bud and in most other
sites (Fig. 6E,G;
Table 1). Bead implantations in
the limb bud showed that the mutant transgene was not BMP responsive
(Fig. 6J). This profound change
in the expression of the 52bpMsx2 transgene led us to test the role
of the TTAATT element in a larger genomic context. We used the 560 bp fragment
because it contained all or most of the cis-regulatory information needed for
expression and BMP responsiveness of Msx2
(Kwang et al., 2002).
In 10T1/2 cells, the homeodomain site mutation resulted in near-complete loss of BMP-inducibility of a transfected 560 bp construct (Fig. 6K), similar to the effect of deleting the entire 52 bp sequence in the 560 bp fragment (Fig. 3I). Analysis of six transgenic lines bearing 560bpMsx2-hdm-hsplacZ mutant transgenes showed that the mutation resulted in a general reduction of transgene expression (compare Fig. 6M with Fig. 3H) as well as a site-selective loss of expression (Table 1). For example, expression was reduced substantially in limb bud mesenchyme but retained in the AER (Fig. 6M, arrow). Bead implantations showed that the mutation abolished BMP-responsiveness in limb bud mesenchyme of E11.5 embryos (Fig. 6O). We conclude that the TTAATT element is crucially important for BMP responsiveness of Msx2 both in cultured cells and in a subset of sites in murine embryos.
The Msx2 BMP-responsive element can interpret Dpp signals accurately in Drosophila
That the homeodomain and Smad consensus sites are required coordinately for
the function of the Msx2 BMPRE in murine embryos, and that the
sequence of the BMPRE is highly conserved among mammalian groups, raised the
question of whether this combination of homeodomain and Smad consensus sites
reflects an ancient mechanism for BMP-dependent transcriptional activation.
Cis-regulatory elements can undergo mutational turnover yet maintain their
function (Ludwig et al.,
2000). Thus, despite the apparent lack of sequence conservation of
the BMPRE outside mammals, the possibility remained that it might function in
a more diverse group of organisms.
To test this idea, we asked whether the Msx2 BMPRE was capable of responding to Dpp signals in Drosophila. We chose Drosophila because its distant relation to mouse would provide a stringent test of the hypothesis that the BMPRE can function over a large phylogenetic distance. In addition, a large number of mutants in components of the Dpp pathway are available, enabling us to rigorously test whether the murine element was responding appropriately to Dpp signals.
We produced transgenic flies bearing the 560, 480, 220 and 52 bp sequences driving nuclear-localized lacZ. We examined expression in imaginal discs and embryos, both in wild-type flies and in mutants in which Dpp signaling was perturbed. Examples of transgene expression are shown in Fig. 7. The 480bpMsx2-lacZ transgene was expressed in wing imaginal discs in a pattern that closely resembled that of vestigial, a known Dpp target (Fig. 7B,C). Ectopic activation of Dpp signaling with A9Gal4>TkvA resulted in a corresponding expansion of Msx2 transgene expression (Fig. 7D). In stage 13 embryos, the 220bpMsx2-lacZ transgene was expressed in parasegments (ps) 3 and 7 of embryo visceral mesoderm in a pattern closely matching that of dpp (Fig. 7E,G). This pattern was expanded throughout the gut in embryos in which dpp expression was driven ectopically by a heat shock promoter (Fig. 7I). In dpp (S11/S22) regulatory mutants, in which dpp signaling is lost specifically in ps3 (Fig. 7F, arrow), the 220bpMsx2-lacZ transgene was also downregulated in ps3 (Fig. 7H, arrow).
|
When we tested the expression of 52bpMsx2-lacZ transgenes bearing the same GCCG or TTAATT site mutations used for transgenic mice, we found that each mutation resulted in a profound loss of transgene expression (Fig. 7P,Q) (five independent lines each). Similar results were apparent in stage 13 embryos bearing these mutant transgenes (data not shown). Together, these data show: (1) that sequences within the Msx2 promoter can respond to Dpp signaling in Drosophila embryos and wing imaginal discs; and (2) that both TTAATT and GCCG sites are crucial for expression of the Msx2 BMPRE in Drosophila embryos as they are in mouse embryos.
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Discussion |
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Distinct cis-regulatory elements are required for BMP-dependent activation of Msx2 transgenes in different developmental settings
A long-term goal in the analysis of the Msx2 BMPRE has been to
identify trans-regulators that cooperate with Smads to modulate BMP
responsiveness in different developmental settings. We provide evidence here
that a homeodomain consensus site is required, together with Smad consensus
sites, for BMP responsiveness of Msx2 transgenes, and that these
sites function together with other cis-regulatory elements to control
differential BMP responsiveness and expression in subregions of limb
mesenchyme and in other structures in the developing embryo. That the
consensus homeodomain site plays such a major role in the BMP responsiveness
of the Msx2 promoter was unexpected. Although Smad sites are
typically found to be functionally crucial in vertebrate BMP responsive
elements (Lopez-Rovira et al.,
2002; Park and Morasso,
2002
), similar crucial roles for homeodomain sites have not been
documented. We note, however, that Smad1 interacts with Hoxc8 during
activation of the osteopontin promoter
(Shi et al., 1999
) and that in
Xenopus embryos, the Xvent-2 homeodomain protein can act as a
Smad1-specific co-activator during maintenance of its own transcriptional
regulation (Henningfeld et al.,
2002
).
It is clearly of interest to know the identities of proteins that interact
with the TTAATT site in the Msx2 BMPRE. Homeodomain-containing
proteins of the Nk (including Msx), paired and Hox classes are capable of
binding this element in vitro (Fig.
6; S.M.B. and R.M., unpublished). Representatives of each class
are expressed in patterns consistent with a role in the regulation of
Msx2, though genetic proof that any such proteins participate in the
BMP-dependent activation of Msx2 is lacking. Interestingly,
Xenopus Xmsx-1 has been shown to physically associate with
pathway-restricted Smads and Smad4
(Yamamoto et al., 2001),
suggesting that like Xvent2, Msx proteins may participate in an autoregulatory
loop.
In addition to homeodomain-containing proteins, members of the Fox family
of forkhead/winged helix transcription factors have emerged as candidate
regulators of the BMP-responsiveness of Msx2. Foxc1 is required for
the BMP-dependent induction of Msx2 in calvarial tissues of murine
embryos (Rice et al., 2003).
Inspection of the sequence around the TTAATT site revealed a close match (5/7)
to a consensus derived for Hnf3/Fox class proteins
(Fig. 2) (Gao et al., 2003
). Whether
Fox proteins can bind this sequence, and, if so, whether such binding is
disrupted by mutations in the TTAATT site are unanswered questions. It is
intriguing that Fox proteins can form heterodimers with homeodomain-containing
proteins (Foucher et al.,
2003
), and that the Fox protein Fast1 can interact with Smads and
modulate the activity of TGFß-responsive promoters
(Chen et al., 1996
;
Chen et al., 1997
;
Weisberg et al., 1998
).
Deletion analysis showed sequences flanking the 52 bp element control the
location, sensitivity and timing of BMP responsiveness in limbs and other
sites in mouse embryos. Within these sequences are consensus binding sites for
Creb, OAZ and Tcf/Lef1 (Fig.
2), each implicated in BMP signaling in widely divergent organisms
(Hata et al., 2000;
Ionescu et al., 2004
;
Theil et al., 2002
). In the
Drosophila Ubx promoter, for example, Dpp responsiveness in
parasegments 3 and 7 of the embryonic midgut depends on a CRE site acting
together with Mad sites (Waltzer and
Bienz, 1999
). In addition, Tcf/Lef1 sites located 270 and 420 bp
upstream (at positions 4130 and 4280) of the
4Msx2 fragment, participate in BMP responsiveness in ES cells
(Hussein et al., 2003
).
Mutation of these sites in mouse embryos will provide insight into their roles
in modulating the BMP responsiveness of Msx2 in different tissues and
cell types. We showed previously that Pax3 acts through a site in the 560 bp
fragment to repress Msx2 expression in the dorsal neural tube
(Kwang et al., 2002
). We have
not yet identified sequences required for upregulated neural tube expression
of Msx2 in the absence of Pax3 function, but it is possible
that these sequences include the Msx2 BMPRE. Finally, we note that
YYI has been implicated in the activation of Msx2 expression in
craniofacial structures, but in a manner independent of the BMP pathway
(Tan et al., 2002
).
In Drosophila, brinker and schnurri are crucial for the
Dpp-dependent expression of a number of genes. Brinker (Brk) acts through
GC-rich sites to repress target gene expression
(Rushlow et al., 2001;
Sivasankaran et al., 2000
).
Schnurri represses brinker expression in a Dpp-dependent manner
(Marty et al., 2000
;
Torres-Vazquez et al., 2000
).
That Msx2 transgene expression in Drosophila embryos expands
in a brinker mutant is consistent with results showing that Brk acts
through Mad-like binding sites for its repressive function
(Kirkpatrick et al., 2001
;
Saller and Bienz, 2001
), and
with our findings that the Msx2 BMPRE is probably a direct target of
Smad1 (Fig. 5). Our
demonstration that mutations in Mad/Smad1-binding sites cause loss of
transgene expression in Drosophila, together with the expansion of
transgene expression in the brinker mutant, suggest that the
Msx2 BMPRE is subject to positive and negative regulation in
Drosophila, as expected for direct targets of Mad.
Whether Msx2 transgenes are similarly regulated by positive and
negative inputs in mouse embryos is not clear. Orthologs of brinker
have not been found in vertebrates. However, Zeb-2, a zinc-finger
transcription factor, has been identified as a negative regulator of BMP
signaling (Postigo, 2003).
This protein binds to a GC-rich sequence and can interact with Smads
(Verschueren et al., 1999
), as
well as inhibit expression of the BMP target, Xmsx1, in
Xenopus (Postigo et al.,
2003
).
Evolution of the Msx2 BMP-responsive element
Despite the lack of demonstrable sequence homology between the
Msx2 BMPRE and Dpp-responsive enhancers in flies, our transgenic
experiments show that the Msx2 element provides an accurate readout
of Dpp activity in Drosophila. This is not merely a result of the
activity of Mad, as a mutant transgene bearing a disabled TTAATT sequence but
intact GCCG consensus sites was not expressed in Drosophila embryos.
We note that the requirement of the TTAATT site for BMP responsiveness of
Msx2 transgenes in mice and flies is reminiscent of the dependence of
the labial, tinman and even-skipped Dpp-activated enhancers
of Drosophila on the homeodomain proteins Labial and Tinman
(Knirr and Frasch, 2001;
Marty et al., 2001
;
Xu et al., 1998
).
That several BMP-responsive enhancers spanning a wide phylogenetic distance exhibit a dual requirement for homeodomain and Smad sites suggests synergistic interactions between homeodomain and Smad proteins is an ancient feature of the BMP/Dpp pathway. Such interactions may represent a general mechanism for integrating BMP/Dpp signaling inputs with tissue-specific transcription factors.
Few cis-regulatory elements are known to function over an evolutionary
distance comparable with that separating mammals and insects. One such element
is the eye enhancer of the Drosophila eyeless gene, a Pax6
ortholog. In transgenic mice, this enhancer directs expression that partially
recapitulates that of endogenous Pax6
(Xu et al., 1999). Another is
the murine Hoxa2 rhombomere 2-specific enhancer, which is expressed
in head segments of transgenic flies, paralleling the expression of the
Hhoxa2 ortholog, proboscipedia
(Frasch et al., 1995
).
Similarly, a Hox4b cis-regulatory element mediates correct spatial
expression in Drosophila (Malicki
et al., 1992
), and the orthologous Drosophila
autoregulatory element from the homeotic gene, Deformed, functions
appropriately in mice (Awgulewitsch and
Jacobs, 1992
).
These examples notwithstanding, careful comparative analyses within
nematode and echinoderm lineages suggest that cis-regulatory elements usually
evolve rapidly, becoming non-alignable within 35-50 million years
(Ruvinsky and Ruvkun, 2003;
Wray et al., 2003
).
Intriguingly, such divergent promoter elements frequently maintain their
function, suggesting they are subject both to drift and stabilizing selection,
which promote rapid sequence divergence while maintaining regulatory function
(Ludwig et al., 2000
;
Ruvunski and Ruvkun, 2003
;
Wray et al., 2003
). It seems
probable that the ability of the Msx2 BMPRE to function in flies,
despite its lack of significant sequence identity to known Dpp-responsive
enhancers, can be explained by a similar process of drift and stabilizing
selection. We suggest this process differs from that operating on a typical
promoter element only in its slow pace, reflecting the fundamental and
conservative role of the machinery that controls the transcription of crucial
effectors of the BMP pathway.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Arora, K., Levine, M. S. and O'Connor, M. B. (1994). The screw gene encodes a ubiquitously expressed member of the TGF-beta family required for specification of dorsal cell fates in the Drosophila embryo. Genes Dev. 8,2588 -2601.[Abstract]
Awgulewitsch, A. and Jacobs, D. (1992). Deformed autoregulatory element from Drosophila functions in a conserved manner in transgenic mice. Nature 358,341 -344.[CrossRef][Medline]
Bell, J. R., Noveen, A., Liu, Y. H., Ma, L., Dobias, S., Kundu, R., Luo, W., Xia, Y., Lusis, A. J., Snead, M. L. et al. (1993). Genomic structure, chromosomal location, and evolution of the mouse Hox 8 gene. Genomics 16,123 -131.[CrossRef][Medline]
Benchabane, H. and Wrana, J. L. (2003). GATA-
and Smad1-dependent enhancers in the Smad7 gene differentially interpret bone
morphogenetic protein concentrations. Mol. Cell. Biol.
23,6646
-6661.
Chen, X., Rubock, M. J. and Whitman, M. (1996). A transcriptional partner for MAD proteins in TGF-beta signalling. Nature 383,691 -696.[CrossRef][Medline]
Chen, X., Weisberg, E., Fridmacher, V., Watanabe, M., Naco, G. and Whitman, M. (1997). Smad4 and FAST-1 in the assembly of activin-responsive factor. Nature 389, 85-89.[CrossRef][Medline]
Daluiski, A., Engstrand, T., Bahamonde, M. E., Gamer, L. W., Agius, E., Stevenson, S. L., Cox, K., Rosen, V. and Lyons, K. M. (2001). Bone morphogenetic protein-3 is a negative regulator of bone density. Nat. Genet. 27, 84-88.[CrossRef][Medline]
Davidson, D. (1995). The function and evolution of Msx genes, pointers and paradoxes. Trends Genet. 11,405 -411.[CrossRef][Medline]
de Jong, R., van der Heijden, J. and Meijlink, F. (1993). DNA-binding specificity of the S8 homeodomain. Nucleic Acids Res. 21,4711 -4720.[Abstract]
Florence, B., Handrow, R. and Laughon, A. (1991). DNA-binding specificity of the fushi tarazu homeodomain. Mol. Cell. Biol. 11,3613 -3623.[Medline]
Foucher, I., Montesinos, M. L., Volovitch, M., Prochiantz, A.
and Trembleau, A. (2003). Joint regulation of the MAP1B
promoter by HNF3beta/Foxa2 and Engrailed is the result of a highly conserved
mechanism for direct interaction of homeoproteins and Fox transcription
factors. Development
130,1867
-1876.
Frasch, M., Chen, X. and Lufkin, T. (1995).
Evolutionary-conserved enhancers direct region-specific expression of the
murine Hoxa-1 and Hoxa-2 loci in both mice and Drosophila.
Development 121,957
-974.
Furuta, Y. and Hogan, B. L. (1998). BMP4 is
essential for lens induction in the mouse embryo. Genes
Dev. 12,3764
-3775.
Gao, N., Zhang, J., Rao, M. A., Case, T. C., Mirosevich, J.,
Wang, Y., Jin, R., Gupta, A., Rennie, P. S. and Matusik, R. J.
(2003). The role of hepatocyte nuclear factor-3 alpha (Forkhead
Box A1) and androgen receptor in transcriptional regulation of prostatic
genes. Mol. Endocrinol.
17,1484
-1507.
Gehring, W. J., Qian, Y. Q., Billeter, M., Furukubo-Tokunaga, K., Schier, A. F., Resendez-Perez, D., Affolter, M., Otting, G. and Wuthrich, K. (1994). Homeodomain-DNA recognition. Cell 78,211 -223.[Medline]
Graham, A., Francis-West, P., Brickell, P. and Lumsden, A. (1994). The signalling molecule BMP4 mediates apoptosis in the rhombencephalic neural crest. Nature 372,684 -686.[CrossRef][Medline]
Gurdon, J. B. and Bourillot, P. Y. (2001). Morphogen gradient interpretation. Nature 413,797 -803.[CrossRef][Medline]
Hata, A., Seoane, J., Lagna, G., Montalvo, E., Hemmati-Brivanlou, A. and Massague, J. (2000). OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signaling pathways. Cell 100,229 -240.[Medline]
Heldin, C. H., Miyazono, K. and ten Dijke, P. (1997). TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 390,465 -471.[CrossRef][Medline]
Henningfeld, K. A., Rastegar, S., Adler, G. and Knochel, W.
(2000). Smad1 and Smad4 are components of the bone morphogenetic
protein-4 (BMP-4)-induced transcription complex of the Xvent-2B promoter.
J. Biol. Chem. 275,21827
-21835.
Henningfeld, K. A., Friedle, H., Rastegar, S. and Knochel,
W. (2002). Autoregulation of Xvent-2B; direct interaction and
functional cooperation of Xvent-2 and Smad1. J. Biol.
Chem. 277,2097
-2103.
Hogan, B., Beddington, R., Constantini, F. and Lacy, E. (1994). Manipulating the mouse embryo, a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Hollnagel, A., Oehlmann, V., Heymer, J., Ruther, U. and
Nordheim, A. (1999). Id genes are direct targets of bone
morphogenetic protein induction in embryonic stem cells. J. Biol.
Chem. 274,19838
-19845.
Hoodless, P. A., Haerry, T., Abdollah, S., Stapleton, M., O'Connor, M. B., Attisano, L. and Wrana, J. L. (1996). MADR1, a MAD-related protein that functions in BMP2 signaling pathways. Cell 85,489 -500.[Medline]
Huang, J. D., Schwyter, D. H., Shirokawa, J. M. and Courey, A. J. (1993). The interplay between multiple enhancer and silencer elements defines the pattern of decapentaplegic expression. Genes Dev. 7,694 -704.[Abstract]
Hullinger, T. G., Pan, Q., Viswanathan, H. L. and Somerman, M. J. (2001). TGFbeta and BMP-2 activation of the OPN promoter, roles of smad- and hox-binding elements. Exp. Cell Res. 262,69 -74.[CrossRef][Medline]
Hussein, S. M., Duff, E. K. and Sirard, C.
(2003). Smad4 and beta-catenin co-activators functionally
interact with lymphoid-enhancing factor to regulate graded expression of Msx2.
J. Biol. Chem. 278,48805
-48814.
Ionescu, A. M., Drissi, H., Schwarz, E. M., Kato, M., Puzas, J. E., McCance, D. J., Rosier, R. N., Zuscik, M. J. and O'Keefe, R. J. (2004). CREB Cooperates with BMP-stimulated Smad signaling to enhance transcription of the Smad6 promoter. J. Cell Physiol. 198,428 -440.[CrossRef][Medline]
Ishida, W., Hamamoto, T., Kusanagi, K., Yagi, K., Kawabata, M.,
Takehara, K., Sampath, T. K., Kato, M. and Miyazono, K.
(2000). Smad6 is a Smad1/5-induced smad inhibitor.
Characterization of bone morphogenetic protein-responsive element in the mouse
Smad6 promoter. J. Biol. Chem.
275,6075
-6079.
Ishii, M., Merrill, A. E., Chan, Y. S., Gitelman, I., Rice, D.
P., Sucov, H. M. and Maxson, R. E., Jr (2003). Msx2 and Twist
cooperatively control the development of the neural crest-derived skeletogenic
mesenchyme of the murine skull vault. Development
130,6131
-6142.
Jazwinska, A., Kirov, N., Wieschaus, E., Roth, S. and Rushlow, C. (1999). The Drosophila gene brinker reveals a novel mechanism of Dpp target gene regulation. Cell 96,563 -573.[Medline]
Johnson, K., Kirkpatrick, H., Comer, A., Hoffmann, F. M. and
Laughon, A. (1999). Interaction of Smad complexes with
tripartite DNA-binding sites. J. Biol. Chem.
274,20709
-20716.
Jonk, L. J., Itoh, S., Heldin, C. H., ten Dijke, P. and Kruijer,
W. (1998). Identification and functional characterization of
a Smad binding element (SBE) in the JunB promoter that acts as a transforming
growth factor-beta, activin, and bone morphogenetic protein-inducible
enhancer. J. Biol. Chem.
273,21145
-21152.
Kim, J., Johnson, K., Chen, H. J., Carroll, S. and Laughon, A. (1997). Drosophila Mad binds to DNA and directly mediates activation of vestigial by Decapentaplegic. Nature 388,304 -308.[CrossRef][Medline]
Kirkpatrick, H., Johnson, K. and Laughon, A.
(2001). Repression of dpp targets by binding of brinker to mad
sites. J. Biol. Chem.
276,18216
-18222.
Knirr, S. and Frasch, M. (2001). Molecular integration of inductive and mesoderm-intrinsic inputs governs even-skipped enhancer activity in a subset of pericardial and dorsal muscle progenitors. Dev. Biol. 238,13 -26.[CrossRef][Medline]
Kothary, R., Clapoff, S., Darling, S., Perry, M. D., Moran, L. A. and Rossant, J. (1989). Inducible expression of an hsp68-lacZ hybrid gene in transgenic mice. Development 105,707 -714.[Abstract]
Kretzschmar, M., Liu, F., Hata, A., Doody, J. and Massague, J. (1997). The TGF-beta family mediator Smad1 is phosphorylated directly and activated functionally by the BMP receptor kinase. Genes Dev. 11,984 -995.[Abstract]
Kusanagi, K., Inoue, H., Ishidou, Y., Mishima, H. K., Kawabata,
M. and Miyazono, K. (2000). Characterization of a bone
morphogenetic protein-responsive Smad-binding element. Mol. Biol.
Cell 11,555
-565.
Kwang, S. J., Brugger, S. M., Lazik, A., Merrill, A. E., Wu, L. Y., Liu, Y. H., Ishii, M., Sangiorgi, F. O., Rauchman, M., Sucov, H. M. et al. (2002). Msx2 is an immediate downstream effector of Pax3 in the development of the murine cardiac neural crest. Development 129,527 -538.[Medline]
Lagna, G., Hata, A., Hemmati-Brivanlou, A. and Massague, J. (1996). Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways. Nature 383,832 -836.[CrossRef][Medline]
Laughon, A. (1991). DNA binding specificity of homeodomains. Biochemistry 30,11357 -11367.[Medline]
Liberatore, C. M., Searcy-Schrick, R. D., Vincent, E. B. and Yutzey, K. E. (2002). Nkx-2.5 gene induction in mice is mediated by a Smad consensus regulatory region. Dev. Biol. 244,243 -256.[CrossRef][Medline]
Lien, C. L., McAnally, J., Richardson, J. A. and Olson, E. N. (2002). Cardiac-specific activity of an Nkx2-5 enhancer requires an evolutionarily conserved Smad binding site. Dev. Biol. 244,257 -266.[CrossRef][Medline]
Liu, Y. H., Ma, L., Wu, L. Y., Luo, W., Kundu, R., Sangiorgi, F., Snead, M. L. and Maxson, R. (1994). Regulation of the Msx2 homeobox gene during mouse embryogenesis, a transgene with 439 bp of 5' flanking sequence is expressed exclusively in the apical ectodermal ridge of the developing limb. Mech. Dev. 48,187 -197.[CrossRef][Medline]
Liu, F., Hata, A., Baker, J. C., Doody, J., Carcamo, J., Harland, R. M. and Massague, J. (1996). A human Mad protein acting as a BMP-regulated transcriptional activator. Nature 381,620 -623.[CrossRef][Medline]
Liu, F., Pouponnot, C. and Massague, J. (1997).
Dual role of the Smad4/DPC4 tumor suppressor in TGFbeta-inducible
transcriptional complexes. Genes Dev.
11,3157
-3167.
Lopez-Rovira, T., Chalaux, E., Massague, J., Rosa, J. L. and
Ventura, F. (2002). Direct binding of Smad1 and Smad4 to two
distinct motifs mediates bone morphogenetic protein-specific transcriptional
activation of Id1 gene. J. Biol. Chem.
277,3176
-3185.
Ludwig, M. Z., Bergman, C., Patel, N. H. and Kreitman, M. (2000). Evidence for stabilizing selection in a eukaryotic enhancer element. Nature 403,564 -567.[CrossRef][Medline]
Ma, H., Shang, Y., Lee, D. Y. and Stallcup, M. R. (2003). Study of nuclear receptor-induced transcription complex assembly and histone modification by chromatin immunoprecipitation assays. Methods Enzymol. 364,284 -296.[Medline]
Malicki, J., Cianetti, L. C., Peschle, C. and McGinnis, W. (1992). A human HOX4B regulatory element provides head-specific expression in Drosophila embryos. Nature 358,345 -347.[CrossRef][Medline]
Marty, T., Muller, B., Basler, K. and Affolter, M. (2000). Schnurri mediates Dpp-dependent repression of brinker transcription. Nat. Cell Biol. 2, 745-749.[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]
Massague, J. (1998). TGF-beta signal transduction. Annu. Rev. Biochem. 67,753 -791.[CrossRef][Medline]
Massague, J. and Chen, Y. G. (2000).
Controlling TGF-beta signaling. Genes Dev.
14,627
-644.
Maxson, R. E., Ishii, M. and Merrill, A. (2003). Msx genes in organogenesis and human disease. In Advances in Developmental Biology and biochemistry Vol. 13, Murine Homeobox Gene Control of Embryonic Patterning and Organogenesis (ed. T. Lufkin), pp. 43-68. Amsterdam, The Netherlands: Elsevier Science.
Norris, R. A. and Kern, M. J. (2001). The
identification of Prx1 transcription regulatory domains provides a mechanism
for unequal compensation by the Prx1 and Prx2 loci. J. Biol.
Chem. 276,26829
-26837.
Park, G. T. and Morasso, M. I. (2002). Bone
morphogenetic protein-2 (BMP-2) transactivates Dlx3 through Smad1 and Smad4,
alternative mode for Dlx3 induction in mouse keratinocytes. Nucleic
Acids Res. 30,515
-522.
Pollard, S. L. and Holland, P. W. (2000). Evidence for 14 homeobox gene clusters in human genome ancestry. Curr. Biol. 10,1059 -1062.[CrossRef][Medline]
Postigo, A. A. (2003). Opposing functions of
ZEB proteins in the regulation of the TGFbeta/BMP signaling pathway.
EMBO J. 22,2443
-2452.
Postigo, A. A., Depp, J. L., Taylor, J. J. and Kroll, K. L.
(2003). Regulation of Smad signaling through a differential
recruitment of coactivators and corepressors by ZEB proteins. EMBO
J. 22,2453
-2462.
Raftery, L. A. and Sutherland, D. J. (1999). TGF-beta family signal transduction in Drosophila development, from Mad to Smads. Dev. Biol. 210,251 -268.[CrossRef][Medline]
Rice, R., Rice, D. P., Olsen, B. R. and Thesleff, I. (2003). Progression of calvarial bone development requires Foxc1 regulation of Msx2 and Alx4. Dev. Biol. 262, 75-87.[CrossRef][Medline]
Rosen, V., Nove, J., Song, J. J., Thies, R. S., Cox, K. and Wozney, J. M. (1994). Responsiveness of clonal limb bud cell lines to bone morphogenetic protein 2 reveals a sequential relationship between cartilage and bone cell phenotypes. J. Bone Miner. Res. 9,1759 -1768.[Medline]
Rushlow, C., Colosimo, P. F., Lin, M. C., Xu, M. and Kirov,
N. (2001). Transcriptional regulation of the Drosophila gene
zen by competing Smad and Brinker inputs. Genes Dev.
15,340
-351.
Ruvinsky, I. and Ruvkun, G. (2003). Functional
tests of enhancer conservation between distantly related species.
Development 130,5133
-5142.
Saller, E. and Bienz, M. (2001). Direct
competition between Brinker and Drosophila Mad in Dpp target gene
transcription. EMBO Rep.
2, 298-305.
Satokata, I. and Maas, R. (1994). Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. Nat. Genet. 6, 348-356.[Medline]
Satokata, I., Ma, L., Ohshima, H., Bei, M., Woo, I., Nishizawa, K., Maeda, T., Takano, Y., Uchiyama, M., Heaney, S. et al. (2000). Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat. Genet. 24,391 -395.[CrossRef][Medline]
Shi, Y. and Massague, J. (2003). Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113,685 -700.[Medline]
Shi, Y., Wang, Y. F., Jayaraman, L., Yang, H., Massague, J. and Pavletich, N. P. (1998). Crystal structure of a Smad MH1 domain bound to DNA, insights on DNA binding in TGF-beta signaling. Cell 94,585 -594.[Medline]
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.
Shim, S., Bae, N. and Han, J. K. (2002). Bone
morphogenetic protein-4-induced activation of Xretpos is mediated by Smads and
Olf-1/EBF associated zinc finger (OAZ). Nucleic Acids
Res. 30,3107
-3117.
Sivasankaran, R., Vigano, M. A., Muller, B., Affolter, M. and
Basler, K. (2000). Direct transcriptional control of the Dpp
target omb by the DNA binding protein Brinker. EMBO J.
19,6162
-6172.
Suzuki, A., Chang, C., Yingling, J. M., Wang, X. F. and Hemmati-Brivanlou, A. (1997). Smad5 induces ventral fates in Xenopus embryo. Dev. Biol. 184,402 -405.[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.
Tan, D. P., Nonaka, K., Nuckolls, G. H., Liu, Y. H., Maxson, R.
E., Slavkin, H. C. and Shum, L. (2002). YY1 activates Msx2
gene independent of bone morphogenetic protein signaling. Nucleic
Acids Res. 30,1213
-1223.
Theil, T., Aydin, S., Koch, S., Grotewold, L. and Ruther, U.
(2002). Wnt and Bmp signalling cooperatively regulate graded Emx2
expression in the dorsal telencephalon. Development
129,3045
-3054.
Torres-Vazquez, J., Warrior, R. and Arora, K. (2000). schnurri is required for dpp-dependent patterning of the Drosophila wing. Dev. Biol. 227,388 -402.[CrossRef][Medline]
Vainio, S., Karavanova, I., Jowett, A. and Thesleff, I. (1993). Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell 75,45 -58.[Medline]
Verschueren, K., Remacle, J. E., Collart, C., Kraft, H., Baker,
B. S., Tylzanowski, P., Nelles, L., Wuytens, G., Su, M. T., Bodmer, R. et
al. (1999). SIP1, a novel zinc finger/homeodomain repressor,
interacts with Smad proteins and binds to 5'-CACCT sequences in
candidate target genes. J. Biol. Chem.
274,20489
-20498.
Waltzer, L. and Bienz, M. (1999). A function of
CBP as a transcriptional co-activator during Dpp signalling. EMBO
J. 18,1630
-1641.
Weisberg, E., Winnier, G. E., Chen, X., Farnsworth, C. L., Hogan, B. L. and Whitman, M. (1998). A mouse homologue of FAST-1 transduces TGF beta superfamily signals and is expressed during early embryogenesis. Mech. Dev. 79, 17-27.[CrossRef][Medline]
Wiersdorff, V., Lecuit, T., Cohen, S. M. and Mlodzik, M.
(1996). Mad acts downstream of Dpp receptors, revealing a
differential requirement for dpp signaling in initiation and propagation of
morphogenesis in the Drosophila eye. Development
122,2153
-2162.
Wrana, J. L. (2000). Regulation of Smad activity. Cell 100,189 -192.[Medline]
Wray, G. A., Hahn, M. W., Abouheif, E., Balhoff, J. P., Pizer,
M., Rockman, M. V. and Romano, L. A. (2003). The evolution of
transcriptional regulation in eukaryotes. Mol. Biol.
Evol. 20,1377
-1419.
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.
Xu, P. X., Zhang, X., Heaney, S., Yoon, A., Michelson, A. M. and
Maas, R. L. (1999). Regulation of Pax6 expression is
conserved between mice and flies. Development
126,383
-395.
Yamamoto, T. S., Takagi, C., Hyodo, A. C. and Ueno, N. (2001). Suppression of head formation by Xmsx-1 through the inhibition of intracellular nodal signaling. Development 128,2769 -2779.[Medline]
Zawel, L., Dai, J. L., Buckhaults, P., Zhou, S., Kinzler, K. W., Vogelstein, B. and Kern, S. E. (1998). Human Smad3 and Smad4 are sequence-specific transcription activators. Mol. Cell 1,611 -617.[Medline]
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