Department of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195-7350, USA
* Author for correspondence (e-mail: kimelman{at}u.washington.edu)
Accepted 21 April 2004
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SUMMARY |
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Key words: Bmp, Wnt, Posterior mesoderm, T-box genes, Zebrafish
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Introduction |
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Three bmps are expressed in the mesoderm of the gastrula-stage
embryo and their expression continues at the most posterior end of the embryo
throughout somitogenesis (Martinez-Barbera
et al., 1997; Nikaido et al.,
1997
). As in Xenopus, a large body of evidence
demonstrates that the Bmps are key regulators of the posterior mesoderm
patterning in zebrafish (reviewed by
Hammerschmidt, 2002
). For
example, bmp2b/swirl is expressed in a gradient along the
dorsoventral axis (Barth et al.,
1999
; Wagner and Mullins,
2002
) and zebrafish mutants lacking bmp2b/swirl do not
form tails (Mullins et al.,
1996
; Kishimoto et al.,
1997
). Like the bmps, zebrafish wnt8 is also
expressed in the ventrolateral mesoderm during gastrulation and at the
posterior end of the embryo during somitogenesis
(Kelly et al., 1995
). In
mutants lacking wnt8, the formation of the ventrolateral and
posterior mesoderm is severely disrupted, demonstrating a requirement for Wnt
signaling in the development of these mesodermal territories
(Lekven et al., 2001
).
Two studies have suggested that the Wnt and Bmp pathways cooperate to form
the ventral and posterior mesoderm in Xenopus
(Hoppler and Moon, 1998;
Marom et al., 1999
). The
importance of such a cooperative interaction between these pathways has been
further highlighted in a recent zebrafish study, demonstrating that combined
overexpression of Wnt8 and Bmp is capable of inducing ectopic tail formation
(Agathon et al., 2003
). How
these pathways integrate to regulate mesodermal gene expression is
unknown.
In order to understand mechanistically how these two pathways interact, we
sought a posterior mesodermal gene whose expression required input from both
the Bmp and Wnt pathways. The T-box transcription factor tbx6
(Hug et al., 1997) was found
to meet this criterion, and we therefore identified a minimal region of the
tbx6 gene that reproduces the endogenous gene expression pattern.
Analysis of the tbx6 promoter shows that Bmp and Wnt8 regulate
tbx6 expression through different domains within the promoter,
demonstrating that these pathways intersect by regulating transcription of
specific target genes. Elimination of regions that respond to Bmp or Wnt
signaling revealed that the promoter is able to respond to overexpression of a
single pathway, whereas normally the promoter integrates responses to both
pathways to provide normal tbx6 expression. Analysis of the
expression of the endogenous gene under conditions in which either Bmp or Wnt
signaling is blocked together with analysis of the tbx6 promoter has
allowed us to put forth a model in which submaximal levels of Bmp and Wnt
signals cooperate to regulate the formation of the posterior mesoderm.
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Materials and methods |
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Plasmids and constructs
Various gfp reporter constructs were made by cloning the
tbx6 promoter fragments into the BamHI restriction site of
the gfp reporter plasmid vector. The dual fluorescence system was
created by inserting the pXex-bfp DNA (gift of Stephen C. Ekker) into
the existing gfp reporter constructs. The luciferase
reporter constructs were made by cloning the tbx6 promoter fragments
into the multiple cloning sites of the PGL3 promoter vector (Promega).
Site-directed mutagenesis
The two proximal Tcf site mutations in the tbx6 promoter were
generated using the QuikChange procedure (Stratagene). Mut1-2.0 was
constructed by using primers:
5'-gtgcacatacacacctctggcccccctcgaggtgatggaagaaaggtagaagcctag-3'
and
5'-ctaggcttctaccttcttccatcacctcgaggggggccagaggtgtgtatgtgcac-3'.
Mut2-2.0 was constructed by using primers:
5'-gtgggctggctggagacaaaagacaggttaacgaggagactgatgttttgacagaag-3'
and
5'-cttctgtcaaaacatcagtctcctcgttaacctgtcttttgtctccagccagcccac-3'.
Mut1,2-2.0 construct was generated by combining the two mutated Tcf
sites.
DNA and RNA injections
DNA was isolated with a Qiagen midi kit and resuspended in RNase-free
water. DNA at a concentration of 0.1 mg/ml was used for injection. RNAs were
synthesized from SalI linearized SP64T-Xbmp4 (gift of Jim
Smith), Asp718 linearized CS2-zwnt8 (gift of Randy Moon) and
NotI linearized CS2-TVGR (gift of Paul Wilson) templates
using the mMessage Machine Kit (Ambion) and dissolved in RNase-free sterile
water. RNA (at concentrations indicated in the text) was injected in the
presence or absence of 0.1 µg of reporter DNA into one-cell zebrafish
embryos. The expression of the reporter gene was analyzed at the appropriate
stages using bright field or fluorescence microscopy.
In situ hybridization
Whole-mount in situ hybridization was performed using digoxigenin-labeled
antisense RNA probes and visualized using anti-digoxigenin Fab fragments
conjugated with alkaline phosphatase (Roche Molecular Biochemicals) as
described (Griffin et al.,
1998). Riboprobes were made from DNA templates, which were
linearized and transcribed with either SP6 or T7 RNA polymerases. Embryos were
processed and hybridized as described
(Griffin et al., 1998
), except
that 5 µg/ml of Proteinase K in PBS/0.1% Tween-20 was used for 5 to 10
minutes depending on the age of the collected embryos.
Xenopus and zebrafish transgenesis
PAC DNA was isolated with the Qiagen midi kit. To generate Xenopus
transgenic embryos, we used 200 pg of PAC DNA for each injection as described
(Amaya and Kroll, 1999) with
the exception that no restriction enzyme was used. To generate stable
transgenic zebrafish lines, reporter plasmid DNA was digested with
BssHII to completion, and then separated on a 1% agarose preparative
gel overnight. The gel slice containing the DNA insert was isolated and DNA
was electroeluted using a Schleicher and Schuell ELUTRAP. DNA was extracted
once with equal volume of phenol and once with an equal volume of chloroform.
After extraction, DNA was ethanol precipitated and then resuspended in
distilled water. For injection, 80 mg/ml of purified DNA insert was used to
inject one-cell stage zebrafish embryos. Injected embryos were examined at the
15- to 18-somite stage for the presence of GFP fluorescence. Embryos showing
specific GFP fluorescence in the tail region were collected and raised to
sexual maturity as founders. Different combinations of founder crosses were
set up for the identification of F1 embryos showing non-mosaic GFP
fluorescence in the tail region at the 15- to 18-somite stage.
Luciferase assays
Injected embryos were collected at the shield stage and then separated into
three pools of 10 embryos each for assay in triplicate. Experiments were
repeated at least three times. Excess zebrafish embryo medium was removed,
embryos were homogenized in 100 µl of 1xCell Culture Lysis Reagent
(Promega), and cleared by 10 minutes' microcentrifugation at room temperature.
Fifty microliters of the resulting supernatant was used for luciferase
activity assays that were performed according to the Promega protocol with a
Berthold luminometer.
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Results |
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Identification and characterization of the tbx6 promoter
In order to understand how these two pathways regulate tbx6
expression, we screened a zebrafish PAC genomic library and obtained three
tbx6 positive clones. Using the frog transgenesis approach
(Amaya and Kroll, 1999), we
tested one of these tbx6 positive PAC clones (PAC-tbx6) for
its ability to target expression of zebrafish tbx6 to the posterior
mesoderm of developing Xenopus embryos. The advantage to using
Xenopus is that we could examine the expression of the zebrafish
tbx6 gene using in situ hybridization because the fish and frog
tbx6 genes are sufficiently divergent
(Uchiyama et al., 2001
),
without the need to make a reporter gene fusion in the PAC DNA. Approximately
20% of the transgenic embryos (7/36) showed zebrafish tbx6
transcripts in the posterior paraxial mesoderm (see Fig. S1A,B at
http://dev.biologists.org/supplemental),
whereas the remainder of the embryos failed to express zebrafish tbx6
from the injected PAC (see Fig. S1C at
http://dev.biologists.org/supplemental).
This expression pattern matches the endogenous Xenopus tbx6
(Uchiyama et al., 2001
) as
well as that of the ntl ortholog Xbra
(Smith et al., 1991
) (see Fig.
S1D at
http://dev.biologists.org/supplemental).
These results demonstrate that PAC-tbx6 has all of the regulatory
elements necessary for expression within the posterior mesoderm, and that
these regulatory sites are conserved between fish and frogs.
Having determined that PAC-tbx6 targets expression to the posterior mesoderm in developing Xenopus embryos, we subcloned smaller fragments and characterized them in zebrafish. We initially focused on two constructs fused in frame to GFP, ptbx6-1.7-gfp and ptbx6-2.0-gfp, which contain 1.7 and 2.0 kb of DNA upstream from the start of translation, respectively, and the first intron (Fig. 3A). These constructs were injected into one-cell stage zebrafish embryos and then analyzed for GFP fluorescence at different stages of development. At 60-75% epiboly, we observed GFP fluorescence at the margin where endogenous tbx6 is normally expressed (Fig. 3B,C, Table 1). At the 15- to 18-somite stage, GFP fluorescence was detected in the tail mesoderm, again matching the endogenous pattern of expression (Fig. 3D,E, Table 1). Although both reporter constructs were expressed in the same pattern, we noticed that ptbx6-2.0-gfp produced consistently higher fluorescence levels than ptbx6-1.7-gfp, suggesting the presence of a general enhancer between -1.7 and -2.0 kb. The fluorescence results were confirmed by in situ hybridization with a gfp probe on zebrafish embryos injected with either of the two reporter constructs (see Fig. S2 at http://dev.biologists.org/supplemental).
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Regulation of the tbx6 promoter by Wnt and Bmp
During the development of the posterior mesoderm, the endogenous
tbx6 promoter must be able to integrate inputs from both the Wnt and
Bmp pathways, as shown above. We therefore tested the ability of
ptbx6-1.7-gfp to respond to these signaling pathways by injecting the
promoter with wnt8 or Xbmp4 and compared the response of the
endogenous gene with the same levels of exogenous Wnt and Bmp signaling
inputs. Similar to the response of the endogenous tbx6 gene in the
presence of Wnt8 overexpression, we observed ectopic GFP fluorescence in
embryos coinjected with wnt8 RNA and ptbx6-1.7-gfp
(Fig. 4A-D), showing that the
tbx6 promoter is capable of responding to Wnt8. Ectopic expression of
Xbmp4 RNA also induced widespread expression of the
ptbx6-1.7-gfp as in the case of the endogenous tbx6
gene (Fig. 4E-H). These results
show that the promoter is activated by either Wnt or Bmp signals, similar to
that of the endogenous tbx6 gene.
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Using our dual fluorescence system, we asked whether the activation of ptbx6-2.0-gfp in the posterior mesoderm required these Tcf DNA binding sites. To do this, we mutated the two proximal Tcf sites (Tcf-1 and Tcf-2) (Fig. 3A), generating mut1-2.0-gfp/Xex-bfp containing a mutation in the first Tcf site, mut2-2.0-gfp/Xex-bfp containing a mutation in the second Tcf site, and mut1,2-2.0-gfp/Xex-bfp containing both mutations. We found that either single mutation alone did not dramatically affect the expression of the tbx6 promoter in the developing posterior mesoderm as indicated by the presence of strong GFP fluorescence (Fig. 6A-F, Table 1). In contrast, zebrafish embryos injected with the double Tcf site mutation construct showed almost no detectable GFP fluorescence in the majority of the embryos and the remainder had only occasional BFP-positive mesodermal cell expressing GFP (Fig. 6G-L, Table 1). The double Tcf site mutant promoter was still functional, however, because overexpression of the constitutively active Tcf was able to activate transcription from this promoter, probably through the remaining Tcf site (Table 1). In addition, this promoter was activated when coinjected with Xbmp4, as was the wild-type promoter (Table 1). These results demonstrate that either of the two most proximal Tcf sites are required for normal tbx6 expression, showing that Wnt signaling directly activates the tbx6 gene.
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A 900 bp tbx6 promoter fragment is insufficient for normal expression
Because all three Tcf DNA binding sites are located in the 900 bp proximal
promoter region, we wanted to know if this region of the promoter is
sufficient to correctly target expression to the developing posterior
mesoderm. To answer that, we constructed the reporter gene,
ptbx6-0.9-gfp/pXex-bfp, by placing the sequence of the 900 bp
proximal region of the tbx6 promoter in front of the gfp
gene, together with pXex-bfp. We observed no detectable GFP
fluorescence in any of the embryos injected with
ptbx6-0.9-gfp/pXex-bfp, even though these embryos had robust BFP
expression (Table 1). In
support of this, when the ptbx6-0.9 was fused to luciferase,
we found that it was only 6% as active as ptbx6-2.0
(Table 1).
To determine if the Tcf sites were still functional in this construct, we examined the responsiveness of ptbx6-0.9-gfp to the constitutively active Tcf and to ectopic Wnt8. In both cases we observed ectopic GFP fluorescence in the embryos, showing that ptbx6-0.9 is functional and capable of responding to Tcf activation (Table 1). Thus the 900 bp proximal region of the promoter is capable of responding to Tcf activation, but it is not sufficient to drive the expression of gfp in the developing posterior mesoderm, possibly because it lacks the ability to respond to Bmp signaling.
Identification of a Bmp responsive domain within tbx6 promoter
To determine whether ptbx6-0.9-gfp can respond to Bmp, we
coinjected either ptbx6-1.7-gfp or ptbx6-0.9-gfp with
Xbmp4 RNA into one-cell stage embryos and examined GFP fluorescence.
At 50% epiboly, we observed no GFP fluorescence in embryos coinjected with
ptbx6-0.9-gfp and Xbmp4 RNA
(Table 1). In contrast, we
observed ectopic GFP fluorescence in embryos coinjected with
ptbx6-1.7-gfp and Xbmp4 RNA
(Fig. 4G,H,
Table 1). These results show
that ptbx6-0.9-gfp has a Wnt response element but lacks a promoter
element necessary for the Bmp response.
Two regions were deleted in ptbx6-1.7 to produce ptbx6-0.9, the distal 800 bp region (dr800) and the first intron. To ask whether either of these regions can mediate Bmp responsiveness, we placed them in front of the SV40 minimal promoter driving the luciferase gene, generating dr800-SV40-luc and intron-SV40-luc. These reporter constructs were coinjected with and without Xbmp4 RNA into one-cell stage zebrafish embryos and luciferase activity was assayed at the shield stage. Addition of dr800 to SV40-luc enhanced the promoter activity approximately 3-fold, probably because of the endogenous Bmp present in the embryo (Fig. 7A). Coinjection of Xbmp4 RNA and dr800-SV40-luc resulted in a further 3-fold increase in activity. In contrast, with intron-SV40-luc the promoter activity was the same with or without the addition of Xbmp4 RNA (Fig. 7A). These data demonstrate that the Bmp response element is located in the 800 bp distal region of the tbx6 promoter.
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Discussion |
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Although the normal expression of tbx6 is dependent on Wnt and Bmp signaling, we find that inhibition of either one of these signaling pathways does not lead to a complete loss of tbx6 expression in the ventrolateral mesoderm. Interestingly, wnt8 morphants show a graded reduction of tbx6 expression with the strongest effect in the lateral domains of the ventrolateral mesoderm. In embryos injected with wnt8MOs, bmp expression becomes ventrally restricted even at the shield stage (see Fig. S3A-D at http://dev.biologists.org/supplemental), and tbx6 expression is restricted to this region. In contrast, loss of Bmp function causes a strong uniform reduction in tbx6 expression, but does not significantly alter the expression of wnt8 (see Fig. S3E-H at http://dev.biologists.org/supplemental). These findings show that the Wnt and Bmp signaling pathways have distinct impacts on the overall expression of tbx6 in the posterior mesoderm.
Although the contribution of Bmp and Wnt8 signals has a qualitative and quantitative difference in their ability to activate tbx6 expression, their cooperation is essential to give the normal expression pattern during posterior mesoderm formation. This raises the question, why is combinatorial signaling used to regulate tbx6 and other mesodermal genes? The expression patterns of the bmp genes and wnt8 are very dynamic during early embryogenesis, yet tbx6 expression is maintained in the ventrolateral mesoderm throughout somitogenesis. Combinatorial signaling not only restricts the domain in which tbx6 is expressed to regions where both signals are present, but it permits tbx6 to be expressed in regions where Bmp signaling is very low (the lateral regions) and where Wnt8 signaling is very low (the ventral regions from 90% epiboly onwards). Thus as the levels of the signals vary, tbx6 expression is maintained.
Interestingly, a family of three Drosophila genes,
Dorsocross1-3, encode T-box genes most closely related to
tbx6 (Reim et al.,
2003). Although the expression patterns of these genes is complex,
the expression of all three genes in the dorsal ectoderm and mesoderm requires
both Wnt and Bmp signals (Reim et al.,
2003
). Whether this represents conservation of regulation or
convergent evolution awaits further analysis, but it is an intriguing
parallel.
Analysis of the tbx6 promoter during posterior mesoderm development
Two models can be used to account for the required input of both Wnt and
Bmp signals for the normal tbx6 expression in the posterior mesoderm.
One is that these pathways may signal independently of each other, but
function together in a cooperative fashion to activate the expression of
tbx6. Alternatively, these pathways may function in a linear pathway
such that one signal is upstream of the other as was suggested from
Xenopus studies (Hoppler and
Moon, 1998; Marom et al.,
1999
) and a recent zebrafish study
(Agathon et al., 2003
).
To delineate the interplay between Wnt8 and Bmp signaling pathways in regulating the expression of tbx6 during posterior mesoderm development, we isolated and characterized the promoter of tbx6. We found that a genomic fragment including sequences from 1.7 kb upstream of the start of translation through the second exon recapitulated the normal tbx6 expression pattern in both zebrafish and Xenopus embryos, demonstrating that the regulatory elements are conserved between these species. The normal domain of tbx6 expression was observed when the intron was removed from the promoter; however, we also observed some ectopic expression outside of the mesodermal domain, indicating that the intron contains a repressor of non-mesodermal expression (D.P.S. and D.K., unpublished).
Analysis of the tbx6 promoter revealed independent elements that
respond cooperatively to Wnt and Bmp signals. In the proximal 900 bp, we
discovered three Tcf sites and demonstrated that mutation of both of the two
most proximal sites prevents normal tbx6 expression. Because Tcf is
activated by Wnt signaling, these results demonstrate that tbx6 is a
direct target of Wnt signaling. We located the Bmp response element to a
distinct region of the tbx6 promoter, located between 1.7 kb and 0.9
kb (dr800) from the start of translation. In the presence of high levels of
Bmp signal, this region alone can activate transcription when attached to a
SV40 minimal promoter, but the activity is much weaker than that of the full
1.7 kb promoter. Within this dr800 region, we identified a 65 bp domain, which
is capable of responding to Bmp activation. Smaller fragments did not respond
to Bmp signaling, suggesting that the Bmp response element is composed of more
than one transcription factor binding site. How this element is activated by
Bmp signaling is not yet known. A transcription factor required for the
function of Bmp-type Smads called OAZ was shown to be required for the
activation of a key Bmp target gene in Xenopus
(Hata et al., 2000), but no
OAZ site was found in the tbx6 promoter. Moreover, during
somitogenesis OAZ is expressed anteriorly
(Hata et al., 2000
), whereas
tbx6 is expressed at the most posterior end of the embryo
(Hug et al., 1997
), suggesting
that a novel transcription factor regulates the Bmp response of
tbx6.
Regulation of tbx6 by combinatorial Wnt and Bmp signaling
Because the normal regulation of tbx6 requires input from both the
Wnt and Bmp pathways, we were surprised to find that ectopic expression of
either signal at the level used to produce tails
(Agathon et al., 2003) resulted
in strong activation of the endogenous tbx6 gene and of the
tbx6 promoter. Using inhibitors of Bmp and Wnt8 signaling, we found
that overexpression of each signaling pathway alone is able to activate
tbx6 expression when the other pathway is blocked, demonstrating that
Bmp and Wnt8 have the potential to activate tbx6 without the
contribution of the other signaling pathway. Because the endogenous Bmp and
Wnt8 can not activate tbx6 when the other pathway is inhibited, our
results suggest that Bmp and Wnt signals are present in the embryo at
relatively low levels such that they can combinatorially activate
tbx6 only when both are present. This mechanism ensures that
tbx6 is only expressed in the regions where these two factors
overlap. How this works in regulating the promoter remains to be determined.
At endogenous levels of signaling, it could be that the Wnt and Bmp response
sites are only active transiently when a single signal is present, and only
when both signals are present does a stable transcription complex form. At
higher levels of signaling through one factor, the sites are occupied
continuously and this promotes transcription of the tbx6 gene.
From all of this data we have put together a model to explain how tbx6 is regulated in the normal embryo and in mutants. In the normal embryo, wnt8 is expressed at the margin in the gastrula-stage embryo and bmps are expressed in a ventral to dorsal gradient (Fig. 8A). Together they combine to activate tbx6 at the margin, with both signals working at submaximal levels. In bmp mutants (and noggin-injected embryos), Wnt8 causes a low level of tbx6 expression in the lateral and ventral regions (Fig. 8B). In wnt8 mutants (or in embryos injected with wnt8MOs), Bmps activate tbx6 in the ventral region where they are most abundant (Fig. 8C).
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ACKNOWLEDGMENTS |
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Footnotes |
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