1 The Salk Institute for Biological Studies, Gene Expression Laboratory, 10010
North Torrey Pines Road, La Jolla, CA 92037-1099. USA
2 Instituto Gulbenkian de Ciencia, Rua Da Quinta Grande n 6, 2780-901 Oeiras,
Portugal
3 Cardiovascular Research Center, Massachusetts General Hospital, 149
13th Street, Charlestown, MA 02129, USA
Author for correspondence (e-mail:
belmonte{at}salk.edu)
Accepted 21 August 2002
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SUMMARY |
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Key words: T-box, Tbx5, Wnt2b, Fgf10, Pectoral fin, Limb, Zebrafish, Chick, heartstrings
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INTRODUCTION |
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Despite the orchestrated interactions needed between outgrowth and identity
during development of tissues and organs, these phenomena, for ease of
analysis, are often treated as distinct processes. The decision to become
either a forelimb or a hindlimb is also made at the earliest stages of limb
initiation. Several studies suggest the intriguing possibility that limb
identity could be determined by the specific expression of a single
transcription factor. Two members of the T-box family of transcription
factors, Tbx4 and Tbx5
(Gibson-Brown et al., 1996;
Gibson-Brown et al., 1998
;
Isaac et al., 1998
;
Logan et al., 1998
;
Ohuchi et al., 1998
;
Tamura et al., 1999
;
Begemann and Ingham, 2000
)
(reviewed by Ohuchi and Noji,
1999
), and a member of the OTX-related subclass of paired-type
homeodomain proteins, Pitx1
(Lanctot et al., 1997
;
Logan et al., 1998
), show a
restricted forelimb and hindlimb distribution in the LPM prior to limb
budding. Tbx5 transcripts are expressed in the presumptive forelimb
area in all tetrapods studied so far, and conversely, Tbx4 and
Pitx1 transcripts are restricted to the presumptive hindlimb region.
Furthermore, gain- and loss-of-function experiments in chick and mice lend
support to the idea of limb identity being determined by a discrete set of
molecular determinants (Logan and Tabin,
1999
; Rodriguez-Esteban et
al., 1999a
; Szeto et al.,
1999
; Takeuchi et al.,
1999
).
The co-localization of factors shown to be capable of inducing limb outgrowth (WNT2b and WNT8c) and factors involved in limb identity (Tbx5 and Tbx4), raises the question of whether both processes are linked or take place independently of one another. In this study, we combine gain- and loss-of-function approaches in both zebrafish and chick embryos to analyze the molecular relationships occurring among Fgf10, Wnt2b and Tbx5 during initiation and specification of forelimb identity. Our results demonstrate that, in addition to its role governing forelimb identity, Tbx5 is both necessary and sufficient for forelimb initiation. We also demonstrate that Tbx5 is directly upstream of Fgf10 in the signaling cascade that directs limb outgrowth. In turn, a feedback loop is uncovered in which FGF signaling is required to maintain Tbx5 expression. This study also extends our previous results in chick, showing that wnt2b is necessary for pectoral fin initiation in zebrafish. Finally, our results indicate that two key events of limb development, namely limb identity determination and limb initiation are not independent processes, and that Tbx5 and Wnt2b function together to initiate and specify forelimb identity. Altogether, our results unveil the existence of a complex network of molecular interactions that establishes, propagates and maintains the expression of signaling molecules and transcription factors responsible for limb outgrowth and identity.
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MATERIALS AND METHODS |
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Morpholino injections
Morpholino oligonucleotides were designed by and obtained from GeneTools
LLC (Eugene, Oregon). The zebrafish tbx5 morpholino lies from
nucleotide position -5 to +18:
The standard control oligonucleotide available from Gene Tools was used. The morpholinos were solubilized in 1x Danieau's solution and injected into one-cell stage zebrafish embryos at a range of 2-10 ng/embryo.
heartstringsm21 (hst) mutant lines
The hst mutant was identified in an ENU-induced mutagenesis screen
for perturbation of cardiac function in zebrafish. The mutation has been
mapped to the tbx5 gene and introduces a nonsense mutation at codon
316 (Garrity et al.,
2002).
RNA injections
The open reading frame, excluding the 5' and 3' untranslated
sequences, of zebrafish fgf10, wnt2b and tbx5 were cloned
into the pCS2 vector. Capped RNAs were synthesized from these constructs using
the mMessage mMachine kit (Ambion). Seventy picogram of fgf10 mRNA
and 100 pg of tbx5 and wnt2b mRNA was injected into one-cell
stage zebrafish embryos.
Whole-mount in situ hybridization and Alcian Blue cartilage
staining
Injected zebrafish embryos were scored for pectoral fin phenotypes at 30
hours post-fertilization (hpf) to 5 days post-fertilization (dpf) using a
stereomicroscope. Further analysis was conducted at 24-48 hpf by whole-mount
in situ hybridization, as described previously
(Hammerschmidt et al., 1996),
and at 5 dpf by Alcian blue staining as described previously
(Schilling et al., 1996
).
Zebrafish riboprobes used were fgf8
(Furthauer et al., 1997
),
tbx5 (Tamura et al.,
1999
) and shh (Ekker
et al., 1995
). The zebrafish fgf10 riboprobe spans the
entire ORF, and the wnt2b riboprobe contains 900 bp of the coding
sequence corresponding to the N-terminal region. Viral injected and bead
implanted chick embryos were examined by whole-mount in situ hybridization and
Alcian Blue cartilage staining as described by Vogel et al.
(Vogel et al., 1996
). For the
zebrafish studies, a minimum of 100 embryos was examined for each in situ
hybridization.
Viral production and injections into chick embryos
Adenovirus expressing the mouse Axin gene was produced and
injected as previously described (Kawakami
et al., 2001). The full-length mouse Tbx5 cDNA and a
truncated form of chick Tbx5 (amino acids 62-521), lacking the
N-terminal region upstream of the T-box, were cloned into an RCAS
retroviral vector to produce RCAS-Tbx5 and RCAS-Tbx5
N
constructs, respectively. Subsequent transfection into chick embryonic
fibroblasts and retroviral production were performed as described previously
(Vogel et al., 1996
).
RCAS-Tbx5 was injected into stage 5-8 chick embryos in the LPM.
RCAS-Tbx5
N was injected into stage 8-10 chick in the LPM.
Staging of chick embryos was according to Hamburger and Hamilton
(Hamburger and Hamilton,
1951
). An RCAS-alkaline phosphatase virus was injected as
a control and no phenotypic changes in gene expression or limb morphology were
observed.
Bead implantation
Beads soaked with the FGF receptor tyrosine kinase inhibitor SU5402
(Calbiochem), used at 1 mg/ml in DMSO, were implanted into stage-14 to -18
chick embryos as described previously
(Rodriguez Esteban et al.,
1999b). Control beads were implanted at the same stage and no
change in gene expression was observed.
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RESULTS |
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|
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The results of the tbx5 loss-of-function experiments suggest that,
in addition to its role in controlling limb identity, tbx5 plays a
role in limb initiation and outgrowth. The range of phenotypes obtained also
indicates that the function of tbx5 is dosage dependent. These
results are in agreement with the recent Ahn et al. study which also showed
that tbx5 is required for pectoral fin formation
(Ahn et al., 2002).
The FGF family of signaling molecules is known to be required and
sufficient for limb initiation (reviewed by
Martin, 1998;
Martin, 2001
). Given the known
role of T-box proteins as transcriptional regulators, we reasoned that
tbx5 might function by regulating the expression of fgf10.
To start addressing this, we screened several zebrafish cDNA libraries using
chick fgf10 as a probe and cloned zebrafish fgf10
(Fig. 2A). As has been
previously reported in mouse and chick embryos
(Ohuchi et al., 1997
), at 24
hpf, prior to initial budding of the pectoral fins, zebrafish fgf10
can be detected in the LPM of the presumptive pectoral fin field, in a pattern
temporally and spatially similar to tbx5 (compare
Fig. 2B,C). In later stages,
zebrafish fgf10 is observed in the mesenchyme of the pectoral fin
buds, overlapping tbx5 expression (compare
Fig. 2D,F with 2E,G).
Expression is also observed in the branchial arches, otic vesicle, heart
primordium and tail bud (data not shown), as in the corresponding structures
of other vertebrates (Ohuchi et al.,
1997
).
|
Given the aforementioned tbx5 loss-of-function phenotypes, we decided to examine the expression of mesodermal [fgf10 and sonic hedgehog (shh)] and ectodermal (fgf8) markers involved in the early stages of limb development. As shown in Fig. 3A,B, injection of tbx5 morpholino, but not control morpholino, resulted in complete loss of fgf10 expression in the pectoral fin bud forming region of the LPM at 26 hpf, while expression in other regions remained unaffected. We could also not detect shh (data not shown) or fgf8 (a marker for the apical fold; compare Fig. 3C with 3D).
|
To complement the morpholino studies, we made use of the novel zebrafish
heartstrings (hst) mutant. The hst mutation has
been very recently mapped to a point mutation in the open reading frame of
zebrafish tbx5, and generates an early terminated protein
(Garrity et al., 2002). As
with tbx5 morphant embryos, homozygous hst embryos do not
develop pectoral fins. We examined the expression of fgf10 in
hst mutant embryos, at the early stages of fin development, and
observed a complete loss of expression
(Fig. 3E). We have also
examined whether the Fgf10 gene is a direct target of Tbx5 in vitro.
A putative Brachyury/Tbx5 binding site is located approximately 400 base pairs
upstream of the start site that is conserved in both human and mouse
Fgf10 genes. Deletion of this site results in complete loss of
activation of the fgf10 promoter by Tbx5, indicating that Tbx5 has
the capacity to directly regulate fgf10 expression (data not shown).
Taken together, our data indicate that tbx5 is required for fin
initiation and suggest that it functions upstream of the FGF pathway that
directs the early stages of fin outgrowth.
wnt2b is necessary for limb initiation and regulates tbx5
We recently reported that the WNT signaling pathway is required for normal
limb development in chick embryos, regulating very early stages of limb
induction (Kawakami et al.,
2001). We demonstrated that Wnt2b, which is expressed in
the LPM of the forelimb field, regulates limb initiation through induction of
Fgf10. Given the lack of Fgf10 expression in the
Tbx5 loss-of-function experiments, we hypothesized that Tbx5
might interact with the WNT signaling pathway. To address this issue, we
cloned the zebrafish wnt2b homologue and performed loss-of-function
experiments.
We screened several zebrafish genomic and cDNA libraries using chick
Wnt2b as a probe and obtained a putative Wnt2b clone with a
full-length open reading frame. Sequence comparison demonstrated that our
positive clones represented zebrafish wnt2b
(Fig. 4A). As reported in chick
embryos (Jasoni et al., 1999;
Kawakami et al., 2001
), at 22
hpf, zebrafish wnt2b is expressed in the developing eye, and a
localized mesodermal region medial to the LPM at the somite level where the
pectoral fin buds will form (Fig.
4B-D). In later stage embryos, when the fin bud started to form,
we could no longer detect wnt2b (data not shown). Thus, the
expression pattern of zebrafish wnt2b appears to be conserved between
zebrafish and chick embryos.
|
wnt2b loss-of-function experiments were performed using a wnt2b morpholino to downregulate endogenous wnt2b (10 ng per injection). This loss-of-function study resulted in a large portion (75%, n=230; Table 1) of embryos that lacked pectoral fins (Fig. 5B), indicating that wnt2b plays an important role during pectoral fin development. Cartilage staining confirmed that these embryos lacked pectoral fin structures and the shoulder girdle (Fig. 5E). By reducing the amount of wnt2b morpholino injected (to a minimum of 2 ng), we were able to obtain a broad range of fin phenotypes very similar to those described for the tbx5 morphants (data not shown). In 5 dpf wnt2b morphants, structures other than the pectoral fins developed similarly to wild type embryos (Fig. 5A,B,D,E). Thus, we exclude the possibility that the phenotypes observed were caused by general developmental arrest. These results indicate that the function of wnt2b is necessary for normal fin initiation, and is affected by gene dosage. Embryos injected with a control morpholino did not display any obvious abnormal phenotype (Fig. 5A,D).
|
To confirm the specificity of the morpholino antisense experiments, we attempted to rescue the wnt2b loss-of-function phenotypes by co-injecting wnt2b morpholino with wnt2b RNA. As shown in Table 1, co-injection of the wnt2b RNA completely rescued the fin phenotypes in more than half of the wnt2b morphants (n=60).
Subsequent to morpholino injection, we analyzed the expression of early
markers of limb development, as in the tbx5 morphants. We could not
detect fgf10, fgf8 or shh expression in the fin field of
embryos injected with the wnt2b morpholino (compare
Fig. 5L with 5M; and data not
shown). These results are consistent with the observed phenotypes and our
previous studies in chick embryos
(Kawakami et al., 2001).
The loss of fgf expression in both tbx5 and
wnt2b morphant experiments suggested that tbx5 and
wnt2b function in a common pathway with respect to limb development,
and that this pathway lies upstream of the FGF signaling network that
regulates limb initiation. The notion of a common pathway is further supported
by the observation that tbx5 expression was significantly
downregulated in the wnt2b morphants (compare
Fig. 5G,I with 5H,J,K). We
reasoned that a key function of wnt2b in the limb initiation process
might be to regulate tbx5 expression. If this were the case, then
forced expression of tbx5 in the wnt2b morphants could
rescue the fin phenotypes. To address this, we co-injected embryos with the
wnt2b morpholino and tbx5 RNA. As shown in
Fig. 5C,F and
Table 1, co-injection of the
tbx5 RNA could rescue the wnt2b morphant phenotypes, such
that 18% fewer embryos had a nofin phenotype (57% as compared with 75% of
embryos injected with morpholino alone). Conversely, we could not rescue the
tbx5 morphant phenotypes with wnt2b RNA (data not shown). We
note that LPM expression of wnt2b at the time of fin initiation was
normal in the tbx5 morphants (Fig.
5N). These results advocate a fin initiation pathway that is
co-regulated by wnt2b and tbx5, and suggest that
tbx5 lies downstream of wnt2b in this process. We have also
observed that a consensus binding site for Lef1, a transcription factor that
interacts with ß-catenin (Roose and
Clevers, 1999), is conserved in both human and mouse Tbx5
genomic sequences, approximately 7.3 kb upstream of the ATG. Deletion of this
site results in a nearly 50% decrease in the activation of this promoter by
Wnt2b, indicating that WNT signaling through the canonical
ß-catenin pathway has the capacity to activate the Tbx5 promoter
(data not shown).
Tbx5 is necessary and sufficient for limb induction
The above results indicate that tbx5 and wnt2b function
together to regulate limb initiation and outgrowth. We have previously shown
that the WNT2b/ß-catenin pathway is required and sufficient for limb
initiation, and that downregulation of this pathway is able to inhibit limb
formation (Kawakami et al.,
2001). To further examine the relationship between the WNT
signaling pathway and Tbx5, as well as the evolutionary conservation
of this process, we made use of chick embryos, which permit misexpression
experiments in a temporally and spatially restricted manner. An adenovirus
expressing Axin was injected into the LPM of stage 8 chick embryos.
Axin is a potent, well-characterized negative regulator of WNT/ß-catenin
pathway (Peifer and Polakis,
2000
). An adenovirus expressing EGFP was co-injected to
assess the spatial distribution of the adenovirus as well as tissue integrity.
As shown in Fig. 6A, B, the
regions of injected embryos expressing Axin displayed a significant
downregulation of Tbx5 (53%, n=60). Injection of control
adenovirus expressing EGFP alone did not result in any obvious
phenotypic alterations (data not shown). This result indicates that an active
WNT/ß-catenin pathway is required for normal expression of Tbx5
in the LPM, and that this regulation is conserved between chick and fish.
|
To further investigate the conservation of Tbx5 function, we
generated a retrovirus expressing a truncated form of Tbx5 that maintains the
DNA-binding T-box domain but lacks the amino terminus. Injection of this
construct into the presumptive wing field of stage 8-10 embryos led to a
significant truncation of the wings (87%, n=70). Embryos injected
with a control retrovirus did not display any obvious phenotype. Examination
of the morphology of the truncated wings after cartilage staining indicated
limb elements were truncated at later stages of development. The most common
phenotypes were hypoplasia or disappearance of zeugopodal elements, typically
the radius, and the absence of some anterior digits (compare
Fig. 6D with 6E,F). Importantly, the extent of the limb truncations observed was comparable to the
zebrafish fin truncations obtained with low levels of the tbx5
morpholino. We also observed that injection of the truncated Tbx5
construct led to a downregulation of Fgf10 expression in the treated
embryos (Fig. 6C) (80%,
n=50). Given these results, we postulate that since T-box
transcription factors interact with other members of the transcription
machinery to activate target genes (Bruneau
et al., 2001; Hiroi et al.,
2001
), removal of the potential interaction domain, but not the
DNA-binding domain, may have resulted in a dominant negative form of Tbx5.
Taken together, these results suggest that, as in zebrafish, downregulation of
Tbx5 function in chick embryos inhibits limb outgrowth and that
Tbx5 functions upstream of Fgf10.
The combined results from our zebrafish and chick experiments indicate that normal Tbx5 function is necessary for proper limb initiation. To determine if Tbx5 is sufficient for this process, we injected a retrovirus expressing Tbx5 in the LPM of chick embryos at stage 5-8. While embryos injected with a control virus did not display any obvious abnormal phenotype (data not shown), embryos injected with the Tbx5 retrovirus had additional limb bud-like structures (40%, n=89). These embryos were left to develop further, and the morphological features of the ectopic limb-like structures were examined after cartilage staining. As shown in Fig. 6G,H, Tbx5 overexpression induced additional cartilaginous elements. Stylopodal elements of the ectopic limb-like structure appear to be shared with the endogenous leg, whereas we obtained a range of extra zeugopodal and autopodal elements. The identity of the ectopic structures is difficult to determine because they appear as hybrid structures. However, our findings clearly demonstrate the inductive capacity of Tbx5 during limb outgrowth. We also observed that during the process of ectopic induction, Tbx5, Fgf10, and Wnt2b are induced (data not shown), suggesting a de novo deployment of the limb initiation program.
Fgf10 maintains Tbx5 expression during limb
initiation
The WNT and FGF signaling pathways interact and regulate each other to
transfer inductive signals between tissues involved in limb initiation
(Kawakami et al., 2001). Also,
FGFs are capable of activating the expression of Tbx5, as
demonstrated by the ability of FGF applied to the flank LPM to induce
Tbx5 expression (Isaac et al.,
2000
). To further our understanding of the regulatory network
governing limb initiation, and the role Tbx5 plays in that process,
we blocked FGF signaling using a potent inhibitor of the FGF receptor tyrosine
kinases (SU5402) (Mohammadi et al.,
1997
), and monitored the expression of Tbx5. Beads soaked
in this inhibitor were applied to the LPM of stage 15-18 chick embryos. As
shown in Fig. 7A, a significant
downregulation of Tbx5 was observed in a broad area of tissue
surrounding the bead (88%, n=43). Beads soaked in DMSO did not result
in any alterations (data not shown). This indicates that FGF signaling is
required to maintain expression of Tbx5 during limb outgrowth.
|
In agreement with the above results, the pattern of tbx5 expression in zebrafish hst mutants is also altered at 26 hpf, when fgf10 is normally expressed in the presumptive fin mesenchyme. In wild-type embryos, tbx5 is expressed in the presumptive fin field of the LPM by 26 hpf, when fin outgrowth initiates (Fig. 7B), and continues in the mesenchyme of the developing fin. In hst mutant embryos, however, tbx5 expression in the LPM is significantly decreased at 26 hpf (Fig. 7C) and is not detected by 48 hpf. Downregulation of tbx5 was also seen in tbx5 morphants (data not shown). Therefore, we examined the ability of fgf10 to maintain tbx5 expression, given the early loss of tbx5 expression in hst mutants. As shown in Fig. 7D, injection of 70 pg of zebrafish fgf10 mRNA into one-cell stage hst embryos resulted in a maintenance of tbx5 expression in embryos examined at 36 hpf (100%, n=213). However, we were unable to rescue fin outgrowth (data not shown), suggesting that functional Tbx5 is required for continuous outgrowth of the pectoral fins. These data further support our hypothesis that fgf10 is involved in maintaining tbx5 expression.
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DISCUSSION |
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In contrast to limb initiation, the process of limb identification appears
to be regulated, at least in part, by the T-box family of transcription
factors. Tbx5 plays a role in determining the identity of forelimbs,
while Tbx4 determines the identity of hindlimbs
(Rodriguez-Esteban et al.,
1999a; Takeuchi et al.,
1999
). Prior to this study, two key lines of evidence suggested
that Tbx5 might also be involved in the limb initiation process.
First, the expression patterns of Fgf10 and Tbx5 partially
overlap in the LPM at the time of limb initiation and before any morphological
limb structures are visible. Second, Tbx5 is quickly induced in
ectopic limb induction experiments (Isaac
et al., 2000
). When beads soaked in FGF protein are implanted to
induce an ectopic limb, Tbx5 expression is detected in the LPM within
1 hour. This induction occurs earlier than that of other Fgf genes
that are essential to establish limb buds
(Isaac et al., 2000
). These
observations indicate that limb type specification and Tbx gene
expression are very early events and also suggest that Tbx5 may play
a role in limb initiation. Here, through loss-of-function experiments in
zebrafish, we demonstrate that tbx5 is necessary for fgf10
expression in the LPM. In agreement with its ability to activate
Fgf10 expression, we show that Tbx5 is also sufficient to
induce limb-like structures. Two key results also highlight a reciprocal
regulation of tbx5 by FGFs. First, inhibition of FGF receptor
tyrosine kinase activity in the chick embryo resulted in downregulation of
tbx5 expression in the LPM at the initiation of limb budding. Second,
injection of fgf10 mRNA into the zebrafish hst mutant
resulted in a maintenance of tbx5 expression beyond what is normally
observed in those embryos. It is worth noting that fgf10, although
able to maintain the expression of tbx5, could not rescue the loss of
pectoral fins in the hst mutant. This result further supports the
notion that tbx5 activity is necessary not only for limb initiation,
but also to maintain outgrowth. Our molecular and genetic analyses with the
tbx5 morphants and the hst mutant extend the recent report
by Ahn et al. (Ahn et al.,
2002
). The authors demonstrated that tbx5 is required for
pectoral fin formation and the cell movement in the LPM that contributes to
pectoral fin budding.
Analysis of Tbx5 expression in Fgf10-/- mice
also suggests that Fgf10 regulates Tbx5 expression. In these
mice, Tbx5 expression is initially detected in the presumptive
forelimb mesoderm, but later its expression is clearly downregulated
(Sekine et al., 1999). Thus,
Fgf10 is not required for induction of Tbx5 expression in
the LPM, but does appear to play a role in the maintenance of its expression.
Such a regulatory interaction between T-box genes and Fgfs also takes
place in the Xenopus blastula. Here, Brachyury, the founding member
of the T-box family, not only activates eFgf expression, but also
forms a regulatory loop with eFGF, in which eFGF maintains Brachyury
expression in isolated gastrula (Isaacs et
al., 1994
; Casey et al.,
1998
).
We have previously shown that Wnt2b functions upstream of
Fgf10 in the LPM. The results presented here are consistent with the
notion that wnt2b also functions upstream of tbx5. First and
foremost, tbx5 mRNA can rescue the fin outgrowth phenotype of
wnt2b loss-of-function morphants. In contrast, wnt2b mRNA
cannot rescue the related phenotype of the tbx5 loss-of-function
morphants. Second, although tbx5 expression is downregulated in the
wnt2b morphants, wnt2b expression is unaffected in the
tbx5 morphants. Third, a requirement for WNT/ß-catenin signaling
for Tbx5 expression was demonstrated in chick embryos. Specifically,
Axin, an inhibitor of WNT/ß-catenin signaling, blocks Tbx5
expression in the LPM. Not surprisingly, we identified a highly conserved Lef1
binding site in the Tbx5 promoter, a known element required for
ß-catenin-dependent transcription
(Roose and Clevers, 1999)
(data not shown).
Our data suggest that the roles of Wnt2b, Tbx5 and Fgf10
are conserved in chick and zebrafish during limb initiation and identity. Yet,
recent findings in mouse suggest the existence of a more complex WNT signaling
network mediating limb initiation. We have generated mice bearing
loss-of-function mutations in the Wnt2b gene and have observed no
alteration in limb patterning or outgrowth. Further, we could not detect
Wnt2b expression in the LPM of mouse embryos at a time when limb
initiation begins (A. R. and J. C. I. B., unpublished data). Additionally,
results from others show that
Tcf1-/-/Lef1-/- mice lack a reduction
in Tbx5 expression (B. Bruneau, Hospital for Sick Children, Toronto,
personal communication). However, it is possible that other Tcf family members
could compensate for loss of the targeted genes. It should be noted that some
other mouse Wnt genes are not expressed in comparable structures to
those in chick and zebrafish. For example, wnt3a, a gene that is
expressed in the apical ectoderma ridge (AER) during the process of limb
budding in zebrafish (Y. K. and J. C. I. B., unpublished data) and chick
(Kengaku et al., 1998), and
whose function is necessary for limb outgrowth in those organisms, is not
expressed in the AER of mice (Parr et al.,
1993
). Thus, it is apparent that although the programs of limb
initiation and identification are conserved in tetrapods, the molecular action
of specific Wnts has diverged. As such, a different, uncharacterized
WNT molecule might be expressed in the LPM, comparable to the expression
pattern of Wnt2b in chick and zebrafish. Efforts are currently
underway to identify and characterize this signaling molecule in mouse.
The results presented here lead us to propose that WNT/ß-catenin
signaling controls Tbx5 expression in the LPM. Tbx5, in turn,
regulates the expression of Fgf10, leading to limb initiation. Last,
Fgf10 appears to play a role in maintaining Tbx5 expression,
indicating the existence of a feedback loop between these two factors. We
cannot exclude the possibility that Tbx5 also regulates WNT signaling
via a feedback loop. The interaction of Tbx5 and Fgf10 is
well conserved in vertebrate limb development. In mice, loss of Tbx5
function results in both loss of Fgf10 expression and loss of
forelimbs (B. Bruneau and M. Logan, National Institute for Medical Research,
London, personal communications). While the forelimbs and hindlimbs of all
tetrapods share many of the signaling pathways required for their outgrowth
and patterning, so far no single transcription factor has been positioned in a
molecular cascade that is specifically required for limb outgrowth. Our
observations that Tbx5, currently regarded as a limb identity
determination gene (Rodriguez-Esteban et
al., 1999a; Takeuchi et al.,
1999
), is involved in the limb initiation process, provide
significant insight into the tight linkage observed between limb initiation
and limb identity. This may help us to further understand the orchestrated
interactions needed during embryogenesis for the outgrowth and identity of
other tissues and organs where T-box genes and the WNT and FGF signaling act
in concert.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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