Graduate School of Biological Sciences, Nara Institute of Science and
Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan
* Present address: Cardiovascular Research, The Hospital for Sick Children, 555
University Avenue, Toronto, ON M5G 1X8, Canada
Author for correspondence (e-mail:
ogura{at}bs.aist-nara.ac.jp).
Accepted 24 February 2003
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SUMMARY |
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Key words: Tbx5, Tbx4, Limb initiation, Wnt, Fgf, Chick
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INTRODUCTION |
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Recent studies have shown that members of the fibroblast growth factor
(Fgf) family play key roles in the limb initiation
(Martin, 1998;
Martin, 2001
). When applied
locally in the lateral plate mesoderm, several Fgfs induce an ectopic limb
(Cohn et al., 1995
;
Ohuchi et al., 1995
;
Crossley et al., 1996
;
Vogel et al., 1996
;
Ohuchi et al., 1997
). In a
current model, signaling of two different Fgfs, Fgf8 and Fgf10, is a key for
the limb outgrowth (Martin,
2001
). Although data highlight the pivotal roles of Fgfs, limb bud
formation is initiated in Fgf10 knockout mice, suggesting that
another factor(s) may induce limb initiation
(Sekine et al., 1999
;
Min et al., 1998
).
Interestingly, limb buds of the Fgf10-null mice exhibit robust
expression of Tbx5 and Tbx4 in the limb fields, although the
limb buds cease to grow and remain flat
(Sekine et al., 1999
;
Min et al., 1998
). This
observation indicates that Tbx5 and Tbx4 may be constituents
of the a priori genetic program that acts upstream of the Fgf signaling
cascade.
Recently, tight crosstalk between Fgf and Wnt proteins, another family of
secreted factors, has been shown to control limb initiation in the chick
embryo (Kawakami et al.,
2001). Wnt2b and Wnt8c (which are expressed in
the chick forelimb and hindlimb, respectively) are capable of inducing ectopic
limbs in the flank. This additional limb formation is mediated through a
ßcatenin-dependent process and subsequent Fgf10 induction,
placing Wnt proteins upstream of Fgf signaling. However, the intracellular
events that control expression and signaling of these extracellular factors
have not been elucidated. To date, Hox9 genes are known to be expressed in the
lateral plate mesoderm and to demarcate the normal limb fields and the
Fgf-induced additional limbs. This indicates that the Hox code, along the
anteroposterior axis of the embryo, is one of the key determinants for the
limb bud fields, implying that Hox genes act upstream of these signaling
molecules (Cohn et al., 1997
).
Nonetheless, there is a gap between the Hox genes and the Wnt/Fgf network, in
which putative transcription factor(s) act as initiator(s) of limb bud
development. To explore this, we performed a series of experiments using both
loss-of-function and gain-of-function approaches. Our data highlight the
pivotal roles played by Tbx5 and Tbx4 during initiation of
limb bud outgrowth.
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MATERIALS AND METHODS |
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Plasmids construction
The coding regions of chick Tbx5 and Tbx4 were amplified
by the PCR, and ligated into a pSLAX 12 Nco vectors (pSLAX-Tbx5 and
Tbx4, respectively) (Mogan and
Fekete, 1996). To construct the dominant-negative forms, a
repressor domain of the Drosophila Engrailed gene
(Jaynes and O'Farrell, 1991
)
was ligated into the pSLAX plasmid (pSLAX-En). Entire sequences of
chick Tbx5 and Tbx4 were amplified by the PCR with primers,
then ligated to pSLAX-En to create fusion genes
(pSLAX-EnTbx5 and pSLAX-EnTbx4). In both cases, En
gene was placed at the N termini. These fusion cDNAs were isolated and
subcloned into a RCASBP retroviral vector and a modified pCAGGS vector
(Niwa et al., 1991
;
Koshiba-Takeuchi et al.,
2000
).
Retrovirus preparation and injection
RCASBP-EnTbx5 and EnTbx4 plasmids were transfected into
chick embryonic fibroblast cells isolated from SPF chick embryo (line c/o)
trunks, then cultured for 1 week to allow the virus to spread. Cells were
replaced with low-serum media, and incubated further for virion production.
Then, media were collected and spun to concentrate virus particles as
previously described (Morgan and Fekete,
1996).
The concentrated virus solution was mixed with an indicator dye (Fast Green), and injected into the prospective wing and leg fields.
In ovo electroporation
In ovo electroporation was carried out as previously described
(Takeuchi et al., 1999;
Momose et al., 1999
). We
modified our in ovo electroporation techniques to obtain efficient expression
of the transgenes in the limb buds. Briefly, a CUY-21 electroporator (Gene
System, Osaka, Japan) was used. Two platinum electrodes (Gene System, Osaka,
Japan) were used. An anode was inserted beneath the embryonic endoderm and a
cathode was placed on the ectoderm surface. Then, a DNA solution was injected
by a sharp glass pipette into the embryonic tissues. Electric pulses were
applied (7-9 V, 60 mseconds pulse-on, 50 mseconds pulse-off, three to five
times) during injection of the DNA solution.
Whole-mount in situ hybridization and probe isolation
In situ hybridization was performed as previously described
(Wilkinson, 1993). Probes for
chick Wnt8c, Fgf10 and Fgf8 were kindly provided by Drs Jane
Dodd, Sumihare Noji and Juan Carlos Izpisua Belmonte
(Hume and Dodd, 1993
;
Vogel et al., 1996
;
Ohuchi et al., 1997
). Chick
Pea3 was amplified by RT-PCR utilizing the published sequence, then
subcloned into the pKRX vector (Schutte et
al., 1997
). The DNA fragment encoding the Env gene was
isolated from the RCASBP retrovirus vector
(Mogan and Fekete, 1996
), and
then subcloned into pBluescript SKII(+) vector. Chick Wnt2b was also
amplified by RT-PCR, then subcloned into the pKRX vector.
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RESULTS |
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When the EnTbx5 retrovirus was infected into the right prospective wing field at stages 7 to 10, a completely wingless phenotype arose at E12 (Fig. 1A). As expected, wing formation was completely disturbed at the shoulder level (Fig. 1A'): the right scapula is missing, leaving hypomorphic ribs underneath (red arrowheads in Fig. 1A'). In some cases, severe distal truncation was observed at E9 (Fig. 1B). Embryo skeletal preparations showed truncated wings (W') with hypoplastic scapular (s') and clavicular bones (c') (Fig. 1B').
|
When the EnTbx4 viral infection was performed at later stages (stage 11 to 13), the legless phenotype was not obtained, instead leg structure truncation occurred (red arrowheads in Fig. 1D). In this case, the right leg was small and severely distorted, as observed by Alcian Blue staining (red arrowhead in Fig. 1D'): a short and thin femur (f' in Fig. 1D') and hypomorphic distal structures (red arrowheads in Fig. 1D'). In addition, the right ilium and ischium were small and deformed (il' and is' in Fig. 1D', respectively), and the pubis was missing from the pelvis right side (Fig. 1D'). These results indicate that Tbx5 and Tbx4 are directly involved in limb formation processes.
To examine the specificity of EnTbx5 and EnTbx4, we misexpressed these genes in leg and wing buds, respectively. Misexpression of EnTbx4 in the wing bud and EnTbx5 in the leg did not induce any morphological abnormality at E8 (Fig. 1E,F, respectively). Whole-mount in situ hybridization for the Env gene encoded in the vector revealed expression of transgenes in limbs (Fig. 1E,F). Although signals were observed only on the limb surface, expression of transgenes and the resultant normal morphology suggest that EnTbx5 and EnTbx4 specifically abrogate development of the wing and leg buds, respectively.
Stage-dependent action of the dominant-negative Tbx5 and
Tbx4
In a series of misexpression studies, the resultant phenotypes are
dependent on viral infection timing. To explore further, we injected
dominant-negative Tbx viruses at various developmental stages that included
stages 7 to 13 (Table 1).
|
As observed in Fig. 1E,F, misexpression of EnTbx5 and EnTbx4 in the leg and wing, respectively, did not induce the limbless or the limb truncation phenotype (Table 1). This again supports the specific actions of EnTbx5 and EnTbx4 in the developing limb buds.
Misexpression of the dominant-negative Tbx5 and
Tbx4 represses the Wnt/Fgf signaling
Our data strongly suggest that Tbx5 and Tbx4 directly
control limb initiation processes. To confirm this further, the expression of
several genes in the EnTbx5- and EnTbx4-misexpressed limb
buds was examined. In this case, the dominant-negative forms of Tbx5
and Tbx4 were misexpressed by in ovo electroporation, as the
electroporation enables us to express two plasmids simultaneously.
Consequently, we monitored the domains of transgene expression by
coelectroporation of an EGFP (enhanced green fluorescent
protein) expression plasmid (pCAGGS-EGFP)
(Takeuchi et al., 1999;
Momose et al., 1999
).
When the dominant-negative EnTbx5 was misexpressed in the right
prospective wing field, Wnt2b was downregulated in a region where GFP
signals were evident (Fig.
2A,A').
Normal expression of this gene was observed in the lateral plate mesoderm of
the left wing field (Kawakami et al.,
2001). By contrast, when electroporation was performed in the
right prospective leg field, Wnt8c expression was normal in a
GFP-positive area (Fig.
2B,B')
(Hume and Dodd, 1993
),
implying that EnTbx5 misexpression specifically affects
Wnt2b in the wing field, but not Wnt8c in the leg. When
Fgf8 expression was analyzed, this gene was repressed in the right
limb buds (red arrowheads in Fig.
2C). Normal expression of Fgf8 was evident in the AER on
the contralateral side (Fig.
2C). As the EnTbx5 fusion gene was inserted in the RCAS
retrovirus vector, in situ hybridization utilizing an Env probe
visualizes the transgene expression domain. Nineteen hours after
electroporation, robust Env expression was detected at the
prospective wing field (arrowheads in Fig.
2D), indicating successful EnTbx5 misexpression in this
area. As shown in serial sections, Fgf10 expression was downregulated
in the same region of the embryo (arrowheads in
Fig. 2E). Next, chick
Pea3 expression was examined, as Pea3 encodes an Ets-type
transcription factor that acts downstream of Fgf signaling
(Roehl and Nusslein-Volhard,
2001
; Raible and Brand,
2001
). As expected, Pea3 expression was repressed (red
arrow in Fig. 2F), whereas
normal expression was visible in the entire prospective left wing region.
These observations are in accordance with the hypothesis that Tbx5 is
involved in early processes before the onset of the Wnt and Fgf protein
interaction.
|
Dominant-negative Lef1 does not affect Tbx5 and
Tbx4 expression
As previously reported, Wnt signaling regulates Fgf10 expression
in both the presumptive wing and leg buds, placing Wnts signaling upstream of
Fgf10 (Kawakami et al.,
2001). Although our data suggest that both Tbx5 and Tbx4 control
expression of Wnt2b/Wnt8c, Fgf8 and Fgf10, misexpression
data obtained from the EnTbx5 and EnTbx4 constructs do not
clarify whether Tbx genes lie upstream of Wnt protein signaling, or vice
versa. To elucidate this, an expression construct (CAGGS-dnLef1) for
a dominant-negative form of Lef1
(Kengaku et al., 1998
), the
direct nuclear target of Wnt signaling, was constructed. In this experiment, a
pCAGGS vector that has been shown to drive rapid and robust expression of
transgenes in tissues was used (Niwa et
al., 1991
; Koshiba-Takeuchi et
al., 2000
). This construct was electroporated into both the
prospective wing and leg fields at stages 9 to 11, and Tbx5 and
Tbx4 expression was then examined. Twenty-four hours after
electroporation, both Tbx5 and Tbx4 were normally expressed
in the prospective wing and leg fields, respectively
(Fig. 2M). At this stage,
Fgf10 expression was evident in both the wing and leg fields,
although weak repression was observed (Fig.
2M). Repression of Fgf10, Tbx5 and Tbx4 became
evident 30 hours after electroporation
(Fig. 2N). Thirty-six hours
after electroporation, repression of Tbx5 in the wing and
Tbx4 in the leg became obvious
(Fig. 2O). As the Wnt signaling
controls Fgf10 and Fgf8 expression, we speculate that the
dnLef1 repressed Fgf10 and Fgf8, thereby indirectly
downregulating Tbx5 and Tbx4 expression. Taken together, our
data again suggest that Tbx5 and Tbx4 lie upstream of both
the Wnt and Fgf signaling in limb induction. In these experiments,
pCAGGS-EGFP was co-electroporated to visualize the domain of
misexpression and GFP signals in the electroporated areas (data not
shown).
Tbx5 misexpression in the flank induces an additional wing-like
limb
If Tbx5 and Tbx4 are involved in the early processes of
limb initiation, forced misexpression of these genes in the flank would be
expected to induce the formation of additional limb buds, as observed in
implantation of Fgf- or Wnt-expressing cells. As Tbx5 and
Tbx4 specify the wing and leg identity of limb buds, respectively,
Tbx5 and Tbx4 misexpression should induce an additional wing and leg,
respectively. Chick wing is covered by feathers, whereas leg has scaled digits
with claws and feathers in the proximal part. Based upon these morphological
differences, we can identify which type of limb is formed by misexpression.
Implantation of Wnt-expressing cells, for example, Wnt2b in the restricted
flank region, induces a mosaic additional limb (Fig.
3A,A')
with wing-like morphology on the anterior side (W') and leg-like
structure on the posterior (L') (Fig.
3A'). This additional limb formation is accompanied by the
expression of Tbx5 (red arrow) and Tbx4 (blue arrow) on the
anterior and posterior sides, respectively
(Fig. 3B).
|
For the misexpression experiments, Tbx5-EGFP and
Tbx4-EGFP fusion genes were inserted into the pCAGGS expression
vector (CAGGS-Tbx5-EGFP and CAGGS-Tbx4-EGFP). As previously
reported, Tbx5-EGFP and Tbx4-EGFP fusion genes exhibit the
same biological functions as the unmodified Tbx5 and Tbx4,
respectively. In addition, the pCAGGS vector drives rapid transgene
expression, making this system ideal for analyzing early developmental stages
(Niwa et al., 1991;
Koshiba-Takeuchi et al.,
2000
). When the CAGGS-Tbx5-EGFP expression construct was
electroporated into the flank, a wing-like limb was formed (red arrowhead in
Fig. 3E). Although this
additional limb is smaller than the normal wing, its digits were extensively
covered by feather buds. When this embryo was stained with Alcian Blue,
skeletal patterns of this additional wing were similar to the normal wing
(data not shown).
When Wnt2b expression was examined, induction of this gene was clearly observed in the flank, where the GFP was evident (Fig. 3F,G). Fgf10 gene was induced in the small additional limb bud (red arrowhead in Fig. 3H). Sixty hours after electroporation, an additional limb was clearly observed at the posterior side of the normal wing (red arrowhead in Fig. 3I). In this additional limb, distinct expression of Fgf8 was detected in the AER (red arrowhead in Fig. 3I).
To confirm the wing identity of this additional limb, we checked expression of Hox9 genes. In the additional limb, expression of leg-specific Hoxb9 was observed only in the posterior margin (Fig. 3J). Expression of another leg-specific Hoxc9 became very faint (Fig. 3K). By contrast, wing-specific Hoxd9 was expressed (Fig. 3L). These results indicate that the Tbx5-induced additional limb predominantly possesses the wing identity, with the wing-like expression of Hox9 genes, albeit partially.
Tbx4 misexpression in the flank induces an additional leg-like
limb
When the expression plasmid CAGGS-Tbx4-EGFP was electroporated
into the flank at stage 10, an additional limb was induced at E10 (red
arrowheads in Fig. 3M).
Contrary to the Tbx5 misexpression, this limb showed leg-like morphology in
its distal half: separated digits were covered by scales, and claw formation
on the digit tips and suppression of feather formation were evident in the
distal part (Fig. 3M). Unlike
the Wnt2b-induced additional limb (Fig.
3A,A'),
this additional limb has a leg-like appearance on both the anterior and
posterior sides, despite its proximal region showing wing-like morphology.
When the expression of several markers was examined, Wnt8c induction
was evident (Fig. 3N) in an
area where EGFP was observed (Fig.
3O). Fgf10 and Fgf8 were induced in the
electroporated parts (Fig.
3P,Q,
respectively). Collectively, these data indicate that Tbx5 and
Tbx4 act at the early stages of limb initiation, controlling the
downstream Wnt and Fgf signaling cascades.
When expression of Hox9 genes was examined, leg-specific Hoxb9 and Hoxc9 genes were induced in this additional limb (Fig. 3R,S, respectively). By contrast, expression of wing-specific Hoxd9 was suppressed, especially in the posterior half. (Fig. 3T). These results suggest that the Tbx4-induced additional limb mainly possesses the leg identity, with partial wing-like appearance.
Stage-dependent action of Tbx5 and Tbx4
As observed in Table 1, the
actions of dominant-negative Tbx genes and the resultant phenotypes are
dependent on the developmental stages, where stages 7 to 10 are highly
susceptible phases. To complement these loss-of-function approaches,
stage-dependent actions of Tbx5 and Tbx4 in gain-of-function
experiments were examined. For this purpose, we electroporated
pCAGGS-Tbx5-EGFP and pCAGGS-Tbx4-EGFP plasmids at
various stages (summarized in Table
2). Interestingly, in both cases, induction of additional limb
formation was obtained only when these expression plasmids were electroporated
between stages 8 and 12. From stage 13 onwards, complete induction of
additional limb formation was never observed, although we observed robust
fluorescent signal derived co-electroporated pCAGGS-EGFP (data not
shown). This is in clear contrast to the Fgf-induced additional limb buds in
the flank, which can be induced when Fgf is applied during stages 13 to 15
(Crossley et al., 1996;
Vogel et al., 1996
). Local
application of Fgf at earlier or later stages never induces additional limb
buds, setting up a brief sensitive window. This also suggests that competence
to the Fgf signaling is strictly controlled in a stage-dependent way. Contrary
to this, our misexpression experiments have shown that the formation of
additional limb buds is observed only when Tbx genes are misexpressed in the
flank between stages 8 and 12, before the mesoderm cells become competent to
respond to Fgf signaling. Thus, the Tbx-sensitive period begins and ends
before the stages at which Wnt-expressing cells can induce additional limb
buds. These lines of evidence again indicate that Tbx5 and
Tbx4 act upstream of Wnt/Fgf signaling
(Fig. 4).
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DISCUSSION |
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When the dominant-negative forms of Tbx5 and Tbx4 were
misexpressed in the prospective limb fields, complete limbless phenotypes
arose with disruption of shoulder and pelvis formation
(Fig. 1). Scapula was not
formed, and ribs were hypomorphic. In the leg region, ilium and ischium were
hypoplastic without pubis formation, implying that scapula is part of the
limb. These results indicate that suppression of functions of Tbx genes affect
not only the limb development, but also the formation of shoulder and pelvis.
Similar morphological alterations were observed in the Fgf10-knockout
mouse (Sekine et al., 1999;
Min et al., 1998
). In such
mutant limbless mouse, posterior scapula was missing and iliac bone was
rudimentary. As miexpression of the dominant-negative Tbx genes represses the
expression of Fgf10 and Fgf8, morphological changes observed
in chick and mouse are alike. Nonetheless, induced alterations were severer in
chick embryos, probably because the dominant-negative Tbx genes affect both
the Wnt and Fgf cascades.
To construct the dominant-negative forms of Tbx genes, we used the
Engrailed suppressor domain. It has been reported that the repressor
activity of this domain is mediated through the interaction with Groucho
(Jimenez et al., 1997;
Tolkunova et al., 1998
).
Although vertebrate Groucho-related genes are widely expressed in
developing embryos (Fisher and Caudy,
1998
; Chen and Courey,
2000
), their activities in the limb are unclear. Nonetheless, when
analyzed in primary cultures of chick limb mesenchyme cells, both
EnTbx5 and EnTbx4 abrogate efficiently the transcriptional
activation of the Anf and Fgf10 promoters by Tbx5 and Tbx4,
respectively (data not shown). This suggests that the Engrailed acts as an
efficient suppressor in the chick limb buds. Recently, it has been reported
that functional knockdown of zebrafish tbx5 resulted in a failure of
fin bud initiation and the complete loss of pectoral fins
(Ahn et al., 2002
), indicating
that the antisense oligonucleotide-mediated knockdown and misexpression of
EnTbx5 resulted in similar morphological alterations. Hence,
exploiting different techniques in different species is important to
understand the common mechanism of limb initiation.
Although the extracellular events have been analyzed extensively,
intracellular mechanisms that regulate gene expression and limb initiation
have remained unsolved. To date, Hox9 genes are known to be expressed in the
lateral plate mesoderm. Expression of these genes demarcates the fields of
both the normal limb and the additional limbs induced in the flank. This
indicates that Hox genes act upstream of these signaling molecules
(Cohn et al., 1997). However, a
gap exists between the Hox genes and the Wnt/Fgf network. Our data strongly
indicate that Tbx5 and Tbx4 operate in this gap. We also
have shown that Hox9 genes are controlled by Tbx5 and Tbx4
during wing/leg specification (Takeuchi et
al., 1999
). These lines of evidence suggest that a regulatory loop
between Hox9 and Tbx genes is essential for both limb initiation and the
wing/leg identity specification, highlighting the feedback and feed-forward
mechanisms in both extracellular and intracellular signaling cascades.
As shown in Fig. 3, misexpression of Tbx5 and Tbx4 in the flank induces wing-like and leg-like limbs, respectively. Nonetheless, these additional limbs did not show the complete wing or leg appearance. Rather, Tbx5- and Tbx4-induced limbs seem to be mosaic with one type predominating over another. This is consistent with the mixed expression patterns of Hox9 genes (Fig. 3). To obtain rapid and robust expression of Tbx genes, we used pCAGGS expression vector. As pCAGGS induces transient expression of transgene, expression of Tbx genes in the flank might have faded out after triggering expression of Wnt/Fgf genes. This would suggest that induced Wnt/Fgf proteins might initiate limb formation even in mesenchyme cells that were not electroporated. In such case, additional limb buds can be composed of mixed cell populations; electroporated Tbx-positive cells and non-electroporated Tbx-negative cells thereby have a mosaic appearance.
Tbx genes regulate pattern formation in both vertebrate and invertebrate
embryos. From a combination of embryological and genetic approaches, a picture
is emerging that Tbx genes belong to a highly conserved genetic network
involving inductive signals (Papaioannou
and Silver, 1998; Smith,
1999
; Ruvinsky and
Gibson-Brown, 2000
; Tada and
Smith, 2001
). In Xenopus, another T-box transcription
factor, Brachyury (Xbra), directly regulates embryonic fibroblast growth
factor (eFgf) and creates a tight feedback loop. Hence, eFgf
expression maintains Xbra expression, and Xbra maintains
eFgf expression in vivo. Conversely, eFgf inhibition
represses Xbra expression and Xbra inhibition represses
eFgf expression (Isaacs et al.,
1994
). In addition, Xbra has been found to regulate the
Xenopus Wnt11 gene directly, making another connection to Wnt
signaling (Tada and Smith,
2000
). In Drosophila, functions of T-box transcription
factor optomotor blind (omb) are closely related to Dpp
(Decapentaplegic) and Wg (Wingless) signaling cascades.
(Pflugfelder et al., 1992
;
Maves and Schubiger, 1998
).
These observations strongly suggest that Tbx genes are central in the highly
conserved signaling cascades of these inductive signals in both vertebrates
and invertebrates.
Although our data indicate that Tbx5 and Tbx4 control Wnt
and Fgf genes, how these signals are transduced to the nucleus to control
expression of target genes remains to be elucidated. In this sense, we do not
exclude the possibility that Tbx proteins act cooperatively with the Wnt and
Fgf signaling systems, i.e. in parallel or cooperatively with these cascades.
This is compatible with the data published previously
(Ng et al., 2002). Because
another T-box protein, Tbr1, interacts with CASK, a member of the
membrane-associated guanylate kinases (MAGUKs)
(Hsueh et al., 2000
), Tbx
proteins may be targets of extracellular signaling that could change their
transcriptional properties depending on the signaling context. This could be
related to the stage-dependent action of Tbx genes during limb initiation. As
described above, induction of additional limb formation was obtained only when
Tbx genes were electroporated between stages 8 and 12. This is in clear
contrast to the Fgf-induced additional limb buds in the flank, which can be
induced when Fgf is applied during stages 13 to 15
(Crossley et al., 1996
;
Vogel et al., 1996
;
Ohuchi et al., 1997
;
Kawakami et al., 2001
). This
also strongly suggests that competence to the Tbx misexpression and to the Fgf
signaling is strictly controlled in a stage-dependent way. Although we do not
know how these differences in the competence are controlled in vivo, the
signaling context could modulate Tbx proteins and regulate this critical
factor during limb development.
Recently, Anf was shown to be a direct target of Tbx5
(Hiroi et al., 2001;
Bruneau et al., 2001
). Tbx5
alone activates the Anf promoter efficiently. However, in the
presence of another transcription factor, Nkx2.5, Tbx5 is a stronger
activator. In the early stages of limb initiation, Tbx5 alone could induce Wnt
genes, whereas in the later stages, activated Wnt signaling might modulate
Tbx5 activity or induce another transcription factor to act
synergistically, forming a positive feedback loop and upregulating
Fgf10 to maintain limb outgrowth. To examine this hypothesis, precise
biochemical analyses needs to be performed.
Our data reveal that Tbx5 and Tbx4 specifically regulate
Wnt2b and Wnt8c, respectively, to initiate limb outgrowth in
the early stages of development. In the later stages, Tbx5 and
Tbx4 exert different actions to form distinct forelimb and hindlimb
structures, respectively. These indicate that these genes play distinct roles
with distinct specificity. Nonetheless, Tbx5 and Tbx4 are
derived from the same ancestral gene
(Agulnik et al., 1996;
Ruvinsky and Silver, 1997
).
During evolution, these genes have diversified their biological functions to
regulate different Wnt genes and make different limb structures. This is
related to our observation that EnTbx5 and EnTbx4 failed to
repress Wnt8c in the leg and Wnt2b in the wing. As expected,
misexpression of EnTbx5 in the leg and EnTbx4 in the wing
did not affect limb development (Fig.
1E,F).
This suggests that Tbx5 and Tbx4 have acquired different target specificities
during evolution. Although we are still far from a complete understanding of
these processes, our data shed further light on vertebrate limb evolution of
vertebrate limbs, the functions and evolution of the Tbx gene family.
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ACKNOWLEDGMENTS |
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