1 Programmes in Cardiovascular Research and Developmental Biology, The Hospital
for Sick Children, Toronto, ON M5G 1X8, Canada
2 Department of Molecular and Medical Genetics, University of Toronto, Toronto,
ON M5S 1A8, Canada
3 Gene Center and Institute of Biochemistry, University of Munich, 81377 Munich,
Germany
4 Laboratory of Genetics of Development and Diseases Branch, National Institute
of Diabetes, Digestive and Kidney Diseases, 10/9N105, National Institutes of
Health, Bethesda, MD 20892, USA
Author for correspondence (e-mail:
bbruneau{at}sickkids.ca)
Accepted 3 October 2002
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SUMMARY |
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Key words: Limb, Mouse, T-box, Tbx5, Wnt, FGF, Mouse
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INTRODUCTION |
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The definition of the fields of LPM that will give rise to limb buds occur
possibly as a result of patterning by Hox gene activity along the AP axis of
the vertebrate embryo (Cohn et al.,
1997; Cohn and Tickle,
1999
; Popperl et al.,
2000
; Rancourt et al.,
1995
), but it not known what signals are established by these
patterning events that lead to limb bud outgrowth. Experiments in chicken
embryos have suggested that fibroblast growth factors (FGFs) or other
molecules secreted from the intermediate mesoderm (IM), which will give rise
to the nephrogenic mesenchyme (NM), may be responsible for initiating limb
outgrowth (reviewed by Martin,
1998
). These signals are thought to initiate expression of
Fgf10, which is required for limb bud outgrowth, in the limb field
mesenchyme (Min et al., 1998
;
Sekine et al., 1999
). FGF10 in
turn activates Fgf8 expression in the ectoderm overlying the limb
field mesoderm (Min et al.,
1998
; Ohuchi et al.,
1997
; Sekine et al.,
1999
). FGF8 and other FGFs secreted from the apical ectodermal
ridge (AER) subsequently promote limb bud outgrowth, in part by maintaining
Fgf10 expression (Crossley et
al., 1996
; Lewandoski et al.,
2000
; Moon and Capecchi,
2000
; Ohuchi et al.,
1997
; Sun et al.,
2002
; Vogel et al.,
1996
; Xu et al.,
1998
).
FGFs are expressed in the IM and LPM along the entire length of the embryo,
so it follows that there must be a responsive intermediate localized at the
level of the limb buds to transduce the signals from the IM. However,
contradictory information in both chick and mouse embryos in which NM
formation is inhibited has cast doubt on the role of the IM in limb induction,
and it has been suggested that there is no requirement for axial signaling to
the LPM in limb bud initiation
(Fernandez-Teran et al., 1997;
Bouchard et al., 2002
).
Recently, Wnt molecules expressed in the early limb field have been implicated
as key regulators of the FGF loop required for limb bud outgrowth
(Kawakami et al., 2001
).
However, mice lacking transcriptional regulators of ß-catenin dependent
Wnt signaling pathway initiate limb bud growth, although this is not
maintained, perhaps because of lack of AER formation
(Galceran et al., 1999
). What
molecule(s) initiate these signaling cascades and thus limb bud outgrowth at
defined locations along the AP axis of the embryos is not known.
The T-box transcription factor encoding genes Tbx5 and
Tbx4 are early markers of the forelimb and hindlimb fields,
respectively (Bruneau et al.,
1999; Gibson-Brown et al.,
1996
; Gibson-Brown et al.,
1998
; Isaac et al.,
1998
; Logan et al.,
1998
; Ohuchi et al.,
1998
; Saito et al.,
2002
), and have been shown, based on misexpression experiments in
chicken embryos, to be involved in regulating limb type (forelimb versus
hindlimb) identity (Logan and Tabin,
1999
; Rodriguez-Esteban et
al., 1999
; Takeuchi et al.,
1999
). Tbx5 is also required in a dose-dependent manner for
patterning or growth of the forelimbs, as demonstrated by limb defects of
varying degrees caused by dominant TBX5 mutations in humans with
Holt-Oram syndrome (OMIM 142900) or mice that lack one copy of Tbx5
(Basson et al., 1997
;
Bruneau et al., 2001
;
Li et al., 1997
). However, the
role of Tbx5 in early limb development has not been defined.
In order to investigate the role of Tbx5 in early limb formation, we have
analyzed limb bud development in wild-type mice and mice that lack both copies
of Tbx5 (Tbx5del/del mice)
(Bruneau et al., 2001). We show
that Tbx5 is required for the earliest signals that initiate limb bud
outgrowth from the LPM, including establishment of FGF and Wnt signaling. We
propose that Tbx5 initiates limb bud outgrowth following patterning of the LPM
by directly activating Fgf10 in the early limb mesenchyme.
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MATERIALS AND METHODS |
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Fgf10 promoter analysis
Fgf10 genomic sequences were obtained from the mouse ENSEMBL
database
(http://mouse.ensembl.org)
and GenBank
(http://www.ncbi.nlm.nih.gov).
Bacterial artificial chromosomes were identified and obtained from the Centre
for Applied Genomics at the Hospital for Sick Children, and a fragment
comprising 6.5 kb upstream of the coding region was subcloned in a luciferase
expression vector, pXP1. Deletion constructs were made by removing a 600 bp
AflII/NheI fragment (deletion II), a 1.2 kb BlpI
fragment (deletion III), or a 1.9 kb BlpI/NheI fragment
(deletion II/III). Site-directed mutagenesis of TBEa1 was performed by overlap
PCR, replacing the sequences GTGTGA by TATAAA, abolishing the putative TBE and
introducing a SspI restriction site for diagnostic purposes.
Co-transfections of Fgf10-luciferase constructs with a Tbx5
expression construct (Bruneau et al.,
2001) and an activated ß-catenin expression construct
(Miyagishi et al., 2000
) were
performed in COS-7 cells as described
(Bruneau et al., 2001
). The
results shown are the mean±s.d. of at least two independent experiments
performed in triplicate.
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RESULTS |
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Hindlimb markers are not induced in the
Tbx5del/del forelimb field
It has been hypothesized that TBX5 establishes forelimb identity in part by
suppressing expression of genes that define hindlimb identity
(Rodriguez-Esteban et al.,
1999; Takeuchi et al.,
1999
). To determine if the loss of Tbx5 resulted in a conversion
from forelimb to hindlimb identity, we examined the expression of
Pitx1 and Tbx4, which encode hindlimb-specific transcription
factors thought to be involved in establishing hindlimb identity
(Lanctot et al., 1999
;
Logan and Tabin, 1999
;
Rodriguez-Esteban et al.,
1999
; Szeto et al.,
1999
; Takeuchi et al.,
1999
). No induction of Tbx4 or Pitx1 was
observed in the presumptive forelimb field of Tbx5del/del
embryos at E9 (prior to limb bud initiation) or at E9.5 (subsequent to limb
bud initiation), suggesting that endogenous TBX5 does not repress
hindlimb-specific genes in order to direct forelimb identity
(Fig. 2G,H and data not
shown).
Tbx5 is upstream of FGF signaling
Analysis of genes involved in the initiation of limb outgrowth confirmed
that Tbx5 is required for early events in limb bud development. Expression of
Fgf10 and Fgf8 was not detectable in the forelimb field of
Tbx5del/del embryos at E9-9.5
(Fig. 3A-E). The expression of
Fgf10 was never initiated in the LPM at E9
(Fig. 3A), but was intact in
the intermediate mesoderm and hindlimb field of
Tbx5del/del embryos
(Fig. 3A-C). The observation
that hindlimb expression of Fgf10 can be detected in E9.5
Tbx5del/del embryos indicates that undetectable
Fgf10 expression in the forelimb field at this stage is not due to
general growth retardation of the embryos, because hindlimb development is
delayed compared with that of the forelimb. Furthermore, no Fgf10
expression is detected in the limb field of E9 Tbx5del/del
embryos, which are not growth-delayed compared with their littermate controls.
The lack of Fgf10 expression in the forelimb field is therefore a
direct consequence of the loss of TBX5 activity in the limb field. Absence of
Fgf8 expression in of Tbx5del/del forelimbs is
likely to be due to the absence of FGF10 signaling
(Min et al., 1998;
Sekine et al., 1999
).
Similarly, although the expression of the early forelimb field marker
Pea3 was intact in the LPM of Tbx5del/del embryos
at E9, its expression in the putative forelimb bud at E9.5 is absent in
Tbx5del/del embryos
(Fig. 2F), presumably because
of the absence of FGF signaling, which is known to regulate Pea3
transcription in other vertebrate tissues
(Raible and Brand, 2001
;
Roehl and Nusslein-Volhard,
2001
). Expression of the FGF-responsive Snai1 gene,
encoding a transcription factor expressed in the early limb bud
(Ciruna and Rossant, 2001
;
Isaac et al., 2000
;
Nieto, 2002
;
Sefton et al., 1998
), was also
undetectable in Tbx5del/del embryo putative forelimbs
(Fig. 3F), while its expression
elsewhere in the embryo was unaffected. In all cases, although growth
retardation was observed for Tbx5del/del embryos at E9.5,
expression of the genes examined was unaffected in tissues other than those
destined to become forelimb, indicating that developmental delay is not the
cause of the altered gene expression.
Tbx5 expression precedes Fgf gene expression in the LPM
It has been suggested that FGFs from the IM/NM, in particular FGF8, signal
to the LPM to initiate limb bud formation, and perhaps to induce Tbx
gene expression (Crossley et al.,
1996; Gibson-Brown et al.,
1998
; Isaac et al.,
2000
; Isaac et al.,
1998
; Logan et al.,
1998
; Martin,
1998
; Ohuchi et al.,
1999
; Ohuchi et al.,
1998
; Rodriguez-Esteban et
al., 1999
; Vogel et al.,
1996
). However, comparisons of Fgf and Tbx5
expression have been limited to chicken embryos. To establish a potential
hierarchy of regulators of limb development in the mouse, we examined the
temporal and spatial relationship of expression of Fgf8, Fgf10 and
Tbx5. We focused on Fgf8, because Tbx5 expression
does not initially rely on FGF10 (Sekine
et al., 1999
).
Tbx5 is expressed initially at E8.0 in a broad region of the LPM
corresponding to the cardiac crescent
(Bruneau et al., 1999). At E8.5
(5 somites), Tbx5 expression is still confined to the developing
heart (Fig. 4A), but shortly
thereafter, at the eight-somite stage, expression of Tbx5 is robustly
detected additionally in a discrete region of the LPM that corresponds to the
forelimb field (Fig. 4B). This
domain of Tbx5 expression is continuous with the cardiac domain of
Tbx5 expression. At this stage and until E8.5 (eight somites),
Fgf8 expression is rarely or not detectable in the IM
(Fig. 4D)
(Crossley and Martin, 1995
).
Fgf8 at the eight-somite stage is very weakly detectable in the IM,
and this expression is reliably observable in very few embryos at this stage
(Fig. 4E). At subsequent stages
of development, Tbx5 remains expressed in the mesenchyme of the limb
primordium, although not immediately adjacent to the IM expressing
Fgf8 (Fig. 4C,F).
Fgf10 begins to be expressed at E9 in the limb mesenchyme,
overlapping with the domain of Tbx5 expression, as well as in the IM
(Fig. 3A). Therefore,
Tbx5 expression in the LPM destined to become forelimb precedes
expression of Fgf8 in the IM and Fgf10 in the LPM.
|
Tbx5 is upstream of Wnt signaling
Wnt signaling has been implicated in the initiation of limb formation.
Wnt2b in the forelimbs and Wnt8c in the hindlimbs of chicken embryos appear to
be sufficient to initiate limb bud outgrowth via a ß-catenin dependent
pathway, including Fgf10 expression
(Kawakami et al., 2001). We
could not detect expression of Wnt2b or Wnt8c in early mouse
limb buds (data not shown), indicating that these Wnts, unlike in chick
embryos, might not be involved in early stages of mouse limb formation.
Although the identity of homologous Wnts in mouse that might be involved in
limb bud formation is not known, transcription factors downstream of
ß-catenin dependent Wnt signaling, LEF1 and TCF1, play a key role in
mouse limb bud outgrowth (Galceran et al.,
1999
). Lef1 and Tcf1 were expressed in the early
limb field of wild-type embryos, but their transcripts were undetectable in
Tbx5del/del forelimbs
(Fig. 5A,C,E). It is not known
whether Wnt signaling is upstream or downstream of Tbx5, and there is
precedent to suggest that Wnts can directly regulate T-box genes via LEF1
(Galceran et al., 2001
;
Yamaguchi et al., 1999
). To
determine the hierarchy of regulation between Tbx5, Wnt signaling and
Fgf10 expression, we examined Tbx5 and Fgf10
expression in Lef1/Tcf1 double mutant embryos
(Galceran et al., 1999
).
Tbx5 expression at E9.25 was unaffected in
Lef1-/-;Tcf1-/- embryos, while
Fgf10 expression was detectable at weaker levels than wild-type in
these mutants (Fig. 5B,D,F). We
conclude that Tbx5 acts upstream of Wnt signaling in the developing
limb bud, and Wnt signaling via LEF1 and TCF1 are required to maintain normal
levels of Fgf10 expression.
|
Establishing hierarchies of signaling in the limb bud
We have shown that in mice lacking Tbx5, expression of Fgf10,
Lef1, Tcf1, Snai1, Pea3 and Fgf8 in the presumptive limb buds is
abolished. We have further established that Tbx5 is upstream of Wnt signaling.
To examine further the relationship between FGF signaling, Tbx5
expression and expression of potential downstream targets of TBX5, we examined
expression of Tbx5 and Lef1 in
Fgfr2IgIII/
IgIII embryos, in which the
entire immunoglobin-like domain III of the Fgf receptor 2 (Fgfr2)
gene has been deleted (Xu et al.,
1998
). In these mice, the different isoforms of Fgfr2 that are
receptors for FGF8 (FGFR2c) or FGF10 (FGFR2b), and are required for FGF
function in limb bud formation (Martin,
1998
; Xu et al.,
1998
), are deleted. Tbx5 expression was normally
initiated at E9.25 (Fig. 5G),
but not maintained by E10.5, in the presumptive limb bud mesenchyme of
Fgfr2
IgIII/
IgIII embryos (data not
shown). This is similar to what is observed in mice that lack Fgf10
(Sekine et al., 1999
), and
presumably reflects a major role for this receptor in transducing FGF10
signals. Expression of Lef1 was also unaffected in
Fgfr2
IgIII/
IgIII embryos at E9.5
(Fig. 5H).
Tbx5 directly activates the Fgf10 gene
The lack of initiation of Fgf10 expression in the LPM of
Tbx5del/del embryos suggests the possibility that TBX5 may
activate the Fgf10 gene directly. We examined the promoter region of
Fgf10 for potential TBX5 binding sites (TBEs)
(Bruneau et al., 2001;
Ghosh et al., 2001
), by
comparing conserved elements of the 7 kb upstream of Fgf10 in human
and mouse genomic sequences; additional confirmation of conserved sites was
carried out by comparing mouse and rat genomic sequences. The 7 kb upstream of
the Fgf10-coding region have been determined to be sufficient for
Fgf10 transcription in limb buds and elsewhere
(Sasaki et al., 2001
). Three
regions of significant homology between mouse and human sequences could be
identified (Fig. 6A). Within
these regions, three potential TBEs were detected in the genomic sequences of
both species [Fig. 6A,B; based
on the sites described by Bruneau et al.
(Bruneau et al., 2001
)];
additional potential TBEs were found outside the regions of homology. Only one
site was conserved in human, mouse and rat DNA (asterisk in
Fig. 6A). We isolated 6.5 kb of
genomic DNA upstream of the Fgf10 gene, and fused this putative
regulatory region to a luciferase reporter gene. Co-transfections in
COS-7 cells of this Fgf10 reporter gene with a Tbx5
expression construct resulted in powerful (up to 120-fold) activation of the
reporter gene (Fig. 6C),
showing that TBX5 can directly activate the Fgf10 gene.
|
The decreased expression of Fgf10 in
Lef1-/-;Tcf1-/- embryos indicates a
role for Wnt signaling in the regulation of Fgf10 expression. A
single consensus LEF1/TCF1 binding site (TTCAAAG) was identified in mouse and
human Fgf10 regulatory sequences, as shown by `L' in
Fig. 6A. We co-transfected the
Fgf10-luciferase reporter construct with an activated ß-catenin
construct into COS-7 cells, with or without the Tbx5 expression construct.
This activated ß-catenin construct will activate LEF1/TCF1-dependent
transcription (Miyagishi et al.,
2000). Activated ß-catenin at all doses tested activated the
Fgf10-luciferase reporter gene, with a maximum activation of
10-fold (Fig. 6D). In
combination with Tbx5, an additive effect of ß-catenin was
observed (Fig. 6D).
To identify the regions of the Fgf10 promoter that were responsive to TBX5 and ß-catenin-dependent transcription factors, we performed deletion analysis of the Fgf10 promoter (Fig. 6E). We deleted a 0.6 kb region corresponding to most of conserved region II (delII), a 1.2 kb region corresponding to most of conserved region III (delIII) or a 1.9 kb deletion that removes all of region II and most of region III (delII/III) (see Fig. 6A). The first deletion removes the conserved TBE (a1), while the second deletion removes two TBEs (a2 and c), as well as the LEF1/TCF1 binding site. Deletion of region III abolished the response of the Fgf10 promoter to activated ß-catenin, which correlates well with the presence of the LEF1-binding site in this region. Activation by TBX5 was not significantly affected by this deletion. However, deletion of region II abolished activation by TBX5 almost completely (1.5- versus 18-fold). Deletion of both regions II and III completely eliminated activation by either activated ß-catenin or TBX5. To further delineate the contribution of TBEa1, the putative Tbx5 binding sequences GTGTGA of TBEa1 were mutated to TATAAA, and the construct assessed for its response to TBX5. Mutation of TBEa1 (muta1 in Fig. 6E) significantly reduced TBX5 activation of the Fgf10 promoter-luciferase construct to 1.6-fold (compared with 18-fold for the wild-type promoter). The combined data indicate that TBX5 activates the Fgf10 promoter directly mainly via the evolutionarily conserved TBEa1, and that ß-catenin-dependent signaling activates Fgf10 via a conserved LEF1/TCF1-binding site.
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DISCUSSION |
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Tbx5 and forelimb identity
The absence of Tbx5 did not result in acquisition of hindlimb
identity by the forelimb field. Misexpression experiments in chicken embryos
have suggested that forced expression of Tbx5 in the hindlimb and
Tbx4 or Pitx1 in the forelimb can induce a conversion of
limb type identity (Logan and Tabin,
1999; Rodriguez-Esteban et
al., 1999
; Takeuchi et al.,
1999
). Tbx5 misexpression in the hindlimb was shown to
result in the repression of Tbx4, suggesting that the endogenous role
of Tbx5 in the forelimb is to repress Tbx4 expression
(Takeuchi et al., 1999
). Our
data showing that lack of Tbx5 does not lead to expression of
Tbx4 or Pitx1 in the forelimb field does not support this
hypothesis. This is consistent with the observations that limb type identity
in chick embryos is determined at stage 9-12, before the induction of
Tbx5 and Tbx4 in their respective limb fields
(Saito et al., 2002
).
Tbx5del/del embryos do not form forelimb buds, so it may
be argued that expression of Pitx1 and Tbx4 may not be
detected in the absence of limb buds, but these genes are expressed in the
hindlimb LPM field far in advance of limb bud outgrowth (our data)
(Gibson-Brown et al., 1996
;
Gibson-Brown et al., 1998
;
Isaac et al., 1998
;
Logan et al., 1998
;
Ohuchi et al., 1998
;
Saito et al., 2002
), and
expression of Tbx5 in the forelimb is intact in mice that lack limb
buds due to ablation of Fgf10
(Sekine et al., 1999
).
Therefore one would anticipate that a molecular conversion of forelimb to
hindlimb identity in Tbx5del/del embryos would indeed be
detected. In addition, examination of Tbx4 expression in
Tbx5del/del embryos at E9, prior to the initiation of limb
bud outgrowth, did not reveal induction of Tbx4 in the limb field.
Furthermore, conditional deletion of Tbx5 using Prx1-Cre mice, which
deletes the Tbx5 gene in the limb at E9.5, does not result in
forelimb to hindlimb conversion either (C. Rallis, B. G. Bruneau, J. Brough,
C. E. Seidman, J. G. Seidman, C. J. Tabin and M. P. Logan, unpublished). T-box
genes may be involved in later aspects of limb identity, but based on our data
and on explant experiments performed in chicken embryos
(Saito et al., 2002
), they do
not seem to be sole regulators of early forelimb versus hindlimb identity.
Tbx5 and the transition from limb field to limb bud
In Tbx5del/del embryos, the establishment of a
patterned limb field is intact, while all signs of limb bud outgrowth are
absent, including all known regulators of limb bud outgrowth. This is
consistent with data showing that limb bud outgrowth at the proper location
along the AP axis of the LPM is established at stages 13-15 in chicken
embryos, the stage at which Tbx5 begins to be expressed in the LPM
destined to become the forelimb (Kieny,
1969; Pinot, 1970
;
Saito et al., 2002
;
Stephens et al., 1989
). It has
been suggested that evolutionarily conserved Hox functions are essential for
the AP patterning of the LPM (Cohn et al.,
1997
; Cohn and Tickle,
1999
; Popperl et al.,
2000
; Rancourt et al.,
1995
). In particular, in the zebrafish lazarus mutant, in
which all Hox function is abolished, the establishment of the limb fields,
including expression of Tbx5 in the precursors of the pectoral fin,
is absent (Popperl et al.,
2000
). In zebrafish, it has been suggested that Tbx5 controls
mesodermal cell migration into the limb field
(Ahn et al., 2002
). Our
observation that the limb field is intact in mouse embryos lacking
Tbx5 shows that this mechanism is not operative in mammals. Therefore
Tbx5 is not essential for establishment of the limb field.
What, then, is the role of Tbx5 in early limb formation? We
propose that Tbx5 is a primary and direct initiator of limb bud
outgrowth following patterning of the limb field. Supporting this possibility
is the observation that a limb-specific deletion of Tbx5 also results
in an absence of limb bud outgrowth (C. Rallis, B. G. Bruneau, J. Brough, C.
E. Seidman, J. G. Seidman, C. J. Tabin and M. P. Logan, unpublished), and
introduction of a dominant-negative TBX5 molecule in the limb field also
abrogates limb formation (C. Rallis, B. G. Bruneau, J. Brough, C. E. Seidman,
J. G. Seidman, C. J. Tabin and M. P. Logan, unpublished) (J. Takeuchi and T.
Ogura, personal communication). Most importantly, Tbx5 misexpression
can cause the induction of an entire limb from the LPM (J. Takeuchi and T.
Ogura, personal communication), or an additional limb-like structure from the
limb-forming mesoderm (Ng et al.,
2002). Furthermore, this role is evolutionarily conserved in all
tetrapods, from zebrafish to humans (Ahn et
al., 2002
; Basson et al.,
1997
; Garrity et al.,
2002
; Li et al.,
1997
; Ng et al.,
2002
). Therefore Tbx5 is necessary and sufficient for
limb bud outgrowth. Tbx5 encodes a transcription factor, and is
likely to cause limb bud induction by direct activation of downstream targets
such as Fgf10. Indeed, Fgf10 expression is never initiated
in Tbx5-deficient embryos, and we have shown that TBX5 activates the
Fgf10 gene directly via a conserved TBX5-binding site within the
Fgf10 promoter, thus providing a direct and simple mechanism for
induction of limb bud outgrowth.
In contemplating the potential role of TBX5 as initiator of limb bud
induction, one must consider that evidence exists to indicate a role for
signaling from the NM to the LPM to initiate limb bud outgrowth
(Geduspan and Solursh, 1992;
Stephens and McNulty, 1981
;
Strecker and Stephens, 1983
).
FGFs derived from the NM, particularly FGF8, have been proposed to be the
operative molecules in this model
(Crossley et al., 1996
;
Martin, 1998
;
Vogel et al., 1996
). Data
exist that dispute this model, and the pieces of the puzzle have not been
reconciled (Fernandez-Teran et al.,
1997
; Martin,
1998
). Indeed, preventing posterior migration of the NM in chicken
embryos (Fernandez-Teran et al.,
1997
) or disrupting differentiation of the NM in mouse (M.
Bouchard and M. Busslinger, personal communication) does not lead to abnormal
limb formation. We propose that if a role in limb bud initiation exists for
axial signaling, it might be to support Tbx5 expression in
conjunction with patterning of the LPM by Hox genes
(Popperl et al., 2000
) or to
confer competence to the LPM for induction of limb bud outgrowth by Tbx5. The
role of FGF10 in maintenance but not initiation of Tbx5 expression in
the limb bud indicates that FGFs do play a role in supporting Tbx5
expression, although they may not be involved in its initiation
(Sekine et al., 1999
).
Supporting this possibility, we have observed that mice lacking FGFR2b/FGFR2c
also have decreased Tbx5 mRNA levels, but initiate Tbx5
expression normally. A role for FGFs in conferring competence to the LPM would
also be consistent with their expression throughout the LPM and IM.
Hierarchies of signaling in limb bud outgrowth
Tbx5 is clearly upstream of FGF and Wnt signaling in the
developing limb bud, as shown by the decreased expression of Fgf10, Fgf8,
Snai1, Pea3, Lef1 and Tcf1 in the prospective limb field of
Tbx5 knockout embryos, and the intact expression of Tbx5 in
Lef1-/-;Tcf1-/- and
Fgfr2IgIII/
IgIII embryos. It has
additionally been demonstrated that Tbx5 expression is normally
initiated in mice that lack FGF10 (Sekine
et al., 1999
). Furthermore, our genetic and transactivation data
indicates that Wnt signals act in concert with TBX5 to activate Fgf10
fully in the developing limb bud. We have shown in vivo that Fgf10
requires Tbx5 for initiation of its expression, whereas Wnt signaling
via LEF1 and TCF1 is required to sustain high levels of Fgf10
expression. This is supported by our in vitro transactivation data, which show
powerful activation of the Fgf10 promoter by TBX5, and lower levels
of activation by ß-catenin. This is similar to the interaction of VegT
and Wnt signaling in Xenopus organizer formation
(Xanthos et al., 2002
).
However, it is not clear whether decreased Fgf10 expression and
abnormal limb development in
Lef1-/-;Tcf1-/- embryos is due to
direct action of Wnts in the mesenchyme, as previously suggested
(Kawakami et al., 2001
), or
whether it reflects an absence of Wnt signaling from the AER
(Kengaku et al., 1998
), which
is defective in the absence of LEF1 and TCF1
(Galceran et al., 1999
).
Alternatively, FGF signals downstream of Tbx5 may be essential for
mesenchymal activation of Wnt pathways, as shown in the primitive streak of
the mouse (Ciruna and Rossant,
2001
).
Together, these results suggest that Tbx5 is upstream of Wnts and Fgf10 to initiate limb bud outgrowth and initiates the FGF feedback loop required for early limb development by activating expression of Fgf10. Although Wnt signaling is not required for initiation of Fgf10, Wnt signals act additively in concert with TBX5 to maintain appropriate levels of Fgf10 expression in the developing limb. The potential hierarchies regulating limb bud outgrowth are summarized in Fig. 7.
|
Conclusions
In summary, we have shown that Tbx5 is required for the initiation
of limb bud outgrowth after the patterning of the limb field from lateral
plate mesoderm. In the developing mouse heart, Tbx5 is maximally
expressed in the posterior segments that give rise to atrium and left
ventricle (Bruneau et al.,
1999). In Tbx5del/del embryos, normal AP
patterning is maintained in these segments of the developing heart, while
growth of these structures is severely impaired
(Bruneau et al., 2001
).
Therefore, AP patterning of the developing heart is independent of
Tbx5, while early differentiation and subsequent embryonic growth of
specific segments of this organ depends on Tbx5. We have shown that
similar growth dependency exists in the developing limb, where establishment
of the limb field is independent of Tbx5, while initiation of
proximodistal limb bud outgrowth requires intact Tbx5 expression.
Tbx5 appears to initiate limb bud formation at least in part by
direct transcriptional activation of the Fgf10 gene.
In Holt-Oram syndrome, which is caused by TBX5 haploinsufficiency
(Basson et al., 1997;
Li et al., 1997
), limb defects
range in severity: more common manifestations are defects of the thumbs or
carpal bones, but occasionally phocomelia (severe reduction or absence of
zeugopod and stylopod) is observed (Basson
et al., 1994
; Newbury-Ecob et
al., 1996
). In these severe cases, it is likely that limb
outgrowth signals such as FGF10 and FGF8 are affected because of decreased
TBX5 levels. In fact, mice with an AER-specific deletion of
Fgf8 develop limb abnormalities reminiscent of those found in severe
cases of Holt-Oram syndrome (Lewandoski et
al., 2000
; Moon and Capecchi,
2000
). Similar pathways may also be operative in other disorders
caused by T-box gene mutations, such as Ulnar-mammary syndrome (OMIM 181450),
which is caused by dominant mutations in TBX3
(Bamshad et al., 1997
), or
22q11 deletion syndrome (OMIM), which is caused by TBX1
haploinsufficiency (Jerome and
Papaioannou, 2001
; Lindsay et
al., 2001
; Merscher et al.,
2001
). Our results place Tbx5 as the earliest determinant
of limb bud outgrowth, establish a firm molecular basis for the implied
parallels between limb and heart development, and suggest a common pathway for
the differentiation and growth of embryonic structures downstream of T-box
transcription factors.
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ACKNOWLEDGMENTS |
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Footnotes |
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