Division of Developmental Biology, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK
* Author for correspondence (e-mail: mlogan{at}nimr.mrc.ac.uk)
Accepted 14 February 2005
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SUMMARY |
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Key words: Limb development, Limb position, Tbx3, T-box, Gli3, dHand (Hand2), Chick
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Introduction |
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Tbx3 belongs to the Tbx2/3/4/5 subfamily of T-box genes that
originated from a single ancestral gene through gene tandem duplication and
cluster dispersion (Agulnik et al.,
1996; Minguillon and Logan,
2003
; Ruvinsky et al.,
2000
; Wilson and Conlon,
2002
). Tbx3 is expressed in the limb-forming territories
prior to overt limb bud outgrowth. At later stages (st.24 chick, 11.5 dpc in
the mouse), Tbx3 is expressed in two stripes in the anterior and
posterior limb mesenchyme (Gibson-Brown et
al., 1998
; Logan et al.,
1998
; Tumpel et al.,
2002
). Tbx3 is required for normal limb development as
mutations in human TBX3 are associated with Ulnar-Mammary Syndrome
(UMS, OMIM #181450), a dominant disorder characterized by upper(fore) limb
deficiencies (Bamshad et al.,
1997
). Posterior structures of the limb, e.g. the ulna and fifth
digit, are predominantly affected. Tbx3 deletion studies in the mouse
produce phenotypes consistent with the abnormalities observed in UMS
(Davenport et al., 2003
).
Experiments in the chick have shown that the posterior domain of
Tbx3 expression in the limb is positively regulated by Shh
signalling, while the anterior expression domain is repressed by Shh,
suggesting a potential role of Tbx3 in the anteroposterior patterning
of the vertebrate limb (Tumpel et al.,
2002). Furthermore, recent misexpression experiments have
suggested that Tbx3 can alter the identity of posterior digits in the
developing chick hindlimb (Suzuki et al.,
2004
).
Misexpression and gene deletion studies have implicated Tbx3 in limb patterning during limb bud stages. However, Tbx3 is expressed in the limb-forming region prior to overt limb bud outgrowth. To examine a potential early role of Tbx3 in normal limb development, we have misexpressed transcriptional repressor and activator forms of Tbx3 in the developing forelimb region using the avian retroviral system. We provide evidence for a new role for Tbx3 in the genetic network that positions the limb along the rostrocaudal axis of the vertebrate embryo.
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Materials and methods |
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Retrovirus production and embryo infection
Production of retroviral supernatants were carried out as described
previously (Logan and Tabin,
1998). Two full-length Tbx3 viruses were produced; one
includes amino acid residues 1-732 the other amino acid 15-732 of the
predicted protein (AF033669). Both forms produced identical results.
Tbx3EN contains amino acids 15-289 of Tbx3, which spans
the N terminus and DNA-binding T-domain, fused to the engrailed repressor
domain (Jaynes and O'Farrell,
1991
). Tbx3VP16 contains the same residues
fused to two VP16 activation domains
(Ohashi et al., 1994
). The
Gli3ZnF-Vp16 construct contains amino acids 471-636 of the human GLI3
(XP_004833) fused to two VP16 activation domains. The prospective forelimb
territory on the right side of the embryo were infected between stages 8 and
10, as previously described (Logan and
Tabin, 1998
). The left limb served as a contralateral control.
Each virus produced a limb shift phenotype in
30% of infected embryos.
For embryos analyzed before a limb shift phenotype was morphologically
obvious, batches of infected embryos were analyzed. For embryos analyzed at
later stages, embryos with a phenotype were selected.
Whole-mount in situ hybridization
Whole mount in situ hybridizations were carried out essentially as
described (Riddle et al.,
1993). Probes used were Shh
(Riddle et al., 1993
),
Fgf8 (Vogel et al.,
1996
), MyoD, Pax3
(Pourquie et al., 1996
),
Hoxb8, Hoxb9, Hoxc5, Hoxd12 (Burke
et al., 1995
), Wnt26
(Kawakami et al., 2001
),
Tbx5, Tbx2, Tbx3 (Logan et al.,
1998
), dHand (also known as Hand2)
(Fernandez-Teran et al.,
2000
), Gli3
(Schweitzer et al., 2000
) and
Bmp2 (Schlange et al.,
2002
). The Tbx15 probe was produced from a cDNA isolated
from a limb cDNA library (M.P.O.L., unpublished).
Whole-mount immunohistochemistry
Whole-mount immunohistochemistry was performed as previously described
(Kardon, 1998). Axons were
stained using the 3A10 monoclonal antibody (hybridoma supernatant diluted
1/100 from DSHB, Iowa, USA) and detected with a peroxidase-conjugated
anti-mouse secondary (Jackson ImmunoResearch) diluted 1/250.
Cell lines, transfections and luciferase assays
Luciferase assays were performed using COS1 cells. Transfections were
performed using Superfect transfection reagent (Qiagen) following the
manufacturer's protocol. For expression studies, full-length, Engrailed and
VP16 fusion forms were cloned into pcDNA3.1() (Invitrogen). The
reporter pGL3-promoter vector (Promega) containing a single Brachyury
binding site (Kispert et al.,
1995) together with a basal SV40 promoter upstream of the
Firefly Luciferase gene. Luciferase assays were carried out using the
appropriate Reporter Assay System (Promega) according to the manufacturer's
protocol. Normalization of the results was carried out using
ß-Galactosidase Reporter Assay (Promega) according to the manufacturer's
protocol. As an internal control the reporter plasmid was co-transfected with
the ß-galactosidase reporter only. All experiments were performed in
triplicate. Error bars represent the standard deviation over three
experiments.
DiI injection
DiI crystals (Sigma) were diluted in 100% ethanol (5 mg/ml). A 10% working
solution was prepared in 30% sucrose/PBS solution. Misexpression of retrovirus
was performed at HH stage 8-10. Twenty-four hours after retrovirus infection
(stage 14) DiI solution was injected into the embryos at several levels in the
limb-forming region of the LPM and in the adjacent somites, to serve as axial
reference. Equivalent DiI injections were performed in the injected and
control side of the embryo.
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Results |
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Misexpression of Tbx3 can alter limb position
We have investigated the role of Tbx3 in normal limb development
using the chicken retroviral system. Following our targeting strategy
(Materials and methods), we could detect virus broadly in the limb-forming
region from stage 14 onwards (data not shown). Strikingly, misexpression of
full-length Tbx3 shifted the axial position of the injected limb
rostrally (embryos that show rostral limb shift phenotype: n=142;
phenotype frequency 25-30%). Identical results were produced with
Tbx3EN (embryos with phenotype; n=82, phenotype
frequency 25-30%), further suggesting that Tbx3 normally functions as a
transcriptional repressor. Comparison with MyoD, which is expressed
in the dermomyotomal compartment of each somite and serves as an axial
reference, and Shh, which marks the zone of polarizing activity (ZPA)
in the posterior limb, demonstrates the rostral shift in limb position
following Tbx3 misexpression (limb on right), relative to
contralateral control limbs (on left in all cases shown)
(Fig. 2A, n=6/6,
100%). The shift in axial position can extend over the distance of one to
three somites; however, the limb itself is otherwise morphologically normal.
Pax3 is expressed in the dermomyotome of the developing somites and
the migrating myoblasts (Williams and
Ordahl, 1994). Following misexpression of Tbx3 and
mislocation of the limb to a more rostral position along the embryo axis,
myoblasts that migrate into the shifted limb are derived from somites at a
more rostral level. Myoblasts at more caudal levels that normally migrate into
the limb (Fig. 2B, black
arrow), no longer contribute to the limb musculature and remain within the
dermomyotome (Fig. 2B, red
arrow) (n=7/7, 100%). Both delamination and migration of the
myoblasts into the limb depend on the Met receptor and its ligand HGF, also
called scatter factor, produced by non-somitic mesoderm
(Dietrich et al., 1999
).
Following shift of the limb from its normal position, the source of HGF is
presumably also shifted, leading to migration of myoblasts from somites at the
incorrect axial level.
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The limb shifted by Tbx3 misexpression is patterned normally
In a previous study, misexpression of Tbx3 and Tbx2 in
the developing hindlimb, results in changes in anteroposterior patterning of
digits, suggesting these genes have a role in specifying digit identity
(Suzuki et al., 2004). To
determine if patterning of the shifted limb is altered following our
misexpression strategy, we analyzed the expression of genes normally
regionally restricted within the limb. Fgf8 is expressed in the AER
of the injected wing in a pattern indistinguishable from the control limb
(Fig. 2D; n=5/5,
100%). Bmp2, which is expressed in the AER and in the posterior of
the limb as a response to Shh signalling
(Francis et al., 1994
), is
expressed in an identical pattern in injected and uninjected limbs
(Fig. 2E; n=5/5, 100%)
in contrast to other reports (Suzuki et
al., 2004
). This is consistent with the normal distribution of
Shh in Tbx3-injected limbs
(Fig. 2A). Hoxc5,
which is normally expressed in proximal regions of the limb mesenchyme
(Burke et al., 1995
), is
unaffected in the injected limb (Fig.
2F; n=6/6, 100%). Expression of other T-box genes is also
unaffected within the shifted limb despite the rostral mislocation:
Tbx5 is expressed throughout the limb mesenchyme
(Fig. 2G; n=7/7,
100%); Tbx15 is expressed in medial regions of the limb
(Fig. 2H; n=6/6,
100%); and Tbx2 is expressed in anterior and posterior stripes, in a
similar pattern to Tbx3 (Fig.
2I; n=6/6, 100% compare with
Fig. 1H).
We also analyzed skeletal preparations of embryos with a shift phenotype that were allowed to develop to later stages (stage 27). In these examples, the vertebral column of the embryo is normal. However, the limb skeletal elements, including the scapula are mislocated rostrally, although there is no alteration in their morphology (Fig. 2J, n=10/10, 100%). Moreover, the morphology of the digits in the shifted wing (Fig. 2K) is normal compared with those in the contralateral control limb (Fig. 2L). No alterations in digit identity were observed. The same results were obtained following misexpression of Tbx3 in the developing hindlimbs (Fig. 2M; n=10/10, 100%). The axial position of the injected limb is shifted rostrally along the rostrocaudal axis, but hindlimb digit morphology (Fig. 2N) is indistinguishable from that in the contralateral control leg (Fig. 2O). In Tbx3-injected embryos with a shifted wing (n=8) or leg (n=7) analyzed at stage 27, Sox9 expression, which marks the skeletal progenitors, is normal in both forelimbs and hindlimbs, confirming the results obtained from skeletal preparations (data not shown).
Axial Hox gene expression is unaffected following Tbx3 misexpression
To understand the mechanism underlying the rostral shift in limb position,
we investigated the effects of Tbx3 misexpression at stages prior to
morphological limb shifts (stage 13-16). Following our retroviral targeting
strategy, by stage 13, transcripts for Tbx3 are detected at normal
levels on the injected side (right) (Fig.
3A; 15/15, 100%). By stage 16, transcripts are present throughout
the limb-forming territory on the injected side, while on the uninjected side
they are restricted to the posterior (Fig.
3B; 2/12, 17%). Although there is no direct evidence that axial
Hox gene expression controls the position of the limb primordia, the axial
position at which the limbs develop correlates with the expression of several
Hox genes in the LPM (Burke,
2000; Burke et al.,
1995
; Cohn et al.,
1995
; Cohn et al.,
1997
; Rancourt et al.,
1995
). Axial Hox gene expression is not altered in the
Tbx3-injected forelimb area. The rostral expression boundary of
Hoxb8 is not changed (Fig.
3C; n=30/30, 100%). In addition, the expression boundary
of Hoxb9, which is caudal to the region of the LPM that will give
rise to the posterior forelimb mesenchyme
(Burke et al., 1995
;
Cohn et al., 1997
), is at the
same level on the rostrocaudal axis of the embryo in both the control and
injected side (Fig. 3D;
n=27/27, 100%). Further analysis of the expression of several other
Hox genes (Hoxb4, Hoxc4, Hoxb5, Hoxc6, Hoxa9, Hoxc9 and
Hoxd9) produced identical results (data not shown). These results
indicate that the mechanism that shifts limb position in
Tbx3-injected embryos lies downstream of any axial Hox code that may
act to position the limbs.
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To investigate whether cell movement accounts for the phenotype, we performed DiI labelling experiments to follow the fates of cells of the prospective limb-forming region. Following misexpression of Tbx3, cells that normally become part of the posterior limb mesenchyme are instead incorporated into the interlimb flank (Fig. 3H; n=5/5, 100%). Cells in more rostral locations that would not normally contribute to the limb, are recruited to form (anterior) limb. Therefore, the shift in limb position cannot be attributed to migration of cells in the LPM.
Tbx3 and positioning of the ZPA
A shift in limb position is demonstrated by the expression of Shh,
in the posterior of the limb bud, at an inappropriate axial level
(Fig. 2A). We therefore
examined the expression of dHand and Gli3, genes which are
involved in pre-patterning the anteroposterior axis of the limb and
establishing the position of Shh expression in the ZPA
(Charite et al., 2000;
Fernandez-Teran et al., 2000
;
te Welscher et al., 2002
).
During limb induction stages, dHand (also known as Hand2), a
bHLH transcription factor, is expressed throughout the limb-forming region
(Fig. 4A)
(Charite et al., 2000
;
Fernandez-Teran et al., 2000
).
Subsequently, Gli3, a zinc-finger transcription factor, is expressed
throughout almost the entire limb mesenchyme in an anterior-to-posterior
graded fashion (Schweitzer et al.,
2000
). Genetic antagonism between Gli3 and dHand
results in downregulation of dHand expression in the anterior limb
mesenchyme (Fig. 4B). At later
stages, dHand and Gli3 are expressed in the anterior and
posterior limb mesenchyme, respectively, with an overlapping domain of
co-expression in the medial limb (Fig.
4C). The interactions between dHand and Gli3
ultimately position the ZPA prior to Shh signalling
(te Welscher et al., 2002
;
Zuniga and Zeller, 1999
).
Following misexpression of Tbx3, dHand is expressed throughout the
limb-forming region, while in the control side dHand is restricted to
the posterior limb mesenchyme (Fig.
4D; n=7/22, 32%). In addition, there is a downregulation
of Gli3 throughout the injected limb and the caudal border of its
graded expression domain shifts rostrally
(Fig. 4G; n=8/22,
36%). At slightly later stages (stage 19), when the shift phenotype is already
apparent, the domain of dHand expression is shifted to a more rostral
location and is no longer expressed throughout the limb mesenchyme but is
restricted to the posterior (Fig.
4E; n=7/7, 100%). Similarly, at stage 19, Gli3
is expressed in more rostral locations, in an anterior-posterior gradient in
the limb mesenchyme of the shifted limb
(Fig. 4H; n=8/8,
100%). At later stages (stage 21) when the shift phenotype is obvious,
dHand is expressed normally in the posterior mesenchyme of the
shifted limb (Fig. 4F;
n=7/7, 100%) and Gli3 has a normal distribution in the
anterior mesenchyme (Fig. 4I;
n=8/8, 100%). To more accurately define the time course of the limb
shift phenotype, we also analyzed expression of Shh and a downstream
target of Shh, Hoxd12, in Tbx3-injected limbs at stage 19.
Expression domains of both Shh
(Fig. 4J, n=2/2, 100%)
and Hoxd12 (Fig. 4L;
n=2/2, 100%) were shifted to more rostral positions. At later stages
(stage 21), Shh (Fig.
4K, n=2/2, 100%) and Hoxd12
(Fig. 4M; n=2/2, 100%)
were expressed in the normal, posterior domains within the shifted limb. These
data show that following misexpression of Tbx3, the normal
restriction of dHand expression to the posterior limb mesenchyme and
Gli3 to anterior is initially disrupted at early limb-forming stages
prior to Shh expression. However, as soon as Shh is
detectable, its expression domain is shifted to more rostral locations. The
establishment of this altered expression domain serves as the first molecular
evidence of a shift in the rostrocaudal location of the limb. After limb
position has shifted, dHand and Gli3 expression is normal
within the limb mesenchyme.
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Discussion |
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Following misexpression of Tbx3, the expression domains of genes
expressed in the limb are initially expanded rostrally and this is followed by
a shift in limb position. Cells of the flank, rostral to the limb, which would
not normally contribute to the limb, now become incorporated into the limb.
Furthermore, cells that normally form posterior limb mesenchyme now no longer
contribute to the limb. Strikingly, these cells had presumably initially
expressed Tbx5, a gene required and apparently sufficient for limb
initiation (Agarwal et al.,
2003; Ahn et al.,
2002
; Ng et al.,
2002
; Rallis et al.,
2003
; Takeuchi et al.,
2003
). Fate mapping shows that the altered contribution to the
limbs is not simply explained by migration of limb bud precursors. Our data
suggest that once a ZPA is established in a more rostral position, the
positive feedback loop between ZPA and AER now operates at altered axial
levels and the limb develops in an ectopic site. Noticeably, the size of the
ectopic limb is the same as that of the normal limb, suggesting that a
mechanism, yet to be determined, is functioning to regulate limb size.
Other members of the T-box gene family can influence the extent of the
limb. Tbx18 is expressed in lateral plate mesoderm as the limb buds
form and the anterior limit of Tbx18 expression coincides with the
anterior border of the limbs. Following misexpression of Tbx18 in the
presumptive wing bud region, the anterior extent of the limb is expanded
(Tanaka and Tickle, 2004). The
wing bud is extended rather than shifted in position and this extension is
only transient. At later stages, the limb appears to regulate its size and
develops normally. Brachyury is expressed in the LPM at the onset of
limb formation and at later stages of limb development, in the distal limb
mesenchyme that lies underneath the AER. Misexpression of Brachyury
in the chick wing results in anterior expansion of the AER and produces limb
phenotypes consistent with augmented AER extent and function, including
anterior digit duplications and, in rare cases, thickening of the
anterior-most metatarsal (Liu et al.,
2003
).
Tbx3 misexpression does not affect the pattern of axial Hox gene
expression. Hox genes have been implicated in limb position
(Cohn et al., 1997) and a role
for Hox genes in limb positioning is supported by Hoxb5 knockout
mice, which exhibit a unilateral or bilateral rostral shift in axial forelimb
position (Rancourt et al.,
1995
). However, the phenotype in
Hoxb5/ mice differs in several aspects from
that obtained with Tbx3 or Tbx3EN misexpression.
In Hoxb5/ mice, homeotic transformations of
the cervico-thoracic vertebrae from C6-T1 are observed. The clavicle retains a
medial articulation with its normal target, the sternum, resulting in a
V-shaped shoulder girdle. The alteration in limb position in the
Hoxb5/ mice is therefore associated with a
transformation of the entire axial skeleton. The absence of an effect of
Tbx3 on the expression of Hox genes indicates that Tbx3
mediates its effects on limb position independently of any axial Hox code.
Normal limb patterning following misexpression of Tbx3 forms
Roles for Tbx3 and the related gene Tbx2 in specifying
posterior digit identity via Shh and Bmp signalling has been
suggested (Suzuki et al.,
2004; Tumpel et al.,
2002
). Our data implicate Tbx3 in positioning the nascent
limb at pre-bud stages, prior to Shh expression. However, we also
analyzed the shifted limb at later stages. Following misexpression of
full-length Tbx3 and Tbx3EN, the expression pattern of
Bmp2 is unaffected, not expanded, as seen by Suzuki et al., and
ultimately digit identity is unchanged. We also performed injections of
Tbx3 in the hindlimb-forming region and obtained the same result as
seen in forelimbs: the injected hindlimb is shifted rostrally, while the digit
array is unaffected, in contrast to previous results. Our results do not
support a role for Tbx3 in specifying digit identity. One difference
between our experiments and those of Suzuki et al. is the timing of the viral
injections: while we performed our injections at stage 8-10, Suzuki et al.
injected at stage 11-12. Our earlier stage misexpression protocol may account
for our ability to generate limb shifts that were not reported by Suzuki et
al. However, it is not clear why different effects on limb patterning were
observed in these two sets of experiments, as our injection strategy does lead
to broad ectopic expression of Tbx3 at limb bud stages
(Fig. 3B).
A genetic interplay between Tbx3, dHand and Gli3
At limb development stages, prior to Shh expression, genetic
antagonism between dHand and Gli3 establishes an
anteroposterior pre-pattern of the limb that is ultimately responsible for
establishing the position of the ZPA in the posterior limb. At these stages,
Gli3 is acting as a repressor
(Wang et al., 2000), while
dHand is shown to be a transcriptional activator
(Dai and Cserjesi, 2002
;
Dai et al., 2002
).
Misexpression of Tbx3 leads to an expansion, or failure of
repression, of dHand, potentially through an interaction between
Tbx3 and Gli3 (Fig.
7). We predict that Tbx3 is acting to repress
Gli3 expression in the future posterior limb mesenchyme. Repression
by Tbx3 could be responsible for generating the anterior-to-posterior
graded expression of Gli3 in the developing limb primordium. This
model is consistent with the observation that in Tbx3 mutant mice,
dHand is downregulated in the forelimbs and absent in the hindlimbs,
which subsequently leads to a disruption in Shh expression. Our model
would predict that downregulation of dHand is due to high
Gli3 expression that, in the absence of Tbx3, is no longer
restricted to the anterior limb and expands to the posterior.
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Gli3 and limb positioning
We predict that the effects of Gli3ZnF-VP16 on dHand and
Tbx3 are caused by a disruption of endogenous GliR
activity. Misexpression of Gli3ZnF-VP16 leads to the expansion of
Tbx3 and dHand expression domains, rather than the induction
of ectopic patches of expression. This may indicate a non-cell autonomous
action of Gli3 on dHand and presumably also on
Tbx3. A regulatory relationship between Gli3 and
Tbx3 has been demonstrated during lung organogenesis
(Li et al., 2004).
Tbx3 is normally expressed in the mouse lung in the presence of
Shh. In this environment, Gli3 acts as an activator of
Tbx3 transcription. In Shh/
animals, where Gli3 acts as a repressor, Tbx3 transcripts
are significantly reduced. However, in
Shh//Gli3/ animals,
de-repression of Tbx3 is observed and Tbx3 expression is, at
least partially, restored (Li et al.,
2004
). These results, in combination with our own, suggest that
regulatory relationships between Tbx3 and Gli3 may exist
broadly during embryogenesis.
Our data implicate Gli3 in a genetic network that can influence
limb position, yet mice mutant for Gli3 (Extra toes,
XtJ), are not reported to exhibit any shift in axial limb
position (Buscher et al., 1997;
Buscher et al., 1998
;
Hui and Joyner, 1993
;
Litingtung et al., 2002
). In
XtJ/XtJ mice (that lack all Gli3 activity),
dHand is expressed throughout the limb and subsequently Shh
is expressed ectopically in the anterior limb mesenchyme. Misexpression of
dHand alone, at limb bud stages, is capable of inducing Shh
expression, but only in the anterior limb mesenchyme rather than medial
locations (Charite et al.,
2000
; Fernandez-Teran et al.,
2000
). These results suggest that anterior and posterior limb
mesenchyme may express a `licensing factor' required together with
dHand for the induction of Shh and that this factor is
absent from the medial limb mesenchyme. Tbx3 is expressed in two
stripes in the anterior and posterior limb mesenchyme at later stages of limb
development (Fig. 1D) and may
normally act as such a factor.
A requirement for Tbx3 to establish or re-set the domain of
Shh-expressing cells in the ZPA may also explain why no limb shift
phenotype is observed in XtJ/XtJ mice.
Tbx3 expression is not expanded in
XtJ/XtJ mice
(Tumpel et al., 2002). Without
the presence of both Tbx3 and dHand, the position of the
limb is not altered. In Tbx3 misexpression experiments, however,
Tbx3 can repress Gli3 and this leads to an expansion of
dHand. The co-expression of Tbx3 and dHand can, in
turn, `license' ectopic Shh expression, which, in turn, alters the
position of the limb (Fig.
7).
Mice in which Tbx3 is inactivated and humans with UMS who are
haploinsufficient for TBX3 are not reported to exhibit any shift in
axial limb position. This is consistent with the model we propose for the role
of Tbx3 in the early limb bud. Although a limb shift phenotype is not
observed in Tbx3/ mice, the expression
domains of dHand and Shh are downregulated or even
eliminated (Davenport et al.,
2003), consistent with Tbx3 being required for their
normal expression. This finding supports our conclusions that, although not
strictly required to fix limb position, Tbx3 is an important
component of the signals establishing the position of the domain of
Shh-expressing cells that comprise the ZPA in the posterior limb.
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ACKNOWLEDGMENTS |
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