|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 30 November 2006
doi: 10.1242/dev.02622
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Division of Developmental Biology, MRC-National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
* Author for correspondence (e-mail: mlogan{at}nimr.mrc.ac.uk)
Accepted 8 September 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Tbx5, Holt-Oram syndrome (HOS), Limb initiation, Limb outgrowth, Prx1CreERT2
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Experiments in a range of vertebrate model systems have shown that
Tbx5 is required for FL and heart development (e.g.
Agarwal et al., 2003
;
Ahn et al., 2002;
Rallis et al., 2003). Using a
conditional knockout allele of Tbx5, it has been demonstrated that
when the gene is deleted prior to, or during, limb initiation, no FL initiates
(Agarwal et al., 2003;
Rallis et al., 2003).
Similarly, deletion of Tbx4 in the mouse leads to failure of HL
development, although the phenotype is not as profound as that seen in the FL
in the absence of Tbx5 (Naiche
and Papaioannou, 2003).
Although the precise hierarchy remains unclear, and may vary between
species, Tbx5 and Tbx4 play pivotal roles during FL and HL initiation,
respectively, by activating Fgf10 in the limb mesenchyme
(Agarwal et al., 2003;
Logan, 2003;
Naiche and Papaioannou, 2003;
Rallis et al., 2003). Fgf10,
in turn, activates Fgf8 in the apical ectodermal ridge (AER), a
distinct strip at the distal extreme of the overlying ectoderm. Fgf8 signaling
from the AER to the underlying mesoderm is subsequently required for the
maintenance of mesenchymal Fgf10 expression, thereby creating a
positive-feedback loop of FGF signaling between the two tissue layers.
Tbx5 and Tbx4 appear to play analogous roles in the FL and
HL, respectively. Using a gene deletion and replacement strategy, the FL
deficiency defect of the Tbx5 conditional knockout can be rescued by
Tbx4, demonstrating that Tbx4 has the ability to carry out
the functions of Tbx5 in FL initiation
(Minguillon et al., 2005).
Interestingly, Fgf10 mutant mice initiate a limb bud
(Sekine et al., 1999),
suggesting that Tbx5 may also regulate other targets. In addition,
Tbx5 continues to be expressed throughout later stages of limb
outgrowth. Misexpression of a dominant-negative form of Tbx5 in the chick wing
bud leads to the downregulation of Fgf4, Fgf8, Fgf10, Bmp2 and
Wnt3a, and truncated FLs result
(Rallis et al., 2003;
Rodriguez-Esteban et al.,
1999). These observations have led to the proposal that, in
addition to its activity during initiation, Tbx5 also regulates later
FL outgrowth and may be required for the orchestration of the distinct
patterning events taking place in the growing limb
(Logan et al., 1998;
Rallis et al., 2003;
Rodriguez-Esteban et al.,
1999).
In humans, mutations in TBX5 are associated with the dominant disorder
Holt-Oram syndrome (HOS; OMIM 142900)
(Basson et al., 1997;
Li et al., 1997), which leads
to upper(fore)limb and heart deformities. Although haploinsufficiency of TBX5
is fully penetrant, the severity of the limb phenotypes can be variable,
ranging from severe deletion deformities of many of the skeletal elements of
the limb (aplasia), to more subtle phenotypes such as extended phalangeal
elements of the thumbs (Newbury-Ecob et
al., 1996). Similarly, mutations in TBX4 are associated with Small
Patella syndrome (SPS; OMIM 147891), which is characterised by abnormalities
of the lower(hind)limb. The limb skeletal defects in SPS are also
characterised by deletion deformities with variable penetrance; however, they
are generally less severe than those observed in HOS. Although the results
from gene deletion experiments in the mouse are consistent with the phenotypes
observed in HOS and SPS, they do not provide any information on the temporal
requirement for Tbx5 and Tbx4 to form individual elements of
the limb at later stages in development.
Here, we test whether Tbx5 plays a role beyond the initiation stages of FL formation. Using a tamoxifen (TM)-inducible Cre transgenic line (Prx1CreERT2), we have deleted Tbx5 in the nascent FL buds in embryos ranging from 21 somites [early embryonic day (E) 9.5] to E10.5. Surprisingly, although Cre-catalysed deletion of the Tbx5 conditional allele was apparent in the FL, limb outgrowth was not impaired. These results demonstrate that Tbx5 is not required for outgrowth of the limb bud and/or patterning of the FL skeleton. Furthermore, these findings have important implications for our understanding of the aetiology of the limb skeletal defects present in HOS.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Noon on the
day a vaginal plug was observed, was taken to be E0.5. The mouse lines
carrying a conditional allele of Tbx5
(Bruneau et al., 2001),
Rosa26RlacZ (Soriano,
1999) and a Prx1Cre transgene
(Logan et al., 2002) have been
described previously. Construction of the Prx1CreERT2 construct was
carried out using standard ligation procedures. The Prx1Cre backbone
was digested with EcoRI to excise the Cre ORF, which was then
replaced by an EcoRI CreERT2 fragment.
Tamoxifen induction
TM preparation and induction were performed as described on the Joyner
laboratory webpage
(http://saturn.med.nyu.edu/research/dg/joynerlab/protocols.html).
Briefly, mice were gavaged with 6.5 mg (from a 20 mg/ml stock) of TM at the
indicated time points.
Quantitative PCR
To follow the rate of Tbx5 exon 3 recombination, we used primers
flanking this exon and monitored the loss of PCR product resulting from its
recombination. Briefly, E10.5 FL mesenchymal cells were dissected and the
genomic DNA isolated for use in the quantitative PCR analysis. Quantification
of the results was carried out using the ABI Prism 7700 Sequence Detection
System, User Bulletin 2, Comparative CT Method. Primers used were:
Tbx5 exon3 Fwd, 5'-GGCATGGAAGGAATCAAGGT-3'; Tbx5 int 3-4 Rev,
5'-ATTCCCTCCAATGACTGTCC-3'. The amount of template was normalised
using primers that amplify a fragment of the cardiac actin promoter: mCA Fwd,
5'-CCCCCTGGCTGATCCTCTAC-3'; mCA Rev,
5'-TGGTCGCCTTAGCACCAT-3'. All reactions were carried out in
triplicate.
In situ hybridisation
Whole-mount and section in situ hybridisation were carried out essentially
as described previously (Riddle et al.,
1993; Schaeren-Wiemers and
Gerfin-Moser, 1993). A minimum of three mutant embryos were
analysed with each probe at each stage described. Most of the probes used have
been described previously: Shh
(Echelard et al., 1993),
Fgf10 (Bellusci et al.,
1997), Fgf8 (Crossley
and Martin, 1995), Tbx5
(Rallis et al., 2003),
Tbx5 ex3 (kindly provided by C. Minguillon, NIMR, UK), Scx
(Schweitzer et al., 2001),
Sox9 (Morais da Silva et al.,
1996), Col2a (Col2a1 - Mouse Genome Informatics)
(Metsaranta et al., 1991),
Myod (Davis et al.,
1987), Pax3 (Goulding
et al., 1991), Sall4 (a kind gift of S. Harvey, NIMR,
UK), Tbx3 (kindly provided by C. Goding, Marie Curie Research
Institute, UK) and Tbx15 (Singh
et al., 2005).
Histology
The cartilage and bone elements of newborn mouse pups were stained with
Alcian Blue and Alizarin Red, respectively, essentially as described
previously (McLeod, 1980).
Phospho-Histone H3 and TUNEL analysis
Detection of proliferating or apoptotic cells was carried out as previously
described (Rallis et al.,
2003). Briefly, mitotic cells were identified using a rabbit
anti-phosphorylated histone H3 primary antibody (Upstate Biotechnology) and an
HRP-conjugated goat anti-rabbit IgG secondary antibody (Jackson Laboratory).
Apoptotic cell death was assayed by TdT-mediated dUTP nick end labelling
(TUNEL) according to the manufacturer's protocol (Q-Biogene).
Size quantification analysis
The extent of the anteroposterior axis was determined by measuring the
distance between the anterior-most and posterior-most extremes of wild-type
(n=15) and mutant (n=12) limbs at their proximal base.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Rallis et al., 2003). Deletion
of Tbx5 leads to the disruption of FL bud formation and all elements
of the limb fail to form (Rallis et al.,
2003). Such early and profound defects have prevented the study of
potential roles of the gene later in limb development. It therefore remains
unclear whether Tbx5 plays any crucial role in regulating outgrowth
and coordinating patterning of the bud after its initiation. Expression of
Tbx5 is not restricted to the initiation stages of limb bud
development, but rather it is retained throughout the limb outgrowth stages
(Fig. 1 and data not shown).
Serial sections of the FL at different embryonic stages (E11.5, E12.5 and
E15.5) were examined for the expression of Tbx5 along with markers of
the prospective cartilaginous precursors (Sox9 or Col2a),
tendons (Scx) and muscles (Pax3, Myod or muscle myosin). At
E11.5, Tbx5 was expressed in all mesenchymal cells of the limb and
was co-expressed in domains overlapping with bone, tendon and muscle
precursors (Fig. 1A-D). At
E12.5, Tbx5 expression was retained in all mesenchymal cells of the
bud, but not as uniformly as that observed at E11.5
(Fig. 1E). Tbx5
transcripts were detected, but levels appeared reduced along the dorsal and
ventral sides of the limb overlapping with the Myod and Scx
expression domains. However, Tbx5 expression remained elevated in
domains overlapping with Col2a-expressing cartilage precursors in the
centre of the limb (Fig. 1E-H).
At E15.5, Tbx5 expression levels declined and were maintained in only
a few cells at the tip of the digits (not shown).
|
) in combination with transgenic mouse lines
expressing TM-inducible Cre recombinase (CreERT2)
(Feil et al., 1997) under the
influence of the Prx1 promoter, leading to expression of CreERT2 in
the mouse limbs (Prx1CreERT2; Fig.
2). Administration of TM at specific time points and the
consequent activation of Cre activity leads to the deletion of Tbx5
at distinct stages of limb development.
To test the efficacy of the inducible Cre lines that we generated, we
compared their activity with that of the previously described Prx1Cre
transgenic line by crossing Prx1CreERT2 or Prx1Cre mice to
the Rosa26RLacZ reporter line
(Soriano, 1999)
(Fig. 2 and data not shown). To
induce Cre activity from the onset of CreERT2 expression driven by
the Prx1 promoter, we tested oral gavage regimes of a single TM dose
at E8.5 and a double dose at E7.5 and E8.5. The single dose regime of TM
produced comparable levels of recombination as the double dose (data not
shown). Gavage regimes at E7.5 or E6.5 resulted in little or no Cre activity,
respectively. We therefore used a single oral gavage at E8.5 to detect the
earliest Cre activity in the Prx1CreERT2 lines (see Materials and
methods). This comparison reveals that the Prx1Cre transgene has more
robust Cre activity at early stages than does the Prx1CreERT2 line.
Prx1Cre was active in cells of the LPM at the level of the forming
bud in the 14-somite stage embryo and, once a bud was visible, Cre activity
was evident throughout the mesenchymal cells
(Fig. 2A-C). By contrast,
during limb initiation stages, Prx1CreERT2 induced recombination in a
relatively small number of sparsely spaced cells
(Fig. 2D,E,G,H), and only once
the embryo had reached the 21- to 23-somite stage, after initiation of the bud
had occurred, was recombination seen throughout the bud
(Fig. 2F). From this time point
onwards, the Prx1CreERT2 transgene activity in the reporter was
indistinguishable from the Prx1Cre transgene
(Fig. 2I-M and data not shown).
Therefore, the difference between the activities of the Prx1CreERT2
and Prx1Cre deleter transgenes in the FL was the patchy recombination
in the FL prospective region in the 14- to 21-somite stage embryos that
express the TM-inducible Cre.
|
|
;
Rallis et al., 2003), and in
turn Fgf10 induces Fgf8 in the overlying ectoderm. However, despite
the deletion of Tbx5, no differences in Fgf10 expression
were detectable (Fig. 3C,D).
Consistent with normal Fgf10 signaling, Fgf8 was expressed in an
equivalent domain, and at comparable levels throughout the AER, to that of
wild-type or heterozygous littermates (Fig.
3E,F). An orthologue of Drosophila spalt major, Sall4,
has recently been shown in zebrafish and mice to be a target of Tbx5 and to
participate in the FGF signaling feedback loop
(Harvey and Logan, 2006;
Koshiba-Takeuchi et al.,
2006). To test whether Sall4 is also sensitive to
Tbx5 deletion after limb initiation, we performed in situ
hybridisation with Sall4. Tbx5lox/lox-deleted limbs had
reduced Sall4 levels when compared with their wild-type counterparts,
a phenotype most evident in the anterior domain of the limb
(Fig. 3G,H, arrow). To
determine whether the AP patterning of the mutant limbs was normal, we
analysed the expression patterns of a range of markers expressed in nested
domains across the AP axis. Tbx3 was expressed in anterior and
posterior domains, and this pattern was not perturbed in the mutant limb
(Fig. 3I,J). Tbx15 was
expressed in an apparently reciprocal pattern to Tbx3, comprising a
broad medial stripe, and this was unchanged in the
Tbx5lox/lox mutant limb
(Fig. 3M,N). The Shh
expression that marks the ZPA at the posterior of the limb was not disrupted
in Tbx5lox/lox-deleted limbs
(Fig. 3K,L). Therefore, the
initial expression of Tbx5 in embryos up to the 21-somite stage is
sufficient for two key signaling centres in the limb to be established: the
AER and the ZPA. Although the expression of molecular markers of two key signaling centres, Shh (ZPA) and Fgf8 (AER), were established normally, the appearance of the Tbx5lox/lox-deleted limbs was not entirely normal. The mutant limbs were narrower in their anterior to posterior extent (Fig. 2J-M). We compared the number of somites that the limbs spanned by analysing the expression of Fgf8, which marks the AER, and Myod, which marks the somites. Whereas the normal limb spanned approximately five somites (Fig. 3O), the Tbx5lox/lox limbs spanned only four somites (Fig. 3P). Significantly, although both mutant and wild-type limbs were positioned at the equivalent somite level at their posterior extreme, the anterior extent of the mutant limb was reduced. This anterior bias of the limb phenotype in the Tbx5 mutant limbs resembles the HOS phenotype, in which the more anterior structures are most severely affected (see Discussion). Measurement of the mean AP extent of mutant and control limbs at E10.5 indicated that whereas wild-type limbs spanned on average 850 µm, the mutant limbs spanned on average only 700 µm (18% narrower). This reduction in limb size was due to a small, yet detectable, reduction in cell proliferation, and not to an apparent increase in the rate of cell apoptosis in the limb mesenchyme. The number of cells staining positive for phospho-histone H3, a marker for cells undergoing mitosis, was greater in wild-type than in mutant limbs. However, no differences between wild-type and mutant limbs were seen for cells staining positive following TUNEL, a marker of cells undergoing apoptosis (see Fig. S1 in the supplementary material).
|
).
The remaining four digits appeared completely normal. These phenotypes were
not observed in embryos resulting from TM administration at E10.5, where all
the skeletal elements were normal (Fig.
4I). These results are reinforced by the absence of abnormal limb
phenotypes when a Col2a-Cre deleter line
(Ovchinnikov et al., 2000) is
used to delete Tbx5 function in the cartilage progenitors of the limb
skeleton from E11.5 onwards (P.H. and M.P.O.L., unpublished). These results
confirm that Tbx5 is not required for outgrowth of the FL and
demonstrate that Tbx5 is not required for formation or patterning of
the limb skeleton. |
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Ahn et al.,
2002; Ng et al.,
2002; Rallis et al.,
2003), and Tbx5 knockout mice do not initiate a FL
(Agarwal et al., 2003;
Rallis et al., 2003). These
and other experiments have lead to the placement of Tbx5 (and its
paralogue Tbx4 in the HL) at the top of the genetic cascade required
for limb formation. However, Tbx5 (and Tbx4), continue to be
expressed at later stages of limb development
(Fig. 1), and misexpression of
dominant-negative forms of Tbx5 in the chick leads to limb truncations and the
downregulation of genes required for limb outgrowth
(Rallis et al., 2003;
Rodriguez-Esteban et al.,
1999). It has therefore been proposed that Tbx5 might
continue to play a crucial role in limb outgrowth and in later skeletal
patterning events in the growing limb
(Rallis et al., 2003;
Rodriguez-Esteban et al.,
1999). Our results, and those of others
(Naiche and Papaioannou,
2007), suggest that this is not the case, and that Tbx5
and Tbx4 do not have a major role in limb outgrowth and formation of
the limb skeleton.
The deletion of Tbx5 using Prx1Cre results in the
complete failure of FL formation, whereas deletion with Prx1CreERT2
does not lead to the same extreme phenotype and early limb markers are
expressed relatively normally (Fig.
3). As the resulting mutant limbs do not express an active
Tbx5 gene (Fig. 2N,
Fig. 3B), the difference in
phenotypes (i.e. limbless versus limb, respectively) is a consequence of the
timing of the gene deletion. Our analysis shows that Prx1Cre is
active in the LPM of 14-somite stage embryos at the level of the forming bud.
By contrast, Prx1CreERT2, although initially active at the same stage
(starting at 14 somites), leads to recombination in a small and sparse number
of cells in the bud. Only in 21- to 23-somite stage embryos is activity seen
throughout the FL mesenchyme in a manner comparable with that of
Prx1Cre. The temporal disparity in Cre activity between the two Cre
deleter lines, and the distinct phenotypes produced, demonstrate the critical
time window within which Tbx5 is required for FL initiation. This
allows us to identify two distinct phases in early limb development: a limb
initiation phase that is Tbx5 dependent
(Fig. 5, top), followed by a
limb outgrowth phase that is Tbx5 independent
(Fig. 5, bottom). During the
initiation phase, Tbx5 activates Fgf10 expression, and this phase is
complete by the 21-somite stage. At this time, key signaling centres in the
limb, the AER and the ZPA, are established. Deletion of Tbx5 after
the 21-somite stage (TM administration at stages prior to E10.5) does not
impair limb outgrowth, although the limb is not entirely normal. This is
consistent with recent observations showing that, in addition to regulating
Fgf10, Tbx5 also activates Sall4. Sall4 is required for the
expression of mesenchymal FGFs, possibly by facilitating establishment of the
FGF signaling feedback between the mesenchyme and overlying ectoderm
(Harvey and Logan, 2006;
Koshiba-Takeuchi et al.,
2006), thereby enabling maintenance of Fgf8 expression in
the AER. In humans, mutations in SALL4 are associated with Okihiro
syndrome (OS; OMIM 607323) (Kohlhase et
al., 2002), and the limb abnormalities characteristic of OS share
many similarities with those found in HOS. Strikingly, the skeletal
abnormalities resulting from TM administration at E8.5 and E9.5 are
reminiscent of both HOS and OS. It is thus plausible that the phenotypes
observed in mice gavaged at E8.5 and E9.5 are due to the reduction in
Sall4 activation by Tbx5 (e.g.
Fig. 3H)
(Harvey and Logan, 2006;
Koshiba-Takeuchi et al.,
2006). Crucially, deletion of Tbx5 function at later
stages does not produce HOS-like skeletal phenotypes. This defines the
outgrowth phase when the feedback loop between mesenchymal Fgf10 and
ectodermal Fgf8 has been established, can operate and is maintained
independently of Tbx5 (Fig.
5, bottom).
|
), in
which a reduction in anterior expression is also observed.
Skeletal analysis of Tbx5 mutant limbs following TM administration
up to E9.5 revealed additional phenotypes characteristic of HOS. In
individuals with HOS, the left limb is commonly more severely affected than
the right (Newbury-Ecob et al.,
1996). In embryos generated following a gavage regime at E8.5, but
importantly not at later stages of TM administration, the left limb was
consistently more severely affected than the right limb. Furthermore,
following gavage at E8.5 and E9.5, the FLs of the resulting embryos had
anterior abnormalities, in particular an extended digit 1
(Fig. 4), similar to the thumb
abnormalities characteristic of HOS (R. Newbury-Ecob, personal communication)
(Newbury-Ecob et al., 1996).
Significantly, however, when Tbx5 was deleted later (gavage at
E10.5), no skeletal malformations were observed, demonstrating that HOS
skeletal phenotypes are caused by loss of Tbx5 activity at earlier
stages. Furthermore, we did not observe several prominent features of HOS,
such as aplasia of skeletal elements. In combination, these results imply that
different HOS manifestations can be attributed to distinct stages of
Tbx5 activity.
Recently, suggested roles for Tbx4 and Tbx5 in the
specification of limb-type identity have been ruled out
(Minguillon et al., 2005). Our
data and those of others (Naiche and
Papaioannou, 2007) suggest that these genes likewise play no role
in regulating limb outgrowth, despite their expression being maintained during
the outgrowth stages of limb development. It is possible that the activities
of Tbx5 and Tbx4 during these later stages of limb development are controlled
at a post-transcriptional and/or post-translational level. Evidence for
post-translational regulation has come from the description of a PDZ-LIM
protein, Lmp4, which is expressed in the limb. When co-expressed in COS-1
cells, Lmp4 is able to bind to Tbx5 and Tbx4, leading to cytoplasmic
localisation of the complex via association with the actin cytoskeleton
(Krause et al., 2004). It
remains to be determined, however, whether Tbx5 and Tbx4 are regulated in this
manner during limb development. Similarly, in Xenopus, the canonical
T-box protein Xbra has been shown to bind Smad1, a binding that modulates its
activities (Messenger et al.,
2005). Because Smad1 activity is dynamically regulated
(Massague et al., 2005), it is
possible that a similar situation exists for Tbx5 and Tbx4 during limb
outgrowth, and that their activities are controlled via the regulation of
co-factors. Our results for Tbx5, and those of others for Tbx4, demonstrate
that the activities of these proteins are regulated during limb development.
Future work will determine if Tbx5 and Tbx4 are required for other processes
during limb development, and whether their activities are regulated at a
post-transcriptional or post-translational level.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/1/85/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Agarwal, P., Wylie, J. N., Galceran, J., Arkhitko, O., Li, C.,
Deng, C., Grosschedl, R. and Bruneau, B. G. (2003). Tbx5 is
essential for forelimb bud initiation following patterning of the limb field
in the mouse embryo. Development
130,623
-633.
Ahn, D. G., Kourakis, M. J., Rohde, L. A., Silver, L. M. and Ho, R. K. (2002). T-box gene tbx5 is essential for formation of the pectoral limb bud. Nature 417,754 -758.[CrossRef][Medline]
Basson, C. T., Bachinsky, D. R., Lin, R. C., Levi, T., Elkins, J. A., Soults, J., Grayzel, D., Kroumpouzou, E., Traill, T. A., Leblanc-Straceski, J. et al. (1997). Mutations in human TBX5 [corrected] cause limb and cardiac malformation in Holt-Oram syndrome. Nat. Genet. 15,30 -35.[CrossRef][Medline]
Bellusci, S., Grindley, J., Emoto, H., Itoh, N. and Hogan, B. L. (1997). Fibroblast growth factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung. Development 124,4867 -4878.[Abstract]
Bruneau, B. G., Nemer, G., Schmitt, J. P., Charron, F., Robitaille, L., Caron, S., Conner, D. A., Gessler, M., Nemer, M., Seidman, C. E. et al. (2001). A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 106,709 -721.[CrossRef][Medline]
Crossley, P. H. and Martin, G. R. (1995). The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121,439 -451.[Abstract]
Davis, R. L., Weintraub, H. and Lassar, A. B. (1987). Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51,987 -1000.[CrossRef][Medline]
Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J. A. and McMahon, A. P. (1993). Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75,1417 -1430.[CrossRef][Medline]
Feil, R., Wagner, J., Metzger, D. and Chambon, P. (1997). Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem. Biophys. Res. Commun. 237,752 -757.[CrossRef][Medline]
Goulding, M. D., Chalepakis, G., Deutsch, U., Erselius, J. R. and Gruss, P. (1991). Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. EMBO J. 10,1135 -1147.[Medline]
Harvey, S. A. and Logan, M. P. (2006). sall4
acts downstream of tbx5 and is required for pectoral fin outgrowth.
Development 133,1165
-1173.
Kaufman, M. H. (2001). The Atlas of Mouse Development. Cambridge: Academic Press.
Kohlhase, J., Heinrich, M., Schubert, L., Liebers, M., Kispert,
A., Laccone, F., Turnpenny, P., Winter, R. M. and Reardon, W.
(2002). Okihiro syndrome is caused by SALL4 mutations.
Hum. Mol. Genet. 11,2979
-2987.
Koshiba-Takeuchi, K., Takeuchi, J. K., Arruda, E. P., Kathiriya, I. S., Mo, R., Hui, C. C., Srivastava, D. and Bruneau, B. G. (2006). Cooperative and antagonistic interactions between Sall4 and Tbx5 pattern the mouse limb and heart. Nat. Genet. 38,175 -183.[CrossRef][Medline]
Krause, A., Zacharias, W., Camarata, T., Linkhart, B., Law, E., Lischke, A., Miljan, E. and Simon, H. G. (2004). Tbx5 and Tbx4 transcription factors interact with a new chicken PDZ-LIM protein in limb and heart development. Dev. Biol. 273,106 -120.[CrossRef][Medline]
Li, Q. Y., Newbury-Ecob, R. A., Terrett, J. A., Wilson, D. I., Curtis, A. R., Yi, C. H., Gebuhr, T., Bullen, P. J., Robson, S. C., Strachan, T. et al. (1997). Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family. Nat. Genet. 15,21 -29.[CrossRef][Medline]
Logan, M. (2003). Finger or toe: the molecular
basis of limb identity. Development
130,6401
-6410.
Logan, M., Simon, H. G. and Tabin, C. (1998). Differential regulation of T-box and homeobox transcription factors suggests roles in controlling chick limb-type identity. Development 125,2825 -2835.[Abstract]
Logan, M., Martin, J. F., Nagy, A., Lobe, C., Olson, E. N. and Tabin, C. J. (2002). Expression of Cre Recombinase in the developing mouse limb bud driven by a Prxl enhancer. Genesis 33,77 -80.[CrossRef][Medline]
Massague, J., Seoane, J. and Wotton, D. (2005).
Smad transcription factors. Genes Dev.
19,2783
-2810.
McLeod, M. J. (1980). Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S. Teratology 22,299 -301.[CrossRef][Medline]
Messenger, N. J., Kabitschke, C., Andrews, R., Grimmer, D., Nunez Miguel, R., Blundell, T. L., Smith, J. C. and Wardle, F. C. (2005). Functional specificity of the Xenopus T-domain protein Brachyury is conferred by its ability to interact with Smad1. Dev. Cell 8,599 -610.[CrossRef][Medline]
Metsaranta, M., Toman, D., De Crombrugghe, B. and Vuorio, E. (1991). Specific hybridization probes for mouse type I, II, III and IX collagen mRNAs. Biochim. Biophys. Acta 1089,241 -243.[Medline]
Minguillon, C., Del Buono, J. and Logan, M. P. (2005). Tbx5 and Tbx4 are not sufficient to determine limb-specific morphologies but have common roles in initiating limb outgrowth. Dev. Cell 8,75 -84.[CrossRef][Medline]
Morais da Silva, S., Hacker, A., Harley, V., Goodfellow, P., Swain, A. and Lovell-Badge, R. (1996). Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds. Nat. Genet. 14, 62-68.[CrossRef][Medline]
Naiche, L. A. and Papaioannou, V. E. (2003).
Loss of Tbx4 blocks hindlimb development and affects vascularization and
fusion of the allantois. Development
130,2681
-2693.
Naiche, L. A. and Papaioannou, V. E. (2007).
Tbx4 is not required for hindlimb identity of post-bud hindlimb growth.Development
134,93
-103.
Newbury-Ecob, R. A., Leanage, R., Raeburn, J. A. and Young, I. D. (1996). Holt-Oram syndrome: a clinical genetic study. J. Med. Genet. 33,300 -307.[Abstract]
Ng, J. K., Kawakami, Y., Buscher, D., Raya, A., Itoh, T., Koth, C. M., Rodriguez Esteban, C., Rodriguez-Leon, J., Garrity, D. M., Fishman, M. C. et al. (2002). The limb identity gene Tbx5 promotes limb initiation by interacting with Wnt2b and Fgf10. Development 129,5161 -5170.
Ovchinnikov, D. A., Deng, J. M., Ogunrinu, G. and Behringer, R. R. (2000). Col2a1-directed expression of Cre recombinase in differentiating chondrocytes in transgenic mice. Genesis 26,145 -146.[CrossRef][Medline]
Rallis, C., Bruneau, B. G., Del Buono, J., Seidman, C. E.,
Seidman, J. G., Nissim, S., Tabin, C. J. and Logan, M. P.
(2003). Tbx5 is required for forelimb bud formation and continued
outgrowth. Development
130,2741
-2751.
Riddle, R. D., Johnson, R. L., Laufer, E. and Tabin, C. (1993). Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75,1401 -1416.[CrossRef][Medline]
Rodriguez-Esteban, C., Tsukui, T., Yonei, S., Magallon, J., Tamura, K. and Izpisua Belmonte, J. C. (1999). The T-box genes Tbx4 and Tbx5 regulate limb outgrowth and identity. Nature 398,814 -818.[CrossRef][Medline]
Schaeren-Wiemers, N. and Gerfin-Moser, A. (1993). A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes. Histochemistry 100,431 -440.[CrossRef][Medline]
Schweitzer, R., Chyung, J. H., Murtaugh, L. C., Brent, A. E.,
Rosen, V., Olson, E. N., Lassar, A. and Tabin, C. J. (2001).
Analysis of the tendon cell fate using Scleraxis, a specific marker for
tendons and ligaments. Development
128,3855
-3866.
Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, T., Yagishita, N., Matsui, D., Koga, Y., Itoh, N. et al. (1999). Fgf10 is essential for limb and lung formation. Nat. Genet. 21,138 -141.[CrossRef][Medline]
Singh, M. K., Petry, M., Haenig, B., Lescher, B., Leitges, M. and Kispert, A. (2005). The T-box transcription factor Tbx15 is required for skeletal development. Mech. Dev. 122,131 -144.[CrossRef][Medline]
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  L. A. Naiche and V. E. Papaioannou Tbx4 is not required for hindlimb identity or post-bud hindlimb outgrowth Development, January 1, 2007; 134(1): 93 - 103. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
