1 Division of Developmental Biology, National Institute for Medical Research,
Mill Hill, London NW7 1AA, UK
2 Cardiovascular Research and Developmental Biology, The Hospital for Sick
Children and Department of Molecular and Medical Genetics, University of
Toronto, Toronto, ON, Canada
3 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
4 Department of Genetics, Howard Hughes Medical Institute, Harvard Medical
School, Boston, MA 02115, USA
5 Cardiovascular Division, Howard Hughes Medical Institute, Brigham and Women's
Hospital, Boston, MA 02115, USA
* Author for correspondence (e-mail: mlogan{at}nimr.mrc.ac.uk)
Accepted 10 March 2003
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SUMMARY |
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Key words: Limb development, Limb-type identity, Tbx5, T-box genes, Mouse, Chick
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INTRODUCTION |
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Several genes have been identified that are expressed exclusively in either
the developing forelimb or hindlimb. The T-box transcription factor
Tbx5 is first detected in the prospective forelimb mesenchyme prior
to overt limb bud outgrowth and this limb-type restricted expression pattern
is maintained throughout limb development stages
(Gibson-Brown et al., 1996;
Isaac et al., 1998
;
Logan et al., 1998
;
Ohuchi et al., 1998
). A
closely related T-box gene, Tbx4, and a paired-like homeodomain
factor, Pitx1, are both expressed in a reciprocal pattern in the
developing hindlimb mesenchyme
(Gibson-Brown et al., 1996
;
Isaac et al., 1998
;
Lanctot et al., 1997
;
Logan et al., 1998
;
Ohuchi et al., 1998
).
Misexpression experiments in the chick have demonstrated that ectopic
expression of Tbx5 in the leg bud is capable of transforming the
hindlimb to a more forelimb character
(Takeuchi et al., 1999
).
Conversely, misexpression of Tbx4 or Pitx1 in the developing
wing bud is capable of transforming the forelimb to a more hindlimb character
(Logan and Tabin, 1999
;
Rodriguez-Esteban et al.,
1999
; Takeuchi et al.,
1999
). Direct evidence for a role of Tbx5 in forelimb
development has been provided by the discovery that mutations in human
TBX5 cause Holt-Oram Syndrome (HOS, OMIM 142900), a dominant disorder
characterised predominantly by upper(fore) limb defects and heart
abnormalities (Basson et al.,
1997
; Li et al.,
1997
). Targeted deletion of Tbx5 in the mouse has
demonstrated that this gene is essential for normal heart development
(Bruneau et al., 2001
).
Tbx5-null embryos die at or around embryonic day (E) 10 because of
the severity of the heart defects. Diminished TBX5 function in human,
however, does not obviously affect limb-type identity but instead produces
deletion deformities (Basson et al.,
1994
). Therefore, Tbx5 may have roles related to growth
and differentiation of the embryonic limbs that are distinct from, or
intrinsically linked to, its role in defining limb-type identity.
To examine the role of Tbx5 in forelimb development we have undertaken two strategies to disrupt its function in the developing limb bud. We have used a conditional knockout strategy to delete Tbx5 function in the developing limbs while leaving the gene intact in other areas of the developing embryo. This approach avoids the complication of phenotypes arising from Tbx5 loss-of-function in regions of the embryo other than the limb, in particular the heart. The second approach involves using avian retroviruses to misexpress dominant-negative Tbx5 constructs to knock down Tbx5 function in the developing wing bud. As a complementary strategy, we also misexpressed full-length and dominant-active forms of the gene.
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MATERIALS AND METHODS |
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PCR
PCR analysis to genotype pup tail and embryonic material (E10, 30 somites)
was carried out in a single reaction using three primers that identify the
endogenous Tbx5 allele, and both the conditional (floxed) and deleted
(floxed-out) Tbx5 allele (Bruneau
et al., 2001).
Retrovirus production and infection
Cloning of retroviral constructs and production of concentrated retroviral
supernatants were carried out as described previously
(Logan and Tabin, 1998). The
Tbx5
construct contains amino acids 1-274 of the full-length
chick Tbx5 clone (Accession Number AF069396). The
Tbx5en construct contains amino acids 1-274 of the
full-length Tbx5 clone fused to amino acids 2-298 of the
Drosophila Engrailed protein
(Jaynes and O'Farrell, 1991
).
The Tbx5vp16 contains amino acids 1-274 of Tbx5
fused to a duplex of the lambda hinge region and VP16
(Ohashi et al., 1994
). Cells
of the prospective forelimb were infected between HH stages 8-10 with
concentrated viral supernatants as previously described
(Logan and Tabin, 1998
).
Whole-mount in situ hybridisation
Whole-mount in situ hybridisation was carried out essentially as previously
described (Riddle et al.,
1993). A minimum of two mutant embryos were analysed at each stage
described with each probe. Most probes have been described previously: chick
Shh (Riddle et al.,
1993
), mouse Shh
(Echelard et al., 1993
), chick
Msx (Ros et al.,
1992
), chick Hoxc4
(Nelson et al., 1996
), mouse
Fgf10 (Bellusci et al.,
1997
), mouse Pea3
(Chotteau-Lelievre et al.,
2001
), mouse Fgf8
(Crossley and Martin, 1995
),
mouse Tbx4 (Bruneau et al.,
2001
), mouse Pitx1
(Logan and Tabin, 1999
) and
chick Fgf8 (Vogel et al.,
1996
). A fragment of the chick Lhx9 sequence was isolated
from a chick plasmid library (Logan et
al., 1998
) and its identity confirmed by sequencing and comparison
with published sequences. A chick Groucho homologue Grg4 was
generously provided by Johan Ericson (Muhr
et al., 2001
). Section in situ hybridisation was performed on 20
µm paraffin wax-embedded sections of stage 21 chick embryos. Additional
chick Groucho genes were cloned by degenerate PCR from chick limb cDNA
prepared from HH stages 20-23. The degenerate PCR primers lie in the highly
conserved Q domain and WD40 domains of Drosophila, mouse and human
Groucho genes: 5'-AA-RACIGARATGCARMGICAY-3',
5'-IGCYTCICCICCIACDATIAR-3'. PCR using a 45°C annealing
temperature yielded multiple products, including a 1.1 kb fragment that was
cloned into the pGEM-T vector (Promega). Sequencing of this clone revealed
similarity to human TLE3.
Histology, TUNEL analysis and immunofluorescence assays
The cartilage and bone elements of newborn mouse pups were stained with
Alcian Blue and Alizarin Red, respectively, essentially as described
previously (Hogan et al.,
1994). Apoptotic cell death was assayed with TdT-mediated dUTP
nick end labelling (TUNEL). Mouse embryos were fixed overnight in 4%
paraformaldehyde and then processed in whole mount using TUNEL reagents
(Q-BIOgene) following the manufacturer's protocol. Chick embryos were fixed
overnight in 4% paraformaldehyde, washed in PBT and embedded in OCT (BDH,
Merck). Transverse sections (12 µm) were assayed by TUNEL according to the
manufacturer's protocol. To detect cells in mitosis, a rabbit
anti-phosphorylated histone H3 primary antibody (Upstate Biotechnology) and
Cy3-conjugated goat anti-rabbit IgG secondary antibody (Jackson Laboratory)
were used following the protocol described previously
(Yamada et al., 1993
).
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RESULTS |
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Hindlimb markers are not expressed in the forelimb region in the
absence of Tbx5
Previous studies in the chick have suggested that Tbx5 expressed
in the prospective forelimb region may repress expression of the closely
related gene Tbx4 which is normally restricted to the hindlimb
(Takeuchi et al., 1999). We
were therefore interested to determine whether, in the absence of
Tbx5, hindlimb markers would be ectopically expressed in the forelimb
region. In Tbx5lox/lox;Prx1Cre embryos, Tbx4
expression is detectable in the hindlimb but is not detected in the forelimb
region at E9.5 or E10.5 (Fig.
2I; data not shown). Similarly, Pitx1 expression is
restricted to the hindlimb in the mouse
(Fig. 2J). After deletion of
Tbx5 in the forelimb, Pitx1 expression remains restricted to
the hindlimb and is not expressed in the forelimb region at E9.5 or E10.5
(Fig. 2K; data not shown).
Taken together, these data suggests that Tbx5 does not normally
function to repress the expression of hindlimb markers, Tbx4 or
Pitx1, in the forelimb region as, in the absence of Tbx5,
the expression patterns of Tbx4 and Pitx1 are unaffected.
These results are in agreement with similar studies in which Tbx5
function has been deleted in all cells of the developing embryo
(Agarwal et al., 2003
).
Tbx5 is required for limb bud outgrowth
The absence of any morphological forelimb bud led us to suspect a defect at
earlier stages of limb development. Fgf10 is expressed in the lateral
plate mesoderm (LPM) of the prospective limb field prior to the expression of
Fgf8 in the prospective AER
(Ohuchi et al., 1997).
Fgf10 and other members of the Fgf family, are capable of inducing
ectopic limb bud formation when applied to cells in the interlimb flank
(Cohn et al., 1995
;
Martin, 1998
). The functional
importance of Fgf10 in limb formation was further demonstrated by the
observation that mice carrying a null mutation in Fgf10 fail to form
an AER and pups are born with severely truncated limbs
(Min et al., 1998
;
Sekine et al., 1999
). In the
absence of Tbx5 function, Fgf10 is not expressed in the
prospective forelimb bud mesenchyme by E9.5 (21 somites) (Fig.
3B,D).
Pea3 is an Ets-related transcription factor that is
expressed in the prospective limb mesenchyme
(Chotteau-Lelievre et al.,
2001
) in a complementary pattern to Fgf10
(Fig. 3E). It has been proposed
to mediate the nuclear response to Fgf signalling directly
(Raible and Brand, 2001
) and
it therefore provides a molecular read-out of Fgf signalling. Pea3 is
not expressed in the forelimb region of
Tbx5lox/lox;Prx1Cre embryos by E9.5 (21 somites)
(Fig. 3F) consistent with a
failure of Fgf signalling. Pea3 is also expressed in the
intermediate mesoderm lateral and caudal to the forelimb
(Fig. 3E). This expression
domain is not affected in Tbx5lox/lox;Prx1Cre embryos
(Fig. 3F), indicating that the
effect on Pea3 is limited to the cells of the prospective forelimb
and is not affected at other sites in the developing embryo. TdT-mediated dUTP
nick end labelling (TUNEL) analysis demonstrated that by E9.5 (24 somites)
cells in the prospective forelimb region of
Tbx5lox/lox;Prx1Cre embryos were first detected to be
undergoing an increased extent of apoptotic cell death
(Fig. 3H) when compared with
control embryos at a similar stage (Fig.
3G). By E10.5 the domain of cell death in
Tbx5lox/lox;Prx1Cre embryos was extensive throughout the
forelimb forming region (Fig.
3J) compared with control embryos
(Fig. 3I). These results
demonstrate that as a result of Tbx5 inactivation, Fgf10 is
not expressed and cells of the prospective forelimb subsequently undergo
extensive apoptosis.
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DISCUSSION |
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A striking additional observation of conditional knockout of Tbx5
in limb mesenchyme is the complete absence of all the elements of the
appendicular skeleton. Tbx5 is required for the formation of the
clavicle and scapula of the pectoral girdle in addition to the skeletal
elements of the limb proper. This phenotype is more severe than that observed
in the forelimb of Fgf10-null mice. This indicates that Tbx5
has a broader influence on forelimb development than Fgf10 and is
absolutely required for the formation of all elements derived from the
forelimb lateral plate mesoderm. Although all skeletal elements of the limb
proper and most of the pectoral girdle are formed from lateral plate tissue of
the prospective forelimb region, the proximal portion of the scapula blade is
derived from the somites that lie medial and adjacent to the forelimb
(Burke, 2000;
Huang et al., 2000
). After
limb ablation in the chick, the somite-derived hypaxial myoblasts that form
the limb musculature, but do not express Tbx5, are never recruited to
the limb field (Gumpel-Pinot et al.,
1984
). By extension, the most likely explanation for the loss of
the entire scapula in Tbx5lox/lox;Prx1Cre embryos is that,
after deletion of Tbx5 in the limb mesenchyme and the failure of
early limb bud formation, the somite-derived (and at that stage
non-Tbx5-expressing) scapula precursors are not recruited into the
limb field.
Recently, the requirement of tbx5 for the formation of the
pectoral fin in zebrafish has been demonstrated using morpholino antisense
oligonucleotides to knock down tbx5 function
(Ahn et al., 2002;
Garrity et al., 2002
). This
observation is consistent with work in other species by ourselves and others
(Agarwal et al., 2003
;
Ng et al., 2002
). In one
report (Ahn et al., 2002
), the
authors conclude that the function of tbx5 in the development of the
zebrafish forelimb involves the directed migration of lateral plate mesodermal
cells to the future limb-bud-forming region. However, the results of our
experiments in the chick and mouse do not support a model in which
Tbx5 is involved in directing migration of cells of the prospective
forelimb. In higher vertebrates, cells of the prospective forelimb do not
undergo a similar migration (Saunders et
al., 1957
; Saunders et al.,
1959
; Searls,
1967
; Searls and Janners,
1969
) and Tbx5-expressing cells detected in the
prospective forelimb region are not migratory
(Gibson-Brown et al., 1998
;
Isaac et al., 1998
;
Logan et al., 1998
;
Ohuchi et al., 1998
).
Somite-derived cells that contribute to the limb do not express Tbx5
(data not shown). Furthermore, in Tbx5-null embryos, patterning of
the lateral plate mesoderm at limb levels is intact and only limb bud
outgrowth is affected by constitutive loss of Tbx5
(Agarwal et al., 2003
).
Although our results and those of Ahn et al. are phenotypically similar, our
results demonstrate a different mechanism for Tbx5 action. Although
in lower vertebrates tbx5 may have a role in migration of limb
precursor cells, in higher vertebrates Tbx5 is required to regulate
inductive signalling interactions essential for limb bud initiation and
continued limb outgrowth.
Tbx5 is required for continued limb outgrowth
Our misexpression experiments in the chick demonstrate that Tbx5
is not only required for limb initiation but is also required at later stages
of limb development for continued outgrowth. Knock down of Tbx5
function by misexpression of dominant-negative forms of the protein leads to
the disruption of the induction and maintenance of the AER. This role for Tbx5
can be placed within our current models of limb initiation and outgrowth
(Fig. 6K). Fgf10
expressed in the lateral plate mesoderm is initially required for the
induction of Fgf8 in cells of the nascent AER
(Fig. 6K) (Ohuchi et al., 1997;
Yonei-Tamura et al., 1999
).
Fgf8 expressed by cells of the AER is required, in turn, for the
maintenance of Fgf10 in distal mesenchyme underlying the AER. After
AER formation, a positive feedback loop is established between
Fgf8-expressing cells in the AER and cells of the distal mesenchyme
expressing Fgf10 (Fig.
6K) (Ohuchi et al.,
1997
). Our results are consistent with a requirement for
Tbx5 in the AER-mediated maintenance of Fgf10 expression in
the distal mesenchyme at later stages of development. Disruption of this
epithelial-mesenchymal induction loop would lead to severe truncation of limb
outgrowth and could explain the reduction deformities observed in HOS which,
in the most severe cases, results in an almost complete absence of all
elements of the limb (phocomelia). The more commonly observed characteristic
features of HOS phenotypes are deformities of anterior elements of the limb,
such as the thumb, thenar elements or radius. After knock down of
Tbx5 by misexpression of dominant-negative constructs we observed
defects in AER formation and maintenance primarily in the anterior of the
infected wing bud. Loss of Fgf8 expression is observed in the
anterior AER and a concomitant failure of Fgf-mediated signalling to
the underlying mesenchyme is indicated by the loss of expression of various
markers in the anterodistal mesenchyme. Our chick misexpression protocol
generated a phenotype consistent with characteristic abnormalities presented
in individuals with HOS. The downregulation of these anterior markers
therefore provide a molecular context to understand the genesis of HOS
deformities. Unfortunately, we are unable to analyse the skeletal deformities
that would have resulted from the disruption of gene expression at early limb
bud stages, because continued spread of the retrovirus produces heart defects
that lead to embryonic lethality (data not shown). A direct, positive
regulatory relationship between a T-box gene and an Fgf gene has previously
been demonstrated between brachyury and eFgf during mesoderm
induction in Xenopus
(Schulte-Merker and Smith,
1995
). Our results suggest that this regulatory relationship has
been conserved and reused in the context of the limb. This is consistent with
results demonstrating that Tbx5 binding sites are present in the
promoter of Fgf10 (Agarwal et al.,
2003
; Ng et al.,
2002
). Tbx4, a closely related T-box family member
(Agulnik et al., 1996
), may
play an analogous role to Tbx5 in the hindlimb.
Tbx5 acts as a transcriptional activator
Misexpression of both types of dominant-negative Tbx5 constructs
produced essentially identical results and are consistent with defects
observed because of haploinsufficiency of TBX5 in HOS
(Basson et al., 1997;
Li et al., 1997
) and defects
in the Tbx5 heterozygous knockout mouse
(Bruneau et al., 2001
). In the
converse experiment, misexpression of full-length Tbx5 or constructs
containing the T-domain of Tbx5 fused to the VP16 transcriptional activation
produced identical results. Moreover, the phenotypes observed complement those
observed with dominant-negative constructs. Together, these observations
support the conclusion that Tbx5 is acting as a transcriptional
activator in the forelimb. These results are consistent with reports
demonstrating that Tbx5 can transactivate expression from constructs
containing a region of the Fgf10 promoter
(Agarwal et al., 2003
;
Ng et al., 2002
).
Dual functions for Tbx5
The results of loss-of-function studies in mouse, chick and zebrafish,
combined with previous misexpression studies performed in the chick reveal
dual roles for Tbx5 in limb development. Tbx5 is required to
induce and maintain Fgf10 expression that in turn is necessary for
forelimb initiation and continued outgrowth
(Fig. 6K). Tbx5 also
acts to specify forelimb identity by influencing the response of cells of the
forelimb to common patterning cues
(Takeuchi et al., 1999). As
Tbx5 is required for limb initiation and outgrowth, loss-of-function
approaches have not been informative as to the role of Tbx5 in
specifying limb-type identity. Further studies will be required in which the
requirement for Tbx5 in limb outgrowth can be uncoupled from its role
in specifying limb-type identity.
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ACKNOWLEDGMENTS |
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