1 Department of Pediatrics, University of Texas Southwestern Medical Center,
Dallas, TX 75390-9148, USA
2 Department of Department of Molecular Biology, University of Texas
Southwestern Medical Center, Dallas, TX 75390-9148, USA
3 Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi,
Shinjuku-ku, Tokyo 160-8582, Japan
Author for correspondence (e-mail:
deepak.srivastava{at}utsouthwestern.edu)
Accepted 13 August 2004
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SUMMARY |
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Key words: Tbx1, 22q11.2 deletion syndrome, Anterior heart field, Foxa2, Fgf8, Fgf10
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Introduction |
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The pharyngeal apparatus is a transient vertebrate-specific complex that
arises from a series of bilaterally symmetric bulges on the ventrolateral
surface of the head and neck region, namely the pharyngeal arches. The arches
develop in a cranial-caudal sequence under the control of clustered homeobox
(Hox) genes and are composed of a number of different embryonic cell types
(Graham, 2003;
Graham and Smith, 2001
).
Distinct lineages within the pharyngeal arches serve as precursors to a wide
variety of cardiac and craniofacial structures, including those derived from
pharyngeal endoderm, ectoderm, mesoderm and neural crest mesenchyme. Paracrine
signaling between each group of cells reciprocally regulates cell fate,
proliferation and death decisions. Arterial vessels arising from the heart
traverse symmetrically through each pharyngeal arch and are ultimately
patterned along the cranial-caudal axis in an intricate fashion to form the
mature aortic arch and pulmonary artery. Neural crest-derived cells are
involved in the patterning of these vessels and in septation of the single
cardiac outflow tract into two distinct vessels
(Yutzey and Kirby, 2002
).
A more recently recognized lineage involving the pharyngeal arches is the
anterior or secondary heart field
(Mjaatvedt et al., 2001;
Waldo et al., 2001
;
Kelly et al., 2001
). In
contrast to the atrial and ventricular chambers that arise from lateral
cardiac mesoderm progenitors, the most anterior pole of the heart comprising
the outflow tract and part of the right ventricle is added later by mesodermal
cells in the pharyngeal region that lie anterior and dorsal to the heart tube.
The anterior heart field cells are thought to migrate into the outflow tract
through the pharyngeal mesoderm, which also contributes to facial muscles
(Lu et al., 2002
), and
differentiate into myocardium and endocardium. The regulatory processes that
govern its development remain largely unknown, although fibroblast growth
factor 10 (Fgf10) and the transcription factor Islet1 mark these cells and are
involved in their development (Kelly et
al., 2001
; Cai et al., 2004).
Because the cardiac outflow tract is the predominant region affected in
22q11DS, this syndrome may afford an opportunity to understand development of
the anterior heart field and its role in the outflow tract. Tbx1, a member of
the T-box-containing family of transcription factors, is the most likely
candidate gene within the 22q11 locus responsible for the pharyngeal
arch-derived defects observed (Lindsay et
al., 1999; Lindsay et al.,
2001
; Merscher et al.,
2001
; Schinke and Izumo,
2001
). Mice heterozygous for targeted deletion of Tbx1
have a low penetrance (25%) of aortic arch patterning defects, but little
evidence of other features of 22q11DS
(Lindsay et al., 2001
;
Merscher et al., 2001
). By
contrast, homozygous-null mutants of Tbx1 have most features of
22q11DS, including absence of thymus, cleft palate, ear defects and
characteristic cardiac outflow tract anomalies
(Jerome and Papaioannou,
2001
). Correspondingly, Tbx1 is expressed in the
pharyngeal arch endoderm, mesodermal core, anterior heart field and head
mesenchyme, although it is conspicuously absent in the neural crest-derived
mesenchyme (Chapman et al.,
1996
; Garg et al.,
2001
; Yamagishi et al.,
2003
). We have previously shown that Tbx1 is regulated by
a sonic hedgehog signaling cascade that is directly mediated by a conserved
Fox-binding site upstream of Tbx1 that directs Tbx1
expression in the pharyngeal endoderm and head mesenchyme
(Yamagishi et al., 2003
).
However, the cell types through which Tbx1 functions, its downstream targets
and its regulation in the pharyngeal mesoderm/anterior heart field remain
unknown.
As Tbx1 is a dose-dependent gene and the dose can probably be affected by genetic and environmental modifiers, we have generated an allelic series of Tbx1 deficiency in an attempt to recapitulate more accurately the human del22q11 phenotype and to determine the domains of Tbx1 expression that regulate crucial developmental events. The data presented here suggest that development of the cardiac outflow tract is more sensitive to Tbx1 allelic dose than the craniofacial region, and that this may be due to a reinforcing autoregulatory loop involving the transcription factor, Foxa2, in the pharyngeal mesoderm precursors of the outflow tract. Tbx1 transcripts were selectively downregulated in the pharyngeal mesoderm but not endoderm of Tbx1 hypomorphic mutants. We also found that Foxa2 transcription was diminished specifically in the pharyngeal mesoderm of the Tbx1 hypomorphic mutant and that a Fox site was essential for regulation of Tbx1 in the pharyngeal mesoderm, suggesting that a Tbx1-Foxa2 pathway amplifies Tbx1 expression in the mesoderm. A genome-wide search for Tbx1 targets in the heart identified a cardiac outflow tract enhancer sufficient for fibroblast growth factor 8 (Fgf8) expression that was regulated by Tbx1 in vivo. Moreover, misexpression of Tbx1 in the developing heart resulted in expansion of the outflow tract, as marked by the secondary heart field marker, Fgf10. These findings demonstrate an essential role for Tbx1 in regulating the pharyngeal mesoderm through an amplification loop involving forkhead proteins and culminating in regulation of fibroblast growth factors in the cardiac outflow tract, consistent with the increased allelic dose sensitivity to Tbx1 in the outflow tract compared to the craniofacial region.
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Materials and methods |
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RT-PCR
Total RNA was isolated from E10.0 embryos with Trizol reagent (Gibco BRL)
according to the manufacturer's instruction. Total RNA (1 µg) was
reverse-transcribed with M-MuLV reverse transcriptase by a random primer
labeling method (Roche). RT product (2 µl) was amplified with primers
5'-GCGCTGTGGGACGAGTTCAATCAG-3' (from exon 3) and
5'-GCACAAAGTCCATGAGCAGCATGTAGTC-3' (from exon 4) for 36 cycles to
detect Tbx1 cDNA, or with primers
5'-ACCACAGTCCATGCCATCAC-3' and
5'-TCCACCACCCTGTTGCTGTA-3' for 30 cycles to detect GAPDH control
cDNA.
Cartilage and skeletal preparation
Standard methods were followed for Alcian Blue cartilage preparations and
Alizarin Red for skeletal preparations.
Radioactive section in situ hybridization
35S-labeled antisense riboprobes were synthesized with T3, T7 or
SP6 RNA polymerse (MAXIscript in vitro transcription kit, Ambion, Austin, TX)
from capsulin, Dlx2, Foxc1, Foxc2, Bmp7, Hand2, Et1, Fgf10, Foxa2, Nkx2.6,
Pax1, Pax9, Tbx1 or Fgf8 cDNAs. Radioactive section in situ hybridizations
were performed on paraffin wax-embedded sections of E9.25 to E10.5 wild type,
hypomorphic mutant (Tbx1neo/neo), null mutant
(Tbx1-/-) or compound mutant
(Tbx1neo/) mouse embryos as previously described
(Thomas et al., 1998). Sources
of DNAs for making probes were as follows: Foxc1, Foxc2, Foxa2 and Tbx1
(Yamagishi et al., 2003
);
Dlx2, Hand2 and Et1 (Thomas et al.,
1998
); Fgf8 (Meyers and
Martin, 1999
); Fgf10 (Kelly et
al., 2001
); capsulin (Lu et
al., 2002
); Bmp7, Nkx2.6, Pax1 and Pax9 full-length cDNAs were
cloned by RT-PCR amplification from mouse E9.5 RNA. In situ hybridization of
probes shown were performed at least three times on different embryos.
Generation of transgenic mice
A 5.4 kb DNA fragment (5406/18) upstream of the Fgf8 start
codon was amplified from human genomic DNA by PCR using primers
5'-CCTGTGCTGGGTGATGTTTCCCTAG-3' and
5'-ACCGAGAGCCCGGCGGGTCACGC-3'. This fragment and its derivatives
were cloned into an hsp68-lacZ reporter construct
(Kothary et al., 1989).
Mothers were sacrificed at E9.5 and F0 embryos were stained for
ß-galactosidase (ß-gal) activity. Stable transgenic lines were
established with a minimal 0.9kb (5406/4510) fragment. Potential
Tbx-binding sites within this region included the following sequences:
GGTGGGA, 5208/5202; TCAGCACT, 5149/5143; TGTGAGG,
4948/4942; GGTGACA, 4925/4919; GGTGGCC,
4902/4896. Similar studies were performed with genomic DNA
upstream of Tbx1. Site-directed mutagenesis was used to create point
mutations of Fox site upstream of Tbx1 as described previously
(Yamagishi et al., 2003
).
Tbx1 cDNA was cloned downstream of the ß-myosin heavy chain promoter and linearized plasmid was injected into pronuclei to generate F0 transgenic embryos with Tbx1 expression throughout the embryonic myocardium.
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Results |
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Similar to the palate, most derivatives of the pharyngeal arches, other than those described above, were not as sensitive to the dose of Tbx1 as was the cardiovascular system. Cartilage and skeletal preparation from neonatal animals showed that first pharyngeal arch derivatives, including maxillary, zygomatic and temporal bones appeared grossly normal in hypomorphic mutants (Tbx1neo/neo) when compared with wild-type or Tbx1neo/+ littermates (Fig. 3J-L). By contrast, these structures were condensed and abnormal in null mutants (Tbx1-/-). The second, third, fourth and sixth pharyngeal arches form various cartilage structures in the neck region, such as hyoid bone, thyroid cartilage and cricoid cartilage. In Tbx1neo/neo mutants, all neck cartilage structures developed normally with the exception that a component of the hyoid bone was missing. By comparison, the cartilages of the neck were small and fragmentary structures in Tbx1-/- mutants (Fig. 3J-L). Thus, most pharyngeal arch mesenchyme derivatives were not affected by reduction of Tbx1 dose, resulting from the hypomorphic allele.
Tissue-specific effects of Tbx1 alleles on Tbx1 and Foxa2 transcript levels
To understand the mechanisms through which Tbx1 hypomorphic
alleles affected some tissues more than others, we examined the Tbx1
transcript level in specific expression domains, including the head
mesenchyme, pharyngeal mesoderm and pharyngeal endoderm, as previously
described (Garg et al., 2001).
Coronal sections of E9.5 Tbx1neo/neo embryos revealed that
Tbx1 expression was nearly abolished in the outflow precursors of the
pharyngeal mesoderm and reduced in head mesenchyme, but was remarkably
conserved in pharyngeal endoderm. These observations were consistent with the
tissue-specific allelic dose-sensitivity described above and may explain the
more severe cardiovascular dose-sensitivity of Tbx1 hypomorphs. As
expected, Tbx1 expression was undetectable in
Tbx1-/- embryos (Fig.
4A-C).
|
Because Tbx1 expression in the pharyngeal mesoderm was selectively
extinguished in the Tbx1neo/neo mice, we investigated the
effects on other genes expressed in this cell type. Expression of the
pharyngeal mesoderm marker, capsulin 1 (Lu
et al., 2002), indicated that pharyngeal mesodermal cells were
present in the Tbx1 hypomorph and null embryos
(Fig. 4J-L). Upon examination
of numerous other candidate genes, we found that Foxa2, which encodes
a forkhead-containing transcription factor, was expressed at high levels in
the pharyngeal mesoderm in addition to its known endodermal expression.
However, in the Tbx1 hypomorph and in the null mutants,
Foxa2 was selectively downregulated in the pharyngeal mesoderm and
head mesenchyme, but not in the endoderm
(Fig. 4M-O). These results are
consistent with the more prominent cardiac outflow tract phenotype in the
Tbx1 hypomorphic state as the pharyngeal mesoderm cells give rise to
the most anterior pole of the heart comprising the outflow tract.
Foxa2 regulates Tbx1 in the pharyngeal mesoderm
Our group has previously demonstrated that the forkhead-containing
transcription factors Foxc1 and Foxc2 directly regulated Tbx1 in the
head mesenchyme, and that Foxa2 regulated Tbx1 expression in the
pharyngeal endoderm (Yamagishi et al.,
2003). However, we had not isolated any specific cis elements that
consistently regulated Tbx1 in the pharyngeal mesoderm or cardiac
outflow tract. Because Tbx1 expression was selectively downregulated
in the pharyngeal mesoderm of Tbx1 hypomorphic mice, and due to the
importance of this cell type in cardiac outflow tract development, we sought
to determine the mechanism through which Tbx1 regulation might be
selectively affected in the mesoderm. A 12.8 kb genomic fragment extending
14.3 kb upstream of the translational start site of Tbx1 is capable
of recapitulating all of the Tbx1 expression domains including the mesoderm
(Fig. 5A,B)
(Yamagishi et al., 2003
). We
generated a series of genomic fragments within the 12.8 kb region upstream of
a basal promoter and a lacZ reporter and injected these fragments
into pronuclei to search for a Tbx1 mesoderm enhancer
(Fig. 5). Using VISTA software
to compare genomic regions conserved across species, we identified regions of
high homology between human and mouse that contained conserved cis elements.
One of these regions, encompassing 1.5 kb, could direct pharyngeal mesoderm
and cardiac outflow tract expression (Fig.
5E-H), but only in conjunction with the previously described 200
bp fragment containing a Fox site essential for endoderm and head mesenchyme
expression (Yamagishi et al.,
2003
). A point mutation of the Fox site in the context of the 200
bp and 1.5 kb fragments ablated the endodermal and head mesenchyme expression,
and also disrupted pharyngeal mesoderm and outflow tract expression
(Fig. 5). These results suggest
that the Fox site in the distal enhancer is necessary for directing pharyngeal
mesoderm expression, but requires a second enhancer for high-level expression
in this domain. By electromobility shift assay, Foxa2 and Foxc1/2 can bind and
activate transcription through this site
(Yamagishi et al., 2003
).
Because we show here that Foxa2 is expressed in the pharyngeal mesoderm and is
downregulated in the Tbx1 hypomorphic mutant, it may function in a
reinforcing loop that amplifies Tbx1 expression in the pharyngeal
mesoderm but not endoderm, potentially explaining the lower levels of
Tbx1 transcripts in the pharyngeal mesoderm of the hypomorphic
embryos and the more dose-sensitive phenotype in mesodermal derivatives.
Whether Foxc1/2 can play such a role in the outflow tract or even in the
pharyngeal mesoderm remains to be determined.
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Tbx1 regulation of the anterior heart field
Because Tbx1 was expressed in splanchnic mesoderm that gives rise
to anterior heart field cells as early as the cardiac crescent stage
(Yamagishi et al., 2003), and
Tbx1 later regulated Fgf8 in the outflow tract derived from this
domain, we tested whether Tbx1 was required for Fgf8 or
Fgf10 expression in the anterior heart field cells dorsal to the
heart in the pharyngeal region. By sagittal sectioning, we found that
Tbx1 expression was decreased in the anterior heart field in the
Tbx1neo/neo background
(Fig. 7A). Fgf8 and
Fgf10 expression was detected in this domain but was selectively
diminished in the Tbx1neo/neo background
(Fig. 7B,C, arrowheads)
consistent with Tbx1-mediated regulation of Fgf genes in the anterior heart
field and cardiac outflow tract. Similar results were observed in
Tbx1-/- mice (data not shown). To test in vivo whether
Tbx1 was sufficient to activate Fgf gene expression, we mis-expressed
Tbx1 throughout the developing mouse myocardium using the
ß-myosin heavy chain promoter, disrupting its normal restriction to the
cardiac outflow tract myocardium. Tbx1 mis-expressing mice were found
to have an elongated outflow tract at E10.5, suggesting that Tbx1
misexpression resulted in an expansion of outflow tract myocardium or
reprogramming of ventricular cardiomyocytes into outflow tract cells. Analysis
of the elongated outflow tract using Fgf8 and Fgf10, as a marker of the
anterior heart field revealed that this expanded domain was indeed
characteristic of anterior heart field cells
(Fig. 7C; data not shown).
Disruption of cardiac development induced by Tbx1 misexpression resulted in
embryonic death precluding examination of the effects on outflow septation or
alignment.
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Discussion |
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Cardiovascular dose-sensitivity to Tbx1
Based on the near complete penetrance of persistent truncus arteriosus in
Tbx1neo/neo embryos but 0% incidence of cleft palate or
absent ears, the cardiovascular system appeared to be more sensitive to the
dose of Tbx1 than did parts of the craniofacial region. Aortic arch
patterning was even more sensitive, with defects observed in
Tbx1neo/+ mice. This is consistent with clinical
observations where 75% of 22q11DS have cardiac defects but only 30% have
cleft palate. However, our work could suggest that the embryonic regions may
not be differentially sensitive to Tbx1 mRNA levels but rather more
sensitive to some allelic alterations. The evidence for this comes from the
observation that the Tbx1 mRNA levels were not equal in the various
expression domains of Tbx1neo/neo mice but rather was more
severely affected in the pharyngeal mesoderm. This would explain the more
severe phenotype in pharyngeal mesoderm-derived cells, such as the anterior
heart field cells of the cardiac outflow tract. Based on these data, it is
possible that in humans, heterozygosity of TBX1 results in
differential levels of TBX1 mRNA levels in its diverse expression
domains, thereby resulting in varying incidence of disease in specific organs
with or without genetic modifiers or stochastic events.
Pharyngeal mesoderm transcription of Tbx1 is affected by a Fox-mediated autoregulatory loop
The selective mesoderm decrease in Tbx1 mRNA levels described
above was an unexpected but interesting result. The expression of
Foxa2 in the pharyngeal mesoderm was also a surprise as we and others
have considered Foxa2 a more endoderm-specific gene, at least in the
pharyngeal arches. The observation that Foxa2 was downregulated in
the pharyngeal mesoderm but not endoderm of Tbx1neo/neo
mice provided an opportunity to understand the mechanism that might
potentially explain the mesoderm-specific decrease in Tbx1 mRNA in
Tbx1neo/neo mice. Because we show that a Fox site is
necessary for pharyngeal mesoderm and cardiac outflow tract expression of
Tbx1 in transgenic mice, it is possible that Foxa2 may be the
trans-acting factor that regulates Tbx1 in the pharyngeal mesoderm.
Evidence to support this is that Foxa2 can bind the essential Fox site and
activate transcription through it
(Yamagishi et al., 2003). In
addition, Foxa2 expression in the mesoderm overlaps Tbx1
more closely than any other Fox protein to our knowledge. Unfortunately,
Foxa2 mutant mice die very early, prior to a time at which we could
test whether Foxa2 is necessary for Tbx1 transcription in the
pharyngeal mesoderm (Ang and Rossant,
1994
; Weinstein et al.,
1994
). Therefore, we must still consider the possibility that
other forkhead-containing proteins might be involved in Tbx1
regulation in this domain. For example, Foxc2 is expressed in the
cardiac outflow tract (Winnier et al.,
1999
; Kume et al.,
2001
) and could also be regulating Tbx1 in this region as
we have previously shown in the head mesenchyme
(Yamagishi et al., 2003
).
Nevertheless, given the current data, it is a reasonable hypothesis that the
more severe effects on Tbx1 mesoderm transcript level in
Tbx1neo/neo embryos may be due to disruption of an
amplifying loop involving forkhead proteins.
We had previously reported a Fox site that was necessary and sufficient to
direct Tbx1 expression in the pharyngeal endoderm and head mesenchyme
(Yamagishi et al., 2003). Upon
searching for a mesoderm enhancer, we were unable to identify a separate
genomic region that was sufficient for Tbx1 mesoderm expression.
However, we had observed on rare occasion that the endoderm and head
mesenchyme enhancer could direct very low levels of Tbx1 expression
in the pharyngeal mesoderm (H.Y. and D.S., unpublished). The observation here
that the previously reported Fox site was necessary but not sufficient for
pharyngeal mesoderm expression suggests that it is a weak but essential
enhancer for this domain of expression. Because Fox proteins can be involved
in chromatin relaxation events, it is possible that the necessary Fox site
affects access to other critical cis elements
(Cirillo et al., 2002
). We were
able to identify a 1.5 kb conserved fragment of genomic DNA that was alone
insufficient, but was necessary with the Fox site to confer pharyngeal
mesoderm expression. We do not yet know the precise cis element within this
region or the trans-acting factor that is necessary to collaborate with the
Fox site for this purpose, but it will be an important area of future study.
Nevertheless, it is striking that a single Fox site upstream of Tbx1
is necessary for all of the domains of Tbx1 expression directed by
the 12.8 kb upstream genomic DNA.
Tbx1 regulation of Fgfs in the anterior heart field
Our finding that Fgf8 was downstream of Tbx1 was consistent with
previous reports of a connection between these two genes. In particular,
Fgf8 hypomorphs partially phenocopy Tbx1 mutants
(Abu-Issa et al., 2002;
Frank et al., 2002
); mice
trans-heterozygous for Tbx1 and Fgf8 have more severe aortic
arch defects than either alone (Vitelli et
al., 2002
); Fgf8 has been reported to be downregulated in
the pharyngeal endoderm of Tbx1 mutants; and tissue-specific deletion
of Fgf8 in the Tbx1 expressing domain results in cardiac outflow
tract defects (Brown et al.,
2004
). The Fgf8 enhancer we described is the first in
vivo regulatory region reported for Fgf8 and includes an enhancer
that directs Fgf8 expression in cardiac outflow tract cells derived
from the anterior heart field. An Fgf8 intronic region regulated by
engrailed and Pbx1 was previously reported to have activity in vitro and in
embryoid bodies, but its ability to regulate activity in vivo in specific
domains had not been studied (Gemel et
al., 1999
). In our hands, the intronic enhancer was not sufficient
to direct expression of lacZ in transgenic mice.
The observation of outflow tract expression directed by the Fgf8
enhancer was interesting but unexpected, and led to more careful detection of
Fgf8 transcripts in the outflow tract by in situ hybridization. Still, we
considered whether this domain of expression for the enhancer may represent
outflow tract-specific expression of the neighboring gene Npm3.
However, this is unlikely because of the ubiquitous expression pattern of
Npm3 (MacArthur and Schackleford,
1997). Consistent with our data is the observation that
lacZ knocked into the Fgf8 locus is also expressed in the
cardiac outflow tract (E. Meyers, personal communication). In the
Tbx1 mutant background, this enhancer was not fully activated,
suggesting that Fgf8 requires Tbx1 for regulation in the outflow
tract. Together, the evidence supports an interpretation that there are
normally low levels of Fgf8 transcripts in the cardiac outflow tract
and that the enhancer described normally directs gene transcription in this
domain in a Tbx1-dependent fashion.
Somewhat surprisingly, we did not observe any downregulation of
Fgf8 in the pharyngeal endoderm, as previously reported. This is
consistent with the observation that deletion of Fgf8 only in the pharyngeal
endoderm or ectoderm does not cause cardiac outflow tract defects
(Macatee et al., 2003). Thus,
while Tbx1 and Fgf8 do appear to be in common pathways, their collaboration
may be most important in the cardiac outflow tract rather than endoderm.
Unfortunately, we have been unable to definitively determine if Fgf8
is a direct target of Tbx1 in vivo. There are at least five predicted core
Tbx-binding sequences in the 0.9 kb enhancer of Fgf8 (see Materials
and methods for sequences) and mutation of each individually did not affect
lacZ expression in transgenic mice nor did tandem mutation of up to
three of the sites (T.H. and D.S., unpublished). Although Fgf8 may
indeed be a direct target of Tbx1, convincing in vivo evidence remains
elusive.
Finally, a role for Tbx1 regulation of anterior heart field/cardiac outflow tract gene expression was demonstrated by misexpressing Tbx1 in cardiomyocytes that do not normally express Tbx1. By doing so, we found that Fgf10, a marker of the anterior heart field, was upregulated and the outflow tract appeared elongated. This could be due to Tbx1-mediated upregulation of Fgf10 in the ventricular myocytes, or a result of conversion of ventricular myocytes into cells more characteristic of the outflow tract that are derived from the anterior heart field. Although it is difficult to discern between these possibilities, it is clear that Tbx1 plays an essential role in the cardiac outflow tract and, together with its role in the pharyngeal mesoderm, makes it a likely regulator of cardiomyocytes derived from the anterior heart field.
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Note added in proof |
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ACKNOWLEDGMENTS |
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Footnotes |
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