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DEVELOPMENTAL DYNAMICS 229:763–770, 2004
RESEARCH ARTICLE
T-Box Transcription Factor Tbx2 Represses
Differentiation and Formation of the Cardiac
Chambers
Vincent M. Christoffels, Willem M.H. Hoogaars, Alessandra Tessari, Danielle E.W. Clout,
Antoon F.M. Moorman,* and Marina Campione
Specific regions of the embryonic heart tube differentiate into atrial and ventricular chamber myocardium, whereas
the inflow tract, atrioventricular canal, inner curvatures, and outflow tract do not. These regions express Tbx2, a
transcriptional repressor. Here, we tested its role in chamber formation. The temporal and spatial pattern of Tbx2
mRNA and protein expression in mouse hearts was found to be complementary to that of chamber myocardiumspecific genes Nppa, Cx40, Cx43, and Chisel, and was conserved in human. In vitro, Tbx2 repressed the activity of
regulatory fragments of Cx40, Cx43, and Nppa. Hearts of transgenic embryos that expressed Tbx2 in the prechamber
myocardium completely failed to form chambers and to express the chamber myocardium-specific genes Nppa,
Cx40, and Chisel, whereas other cardiac genes were normally expressed. These findings provide the first evidence
that Tbx2 is a determinant in the local repression of chamber-specific gene expression and chamber differentiation.
Developmental Dynamics 229:763–770, 2004. © 2004 Wiley-Liss, Inc.
Key words: heart development; chamber formation; chamber differentiation; Tbx2; transcription factor
Received 8 August 2003; Revised 1 October 2003; Accepted 7 October 2003
INTRODUCTION
During development, specific regions of the primary heart tube initiate a chamber myocardium-specific program of gene expression,
which includes the expression of Natriuretic precursor peptide type A
(Nppa), Connexin (Cx) 40, Cx43,
and Chisel (van Kempen et al., 1996;
Delorme et al., 1997; Christoffels et
al., 2000; Palmer et al., 2001). This
myocardium expands and forms the
ventricular and atrial chambers during and after looping. The remainder
of the heart tube, the inflow tract,
atrioventricular canal (AVC), and
the outflow tract (OFT), connected
at the inner curvatures, does not initiate the expression of the chamberspecific genes and does not expand. Instead, it largely retains the
primary heart tube phenotype and
remains lined by endocardial cushions involved in septation of the
heart (Moorman and Christoffels,
2003). Congenital cardiac defects
occur in more than 0.5% of live births,
and often result from abnormal development of these regions (Samanek, 2000), but little is known
about the mechanisms underlying
their formation.
Previous analyses showed that
Nppa regulatory DNA sequences
mediate the repression of gene activity in the AVC and OFT, requiring a
DNA binding site for T-box factors
(Habets et al., 2002, 2003). T-box
transcription factors form a large
family of structurally related factors
that play a crucial role in several developmental processes (Papaioannou and Silver, 1998; Tada and
Smith, 2001). Tbx2 functions as a repressor of transcription (Carreira et
al., 1998; He et al., 1999; Jacobs et
al., 2000) and is able to repress Nppa
promoter activity in vitro (Habets et
Experimental and Molecular Cardiology Group, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
Grant sponsor: The Netherlands Heart Foundation; Grant number: M96.002.
Drs. Tessari’s and Campione’s present address is National Research Council, Institute of Neurosciences, Department of Biomedical
Sciences, University of Padova, Padova, Italy.
*Correspondence to: Antoon F.M. Moorman, Department of Anatomy and Embryology, Meibergdreef 15, 1105 AZ, Amsterdam, The
Netherlands. E-mail: [email protected]
DOI 10.1002/dvdy.10487
© 2004 Wiley-Liss, Inc.
764 CHRISTOFFELS ET AL.
al., 2002). In chicken and mouse embryonic hearts, Tbx2 is expressed
specifically in the AVC and OFT (Gibson-Brown et al., 1998; Yamada et
al., 2000; Habets et al., 2002). Therefore, Tbx2 is a candidate for repressing chamber-specific genes in the
AVC and OFT in vivo.
To investigate the function of Tbx2
in the heart, the temporal and spatial patterns of Tbx2 mRNA and protein expression in mouse and human
hearts were assessed, and transgenic mouse embryos were generated that express Tbx2 in the myocardium fated to become chamber
myocardium. The findings provide
evidence that Tbx2 is a determinant
in the local repression of chamber
differentiation and chamber formation.
RESULTS AND DISCUSSION
Spatial and Temporal
Expression of Tbx2 and
Chamber Markers in the
Developing Mouse Heart
Tbx2 expression was not detected in
the myocardium until E8.75, when a
linear heart tube has formed (Fig.
1A–C). Tbx2 expression was first detected shortly after the onset of
Nppa expression in the embryonic
ventricles. The myocardial expression domain of Tbx2 was broad and
included the inflow tract (IFT), AVC,
and OFT but excluded the ventricular myocardium that expressed
Nppa. Tbx2 expression in the heart
region was restricted to Mlc2a-expressing myocardium, including the
dorsal pericardium. This region is part
of the anterior heart field that will
contribute to the OFT at embryonic
day (E) 10 (Kelly et al., 2001) and is
continuous with the posterior pole of
the heart. Expression continued into
the premyocardial cells lining the inside of the pericardial cavity.
At E9.5, the IFT, AVC, and OFT,
continuous in the atrial and ventricular inner curvatures, expressed Tbx2
(Fig. 1E and not shown). Furthermore, the AV cushions and OFT
cushions expressed Tbx2 in a gradient from lumen to myocardium (Fig.
1E,N, and not shown). The ventricular chamber myocardium and forming atrial chamber myocardium at
the outer curvatures did not express
Tbx2. At all stages (E9.5–E14.5), expression patterns of Tbx2 and chamber-specific genes Nppa, Cx40,
Cx43, and Chisel were mutually exclusive in the myocardium (Fig. 1D–
M). In E9.5, hearts expression of
Chisel and Cx43 was only weak in
the atrial chamber myocardium, but
from E10.5 onward, expression of
these genes here increased (Fig.
1K,M). The expression of Chisel and
Cx43 is retained in both ventricles, in
contrast to expression of Nppa,
Cx40, and Tbx5, which in mouse
gradually disappear from most of
the right ventricle and compact
myocardium of the left ventricle
(Bruneau et al., 1999; Christoffels et
al., 2000). This finding suggests that,
whereas expression of Nppa and
Cx40 strictly depends on Tbx5 (Bruneau et al., 2001), expression of
Chisel and Cx43 does not. Myocardial Tbx2 expression decreased with
further development and was barely
detectable at E15–E16 (not shown).
Taken together, our results show the
expression of Tbx2 and of chamberspecific genes is mutually exclusive
throughout stages of cardiac chamber formation and suggest that Tbx2
may suppress activation of these
genes in the areas of its expression.
The cause for the late activation of
the chamber-specific genes in the
myocardium, 1–3 days after its differentiation from precardiac mesoderm, is not clear. The strict localization of Tbx2 expression itself may, in
part, be regulated by BMP2 and
BMP4 (Yamada et al., 2000), which
in the mouse heart together overlap
the Tbx2 expression domain (Fig.
1N–P).
Expression of TBX2 mRNA and
Protein in the Human Heart
We analyzed the expression of TBX2
and NPPA in hearts of human embryos of 6 and of 12 weeks of development. Figure 2B shows that, at 6
weeks (approximately mouse E12),
TBX2 mRNA was detected in the
myocardium of the AV junction, including the AV node. Additionally,
TBX2 expression was detected in the
epicardial mesenchyme of the AV
sulcus. At 12 weeks, expression was
mainly detected in the mesen-
chyme of the AV sulcus and the
smooth muscle layer of coronary arteries (Fig. 2D), similar to the pattern
in fetal mouse hearts (not shown).
NPPA expression was restricted to
the atrial chamber myocardium
and the ventricular trabeculae, in a
pattern similar to that in mouse (Fig.
2C). As reported (Hatcher et al.,
2000), TBX5 was found to be expressed in the atria, including the AV
node, and weaker in the ventricles
(Fig. 2A). The AV myocardial areas
expressing both TBX5 and TBX2 did
not express NPPA (Fig. 2A–C). Taken
together, we conclude that the expression patterns of TBX2 and NPPA
are conserved between mouse and
human, suggesting a similar role of
TBX2 in heart development in both
species.
We next assessed the pattern of
protein expression in E12.5 mouse
embryos and in human embryos of 9
and 10 weeks of development. A
polyclonal antibody raised against
human TBX2 was used (Jacobs et al.,
2000). In mouse, this antibody also
specifically detects Tbx2, albeit with
a low sensitivity. In mouse, mRNA
and protein expression largely overlapped (Fig. 2E,F). However, some
tissues, such as the mesenchyme of
the ventral body wall, expressed
mRNA, whereas protein was undetectable. Larger magnification shows
that Tbx2 protein was present in the
nuclei, in agreement with its function
as a transcription factor (Fig. 2G,H).
In the heart, the expression was detected in the AVC myocardium and
in the cushion mesenchyme of the
AVC and OFT. In the human heart,
expression of TBX2 was detected in
the nuclei of the myocardium of the
right AV ring bundle, in the AV sulcus
mesenchyme, and in the smooth
muscle layer of the coronary arteries
(Fig. 2I–K). Taken together, the expression of Tbx2 mRNA and protein in
the mouse and human heart display
a comparable pattern.
Tbx2 Represses Cx40 and
Cx43 Promoter Activity
The putative regulatory sequences
of mouse Cx40 (Seul et al., 1997) and
rat Cx43 (Chen et al., 1995) were
coupled to the luciferase reporter
gene and transfected to Cos-7 or
Tbx2 REPRESSES CHAMBER FORMATION 765
Fig. 1. Developmental expression pattern of expression of Tbx2 and of chamber-specific marker genes. A–C: Serial sections of an
embryonic day (E) 8.75 mouse heart. Indicated by arrows are the dorsal pericardium (green arrows) and cells lining the pericardial cavity
at the inflow tract (black arrow). Red arrows indicate borders of expression in the embryonic ventricle. D–I: Serial sections of an E9.5 mouse
heart. Red arrows indicate the expression borders between atrioventricular canal and left ventricle and between the atrioventricular
canal and left atrium. Green arrows indicate inner curvature of the atrial region and the outflow tract. Cx40 and Cx43 are expressed in
the endocardium of the outflow tract (black arrows). J,K: Serial sections of an E14.5 heart. Red arrows show borders of expression in the
atrioventricular canal and in the transition of the right ventricle and outflow tract. L,M: Whole-mount in situ hybridization of E10.5 mouse
hearts showing complementary expression of Tbx2 and Chisel. N–P: Serial sections of an E10.5 mouse heart showing the correlation
between the localized expression of Tbx2 and of genes for upstream regulating factors BMP2 and BMP4. Arrows indicate borders of
expression in inflow tract/atrioventricular canal (red) and outflow tract (green). avc, atrioventricular canal; ev, embryonic ventricle; ift,
inflow tract; la, left atrium; lv, left ventricle; nt, neural tube; oft, outflow tract; ra, right atrium; rv, right ventricle; sh, sinus horn.
766 CHRISTOFFELS ET AL.
Fig. 2. Expression patterns of Nppa, Tbx2, and Tbx5 are conserved in human. A–C: Serial sections of a human heart of approximately 6
weeks of development hybridized with indicated probes. The red arrows indicate the right atrioventricular ring bundle and atrioventricular
nodal region; the green arrows indicate the mesenchymal atrioventricular sulcus. D: A coronary artery (red arrow) and vein (green arrow)
in the atrioventricular sulcus of a human embryo of 12 weeks. E,F: Tbx2 mRNA (E) and protein (F) expression in comparably staged
embryonic day 12.5 mouse hearts. Green arrows indicate the left atrioventricular junction and the outflow tract cushion. Red arrows
indicate the ventral body wall. G,H: Enlargements of the left atrioventricular junction and outflow tract cushion of F (boxes). Note nuclear
staining in the atrioventricular junction myocardium and forming valve and cushion mesenchyme. I,J: Serial section of a human heart at
10 weeks of development, incubated with antibodies as indicated. Note the perinuclear staining of SERCA2 versus the nuclear staining of
TBX2. K: Arrows indicate the endothelial lining (green) and nuclear TBX2 expressing muscle layer (red) of the coronary artery. ravrb, right
atrioventricular ring bundle; vs, ventricular septum; as, atrial septum; lb, lung bud; rp, rib primordium; tr, trachea. Other abbreviations as
in Figure 1.
HEK cells. In both cell lines, Cx40 promoter activity and to a lesser extent
Cx43 promoter activity were repressed by Tbx2 (Fig. 3) but not by a
mutated isoform unable to bind to
DNA (Habets et al., 2002). Tbx2 from
which the putative repressor domain
was deleted was unable to repress
promoter activity, whereas Tbx2
fused to the VP16 activation domain
activated both Cx40 and Cx43 promoter fragments (Fig. 3B). These
data, together with our previous re-
sults that Tbx2 can repress Nppa promoter activity (Habets et al., 2002),
provide in vitro evidence for a role of
Tbx2 in repressing chamber-specific
genes.
Inhibition of Chamber
Formation by Tbx2
The ␤-Mhc promoter, which is specifically active throughout the embryonic heart tube from E8 onward
(Colbert et al., 1997), was used to
drive Tbx2 expression in cardiac cells
that are fated to become chamber
myocardium. After pronuclear microinjection, 11 E9.5 transgenic embryos were identified by polymerase
chain reaction (PCR). Of these, five
embryos were indistinguishable from
nontransgenic littermates and normally expressed Nppa, whereas
they did not express transgenic Tbx2
(n ⫽ 4). The other six embryos were
smaller, their hearts were unlooped
and had the appearance of large
Tbx2 REPRESSES CHAMBER FORMATION 767
Fig. 3. Tbx2 represses the activity of the promoters of the Cx40 and Cx43 genes.
A: Dose-dependent (10 – 400 ng) repression of the Cx40 promoter (4 ␮g) but not of the
CMV promoter (4 ␮g). Tbx2 mutants that have a mutation in the T-box and cannot bind to
the DNA (TBX2-DB) or lack the repression domain (TBX2-RD) do not repress Cx40 expression. Wild-type and mutant TBX2 are equally well expressed (Habets et al., 2002).
B: Promoters of Cx40 and Cx43 are repressed by TBX2 and stimulated by VP16-TBX2 fusion
protein. DNA binding mutants of both TBX2 and VP16-TBX2 do not affect promoter activity.
Results are from a representative experiment of three done in duplicate. Bars show the
difference between duplicates.
linear heart tubes of E8.5 embryos,
with a putative common atrium
caudal to a single putative ventricle
(Fig. 4A,D). The cardiac cavity was
enlarged, indicative for hemodynamic failure. The hearts were much
more affected than other structures
such as the neural tube and forelimb
(Fig. 4A). Even though the heart
tubes were small, the anterior ventricular portion of the heart tube was
much thicker than the future ventricular portion of an E8.5 heart, indicating that proliferation of the myocytes of the ventricular wall had
occurred. Freshly isolated fetal cardiomyocytes expressing Tbx2 from a
transfected CMV promoter driven
expression vector divided and incorporated bromodeoxyuridine, but
did not go into apoptosis (not
shown), indicating that high levels of
Tbx2 allow proliferation but do not
induce apoptosis in cardiac myocytes.
Expression analysis on serial sections showed that the affected embryos expressed Tbx2 in the heart
tube (Fig. 4; n ⫽ 5). Mlc2a was expressed in the entire tubular heart
(n ⫽ 3) (Fig. 4B), whereas ␣-Mhc,
␤-Mhc, and Mlc2v were expressed in
an anteroposterior pattern (n ⫽ 3)
that is normally found in E9.5 em-
bryos (Fig. 4G–I; Lyons et al., 1990;
O’Brien et al., 1993; Christoffels et al.,
2000). These data suggest that cardiomyocyte differentiation was relatively normal and anteroposterior
patterning had occurred. In contrast
Nppa gene expression was not detected in the hearts (Fig. 4K–N; n ⫽
4), except in one embryo in a few
myocytes in a patch where no transgenic Tbx2 was observed (Fig. 4E,F).
Also expression of Cx40 and Chisel
were undetectable in the hearts
(Fig. 4C and not shown; n ⫽ 3). Cx40
expression was observed in the endocardium of the OFT and the endothelium of the pharyngeal arch
arteries and the dorsal aorta (Fig.
4C), indicating that repression of the
Cx40 gene was myocardium specific. Because myocardial Cx43 expression is not detectable before
E9.5 in normal embryos (van Kempen et al., 1996; Delorme et al., 1997;
not shown), its expression was expected to be absent in the cardiac
developmentally retarded transgenic embryos (confirmed in one
embryo), and was not further analyzed. We conclude that Tbx2 specifically represses chamber-myocardial genes and, possibly as a
consequence, inhibits chamber differentiation and formation and
looping in the IFT, AVC, inner curvatures, and OFT in vivo, allowing these
regions to retain their primary phenotype longer and to contribute to
chamber-alignment, valve-formation, septation, and to form conduction system components (Davis et
al., 2001; Rentschler et al., 2001;
Moorman and Christoffels, 2003).
The repression of chamber differentiation may well be temporal. The
primary myocardium by definition is
the precursor for all types of myocardium found in the mature heart and
may differentiate further at any
given time. The observed decrease
of myocardial Tbx2 expression during the fetal period coincides with
the “chamberization” of the embryonic OFT (which is largely incorporated into the right ventricle) and
with the initiation of some chamber
genes such as Cx40 expression in the
bundle branches and AV bundle
late in development. Regions that
retain aspects of the primary myocardial phenotype, including the si-
768 CHRISTOFFELS ET AL.
Fig. 4. Expression of Tbx2 in the embryonic heart tube inhibits chamber formation and chamber-specific gene expression. A: Wild-type
(WT) and transgenic (TG) embryonic day 9.5 littermates. B: Mlc2a is expressed in cardiac myocytes. C: Expression of Cx40 is detected in
endothelium of the large arteries (arrows). D: Enlargement of TG heart from A showing small and unlooped heart and large pericardial
cavity (arrowheads). Lines indicate sections shown in E–J. E–J: Sections were hybridized with probes indicated in the panels. A patch of
Nppa expression is observed in the putative ventricular region (arrowhead, E) that does not express transgenic Tbx2 (arrowhead, F).
Green arrows in D, G, and H indicate the putative common atrium. K–N: Sagittal serial sections of a WT embryo (K,L) and a TG littermate
(M,N) analyzed in the same in situ hybridization experiment. Note the absence of Nppa expression in the entire linear heart tube of the
TG. ca, common atrium; a, atrium; fg, foregut; fl, forelimb; pc, pericardial cavity; nt, neural tube. Other abbreviations as in Figure 1. Scale
bar ⫽ 100 ␮m in K (applies to K–N).
noatrial and atrioventricular node
and atrioventricular junction, express Tbx3 throughout development and in the adult (our unpublished observations), which is the
structural and functional homo-
logue of Tbx2 (Agulnik et al., 1996;
He et al., 1999; Lingbeek et al.,
2002; Coll et al., 2002). Future investigations will be focused on the role
of Tbx2 and Tbx3 in the repression
of chamber differentiation and
conduction system formation, and
on the identification of Tbx2 target
genes in the developing heart that
are candidates for regulation of
chamber differentiation, growth,
and looping.
Tbx2 REPRESSES CHAMBER FORMATION 769
EXPERIMENTAL PROCEDURES
Generation of Transgenic
Mouse Embryos
The mouse Tbx2 coding sequence,
kindly provided by Dr. Jung-Ren
Chen (Chen et al., 2001) and the
human TBX2 coding sequence (Jacobs et al., 2000) were cloned in the
HpaI site between a 5-kb regulatory
fragment of the ␤-Mhc gene and
human growth hormone polyadenylation sequences (kindly provided
by Dr. J. Robbins; Rindt et al., 1993;
Colbert et al., 1997), and vector sequences were removed for microinjection into paternal nuclei of FVB
zygotes. Embryos were isolated at
stage E9.5, fixed in 4% paraformaldehyde in PBS, examined morphologically, and processed further for
in situ hybridization.
Tissue Processing for In Situ
Hybridization and
Immunohistochemistry
Staged mouse embryos obtained
from timed mated FVB mice. Human
embryos were obtained after termination of pregnancy at the Academic Medical Center of Amsterdam, at the “MR70/Rutgersstichting,”
Amsterdam, and at the Postgraduate
Medical School in Budapest. The respective local medical– ethical committees approved the studies. Embryos were fixed overnight in 4%
paraformaldehyde in PBS and processed for nonradioactive section in
situ hybridization or in a cold mixture
of methanol, acetone, and water (40:
40:20) for immunohistochemistry.
In Situ Hybridization and
Immunohistochemistry
Nonradioactive section in situ hybridization was performed on 14-␮m
serial sections as described in (Moorman et al., 2001). In vitro transcribed
RNA probes complementary to
Nppa, Cx40, Cx43, Chisel, Mlc2a,
Tbx2, NPPA, TBX2, TBX5 are described (Chapman et al., 1996; Basson et al., 1999; Christoffels et al.,
2000; Habets et al., 2002). For immunolocalization of Tbx2, 7-␮m sections
were incubated with rabbit antiTBX2 IgG (Jacobs et al., 2000) or rab-
bit anti-SERCA2 (Eggermont et al.,
1990). Bound antibody was detected with alkaline phosphatasecoupled secondary antibody and
NBT/BCIP (Roche).
DNA Constructs, Cell Culture,
and Transfections
A 1.2-kb mouse Cx40 upstream regulatory region, from ⫺1,196 to ⫹62
relative to the transcription start site
(Seul et al., 1997), was obtained from
FVB mouse genomic DNA by PCR. A
1.6-kb rat Cx43 promoter fragment
(position, ⫺1,338 to ⫹281) was kindly
provided by Drs. B. Teunissen and M.
Bierhuizen. Both fragments were
cloned into pGL3-Basic (Promega).
pcDNA3.1 (Invitrogen)-based expression vectors for Tbx2, VP16-Tbx2,
Tbx2-DB, and VP16-Tbx2-DB were described previously (Habets et al.,
2002). Cos-7 cells were cultured in
standard conditions in DMEM/F12
(Gibco) supplemented with 10% fetal calf serum and were plated at
0.15 ⫻ 106 cells/well in six-well plates.
Cells were transfected with 4 ␮g of
Cx40 or Cx43 promoter reporter construct; 400 ng of Tbx2-DB, Tbx2-RD, or
VP16-Tbx2 expression constructs; or
10 – 400 ng of Tbx2 expression construct, using the calcium phosphate
method. Furthermore, 200 ng of
CMV-lacZ was transfected as an internal control. Cells were harvested
48 hr after transfection, and luciferase and ␤-galactosidase assays
were carried out by using a Turner
TD-20/20 luminometer (Promega).
ACKNOWLEDGMENTS
We thank Drs. M. van Lohuizen, V.
Papaioannou, C. Basson, J. Robbins,
J. Chen, B. Teunissen, and M. Bierhuizen for antibody, probes, and plasmids, and the Amsterdam center for
sexual and reproductive health
“MR70/Rutgersstichting” for human
embryonic tissues. We also thank Dr.
M.A. van Roon from the Genetically
Modified Mice facility, AMC, for generating transgenic embryos, and C.
de Gier-de Vries and P.A.J. de Boer
for excellent technical assistance.
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