Developmental Biology 211, 100 –108 (1999)

Article ID dbio.1999.9298, available online at http://www.idealibrary.com on

Chamber-Specific Cardiac Expression of Tbx5 and

Heart Defects in Holt–Oram Syndrome

Benoit G. Bruneau,* ,1 Malcolm Logan,* ,1 Nicole Davis,*

Tatjana Levi,* ,† Clifford J. Tabin,* J. G. Seidman,* ,†
and Christine E. Seidman* ,‡
*Department of Genetics, and †Howard Hughes Medical Institute, Harvard Medical School,
200 Longwood Avenue, Boston, Massachusetts 02115; and ‡Howard Hughes Medical Institute
and Cardiovascular Division, Brigham and Women’s Hospital and Harvard Medical School,
75 Francis Street, Boston, Massachusetts 02115

To further define the role of a T-box transcription factor, Tbx5, in cardiac development, we have examined its
expression in the developing mouse and chick heart and correlated this pattern with cardiac defects caused by human
TBX5 mutations in Holt–Oram syndrome. Early in the developing heart, Tbx5 is uniformly expressed throughout the
entire cardiac crescent. Upon formation of the linear heart tube, Tbx5 is expressed in a graded fashion, stronger near
the posterior end and weaker at the anterior end. As the heart tube loops, asymmetric Tbx5 expression continues; Tbx5
is expressed in the presumptive left ventricle, but not the right ventricle or outflow tract. This pattern of expression
is maintained in more mature hearts. Expression in the ventricular septum is restricted to the left side and is
contiguous with left ventricular free wall expression. Trabeculae, vena cavae (inferior and superior), and the atrial
aspect of the atrioventricular valves also express high levels of Tbx5. These patterns of Tbx5 expression provide an
embryologic basis for the prevalence of atrial septal defects (ostium primum and secundum), ventricular muscular
septal defects, and left-sided malformations (endocardial cushion defects, hypoplastic left heart, and aberrant
trabeculation) observed in patients with Holt–Oram syndrome. © 1999 Academic Press
Key Words: heart development; transcription factor; gene expression; embryo.

INTRODUCTION unique pattern of asymmetric (left predominant) upper limb

The T-box transcription factor Tbx5 has been implicated
as an important participant in cardiac development because
mutations in this gene cause Holt–Oram syndrome (HOS)
(Basson et al., 1997, 1999; Li et al., 1997), a rare autosomal
dominant disorder characterized by upper limb deformities
and cardiac defects (OMIM 142900). Most TBX5 mutations
in HOS patients are predicted to function as null alleles;
thus a dominant phenotype appears to be due to haploin-
sufficiency of the gene product. While variable expression
of phenotype is well recognized in affected patients (Basson
et al., 1994, 1999) clinical findings typically exhibit a
Mouse Tbx5 has been deposited in GenBank under Accession
No. AF140427. The full-length cTbx5 sequence has been deposited
in GenBank under Accession No. AF069396.
1
These authors contributed equally to the work presented here.
malformations and atrial or ventricular septal defects. A
variety of other cardiac defects are also observed, including
conduction disease, patent ductus arteriosus, abnormal
ventricular trabeculation, hypoplastic left heart, mitral
valve prolapse, and anomalous pulmonary return (Basson et
al., 1994; Newbury-Ecob et al., 1996; Sletten and Pierpont,
1996). The mechanisms by which TBX5 mutations produce
these phenotypes remain an important question.
Tbx5 is one of at least 12 T-box genes in the human
genome (Agulnik et al., 1998; Law et al., 1998; Papaioannou
and Silver, 1998; Papapetrou et al., 1999; Wattler et al.,
1998; Yi et al., 1999). Each T-box gene encodes a highly
conserved, 180-amino-acid residue T-box that is involved in
DNA binding and dimerization (Muller and Herrmann,
1997) as well as a nonconserved carboxyl region that is
presumably involved in protein–protein interactions. These
genes are expressed throughout development in a range of

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Tbx5 in Heart Development 101

FIG. 1. Sequence alignment of mouse (m), human (h), chicken (c), and newt (nv) Tbx5 proteins. The T domain is boxed. Amino acid
residues different from the consensus are shaded; missing amino acids are indicated by a dash. The human sequence was compiled from
previously published data (hTBX5, GenBank Accession Nos. U80987 and U89353). The partial newt sequence is under GenBank Accession
No. U64433.

tissues and potentially affect diverse developmental path- METHODS

ways (Papaioannou and Silver, 1998). Tbx5 expression has
been analyzed in developing mouse and chick limbs, in
which it correlates with forelimb identity (Gibson-Brown et
al., 1996, 1998b; Isaac et al., 1998; Logan et al., 1998;
Ohuchi et al., 1998). While its expression has also been
reported in the developing heart (Chapman et al., 1996;
Gibson-Brown et al., 1996, 1998a; Li et al., 1997), its precise
temporal and spatial pattern of cardiac expression has not
been elucidated. To gain further understanding of the func-
tions of Tbx5 in cardiac development and to better under-
stand the prevalence of particular cardiac malformations in
Holt–Oram syndrome, we studied Tbx5 expression in the
developing mouse and chick heart. We demonstrate that
Tbx5 has a unique pattern of expression; transcripts are
abundant in the posterior regions of the embryonic heart
and predominate in the left atria, right atria, and left
ventricle of the mature heart. This restricted pattern of
expression of Tbx5 helps to explain the cardiac phenotypes
observed in Holt–Oram syndrome.
Tbx5 cDNAs
A bacteriophage l embryonic mouse limb cDNA library (kindly
provided by Dr. Philip Leder) was screened with a human TBX5
cDNA clone (Basson et al., 1997). Three overlapping clones were
isolated, released by EcoRI digestion, and subcloned into pBlue-
script (Stratagene). The cloning and partial sequence of chicken
Tbx5 (cTbx5) has been previously described (Logan et al., 1998).
Sequencing was performed with an automated sequencer (ABI).
Animals
Mice were purchased from Taconic Farms. Embryos were ob-
tained from Swiss Webster female mice that had been mated with
C57Bl/6 male mice; noon of the day of detection of a vaginal plug
was considered embryonic day (E) 0.5. Fertilized White Leghorn
chicken eggs were purchased from SPAFAS (Norwich, CT). Mouse
and chick embryos were staged according to Kaufman (1992) and
Hamburger and Hamilton (1951), respectively.

Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
102 Bruneau et al.

FIG. 2. Whole-mount in situ hybridization of Tbx5 in early mouse heart development. (A) Frontal view of a two-somite embryo (E8). (B)
Ventral view of an E8.5 embryo. (C) Ventral view of an E8.5 embryo, slightly more developed than the one in (B). (D) A lateral (right side)
view of an embryo at the same stage as the one pictured in (C). (E–G) E9.0 embryo viewed from the right side (E), ventrally (G), and from
the left side (F). The apparent staining in the rv in (G) is due to the atrial expression behind the translucent rv. The pericardium (p) also
expresses Tbx5. (H) An E9.5 embryo, viewed from the left side. fl, forelimb bud; r, retina. (I, J) Heart dissected from an E11.5 mouse embryo
viewed from the front (I) and from the back (J). la, left atrium; lv, left ventricle; ra, right atrium; rv, right ventricle. (K) Tbx5 expression in
an E11.5 mouse embryo. Arrow indicates forelimb.

In Situ Hybridization
Whole-mount in situ hybridization was performed essentially as
described by Riddle et al. (1993). In situ hybridization on sections
of paraffin-embedded mouse embryos were performed as previously
described (Tessarollo et al., 1992), except that [ 33P]UTP was used
instead of [ 35S]UTP, and 0.1% DEPC in PBS (twice for 15 min) was
used instead of triethanolamine. Nonradioactive in situ hybridiza-
tion with chick embryo cryosections were performed as previously
described (Bao and Cepko, 1997). Tbx5 riboprobes were generated
from plasmids containing the coding sequences of mouse or chick
Tbx5. Radioactive in situ hybridization sections were counter-
stained with 0.1% toluidine blue and were viewed using transmit-
ted light to view the tissue morphology and refracted red light to
view the radioactive signals. All images were digitally captured
using a Sony camera and were directly imported into Adobe
PhotoShop 4.0.

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Tbx5 in Heart Development 103

FIG. 3. Whole-mount in situ hybridization of cTbx5 in early chick heart development. (A) Expression of cTbx5 in a stage 8 embryo viewed
ventrally. (B) A stage 9 embryo, viewed ventrally. (C) Stage 16 embryo, viewed from the right side. OT, outflow tract. Expression at stages
20 (D) and 25 (E) becomes restricted to the atria and the LV. (F–H) Heart dissected from a stage 27 chick embryo, viewed from the right (F),
front (G), and left (H).

Clinical Evaluation of Holt–Oram Syndrome
Patients
Informed consent was obtained from all participants evaluated at
the Brigham and Women’s Hospital in accordance with the Human
Research Committee. Clinical evaluations were obtained without
prior knowledge of genotype status. Physical examination and
echocardiogram were used to assess cardiac status. Autopsy records
were used when available. Extensive clinical evaluation of families
A, B, and F have been reported previously (Basson et al., 1994,
1995); detailed cardiac findings of other study families (Basson et
al., 1997, 1999) are our unpublished data.
RESULTS
The complete cDNA coding sequence of mouse Tbx5 was
obtained from three overlapping cDNA clones. Tbx5 en-

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104 Bruneau et al.

codes a 519-amino-acid protein with 97 and 94% homology
(96 and 88% identity) to its human and chicken counter-
parts, respectively (Fig. 1). The T domain of mouse Tbx5
differs from the human and chicken homologues by only 1
amino acid residue and from the partial newt sequence by
only 2 amino acid residues.
Tbx5 expression in the early developing mouse embryo
was assessed by in situ hybridization (Fig. 2). At E9.5 and
E11.5 Tbx5 is expressed in the heart, eye, and forelimbs as
previously reported (Chapman et al., 1996; Gibson-Brown
et al., 1996) (see Figs. 2H and 2K). Cardiac expression of
Tbx5 was analyzed further. At E8.0, Tbx5 transcripts are
abundantly found throughout the cardiac crescent (Fig. 2A).
At E8.25, when the linear heart tube is forming, Tbx5 is
robustly expressed in the very posterior portion of the heart,
which is destined to become the sinus venosa and atria (Fig.
2B). Weaker expression can be observed in the myocardium
that encircles the endocardial tube. In the linear heart tube,
Tbx5 is localized exclusively in the posterior structures of
the heart that give rise to the atria and sinus venosa (Figs.
2C and 2D). As the heart undergoes looping (E8.5–9.0),
expression of Tbx5 expands anteriorly to encompass the
entire future left ventricle (LV). In looped hearts, when the
left and right ventricles have progressed from an anterior–
posterior arrangement to a left–right arrangement (E9.0),
Tbx5 expression is restricted to the left side of the ven-
tricles (Figs. 2E–2H). This pattern persists; by E11.5 there is
very little expression in the RV or outflow tract but signifi-
cant expression in the left ventricle (Figs. 2I and 2J).
In the developing chicken heart, cTbx5 is initially ex-
pressed in the entire bilateral cardiac primordia (Fig. 3A). As
the bilateral primordia fuse in the midline (stage 9), cTbx5
is expressed in a posterior to anterior gradient (Fig. 3B). In
looped hearts at stage 16, almost the entire heart tube,
except the future outflow tract, expresses cTbx5 (Fig. 3C).
This appears to be the only difference between mouse and
chick development, as cTbx5 is down-regulated in the
future RV and becomes restricted to the atria and LV
shortly thereafter (stage 20, Fig. 3D). By stage 25 the
boundary between Tbx5-expressing cells in the LV and
nonexpressing cells in the RV is sharply defined (Fig. 3E).
With septation, the morphological distinction between
chambers is complete (stages 25 and 27) and cTbx5 is
expressed in both atria and LV, but remains excluded from
the RV and outflow tract (Figs. 3F–3H).
During chamber maturation and septation in the devel-
oping mouse heart, atrial Tbx5 expression becomes more
pronounced relative to the LV (Fig. 4). At E13.5, expression
in the left ventricular free wall is detectable and at higher
levels in the LV trabeculae (Figs. 4A and 4C). RV expression
is limited to the trabeculae. Tbx5 mRNA is present at this
stage in both developing atrial septa (Fig. 4B), but expression
in the ventricular septum is limited to the left aspect of the
septum (Fig. 4A). Expression of Tbx5 is also detected in the
atrioventricular valves, with higher levels on the atrial side
of the valve leaflets (Fig. 4B). The left and right superior
vena cavae, as well as the inferior vena cavae, also express
Tbx5 (Figs. 4A, 4B, and 4C).
At stages 26 and 28, sections of chicken embryos reveal
that as in mouse, cTbx5 is unilaterally expressed in the left
side of the interventricular septum (Figs. 5A, 5B, and 5E).
Expression is also observed throughout the atrial myocar-
dium, in the atrial septa, and in the LV free wall (Figs. 5A,
5C, and 5E). The nascent valve cushions express cTbx5 only
in the atrial aspect (Figs. 5C and 5D), consistent with the
observation in mouse that the AV valves express Tbx5
unilaterally.
We have reviewed the spectrum of cardiac defects re-
ported in the literature or evaluated by us in more than 240
patients with Holt–Oram syndrome (Basson et al., 1994,
1995, 1997, 1999; Newbury-Ecob et al., 1996; Sletten and
Pierpont, 1996). Many patients had multiple cardiac mal-
formations; each defect was recorded and located on a
schematic of the mature human heart (Fig. 6). As previously
recognized, atrial and ventricular septal malformations
were prevalent among 301 Holt–Oram cardiac defects.
Abnormalities of left-sided structures were also frequent:
hypoplastic left heart, trabecular defects, mitral valve pro-
lapse, mitral atresia, the conduction system (atrioventricu-
lar block and conduction defects), persistent left superior
vena cava and total anomalous pulmonary venous return.
These defects colocalize with regions that abundantly ex-
press murine Tbx5 (Fig. 6, shaded). In addition, defects are
recognized in shown regions (ductus arteriosus, aorta, pul-
monary arteries) where Tbx5 expression has not been ascer-
tained.

FIG. 4. In situ hybridization of Tbx5 to transverse sections of an E13.5 mouse embryo. Hybridization of the 33P-labeled Tbx5 probe
produces a red signal. (A) Transverse section showing a four-chamber view of the heart. The asterisk indicates the junction of the left
superior vena cava (LSVC) with the accessory hemizygous vein and the left superior intercostal vein. (B) Close-up of a more posterior section
reveals expression in both the septum primum (sp) and the septum secundum (ss) as well as in the LSVC and the av valves (arrows). (C) A
section more posterior than that in (B) shows Tbx5 expression mainly in the atria and LV, as well as the inferior vena cava (ivc).
FIG. 5. In situ hybridization of cTbx5 to a transverse section of chick embryo heart. (A) Transverse section of a stage 26 chick embryo.
vs, ventricular septum. (B) Close-up of (A), showing a detail of the unilateral septal expression, as indicated by the arrowhead. (C–E) Stage
28 embryos. (C) Parasagittal section. av, atrioventricular cushions. (D) Close-up of the av region in (C). The arrowheads indicate the
predominant expression of cTbx5 in the atrial aspect of the av cushions. (E) Low-magnification view, transverse section. pc, pericardium;
as, atrial septum; A, atrium.

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Tbx5 in Heart Development 105

Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
106
FIG. 6. Schematic representation of the mature heart indicating
regions of Tbx5 expression (shaded areas) and the location of
Holt–Oram cardiac defects in 240 patients. Aortic abnormalities
(AA), atrial septal defect (ASD), conduction system defects (CSD),
double outlet right ventricle (DORV), endocardial cushion defect
(ECD), hypoplastic left heart (HLH), mitral valve disease (MVD),
pulmonary artery abnormalities (PAA), total anomalous pulmo-
nary venous return (TAPVR), tetralogy of Fallot (TOF), trabecular
anomalies (TRAB), tricuspid atresia (TA), truncus arteriosus (TRA),
ventricular septal defects (VSD). The numbers of defects are from
the literature or from our unpublished observations (see text for
details).
DISCUSSION
We demonstrate Tbx5 is expressed throughout develop-
ment in a unique and restricted pattern that is conserved
between species. Expression is maintained in the structures
derived from the posterior domains of the linear heart tube,
the atria and left ventricle, but absent in structures that
evolve from the bulbus and conus cordis (ventricular out-
flow tracts). This pattern of expression correlates with
cardiac malformations caused by human TBX5 mutations
in Holt–Oram syndrome. We conclude that these congeni-
tal heart defects in this disorder are not secondary to
hemodynamic perturbation of the developing heart, but are
the direct result of deficiencies in this transcription factor.
These data indicate a unique and critical role for Tbx5 in
atrial and left ventricular development.
Cardiac chamber formation is emerging as a complex
process that requires the interaction of multiple regulators
expressed in overlapping domains. Tbx5 shares a profile of
expression that is unique among chamber-specific cardiac
genes (Biben and Harvey, 1997; Dunwoodie et al., 1998;
Kelly et al., 1998; Kubalak et al., 1994; Lyons et al., 1990;
Srivastava et al., 1997; Zeller et al., 1987). The pattern of
expression of Tbx5 during cardiac embryogenesis is dis-
tinctly different from other chamber-specific transcription
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.
Bruneau et al.
factors such as HAND1, HAND2, Msg1, and Irx4 (Bao et al.,
1999; Biben and Harvey, 1997; Dunwoodie et al., 1998;
Firulli et al., 1998; Riley et al., 1998; Srivastava et al., 1997;
Thomas et al., 1998). This may indicate that different
molecules, or a combination of factors, regulate chamber-
specific expression of these cardiac genes.
The expression of Tbx5 in the atria and LV throughout
the development and maturation of these chambers sug-
gests that this transcription factor is involved in directing
gene expression in morphogenetic processes associated
with specific chamber formation. However, the expression
profile of Tbx5 exhibits additional features, implying a
unique developmental role. Tbx5 expression is evident on
the atrial aspect of the atrioventricular valves in mouse and
chick hearts, as has been reported in human hearts (Li et al.,
1997), suggesting participation of Tbx5 in valvulogenesis.
Valvular polarity has previously not been observed at a
molecular level, and the expression of Tbx5 in the atrial
aspect of the valves indicates that this may be a regulated
process.
Tbx5 exhibits ventricular expression that is surprisingly
asymmetric (left greater than right). The subdivision of the
primitive ventricles into left and right chambers occurs
during late stages of looping of the heart tube and incorpo-
rates multiple embryonic structures. Throughout this com-
plex event, Tbx5 expression is evident in the primitive left
ventricle and at lower levels, in right ventricular trabeculae.
This pattern may indicate that Tbx5 delineates the conus
cordis and truncus arteriosus (which give rise to the right
ventricular outflow tract) from the more proximal bulbus
cordis, which contributes the trabeculated part of the right
ventricle. The striking left-sided Tbx5 expression within
the interventricular septum is consistent with this model
in that right ventricular portions of the septum evolve from
derivatives of the bulbus cordis (Netter and Van Mierop,
1969). While the precise role of Tbx5 in ventricular matu-
ration remains unclear, our data indicate a role for this
transcription factor in establishing a left–right axis within
the embryonic heart following the morphological rearrange-
ment of an anteroposterior sequence of structures to a
left–right organization. Participation in axis formation is a
recognized function of T-box genes in patterning the limb
(Bamshad et al., 1997; Basson et al., 1997; Li et al., 1997),
and conservation of unilateral (left) ventricular expression
of Tbx5 in mouse and chicken supports a comparable
function within the developing heart.
Understanding the contribution of Tbx5 to cardiac em-
bryogenesis helps to explain how human TBX5 mutations
might cause congenital heart disease. Defects in atrial and
ventricular septation are most prevalent in Holt–Oram
syndrome, but additional malformations are well recog-
nized. Expression of Tbx5 in developing atrioventricular
valves and ventricular trabeculae (which contribute to
chordae tendinae) is likely to account for structural and
functional defects in mitral and tricuspid valves. The dis-
Tbx5 in Heart Development
proportionate severity of left versus right ventricular le-
sions (hypoplastic left heart; defects in isomerism) and
infrequent occurrence of right ventricular outflow tract
lesions in Holt–Oram patients may reflect asymmetric (left)
ventricular expression of this factor. A more difficult ques-
tion is why greater anomalies are not observed, particularly
in the atria in which expression is so marked. We suspect
that particularly severe malformations may be embryonic
lethal. Alternatively, since most of these autosomal domi-
nant mutations are predicted to cause TBX5 haploinsuffi-
ciency, robust atrial expression may allow partial compen-
sation by the nonmutated allele. Production of mice with
Tbx5 mutations should help to answer these questions.
In summary, we have demonstrated that Tbx5 is ex-
pressed in developing chick and mouse heart in a unique
fashion. Either by participating in the establishment of axis
formation or by interpreting these signals, Tbx5 has critical
functions in atrial and left ventricle development. Whether
the same genes at all sites within the heart are activated by
Tbx5 will provide further clues to the role of this transcrip-
tion factor in cardiac development.
ACKNOWLEDGMENTS
We thank Dr. Philip Leder for the mouse embryo limb cDNA
library, Barbara McDonough for assistance in compiling patient
information, and Susanne Bartlett for assistance with graphics.
This work was supported by the Howard Hughes Medical Institute
(J.G.S., C.E.S.), the NIH (C.J.T., J.G.S., C.E.S.), and postdoctoral
fellowships from the American Heart Association (Massachusetts
affiliate) (B.G.B.), the Medical Research Council of Canada (B.G.B.),
and the Human Frontiers Science Organization (M.L.).
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Received for publication March 17, 1999
Revised April 5, 1999
Accepted April 5, 1999