T-box factors determine cardiac design
W.M.H. Hoogaars, P. Barnett, A.F.M. Moorman and V.M. Christoffels*
Heart Failure Research Center, Department of Anatomy and Embryology, Academic Medical Center,
Amsterdam (The Netherlands), Fax: +31206976177, e-mail: email@example.com
Online First 13 February 2007
Abstract. The heart of higher vertebrates is a structur-
ally complicated multi-chambered pump that con-
of distinct integrated components have to be gener-
ated, including force-generating compartments, uni-
directional valves, septa and a system in charge of the
initiation and coordinated propagation of the depola-
rizing impulse over the heart. Not surprisingly, a large
number of regulating factors are involved in these
processes that act in complex and intertwined path-
ways to regulate the activity of target genes respon-
mutations in T-box transcription factor-encoding
genes in humans lead to congenital heart defects has
focused attention on the importance of this family of
regulators in heart development. Functional and
genetic analyses in a variety of divergent species has
demonstrated the critical roles of multiple T-box
factor gene family members, including Tbx1, -2, -3, -5,
-18 and -20, in the patterning, recruitment, specifica-
tion, differentiation and growth processes underlying
formation and integration of the heart components.
Insight into the roles of T-box factors in these
processes will enhance our understanding of heart
formation and the underlying molecular regulatory
Keywords. T-box transcription factor, Tbx, heart, patterning, heart fields, progenitor populations, chamber,
The heart of vertebrates is a unidirectional muscular
pump that propels blood by means of synchronized
contractions of two (fish) or four (mammals, birds)
chambers, while the one-way valves prevent return
flow . In contrast to most other organs, the heart of
and the extra embryonic membranes with blood from
the moment it forms, pumping first in a peristaltic
wave along the length of the primitive heart tube,
before ballooning, looping and transforming into the
complex four-chambered heart with its regular paced
beat contraction. To accomplish this, the morphoge-
netic and functional requirements of the heart have
been integrated into a perfectly coordinated genetic
program underlying heart formation. Several key
transcription factors involved in this genetic program
have been identified, including Nkx2-5 and members
families of factors [2, 3]. Although functional require-
ment of these factors has been demonstrated, their
precise functions and interactions during cardiac
morphogenesis have yet to be defined satisfactorily.
Although a few early studies made mention of T-box
factor genes expressed in the heart [4, 5], it was the
Holt-Oram syndrome, a disease associated with con-
genital heart defects of differing severities, that
initiated a series of studies to unravel the function of
this family of transcription factors in heart develop-
ment [6, 7]. The T-box family presently counts 17
members in mouse and humans . Members of the
subfamily, Tbx2, Tbx3 and Tbx5, have all been found to
play specific roles in heart development [8–10].
The embryonic heart is a relatively simple tube
emerging from two sheets of cardioblasts derived
from visceral mesoderm that fuse at the midline.
* Corresponding author.
Cell.Mol.Life Sci. 64 (2007) 646–660
?Birkh?user Verlag, Basel, 2007
Cellular and Molecular Life Sciences
Despite decades of experimental evidence that the
myocardial cells of the initial heart tube represent
a tubular heart containing antero-posteriorly aligned
been theparadigmfor developmentalpatterning until
recently. Two new insights have invoked a shift in
conception. First, direct lineage evidence has been
provided that non-cardiac progenitor cells peripheral
to the initial heart tube make extensive contributions
to the heart  (Fig. 1). Second, chambers differ-
entiate and expand locally at positions along the heart
tube in a non-segmental fashion, a process requiring
antero-posterior as well as ventro-dorsal patterning
 (Fig. 2). T-box factors play very prominent roles
in both these processes. In this review, we will provide
an update on T-box factor function in heart develop-
ment, and discuss these functions in the context of
novel insights that have significantly increased our
appreciation and understanding of heart develop-
Cardiac T-box proteins and their partners
T-box factors take on a major role in guiding tran-
scription events during cardiac development, and
their combinatorial T-box protein interaction events
must be seen as key to determining the individual fate
each cell ultimately derives. For example, the combi-
natorial activities and interactions of Tbx5, Tbx20,
Nkx2-5 and Gata4 lead to cardiac chamber differ-
entiation of which a molecular signature is the the
upregulation and expression of Nppa (Natriuretic
Precursor Peptide type A, also known as ANF).
Inclusion of Tbx2 and Tbx3 in this network leads to
Nppa suppression, notably in the atrioventricular
canal, signifying an apparent suppression of differ-
entiation [8, 12]. Attempts then to examine the
function of T-box factors at the molecular level often
turn to techniques that seek to identify interacting
protein partners. Perhaps one of the better-known
examples was the use of the yeast two-hybrid system
by Hiroi and co-workers  in conjunction with the
homeodomain-containing transcription factor Nkx2-
5. Using Nkx2-5 as bait in a human complementary
DNA (cDNA) library screen Tbx5 was identified as
interacting partner, the synergistic interaction of
which was shown to be a key event in the differ-
entiation of working myocardium [13, 14]. Mutations
which interfere with either the capacity of Tbx5 to
bind DNA or its ability to interact with Nkx2-5 can
a synergistic interaction between Tbx1 and Nkx2-5
has recently postulated to play a role in another
human congenital disease displaying varying heart
malformations, DiGeorge syndrome . Although
on its own, in vitro, Nkx2-5 appears to function as a
moderate transcriptional activator, its ultimate activ-
ity as an activator or repressor depends on interaction
with the T-box factors present in the same cell .
guidance factor instead of a direct transcriptional
activator or repressor.
Studying congenital defects can itself represent a
?natural? experimental setting to discover and eluci-
date novel protein interactions. One such study
involving isolated septal defects leads to the inclusion
of GATA4 as a new synergistic interacting partner of
Tbx5, an interaction shown to play a role in the
expression of Nppa . In our lab we have also
demonstrated an in vitro interaction between GATA4
Figure 1. Schematic overview of the different cardiac progenitor populations. Precursors of the embryonic ventricle (ev), the future left
atria (left atrium, la; right atrium, ra) are derived from the second heart field (gray). The Tbx18+ caudal heart field (dark gray) forms the
sinus horns (sh). A, anterior; L, left; P, posterior; R, right.
Cell.Mol.Life Sci. Vol.64, 2007
Multi-author Review Article
and Tbx2/3 [unpublished observations], which taken
together with the described interaction of Tbx20 with
GATA4 [20, 21] suggests that in the case of over-
lapping expression of T-box proteins, the stoichiom-
etry of T-box proteins and the restricted presence of
other positive or negative regulatory factors will
ultimately decide timing and direction of cellular fate.
In line with this, several other protein interacting
partners have been assigned to the Tbx5 list [22–24].
The cardiac-enriched MYST family histone acetyl-
transferase TIP60 and Tbx5 were observed to be
mutual interactive cofactors through the TIP60 zinc
finger. In transfection assays, TIP60, Tbx5 and Tbx2
activate an enhancer in the SRF gene required for
expression in the developing heart. The zinc-finger-
containing protein Sall4, as well as being regulated by
Tbx5, takes on a dual role, functioning co-operatively
with Tbx5 and Nkx2-5 to upregulate Cx40 (Gja5) and
Fgf10 and antagonistically to downregulate certain
genes such as Nppa. Notably, the presence of Tbx2/3
on Tbx5, though the dependence for this activity on
DNA binding or protein-protein interaction was not
explored.Further,as is observed for the interaction of
Gata4 and Nkx2-5 [13, 19], Sall4 is also unable to
associate with the Tbx5 mutants Gly80Arg and
Figure 2. Schematic overview of heart development in higher vertebrates. Chamber myocardium (red, ventricular; blue, atrial) expands
from the outer curvatures of the primary heart tube, whereas non-chamber myocardium (gray) of the inflow tract (ift), sinus horns (sh),
atrioventricular canal (avc), outflow tract (oft) and inner curvatures does not expand. Sinus horn myocardium gives rise to the sinoatrial
the left-lateral view. A, anterior; D, dorsal; P, posterior; V, ventral; a, atrium; avb, atrioventricular bundle; avc, atrioventricular canal; ev,
embryonic ventricle; la, left atria; lv, left ventricle; ra, right atrium; rv, right ventricle.
648 W.M.H. Hoogaars et al. T-box factors determine cardiac design
Arg237Trp. This may be indicative of a similar mode
of interaction, though one must be careful of over-
interpreting the effect of point mutations on inter-
actions since even small perturbations in molecular
structure can have significant effects on the overall
fold of a protein.
TAZ, a WW domain protein, also represents a
recently discovered protein interacting partner of
Tbx5, TAZ and a related protein, YAP, are hy-
pothesized to play a role in cardiac development
and hypertrophy. Although expression patterns of
TAZ in the heart are not available, an interesting
feature of TAZ is its ability to associate with the
histone acetyltransferase proteins p300 and PCAF.
Co-transfections of Tbx5, TAZ and p300 or PCAF
were shown to upregulate the Nppa promoter in
vitro. However, the true in vivo significance of this
interaction still needs to be tested. This role of
epigenetics in cardiac development can be postu-
lated from the discovery that T-box factors can
associate with chromatin-modifying proteins. Chro-
matin structure and function have been known to
have a key role in cell lineage patterning for many
years, acetylation or deacetylation, for instance,
increasing or decreasing the accessibility of tran-
scription factor binding sites, respectively. In line
with this, Tbx5 activity is influenced by the presence
of the chromatin remodeling protein BAF60c,
shown to potentiate an interaction between Tbx5
and the Baf complex ATPase Brg1 , thereby
assisting in upregulation of cardiac differentiation.
Likewise, though functioning in a negative fashion,
Tbx2 has been shown to recruit and associate with
the histone deacetylase HDAC1 .
Recently, the PDZ-LIM domain protein LMP4 was
found to interact with Tbx5 and to repress its tran-
scriptional activity [27, 28]. LMP4 is localized in the
cytoplasm, associated with the actin cytoskeleton.
Interaction between Tbx5 and LMP4, which is dy-
namic and depends on undefined signals, leads to the
localization of Tbx5 to actin filaments. LMP4-medi-
to be involved in the transcriptional regulatory
activity of Tbx5.
In a review concerning aspects of T-box proteins in
the protein-protein interaction of Tbx20 with a
marker of the second heart field, Isl1. This Lim/
homeodomain protein has so far only been demon-
strated to interact directly with Tbx20 . This
interaction, together with a synergistic interaction
between Gata4 and Tbx20, plays a role in regulating
the expression of Nkx2-5 and Mef2c in the precursors
of the outflow tract and right ventricle.
Finally, T-box family members Tbx2 and Tbx5, and
Tbx5 and Tbx20, respectively, have also been demon-
demonstrate a synergistic role during heart develop-
ment. Although T-box dimerization has often been
postulated, chiefly on the basis of the Xenopus T
binding characteristics of TBX1 [30, 31], these exam-
heterodimerize, forming a synergistically functional
The function of T-box-factors in the progenitor
populations of the heart
During its development, the embryonic heart tube
itself shows limited proliferation, indicating that the
increase must be attributable to recruitment of
precursors to the myocardial phenotype . Current
estimates in mouse suggest that only the precursors of
thefuture leftventricle,andperhaps partofthefuture
atrioventricular canal and atria, are derived from the
first lineage of myocardial cells that comprise the
embryonic heart tube (Fig. 1). The remainder of the
compartments, the outflow tract, right ventricle and
atria, are derived from undifferentiated precursor
cells present outside the heart tube. This population
has been referred to as the second lineage or second
heart field and contains subpopulations referred to as
secondary heart field and anterior heart field [3, 11].
At 7.5 days of mouse development, this second field is
heart tube (Fig. 1). During and after folding of the
embryo, the second field becomes positioned in the
pharyngeal mesoderm, the pericardial mesothelium
and mesoderm cranially, dorsal and caudal to the
tubular heart. This precursor field progressively
provides contributions to both poles and to the dorsal
this structure is disrupted later during development.
It is currently unclear whether the fields are discrete
entities, or just subpopulations of one large heart
precursor field. However, several transcriptional
regulators, including Tbx1, have been identified that
for a cellular subset of the second heart field. Recent
evidence from our laboratory indicates the presence
of yet another precursor population with a molecular
signature distinct from that of the first and second
heart fields (see below). Therefore, a picture emerges
of one large precursor pool for the heart comprising
subpopulations, such as the first and second heart
fields, each with distinctive gene programs, and
Cell.Mol.Life Sci.Vol.64, 2007
Multi-author Review Article
providing spatially and temporally segregated contri-
butions to the heart tube.
Tbx1 and the second heart field
DiGeorge syndrome, also known as del22q11 syn-
drome based on a 1.5–3 MB genomic deletion at the
22q11.2 region seen in many patients [33, 34], is a
common congenital disorder characterized by a
variety of abnormalities, including absence or hypo-
plasia of the thymus, cleft palate, facial dysmorphism
and cardiovascular anomalies such as aortic arch
malformation (specifically hypoplasia of 4thpharyng-
eal arch artery), outflow tract defects and ventricular
septal defects. Tbx1-deficient mice phenocopy impor-
tant aspects of DiGeorge Syndrome, including out-
flow tract abnormalities [35–37]. Therefore, Tbx1 has
a specific function in outflow tract morphogenesis in
the developing heart.
Expression of Tbx1 can be detected in pharyngeal
endoderm, the mesodermal core of the pharyngeal
arches and the second heart field [38–42]. Genetic
lineage studies have demonstrated that Tbx1-positive
cells in the second heart field provide extensive
contributions between E8.5 and E9.5 to the outflow
tract myocardium, endocardium and mesenchymal
cushions [42–44]. A limited contribution can also be
seen in the atria , suggesting that Tbx1 is also
expressed in the larger second heart field. Tracing of
the Tbx1-positive lineage revealed diminished contri-
bution of these cells to the outflow tract in a Tbx1-
deficient background. Furthermore, conditional dele-
tion of Tbx1 within the second heart field results in
reduced cell proliferation, which may underlie the
reduced contributions of the second heart field to the
outflow tract in Tbx1-deficient mice . Elegant
time-course deletion experiments have further dem-
onstrated that Tbx1 is required for outflow tract
development between E8.5/9 and E9.5, coinciding
with the secondary heart field progenitors contribu-
ting to the outflow tract .
Cardiac defects seen in Tbx1-deficient mice are
similar to those observed in Fgf8 hypomorphs [45,
46], suggesting that both may act along a common
pathway. Fgf8, Fgf10 and Tbx1 are all co-expressed in
the second heart field [40-42]. Tbx1 null mice show
reduced Fgf8 expression in the outflow tract and
reduced expression of both Fgf8 and Fgf10 in the
pharyngeal mesoderm [40-42]. Conversely, overex-
Fgf8 and Fgf10 expression, and increase outflow tract
myocardium . Furthermore, Tbx1 was found to
regulate transcriptional activity of Fgf8 and Fgf10
through association with conserved T-box binding
sites in their promoter sequences [40, 42]. Consistent
with these findings, conditional deletion of Fgf8 in the
Tbx1-positive region resulted in outflow tract defects,
suggesting a genetic interaction . Although Fgf8
signaling is indeed crucial for normal development of
second heart field progenitors and outflow tract
septation, as two recent studies demonstrated [47,
48], another recent study showed that forced expres-
sion of Fgf8 in Tbx1 null mice or conditional deletion
change the Tbx1 outflow tract phenotype. This
indicates that Tbx1 and Fgf8 act independently in
outflow tract development .
Pitx2c-deficient mice have outflow tract defects, and
recently Pitx2c was identified as a putative target of
In Tbx1 null mice the expression of Pitx2 is down-
regulated in this subpopulation, along with the out-
flow tract and splanchnic mesoderm. The phenotype
of Tbx1+/–Pitx2+/–heterozygous mice appeared more
severe when compared with single heterozygous
mutant mice of either gene, suggesting a genetic
interaction. Moreover, Tbx1 binds to a critical Pitx2
enhancer and synergistically induces transcriptional
indicating that Tbx1 and Nkx2-5 act in the same
pathway for outflow tract morphogenesis .
Regulation of Tbx1 in the second heart field subpo-
pulationhas beenshowntodepend uponthepresence
of two enhancers that contain conserved binding sites
for forkhead transcription factors [40, 44, 50]. The
are able to bind to these sites and to induce transcrip-
tional activity of these Tbx1 enhancers. Furthermore,
Foxa2 and Tbx1 co-localize in the second heart field,
and Foxa2 has been demonstrated to be a target of
Tbx1 [40, 44]. Taken together, these data suggest that
Foxa2 and Tbx1 act in a common pathway in the
second heart field critical for normal outflow tract
Roles for T-box factors in recruitment and elongation
of the heart tube
Tbx20, of which several splice variants exist, is ex-
pressed in the first heart field, in a subset of second
heart field progenitors, and in the endocardium and
derived mesenchyme of the atrioventricular and out-
flow tract cushions [20, 21, 51] (Table 1). After loop-
ing, the expression of Tbx20 decreases in chamber
myocardium compared with atrioventricular and
cardiac outflow regions [20, 21]. A recent series of
papers showed that mice deficient for Tbx20 die early
during development, displaying severely underdevel-
650W.M.H. Hoogaars et al.T-box factors determine cardiac design
oped, short heart tubes [20, 52–54]. Failure in the
deployment or recruitment of the second heart field is
likely to be a contributing factor to this phenomenon,
recruitment of the extra-cardiac second heart field
progenitors rather than by proliferation. Defects in
either a cell-autonomous or a non-cell-autonomous
process may underlie this problem, though the ex-
pression pattern of Tbx20 is compatible with both.
In Tbx20 null and RNA interference (RNAi) knock-
down embryos the expression of Nkx2-5 and Mef2c,
both required for anterior heart field formation, was
found to be moderately to severely downregulated in
two studies [20, 54]. RNAi knock-down of Tbx20 in
mice results in hypoplasia of the outflow tract and
right ventricle , derivatives of the anterior heart
Islet1 and Gata4 to activate a Mef2c anterior heart
field enhancer and a Nkx2-5 cardiac enhancer. To-
gether, these data indicate that Tbx20 is directly
involved in controlling the anterior heart field.
Although downregulation of Nkx2-5 and Mef2c
Table 1. Cardiac expression patterns of T-box factors during recruitment and chamber formation (E9.5) and during septation (E12.5).
E9.5Tbx1Tbx2 Tbx3 Tbx5Tbx18 Tbx20Cx40Nppa
2nd HF, posterior
2nd HF, anterior
Pulm. vein myocard.
Pulm. vein mesench.
Primary atrial septum
AV canal myocard.
Intraper. art. trunk
trunk, intrapericardial arterial trunk (non-myocardial); OFT, outflow tract; Pulm., pulmonary. 1, only in small dorsal part; 2, lumen to
myocardium gradient; 3, endocardium; 4, higher in trabecular myocardium; 5, restricted to compact myocardium; 6, higher in compact
myocardium 7, left ventricular component is positive; 8, restricted to trabecular myocardium; 9, very weak expression; 10, high in
epicardium of AV groove.
Cell.Mol.Life Sci.Vol.64, 2007
Multi-author Review Article
expression in Tbx20 mutants was not always found
, ectopic upregulation of Tbx2 was consistently
observed [52–54]. Our lab previously showed that b-
myosin heavy chain promoter-driven expression of
Tbx2 in the heart tube prior to chamber formation
caused a complete block of chamber differentiation
cardiac phenotype of these embryos is very similar to
that of Tbx20 mutant mice, strongly suggesting that
de-repression of Tbx2 can largely account for the
phenotype in Tbx20 mutants. If we assume that
defective recruitment of second heart field progeni-
tors underlies the phenotypes of Tbx2 overexpression
and Tbx20 null mice, the question is how excess Tbx2
deployment of the extra-cardiac progenitors. The
answer to this remains elusive, but leads one to
speculate that the myocardium itself controls progen-
itor recruitment. Soluble factors, such as members of
the bone morphogenic protein (BMP) or fibroblast
growth factor (FGF) families, are involved in differ-
entiation of myocardium and in regulation of the
anterior heart field . Several studies have shown
that BMP and FGF factors are regulated by T-box
gene expression in the heart tube may affect the
production of these soluble factors, leading to defec-
tive intercellular signaling underlying progenitor pro-
liferation or recruitment and differentiation.
Tbx5 mutant heart tubes are similarly short and
hypoplastic. They also display a caudal to cranial
gradient in the severity of the defect and a failure of
looping . Once again, a defect in the process of
caudal end of the heart tube cannot be excluded as an
underlying cause of the mutant phenotype. Tbx5 is
also expressed in the dorso-caudal domain of the
second heart field (Table 1, Fig. 3). Therefore, one
cannot discriminate between direct and indirect (non-
cell-autonomous) functions of Tbx5 in recruitment of
deficient in Tbx2 or Tbx3. Both display outflow tract
defects  [Robert Kelly and V. Papaioannou,
personal communication], and both are expressed in
a subpopulation of the second heart field [60–62]
(Table 1, Fig. 3).
Tbx18 and the caudal heart field
During folding of the embryo, Tbx18 is expressed in a
small subpopulation of cells ventral to the developing
heart tube (Fig. 1, Table 1). This region is spatially
associated with the precursors of the forming septum
transversum, and gives rise to the pro-epicardium and
the mesenchyme that borders the myocardial inflow
tract of the heart . Tbx18 is required for main-
taining antero-posterior polarity in somites .
Tbx18-deficient mice die shortly after birth as a result
Figure 3. Transverse serial sections of an E9.5 mouse embryo showing the expression of T-box genes in the heart. (a) T-box expression
patterns in the inflow tract/dorsal mesocardium region compared with second heart field marker Isl1 and myocardial marker Mlc2a. The
black arrows depict the dorsal posterior region of the Isl1+second heart field, which expresses Tbx5 and Tbx20. The red arrows depict the
red arrows depict the pericardial mesothelium and mesenchyme, respectively, of the anterior region of the second heart field, which
cushion mesenchyme expressing Tbx20, Tbx2 and Tbx3. avc, atrioventricular canal; ep, epicardium; la, left atrium; lccv, left common
cardinal vein; pe, proepicardium; pa, pharyngeal arches; ra, right atrium; rv, right ventricle; st, septum transversum.
652 W.M.H. Hoogaars et al.T-box factors determine cardiac design
of severe skeletal malformations. The absence of any
noticeable defects in the highly Tbx18-positive (pro)
epicardial cells and their derivatives, including coro-
nary arteries, is unexpected. Nevertheless, Tbx18-
deficient mice do develop heart defects.
The sinus horns are the myocardial parts of the
Recent mouse lineage and expression analysis experi-
5- negative, but Tbx18-positive, mesenchymal precur-
sors located in the periphery of the inflow tract .
This reciprocal pattern was found to be conserved in
the chick. Normally, the common cardinal veins are
released from the mesenchymal pericardial wall into
the pericardium, and the wall of the released vein
subsequently differentiates into myocardium. Both
processes fail in mice that lack Tbx18. As a conse-
quence, the right and left superior caval veins run
eventually become myocardialized. Intriguingly, the
expression pattern of Tbx18 in the sinus horns of
mouse and chicken is conserved in zebra fish ,
evolutionary equivalent of the sinus horns.
Isl1 expression was found to be largely excluded from
5, but not Tbx18, defines the sinus horn precursor
population as a genetically distinct field in its own
right. Furthermore, the second heart field is posi-
tioned medially to the cardiac crescent before folding
of the embryo (Fig. 1). In contrast, the septum trans-
versum progenitors, with which the Tbx18-expressing
precursors are associated, are localized laterally and
cranially to the cardiac crescent (Fig. 1). The sinus
horn precursors are thus spatially separated from the
second heart field progenitors. Of interest, the two
fields meet at the inflow tract (Fig. 1), suggesting that
sinus horn myocardium recruited first directly adja-
cent to the atria, such as the sinoatrial node, may
receive contributions from both populations.
Roles for T-box factors in chamber development and
conduction system formation
Some time around E10, shortly after looping, the
under way. While the embryonic heart tube enlarges
by recruiting precursor cells at the poles and via the
dorsal mesocardium, a secondary process of chamber
differentiation is being initiated. At E8–8.5, the
expression of marker genes for ventricular and atrial
chamber myocardium, including Nppa,Chisel (Smpx)
and Cx40, can already be observed at the ventral side
of the heart tube  (Fig. 2). This region will expand
(?balloon?) to form the embryonic left ventricle at the
outer curvature . Somewhat later, right-ventricu-
lar and atrial expression of the chamber markers is
observed at discrete sites of the outer curvature, these
being the regions which will expand to form the
respective chamber compartments. Importantly, the
sinus venosus, atrioventricular canal, inner curvature
and the outflow tract will not initiate expression of
chamber markers and will not expand. These struc-
tures initially retain the original embryonic pheno-
type. The sinus venosus and atrioventricular canal
presumably will give rise to the nodal components of
the conduction system  (Fig. 2). The compartments
do not arise from specific heart fields, but rather to a
varying degree obtain contributions from the first and
second heart fields, although the initially formed left-
ventricular compartment receives the greatest contri-
bution from the first heart field [55, 68] (Fig. 1).
Therefore, local cues rather than distinct lineages
Taking into account the positioning of the differ-
entiating chambers, it is likely that antero-posterior
and dorso-ventral patterning underlies chamber for-
mation or the repression thereof (Fig. 2, 4).
The non-chamber myocardium of the atrioventricular
canal, inner curvatures and outflow tract provide
signals to the underlying endocardium to form cush-
ions , from which subsequently the valves and
major parts of the septa will be formed. Furthermore,
the atrioventricular canal retains its slow conducting
properties, and will serve to delay the propagation of
the impulse from atria to ventricles. The accordingly
acquired configuration of slow conducting and con-
tracting primary myocardium and fast conducting and
contracting atrial and ventricular chambers, with
dominant pacemaker activity always found at the
caudal venous end, is sufficient to obtain a synchro-
nously contracting heart with a functional conduction
system . Obviously, many more morphogenetic
steps will still have to be taken to generate septa,
system before the heart truly reaches its mature form
Tbx5 and establishing the anterio-posterior pattern
the cardiac blueprint. The presence of antero-poste-
rior, or cranio-caudal, patterning in the heart tube is a
well-established phenomenon, believed to guide the
formation of distinct components along the antero-
posterior axis. Retinoic acid plays a determining role
Cell.Mol.Life Sci.Vol.64, 2007
Multi-author Review Article
in antero-posterior patterning, as it provides caudal
cardiac progenitors with positional information, thus
invoking the sinuatrial identity and further develop-
ment of these precursors [70, 71]. Tbx5 is expressed in
a caudal-high antero-posterior gradient in the heart
tube, a gradient regulated by retinoic acid [71, 72].
Tbx5 deficiency results in cardiac developmental
arrest, the formed but unlooped heart tube being
characterized by a hypoplastic caudal end, indicating
ment or expansion) of the sinuatrial precursor pop-
ulation. Forced expression of Tbx5 in the entire heart
causes an arrest in heart development and loss of
Mlc2v expression, an anterior marker gene not
normally expressed in the sinuatrial region .
Furthermore, expression of myosin heavy chain 6, a
gene important for development of the sinoatrial
region, has been shown to be regulated by Tbx5 in
vitro . Thus, Tbx5 may represent a patterning
factor linking positional information provided by
retinoic acid and development of the sinuatrial region
of the heart.
Formation of the interventricular septum is initiated
as early as E9.5–10, concomitant with differentiation
posterior axis, the interventricular septum can be
regarded as an antero-posterior boundary structure
between these two ventricles. Normally, the left
ventricle expresses more Tbx5 than the right ventricle
in early heart development. (a)
Schematic representation of an
E9.5–10.5 heart showing T-box
emerging structures. Tbx2 and
Tbx3 exert their function in the
non-chamber myocardium, Tbx1
in the outflow tract and Tbx18 in
the sinus horns. Yellow bars in-
dicate expression patterns of
Tbx5, Tbx20 and Nkx2-5. Tbx5
is required for antero-posterior
patterningand, alongwith Tbx20
and Nkx2-5, for chamber differ-
entiation. Note the absence of
horns. (b) Working model of a T-
box factor regulatory network
for chamber formation. Tbx2
and Tbx3 act as repressors of
chamber differentiation in pri-
mary myocardium where they
compete with Tbx5, while BMP
signaling stimulates Tbx2/3 (and
myocardium. Tbx20 represses
Tbx2 expression in chamber my-
ocardium and regulates prolifer-
ation. Tbx5 acts as a positive
regulator of chamber genes and
proliferation, thus stimulating
Role of T-box factors
654 W.M.H. Hoogaars et al. T-box factors determine cardiac design
. Ectopic expression of Tbx5 in the developing
ventricles results in an interventricular septal defect
and a single ventricle with left-ventricular identity
. More localized ectopic expression results in a
rightward (=anterior) shift of the interventricular
septum, and upregulation of several transcripts nor-
mally enriched in the left ventricle . These studies
suggest Tbx5 is necessary for left ventricular identity,
thus defining the boundary between the left and right
ventricle and providing cues for the localization of
interventricular septum formation. In mouse, Sall4
may be an effector gene of Tbx5 in this process.
Ventricular Nppa expression is higher in the left
ventricle and excluded from the developing interven-
tricular septum. In both Sall4 and Tbx5 haploinsuffi-
cient embryos, Nppa expression is increased in right
ventricle and the expression boundary is lost .
While Tbx5 is required for both Sall4 and Nppa
expression, Sall4 represses transcriptional activity of
Nppa in the interventricular septum. Thus, Tbx5
activates a repressor of its own target genes at the
interventricular boundary of its expression domain
T-box factors control chamber position and
Tbx5 and Nkx2-5 mutant embryos fail to develop
including Cx40 and Nppa [13, 59, 78, 79]. These
findings have been fundamental to our insights into
the molecular programs that drive chamber differ-
entiation. However, the highly localized differentia-
expression patterns of these factors in chamber and
primary myocardium. Moreover, both Tbx5 and
Nkx2-5 are involved in the formation of, again very
localized, atrioventricular derived components of the
conduction system [80–82].
In search of a possible mechanism for chamber-
specific expression of Nppa, we demonstrated that
both a single TBE (T-box binding site) and adjacent
NKE (Nkx2-5 binding site) present in the Nppa
promoter are required for repression of Nppa in the
atrioventricular canal  and outflow tract .
Tbx2 and Tbx3 were found to interact with the TBE,
to repress Nppa through this site and to effectively
and Cx40 promoter [61, 62, 83]. Expression of Tbx2
and Tbx3 is confined to primary (non-chamber)
Nppa, Cx40, Cx43, Chisel and other chamber-specific
genes [60–62, 83]. These findings seem to dictate a
model in which chamber formation (atria, left and
right ventricle) and differentiation is driven by
broadly expressed factors. An additional layer of
spatially restricted repressors inhibits this process in
tract, atrioventricular canal, inner curvatures and
outflow tract  (Fig. 4). Tbx2 gain and loss of
function experiments have demonstrated that Tbx2 is
indeed able and required to inhibit chamber forma-
and whereas it is able to block chamber formation
when expressed ectopically, its deficiency does not
lead to obvious defects in atrioventricular canal
patterning, indicating functional redundancy with
Tbx2 [our unpublished observations].
How do Tbx2 and Tbx3 exert their functions? Both
factors act as repressors of transcription and share
DNA binding properties and target genes [31, 85–88].
They effectively compete with Tbx5, a transcriptional
activator, for TBE-binding and for Nkx2-5, a cardiac
accessory factor, thus repressing chamber-specific
genes and chamber differentiation [61, 62, 83]. The
finding that Tbx3 inhibits myogenic differentiation
 is compatible with the assumed roles of these T-
box factors in inhibiting differentiation of chamber
muscle. A conspicuous property of primary myocar-
dium is that it retains its low proliferation rate while
the chambers are rapidly proliferating and expanding.
Both Tbx2 and Tbx3 appear able to bypass senes-
cence, and are reported to be amplified and overex-
pressed in various cancers [28, 89–93]. They directly
suppress the tumor suppressor/cell-cycle inhibitors
p19ARF(Arf) and p21Cip1(Cdkn1a) [28, 89–91, 94], the
latter by recruiting HDAC1 to the initiator of the p21
promoter . Moreover, Tbx2 is tightly regulated
during the cell cycle, with highest expression levels
during late S-phase and G2 . These properties
would seem to support a role for Tbx2 and Tbx3 in
regulating proliferation in the primary myocardium,
but fall short of explaining why their functions seem
opposing in myocardium as compared with other
systems. Moreover, p21, p19ARF/p16INK4aand p15INK4b
are not elevated in Tbx2-deficient embryos, and
mutation of p53, upregulated by p19 suppression,
did not rescue the Tbx2 mutant phenotype .
However, the lack of response may be due to
compensating factors participating in this pathway,
such as Tbx3 that is co-expressed in the heart .
Nmyc1 (N-myc) is required for early myocardial
proliferation [96, 97]. Its transcripts are enriched in
the compact, fast-proliferating layer of the chambers,
in a pattern complementary to that of Tbx2 [52, 96].
Evans and co-workers  found that Tbx2 directly
represses Nmyc1 and cyclin A2 (Ccna2), a feature
Cell.Mol.Life Sci.Vol.64, 2007
Multi-author Review Article
implicated in myocyte proliferation, thus linking
localized Tbx2 expression to localized differences in
proliferation through Nmyc1. However, recent ex-
periments do not support this role of Tbx2. When
ectopically expressed in the pre-chamber heart, Tbx2
[L.t Dupays and T. Mohun, personal communication]
or Tbx3 [our unpublished observations] blocks cham-
ber formation and chamber-specific gene expression,
but Nmyc1 expression is not affected. Therefore, it
seems fair to conclude that the mechanism of the
localized regulation ofproliferationstill remainsto be
Although lineage data are lacking, careful morpho-
logical analysis and gene expression studies indicate
that the sinoatrial node develops from primary
myocardium at the junction between the right sinus
horn and the right atrium, whereas the atrioventric-
ular node develops from the atrioventricular canal.
The node precursors consequently express Tbx2 and
Tbx3. During development, Tbx2 becomes down-
regulated, whereas Tbx3 expression is maintained
specifically in the nodes, and is the only known
transcription factor to be so expressed [61, 62]. As
mature nodes display many features that resemble
primary myocardium in the embryo, it is attractive to
hypothesize that their formation is derived by Tbx2
and Tbx3 maintaining the primary phenotype. Cur-
rent efforts to address this issue are ongoing, and hint
at an even more active role of Tbx3 in node formation
[our unpublished results]. As discussed above, both
Tbx5 and Nkx2-5 are required for atrioventricular
conduction system development [80, 81], while their
of Tbx2/3 into the Tbx5-Nkx2-5 pathway in the
atrioventricular conduction system, we may begin to
explain some of the highly localized defects in mice
haploinsufficient for Tbx5 or Nkx2-5.
T-box factors and transcriptional networks for
septation and conduction system function
A strict dosage level of Tbx5 is required for septum
formation and conduction system maturation . A
large fraction of individuals with Holt-Oram syn-
drome have heart defects, including atrial and/or
(muscular) ventricular septal defects, and they are at
risk for progressive atrioventricular block and atrial
fibrillation [15,98].Thedose-dependentregulation of
cycle progression  probably represent the under-
lying cause of these defects. Not surprisingly, perhaps,
proteins working together to coordinate a process
often derive a similar spectrum of phenotypes when
mutated. Mutations in the Tbx5 interacting partners
cause septum defects and, in the case of Nkx2-5,
atrioventricular conduction defects [19, 23, 100].
Tbx20 can interact with all of these factors, and is
required for septation [20, 21, 54]. Careful analysis of
Tbx20 heterozygous mutant mice revealed atrial
septal defects that were more frequent and severe
than in Nkx2-5+/–Tbx20+/–double mutant mice .
Tbx3 is also part of this network. Mutations in TBX3
cause ulnar-mammary syndrome characterized by
defects in breast development, apocrine gland, limb
and genital formation , and, indeed, low pene-
trance ventricular septal defects and pulmonary
stenosis . These genetic and functional data
indicate that septation of the atria and ventricles is
governed by a tightly regulated and often spatially
constrained network of interacting transcription fac-
tors . The sensitivity of this network and the
many manifestations of septum defects seen in
mutants possibly reflects the complexity of formation
of these structures, which involves complex signaling
and coordinated directional growth and apoptosis of
the cushion mesenchyme and myocardial muscle. The
challenge will be to elucidate the requirements,
functions and interaction of the transcription factors
in the distinct tissues during septation.
A regulatory T-box factor network
Recent studies of the regulation of T-box genes and
the identification of some of their target genes have
provided a picture, albeit far from complete, of the
regulatory network that controls the temporally and
spatially resolved formation of the heart components
(Fig. 4). Whereas Tbx2 and Tbx3 suppress chamber-
specific genes, their own regulation is controlled by
Bmp-Smad signaling. Bmp2 is expressed in the
atrioventricular canal from its earliest stages of
formation onward, and is required for Tbx2 expres-
sion [69, 104]. Furthermore, Bmp2-soaked beads
induce Tbx2 and Tbx3 expression . Conditional
inactivation of the type 1 Bmp receptor (Bmpr1a/
Alk3) in the heart leads to reduced Tbx2 and Tbx3
levels . Evidence has been put forward which
suggests that the Tbx3 promoter is directly regulated
by Bmp Smads that interact with a consensus Smad
binding element 1.3 kbp upstream of the transcription
start site . Since our results seem to demonstrate
that a 6 kbp upstream promoter fragment of Tbx3 is
not sufficient to drive cardiac expression in transgenic
of this pathway to Tbx3 regulation in vivo remains to
be verified. Whereas BMP2induces Tbx2 and Tbx3 in
the atrioventricular canal, the expression profile of
656 W.M.H. Hoogaars et al. T-box factors determine cardiac design
primary myocardial sinus venosus and outflow tract.
Expression of Tbx2 is derepressed in hearts of Tbx20-
indicate that Tbx20 may directly interact with the
Tbx2 promoter to suppress its activity . Because
Tbx20 and Tbx2 expression overlap in the atrioven-
tricular canal and outflow tract, the presence of a
counteracting activating pathway relieving Tbx20-
mediated repression has to be conceived. The most
obvious candidatewould be a Bmp2/4-Smadpathway.
However, whereas Tbx2 was consistently found to be
found to be either down- [52, 54] or ectopically
upregulated . Intriguingly, Tbx20 may itself also
be positively regulated by BMPs. BMP2 induces
Tbx20 expression in the undifferentiated pre-cardiac
mesoderm in chick  and inactivation of type 1
Bmp receptor in cardiac progenitor cells results in a
modest downregulation of Tbx20. However, again
inconsistently, inactivation of Bmp2 in the heart
progenitors does not lead to reduced Tbx20 expres-
sion . Tbx3 is not de-repressed in Tbx20 mutants,
and function, Tbx2 and Tbx3 are regulated by distinct
inputs. Similarly, applied BMP2 induces Tbx2, Tbx3
and Tbx20 but not Tbx5 [104, 106]. Furthermore, a
recent genome-wide analysis of Tbx5 target genes
revealed that Tbx3, but not Tbx2, is positively
regulated by Tbx5 .
Together, these findings provide a first glimpse into
the complicated and multi-layered T-box factor net-
work that controls critical steps in the localized
formation of the components of the heart.
T-box transcription factors have multiple and diverse
roles in gene regulation and heart development. They
are critical to patterning, localized proliferation,
differentiation, chamber formation, development of
conduction system components, septation and valvu-
logenesis. Studying their functions has been reward-
ing, bringing our understanding of heart development
to a higher level. The challenge will be to understand
precisely how T-box factors regulate the different
aspects of heart morphogenesis. Current evidence
shows that T-box factors act in genetic cross-regula-
tory networks, that they act together with a variety of
other proteins, that they have overlapping as well as
unique functions, and that their functions depend on
tissue-specific context and stage of development. To
study these functions, new animal models that allow
cell type- and stage-specific activation or inactivation
of multiple T-box factor genes are being constructed
and analyzed, and large-scale screens for T-box factor
target genes and interaction partners in different
animal models and tissues, are being performed.
These studies will reveal novel and important details
of known molecular pathways controlling specific
aspects of heart morphogenesis, which will bring us
closer to understanding congenital heart defects.
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