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.
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