1 Victor Chang Cardiac Research Institute, St Vincent's Hospital, 384 Victoria
Street, Darlinghurst, New South Wales 2010, Australia
2 Faculties of Medicine and Life Sciences, University of New South Wales,
Kensington, New South Wales 2056, Australia
* Author for correspondence (e-mail: r.harvey{at}victorchang.unsw.edu.au)
SUMMARY
T-box transcription factors are important players in the molecular circuitry that generates lineage diversity and form in the developing embryo. At least seven family members are expressed in the developing mammalian heart, and the human T-box genes TBX1 and TBX5 are mutated in cardiac congenital anomaly syndromes. Here, we review T-box gene function during mammalian heart development in the light of new insights into heart morphogenesis. We see for the first time how hierarchies of transcriptional activation and repression involving multiple T-box factors play out in three-dimensional space to establish the cardiac progenitors fields, to define their subservient lineages, and to generate heart form and function.
Introduction
The mammalian heart is a mechanical pump that can be viewed developmentally
as a highly modified muscular vessel. The atria are collecting chambers and
the ventricles pumping chambers. Valves separate chambers and guard the
arteries to prevent reflux, and the conduction system coordinates the timing
and efficiency of chamber filling and ejection. One of the remarkable features
of the mammalian heart is that it begins to function from virtually the moment
it comes into being as a simple muscular tube, before the development of the
valves and specialized conduction components. Henceforth, the functional
development of the heart accompanies its morphological development, and we are
now beginning to realize that, to a large (but unknown) extent, function
dictates form. The formation and maturation of chambers and valves, in
particular, are highly dependent on the forces associated with contraction and
blood flow (Bartman et al.,
2004; Hove et al.,
2003
), and on other epigenetic influences such as hypoxia
(Sugishita et al., 2004
). In
this fashion, the efficiencies implicit in adult heart form
(Kilner et al., 2000
) are
actually molded in the embryo by function itself. The molecular details of the
interplay between patterning and functional feedback are largely unknown, and
this additional dimension to developmental regulation brings with it numerous
challenges as we attempt to unravel regulatory circuits.
We have been aided in this endeavour by the discovery a decade ago that
transcription factor pathways guiding cardiogenesis have been conserved in
evolution (Bodmer and Venkatesh,
1998; Harvey,
1996
). A core transcriptional network that involves homeodomain
factors, zinc finger factors of the GATA family, Mef2 factors of the MADS Box
family and, indeed, T-box factors, appears to guide cardiac specification and
differentiation from Drosophila to man, and many of the details of
molecular mechanisms can now be studied using the powerful genetics of
Drosophila (Cripps and Olson,
2002
).
Nonetheless, mammalian heart development involves many morphological
innovations that are not evident in the simple muscular hearts of flies
(Harvey, 2002). In
evolutionary terms, these modifications are relatively recent, and it may be
partly for this reason that they appear vulnerable to genetic perturbation.
Indeed, almost one live-born human baby in 100 has some form of structural
anomaly of the heart (Hoffman and Kaplan,
2002
), many requiring surgical intervention. The evident
connections between cardiac development and human congenital heart disease
(CHD), and our increasing ability to accurately measure heart function in
animal models, even in utero (Zhou et
al., 2002
), ensures that the heart will continue to be a prominent
platform for understanding the origins, the development and the evolution of
organ systems.
Development proceeds via a series of transcriptional and post-transcriptional switches that build regional complexity and functional diversity. Many transcriptional processes in development and organogenesis involve members of the ancient family of T-box transcription factors, named after its founding member, the T protein, now known as Brachyury (see Box 1). This review examines recent new insights into heart morphogenesis and the involvement of T-box genes in these processes. In particular, we explore the roles of T-box genes in defining cardiac progenitor populations, and in the hierarchical transcription factor pathways that specify cardiac chambers and the conduction system.
Heart morphogenesis: new concepts
The basic morphological steps of mammalian heart development are well described (see Box 2). We focus here on two recent insights that have significantly clarified and extended our view of heart morphogenesis, and that have a key relevance to T-box gene function.
The secondary heart field
The discovery that there is selective deployment of two distinct progenitor
pools to the forming heart represents a major advance in how we view cardiac
morphogenesis, and has many implications for the evolution and patterning of
cardiac structures.
The early linear heart tube encompasses progenitors for parts of the
ventricles, with other compartments formed by the addition of cells to its
cranial and caudal poles (De la Cruz et
al., 1977; Noden,
1991
; Redkar et al.,
2001
). We now know that this growth process is based on a series
of progressive lineage restrictions. An elegant fate-mapping technique termed
`retrospective lineage analysis' has shown that the whole heart is likely to
be derived from a single cardiac progenitor pool
(Meilhac et al., 2004
).
However, an early lineage restriction, occurring prior to heart formation,
creates two distinct pools that show profoundly different behaviors
(Fig. 1).
The `first lineage' (Kelly,
2005; Meilhac et al.,
2004
) comes to occupy what we have classically regarded as the
cardiac crescent (see Fig. 1D).
This lineage undergoes early differentiation, as judged by the expression of
myofilament gene markers at the cardiac crescent stage
(Franco et al., 1998
), and is
subsequently used to build the initial linear heart tube, which (in the mouse)
is composed largely of precursors of the left ventricle (LV), with the inflow
region, including the atrioventricular (AV) canal and parts of the atria,
added progressively. The first lineage contributes minimally, if at all, to
the right ventricle (RV) and outflow tract (OFT).
A `second lineage' [referred to at later stages as the secondary heart
field or anterior heart field (Kelly,
2005)] occupies a position caudally and medially to the first
lineage at the cardiac crescent stage, and its differentiation and deployment
to the heart is delayed. On the basis of evidence from a number of sources,
including gene expression patterns (see
Fig. 1A-C) and lineage tracings
using DiI injection, retroviral tagging and Cre-Lox methods, it is now clear
that the second lineage is deployed exclusively to build the RV and OFT,
including its myocardial, smooth muscle and endothelial investments, as well
as parts of the atria (Cai et al.,
2003
; Kelly et al.,
2001
; Mjaatvedt et al.,
2001
; Waldo et al.,
2001
). It contributes only a small number of cells to the LV, at
least initially (Brown et al.,
2004
). At the time of its deployment to the poles of the growing
heart, beginning around E8.5 in the mouse
(Zaffran et al., 2004
), the
secondary heart field appears to encompass cells positioned dorsally and
anterior to the linear heart tube. These cells, collectively termed pharyngeal
mesoderm (Zaffran et al.,
2004
), equate to the dorsal mesocardium and the dorsal pericardial
mesoderm (see Box 2), as well
as to head mesenchyme occupying the future posterior pharyngeal arches. There
may be subdivisions of the second lineage
(Kelly, 2005
), although this
is still a matter of debate (Abu-Issa et
al., 2004
). Nevertheless, cells of the secondary heart field are
beautifully highlighted by the expression of a number of genes and transgenes,
and their deployment to the heart can be followed using tagging methods
(Cai et al., 2003
;
Dodou et al., 2004
;
Hu et al., 2004
;
Kelly et al., 2001
;
Stanley et al., 2001
;
Xu et al., 2004
)
(Fig. 1A-C).
|
Cardiac chamber formation
The origin of chamber myocardium in the forming heart tube has been a
much-debated topic. Recent analyses have now given us a clearer perspective of
this issue, which is so important for our understanding of CHD.
During the incorporation of the first and second lineages into the heart,
chamber myocardium arises in descendents of both lineages
(Christoffels et al., 2004a;
Christoffels et al., 2000
).
Previously, emphasis was placed on the segmental arrangement of chamber
primordia in the forming heart. However, molecular markers, along with
anatomical and electrophysiological observations, now show that chambers are
specified in discreet zones along the outer curvature of the heart tube: the
ventricles at the original ventral surface of the heart tube, and atria
located more caudally in dorsolateral zones (red and blue regions,
respectively, in Fig. 2B)
(Christoffels et al., 2004a
;
Christoffels et al., 2000
).
Several gene markers highlight the forming chambers among them
Nppa, encoding atrial natriuretic factor (ANF;
Fig. 2E-G), and
Chisel/Smpx and Gja5, which encode cytoskeletal and
connexin proteins, respectively. Chamber myocardium is unlikely to arise from
a distinct lineage; rather, it may be induced within primary myocardium in
response to positional cues. Compared with primary myocardium, chamber
myocardium acquires a more differentiated state that is adapted for a lifetime
of mechanical work.
|
The non-chamber myocardium serves several crucial roles in heart
development. The ability of its myocytes to spontaneously depolarize is
greatest in the caudal region of the heart tube. This creates a dominant
pacemaker-like activity in the caudal heart that initiates the slow
anterior-ward propagation of action potentials and peristaltic contraction
waves. Rapid conduction through chamber myocardium is acquired as chambers
mature. Non-chamber myocardium also provides the signals that induce the
formation of prominent endocardial cushions from endothelium, the precursor
structures of valves and septa (see Box
2). Importantly, non-chamber myocardium develops into the more
specialized elements of the central conduction system, including the
sinuatrial (SA) node, the AV node and the Bundle of His (see
Box 3)
(Christoffels et al., 2004a).
Thus, the specification of chamber and non-chamber myocardium in the forming
heart is one of the crucial early lineage decisions upon which much of the
subsequent heart development is based.
T-box genes
T-box proteins are characterized by the presence of a highly conserved
180-amino acid, sequence-specific, DNA-binding domain termed the T-box. The
T-box transcription factor family, of which there are 18 members in mice, is
divided into five subfamilies (Naiche et
al., 2005). Although the crystal structure of Brachyury shows that
it can form a dimer on a palindromic DNA-binding site
(Muller and Herrmann, 1997
),
studies of the binding sites of Brachyury and other T-box factors in genuine
target promoters show that these proteins bind as monomers to one half of the
palindromic site demonstrated for Brachyury
(Naiche et al., 2005
). T-box
proteins function as transcriptional activators, repressors, or both,
depending on the cellular context (Naiche
et al., 2005
), and can interact with other transcription factors
(Bruneau et al., 2001
;
Garg et al., 2003
;
Habets et al., 2002
;
Hiroi et al., 2001
;
Krause et al., 2004
;
Lamolet et al., 2001
;
Maira et al., 2003
;
Stennard et al., 2003
), as
well as with transcriptional co-activators and co-repressors
(Barron et al., 2005
;
Maira et al., 2003
;
Vance et al., 2005
),
nucleosome assembly proteins (Wang et
al., 2004
) and chromatin-modifying proteins
(Lickert et al., 2004
).
Although the specific function of most T-box genes is largely unknown, they
clearly act in a combinatorial and/or hierarchical fashion within the
progenitor fields that shape the developing embryo, including those of the
early mesoderm (Suzuki et al.,
2004), limbs (Messenger et
al., 2005
) and heart (see below). They can also determine distinct
morphogenetic behaviors (Bruce et al.,
2005
; Kwan and Kirschner,
2003
; Russ et al.,
2000
; Yamomoto et al.,
1998
). T-box genes can be rapidly induced by growth factors, often
in a dose-responsive manner (O'Reilly et
al., 1995
). Indeed, development appears to be exquisitely
sensitive to the level of T-box gene expression
(Hatcher and Basson, 2001
),
with several human congenital anomaly syndromes being linked to T-box gene
haploinsufficiency (reviewed by Packham
and Brook, 2003
). Mice bearing mutations in these genes show many
aspects of the corresponding human diseases, providing valuable models for the
further dissection of disease mechanisms
(Naiche et al., 2005
).
Box 3. The cardiac conduction system
The cardiac conduction system (CCS) is composed of several distinct but
integrated components (reviewed by
Christoffels et al., 2004a
A functional CCS arises in the tubular heart before specialized components
become evident anatomically (Christoffels
et al., 2004a
|
At least seven members of the T-box gene family are expressed in the
embryonic heart in humans and vertebrate models, namely Tbx1-5, Tbx18
and Tbx20, and these genes show overlapping expression patterns in
the first and second heart precursor lineages, the myocardium, the endocardium
and valves, the conduction system and the epicardium (reviewed by
Plageman and Yutzey, 2004).
Table 1 documents these genes
and their known loss-of-function phenotypes.
|
An analysis of the Drosophila Dorsocross complex (Doc),
which contains three linked T-box genes related to mammalian
Tbx4/Tbx5/Tbx6, indicates that these genes function in
association with the Nkx2-5-related homeodomain factor `tinman' and the
Gata4-related factor `pannier' at the very heart of cardiac specification
(Reim and Frasch, 2005).
Indeed, Doc triple mutants have no heart. Other Drosophila
T-box genes, midline and H15, related to mammalian
Tbx20, may also participate in cardiac specification, but their main
roles occur later in determining cardioblast differentiation, polarity and
patterning (Miskolczi-McCallum et al.,
2005
; Qian et al.,
2005
; Reim et al.,
2005
). An essential feature of regulation in the
Drosophila heart is the interaction between different T-box genes.
Midline acts in a pathway that represses Doc in a subset of
cardioblasts, and this is crucial for establishing the morphological and
functional characteristics of the distinct classes of muscle cells, one
functioning in the propulsion of haemolymph, and another acting as valves
(Reim et al., 2005
). The
early, instructive and hierarchical roles for T-box genes in fly heart
development appear to be conserved, at least in outline, in the mammalian
heart, as described below.
Tbx1 regulation and function in the secondary heart field
It is likely, as in Drosophila, that TBX5, the gene
mutated in Holt-Oram syndrome, is involved in cardiac specification in
mammals. However, a muscular heart tube is able to form in Tbx5 null
mice, albeit one that is highly truncated, demonstrating that the proposed
early function is not absolutely essential
(Bruneau et al., 2001).
Tbx5 is also involved in heart tube morphogenesis and chamber
formation, as discussed in detail in sections below. More extensive
information on the role of T-box genes in cardiac progenitor populations has
come from analysis of TBX1, which is also involved in human CHD. In
this section, we describe the role of TBX1 in CHD, and its regulation
and function in the secondary heart field.
DiGeorge syndrome, velo-cardio-facial syndrome and conotruncal anomaly face
syndrome are three human congenital anomaly syndromes that share certain
clinical features, including cardiovascular malformations (OMIM numbers 18840,
192430, 217095). They also share the same 1.5 Mb or 3 Mb monoallelic
microdeletion of chromosome 22q11.2, and are now collectively termed the 22q11
deletion syndrome (22q11DS) (for a review, see
Yamagishi and Srivastava,
2003
). This is the most common genetic deletion syndrome in humans
and, as such, its underlying mechanism has attracted intense scrutiny. Through
heroic chromosomal engineering feats in transgenic and knockout mice, the
T-box gene TBX1 has emerged as the leading candidate for disease
causation (reviewed by Lindsay,
2001
), and, indeed, TBX1 mutations have recently been
found in patients who have 22q11DS-like symptoms but not the deletion
(Yagi et al., 2003
). The
clinical features of 22q11DS include severe cardiac OFT and aortic arch
anomalies, as well as other structural malformations of the pharyngeal complex
and behavioural problems. Cardiovascular defects include tetralogy of Fallot,
caused by mal-partitioning of the OFT vessels, persistent truncus arteriosus,
in which the OFT is not septated, and interruption of the left fourth aortic
arch artery. These abnormalities are similar to those caused by the ablation
of cranial neural crest (CNC) cells, and the syndrome has long been regarded
as a `neurocristopathy' (reviewed by Kirby
and Waldo, 1995
). Tbx1 heterozygous mice show defects of
aortic arch artery four, whereas Tbx1 null mice develop most of the
clinical features of 22q11DS, albeit at the severe end of the spectrum,
causing perinatal death (Jerome and
Papaioannou, 2001
; Lindsay et
al., 2001
; Merscher et al.,
2001
).
Tbx1 is a transcriptional activator
(Altaliotis et al., 2005;
Hu et al., 2004
) and, in
mouse, the gene is expressed in head mesoderm, from as early as E7.5, and in
pharyngeal endoderm (Chapman et al.,
1996
; Yamagishi et al.,
2003
). Cre-based lineage tracking shows that the Tbx1
mesodermal domain includes cells of the secondary heart field
(Xu et al., 2004
). The
finding that Tbx1 is not expressed in neural crest cells revealed,
contrary to expectation, that defects of the pharyngeal complex in 22q11DS
secondarily lead to failure of the cellular mechanisms that support CNC
development. Tbx1 expression in head mesoderm and endoderm is
maintained by the signaling morphogen sonic hedgehog (Shh), which is secreted
from pharyngeal ectoderm (Hu et al.,
2004
; Yamagishi et al.,
2003
). Shh induces expression of the forkhead-class transcription
factors Foxc1 and Foxc2 in mesoderm, and Foxa2 in endoderm, and these factors
act directly on Tbx1. In mesoderm, the expression of Foxc1
and Foxc2 are themselves dependent on Tbx1, showing that a
positive-feedback loop involving Fox proteins maintains mesodermal
Tbx1 expression (Hu et al.,
2004
). Retinoic acid signaling represses Tbx1 expression
in the caudal pharynx (Roberts et al.,
2005
).
Fibroblast growth factors (Fgfs) 8 and 10 are key downstream effectors of
Tbx1 in the pharyngeal region. Fgf8 and Fgf10 are both
expressed in pharyngeal tissues in patterns that overlap that of Tbx1
(Kelly et al., 2001;
Vitelli et al., 2002b
)
(Fig. 1A,C), and both Fgf genes
are downregulated in mesoderm of Tbx1 null and hypomorphic embryos
(Hu et al., 2004
;
Vitelli et al., 2002b
).
Furthermore, embryos hypomorphic or conditionally deleted for Fgf8
display the same cardiac and pharyngeal abnormalities seen in Tbx1
null mice and 22q11DS (Abu-Issa et al.,
2002
; Frank et al.,
2002
; Macatee et al.,
2003
; Trumpp et al.,
1999
). Genetic tests have confirmed that Tbx1 and
Fgf8 act in the same pathway
(Vitelli et al., 2002b
).
The ShhFoxc1/c2/a2
Tbx1
Fgf8/10 pathway has multiple roles
in the pharyngeal region: it maintains cell proliferation and differentiation
in mesenchyme, and is important for patterning the endoderm, as well as for
the survival, differentiation and migration of neural crest cells
(Abu-Issa et al., 2002
;
Frank et al., 2002
;
Hu et al., 2004
;
Macatee et al., 2003
;
Xu et al., 2004
). The
variable penetrance of 22q11DS phenotypes is modelled in mutant mice carrying
various combinations of null, hypomorphic or conditionally deleted alleles
(Abu-Issa et al., 2002
;
Hu et al., 2004
;
Xu et al., 2004
). This
probably reflects the multiplicity and complexity of the developmental
processes involved, and in particular their common dependence upon secreted
factors, such as Fgf8 and Fgf10, that are expressed in multiple tissues.
Interestingly, the infiltration of neural crest into the pharyngeal region is
necessary for the normal deployment of myocardium to the OFT, highlighting the
inter-dependence of tissue development in this zone
(Waldo et al., 2005
).
In the secondary heart field, Tbx1 functions in both growth and
differentiation. Fgf8 and Fgf10 are expressed in secondary
heart field mesoderm and associated endoderm, as well as weakly in the OFT
(Hu et al., 2004;
Kelly et al., 2001
). The OFTs
of hearts mutant for Tbx1 or hypomorphic for Fgf8 are
truncated, due to the reduced deployment of Tbx1-positive cells
(Xu et al., 2004
). For
Tbx1, this effect is cell non-autonomous, confirming the involvement
of secreted factors in cell behaviour. These data are consistent with a role
for the Tbx1
Fgf8/Fgf10 pathway in driving proliferation in secondary
heart field mesoderm, contributing to OFT growth. BMP proteins are also
induced in secondary heart field cells proximal to the inflow and outflow
poles of the heart (Cai et al.,
2003
; Waldo et al.,
2001
). BMPs can induce cardiomyogenic differentiation in
collaboration with Fgf8 (Alsan and
Schultheiss, 2002
) and can moderate the proliferation of secondary
heart field mesoderm (Waldo et al.,
2001
). Therefore, a delicate balance between the levels of Fgf and
BMP factors appears essential for secondary heart field development.
T-box genes in chamber development
Mutations in human TBX5 cause the rare autosomal-dominant
Holt-Oram syndrome, which is characterized by forelimb and cardiac congenital
abnormalities, the latter including atrial and ventricular septal defects,
tetralogy of Fallot, hypoplastic left heart, and conduction anomalies (OMIM
number 142900) (Basson et al.,
1997; Li et al.,
1997
). Similar defects are found in Tbx5 heterozygous
mice (Bruneau et al.,
2001
).
Mouse Tbx5 is expressed in the cardiac crescent, indicating its
involvement in the earliest stages of cardiac induction, then in a graded
fashion along the heart tube with the highest levels in the sinuatrial region
(Bruneau et al., 1999). The
graded pattern is established by retinoic acid signaling, which is known to be
essential for the formation of the sinuatrium
(Liberatore et al., 2000
;
Niederreither et al., 2001
).
Expression of Tbx5 remains high in the caudal heart during subsequent
development, but also occurs at lower levels in the LV, left half of the
interventricular septum and trabeculae of the RV
(Bruneau et al., 1999
). The
graded pattern of Tbx5 expression across the heart appears to play an
instructive role in determining the molecular identity and morphogenesis of
chambers. For example, enforced expression of Tbx5 relatively evenly
throughout the heart tube leads to what is likely to be a `caudalization' of
more anterior regions: the upregulation in the RV of markers that are
typically high in the LV; loss of the inter-ventricular septum and trabeculae;
and downregulation of a ventricle marker in the LV
(Liberatore et al., 2000
;
Takeuchi et al., 2003b
). Tbx5
overexpression inhibits myocyte proliferation
(Hatcher et al., 2001
), which
could also be interpreted in this light. In Tbx5 null embryos, the
left ventricular and sinuatrial regions are severely hypoplastic
(Fig. 3C), and numerous chamber
markers, including Nppa, are not expressed
(Bruneau et al., 2001
).
Known target genes of Tbx5 are few. However, Tbx5 associates directly with
other conserved cardiac transcription factors, including the homeodomain
factor Nkx2-5 and the zinc finger factor Gata4
(Bruneau et al., 2001;
Garg et al., 2003
;
Hiroi et al., 2001
), as well
as with the transcriptional co-activators Tip60, a histone acetyltransferase
(Barron et al., 2005
) and
Baf60c, a component of the Swi/Snf-like BAF chromatin remodeling complex
(Lickert et al., 2004
).
CHD-causing mutations in TBX5 or in its partner factors are known to disrupt
some of these interactions (Bruneau et
al., 2001
; Fan et al.,
2003
; Garg et al.,
2003
; Hiroi et al.,
2001
). Acting in synergy with Nkx2-5 and Gata4, Tbx5 can stimulate
transcription from the promoters of chamber-specific genes such as
Nppa and Gja5 (which encodes connexin 40) in vitro
(Bruneau et al., 2001
;
Habets et al., 2002
;
Hiroi et al., 2001
;
Lickert et al., 2004
).
We can conclude that Tbx5 is a positive transcriptional driver of cardiac
specification, as well as of chamber morphogenesis and differentiation, in the
developing mammalian heart. Other cardiac transcription factors, including
Nkx2-5, are also essential for chamber differentiation
(Lyons et al., 1995;
Palmer et al., 2001
;
Tanaka et al., 1999
).
However, the documented features of Tbx5 and Nkx2-5 fall
well short of explaining the spatial specificity of chamber development. For
example, Tbx5 and Nkx2-5 are not expressed in a
chamber-specific manner, and, paradoxically, both factors are essential for
the differentiation of the central conduction system, a derivative of
non-chamber myocardium (Jay et al.,
2004
; Moskowitz et al.,
2004
; Pashmforoush et al.,
2004
). These factors are likely to act as `selector genes',
defining an organ-specific context for many cardiac processes
(Barolo and Posakony, 2002
). To
understand spatial specificity in heart development, other players are
required, and the dynamics of signal-dependent transcriptional changes need to
be defined.
|
The inclusion of Tbx2 and its close relative Tbx3 in the cardiac regulatory
network has given us a way to visualize at least some of the spatial aspects
of cardiac chamber formation. Tbx2 and Tbx3 are transcriptional repressors
(Carreira et al., 1998;
Habets et al., 2002
;
Hoogaars et al., 2004
;
Jacobs et al., 2000
;
Sinha et al., 2000
), and Tbx2
can bind the co-repressor Hdac1, a histone deacetylase
(Vance et al., 2005
). In the
developing heart, Tbx2 and Tbx3 are expressed in non-chamber myocardium,
mutually exclusively of markers of chamber myocardium such as Nppa
(Habets et al., 2002
;
Hoogaars et al., 2004
)
(Fig. 2E-J). In vitro they act
as potent repressors of the promoters of Nppa and Gja5,
which are normally activated in chamber myocardium by Tbx5 and Nkx2-5
(Habets et al., 2002
;
Hoogaars et al., 2004
).
Indeed, Tbx2 and Nkx2-5 form a repressive ternary complex on Nppa
promoter DNA, and mutagenesis has shown that this complex formation depends on
a T-box binding element (TBE) and, to a lesser extent, on a Nkx2-5-binding
element (NKE). Indeed, by transgenic analysis, the TBE and NKE have been shown
to be crucial for Nppa repression in non-chamber myocardium of the AV
canal, with the mutation of either element causing promiscuous transgene
expression in that region (Habets et al.,
2002
). An attractive model is that Tbx2 competes away the positive
chamber factor Tbx5 from the TBE/NKE element of the Nppa gene in
non-chamber myocardium, forming a repressive complex with Nkx2-5
(Fig. 4). In support of this
model, enforced expression of Tbx2 (or Tbx3) throughout the
developing heart tube completely blocks chamber formation
(Christoffels et al., 2004b
;
Hoogaars et al., 2004
), and,
in Tbx2 knockout mice, several chamber markers are activated
inappropriately across the AV canal, effectively merging the LV with the atria
(Harrelson et al., 2004
).
Tbx2 controls regional proliferation through Nmyc1
The data above show that Tbx2 and Tbx3 participate in
setting or reinforcing the boundaries between atrial and ventricular chambers,
but do they guide morphogenesis directly? In analyzing Tbx20 knockout
mice (see below), Evans and colleagues revealed a direct role for Tbx2 in
repressing the expression of Nmyc1 in non-chamber myocardium
(Cai et al., 2005).
Nmyc1 encodes a basic helix-loop-helix leucine zipper transcription
factor related to Myc (previously known as c-myc) that
heterodimerizes with its partner Max. Myc proteins are thought to `tune' the
expression of many genes involved in metabolism (protein synthesis and/or cell
growth), apoptosis and the cell cycle
(Hipfner and Cohen, 2004
).
Studies in the developing lung and brain show that Nmyc1 drives proliferation
in their precursor populations (Kenney et
al., 2003
; Okubo et al.,
2005
).
Nmyc1 is expressed in the heart tube, with transcripts being
enriched in chamber myocardium in a complementary pattern to that of Tbx2
(Cai et al., 2005;
Moens et al., 1993
)
(Fig. 3G,I). With further
development, expression becomes restricted to the so-called `compact
myocardium', corresponding to the outer cellular layers of the ventricles that
expand massively through proliferation. Nmyc1 is in fact essential
for early chamber growth and expansion of the compact layer, as shown by the
analysis of knockout and hypomorphic embryos
(Charron et al., 1992
;
Moens et al., 1993
;
Sawai et al., 1993
).
|
It should be noted that Tbx3 has anti-apoptotic activity in bladder cells
(Ito et al., 2005), and that
Tbx2 and Tbx3 are amplified or overexpressed in pancreatic, breast and skin
tumours. These T-box genes are suspected to override senescence by directly
repressing the cell-cycle inhibitor p21Cip1 gene
(Vance et al., 2005
). The
specific roles for Tbx2 and Tbx3 in facilitating proliferation in other
systems contrast with those described in the heart, in which Tbx2 inhibits
proliferation via the repression of Nmyc1. The roles of T-box genes
in cell-cycle control may therefore vary in different tissues. A common theme,
nonetheless, is that Tbx2 and Tbx3 mediate the transcriptional repression of
cell cycle-related genes.
Hierarchies of repression: Tbx20 negatively regulates Tbx2
A yet higher tier of T-box regulation in the heart has recently come to
light. Four papers this year reported the genetic or RNAi-mediated
knockout/knockdown of Tbx20 in mice
(Cai et al., 2005;
Singh et al., 2005
;
Stennard et al., 2005
;
Takeuchi et al., 2005
), and
knockdowns of Tbx20 orthologues in fish and frogs have also been
reported (Brown et al., 2005
;
Szeto et al., 2002
).
Tbx20 is an ancient member of the T-box family and belongs to the
same sub-family as Tbx1
(Plageman and Yutzey, 2004
).
In the mouse, it is expressed in the cardiac crescent, with some possible
overlap with cells of the secondary heart field
(Kraus et al., 2001
;
Takeuchi et al., 2005
). In
the heart tube, Tbx20 is expressed across the myocardium and also
strongly in endothelial cells associated with endocardial cushions
(Stennard et al., 2003
;
Stennard et al., 2005
;
Takeuchi et al., 2005
). With
further development, expression is turned down in chamber myocardium, but
remains high in endocardial cushions
(Stennard et al., 2005
).
Tbx20 carries strong transcriptional repression and activation domains, and
interacts directly with other cardiac transcription factors, including Tbx5,
Nkx2-5, Gata4/5 and Isl1 (Brown et al.,
2005
; Stennard et al.,
2003
; Takeuchi et al.,
2005
).
The consequences of Tbx20 loss in the mouse are catastrophic for heart
development (Cai et al., 2005;
Singh et al., 2005
;
Stennard et al., 2005
;
Takeuchi et al., 2005
). The
mutant heart tube is small, does not loop, and the deployment of progenitor
cells is impaired (Fig. 3A,B).
Whilst some details of characterization differ between groups, several clear
findings emerge. The expression of the transcription factors genes Nkx2-5,
Gata4 and Mef2c, and the cardiac inducers Bmp2/Bmp5, is
downregulated or delayed, suggesting some positive, albeit redundant,
involvement of Tbx20 in inducing and supporting the core cardiac
transcription factor network, as has been suggested for Tbx20
orthologues in Drosophila
(Miskolczi-McCallum et al.,
2005
; Qian et al.,
2005
). This is consistent with the ability of Tbx20 to activate
cardiac enhancers of Nkx2-5, Mef2c and other heart regulatory genes
in vitro (Takeuchi et al.,
2005
). Small ventricle-like chambers are formed in null mutants,
and these appear to represent precursor pools for the left and right
ventricles, indicating that both first and second heart lineages contribute to
the mutant heart tube. However, chamber myocardium does not differentiate, as
seen by the lack of chamber-restricted markers Nppa, Smpx, Cited1 and
Hand1.
A key finding is that Tbx2 expression is dramatically upregulated
in probably all myocardial progenitor cells in Tbx20 knockout embryos
(Cai et al., 2005;
Singh et al., 2005
;
Stennard et al., 2005
)
(Fig. 3D). As discussed, Tbx2
is a transcriptional repressor that normally inhibits cell proliferation and
chamber differentiation in non-chamber myocardium. Tbx2
overexpression might therefore explain both the complete block of chamber
differentiation, and the severely hypoplastic state of myocardium in
Tbx20 mutant hearts. Hypoplasia is evident after staining in
wholemount or on sections for phospho-histone H3
(Fig. 3E,F). Nmyc1
expression, normally high in chamber myocardium, is virtually eliminated in
the mutant hearts (Fig.
3G-J).
An important mechanistic detail is whether repression of Tbx2 by
Tbx20 is direct or indirect. A pair of TBEs was found 680 bp upstream of
the Tbx2 transcriptional start site, and ChIP analysis shows that
these are occupied by Tbx20 (Cai et al.,
2005
). Tbx20 can repress this portion of the Tbx2 promoter in
vitro in a TBE-dependent manner. We can conclude that Tbx20 represses
Tbx2 directly, and one of its principal roles is to keep Tbx2, a
repressor of chamber differentiation, off in the developing chamber myocardium
(Fig. 4). This seems to fit the
concept of `default repression', a feature of virtually all conserved
signal-responsive transcriptional pathways acting in development
(Barolo and Posakony, 2002
). In
essence, genes that are required to affect an important developmental switch
need to be actively repressed so that other positive drivers of their
transcription, particularly `selector genes', do not cause cryptic activation.
When activated by an external signal, the repressor is displaced or converted
to an activator.
In non-chamber myocardium, the expression of Tbx20 and
Tbx2 overlap. Thus, the inhibitory role of Tbx20 on Tbx2,
clearly evident in chambers, must itself be somehow inhibited in non-chamber
myocardium. We ascribe this function to factor X in
Fig. 4, and because Bmp2 and
Bmp4 are expressed in non-chamber myocardium of the looping heart, and because
they have been implicated in the induction of Tbx2 in vitro
(Yamada et al., 2000), they
are good candidates for this role. Factor X is presumably a signal that
converges on Tbx20 to release the default repression of Tbx2.
Other repressive roles for Tbx20 are also evident in heart development.
Expression of Myl2, a ventricle region marker, is downregulated in
Nkx2-5 mutant embryos, but was `re-expressed' in doubly homozygous
Nkx2-5/Tbx20 mutants, inextricably implicating Tbx20 as a repressor
in the pathway leading to Myl2 expression
(Stennard et al., 2005).
Role of Tbx20 in heart disease
Heterozygous Tbx20 mutant mice appear to be healthy and are
fertile. However, several findings suggest that we should be looking for the
involvement of TBX20 in human CHD and in adult cardiac pathologies.
It is well known that mutations in the cardiac transcription factors TBX5,
NKX2.5 and GATA4 cause a range of CHDs, including atrial septal defect (ASD)
(Prall et al., 2002). TBX20
associates directly with all of these factors
(Brown et al., 2005
;
Stennard et al., 2003
). As in
Nkx2-5 heterozygous mice (Biben
et al., 2000
), there is an increased prevalence of mild atrial
septal anomalies, including atrial septal aneurysm (ASA) and patent foramen
ovale (PFO), in Tbx20 heterozygous mice
(Stennard et al., 2005
). Our
previous work has suggested that ASD, ASA and PFO are degrees of the same
anatomical and pathological continuum
(Biben et al., 2000
), raising
the possibility that the Tbx20 heterozygotes are actually genetically
sensitized to ASD. Indeed, 16% of doubly heterozygous
Nkx2-5/Tbx20 mutants show fully frank ASD
(Stennard et al., 2005
).
Echocardiography on these mice has also shown that they have dilated
cardiomyopathy, with reduced ejection fraction, increased end-systolic LV wall
dimensions, and occasional gross dilation of the RV with myocyte disarray
(Stennard et al., 2005
).
These features are similar to those seen during heart failure in humans.
Intriguingly, no cardiomyocyte hypertrophy was seen in these mice at an
anatomical or molecular level, suggesting that hypertrophy, which is normally
seen as a compensatory response to cardiac pathology, is blocked if
Tbx20 levels are compromised
(Stennard et al., 2005
).
Other developmental transcription factors, including Gata4 and Mef2c, are
directly involved in the hypertrophic response
(Liang and Molkentin, 2002
),
and Tbx20 may be an important partner for these factors.
|
Perspectives
There is much more to learn about cardiac development and its relationship
to CHD. Understanding T-box gene function in both first and second heart
lineages, and during chamber formation, is clearly at the centre of this
endeavour. Our view of T-box factor involvement in cardiac chamber formation
(Fig. 4) deliberately places
Tbx2 in a nodal position. This is because it is a key regulator of the
chamber/non-chamber lineage separation upon which all subsequent cardiac
morphogenesis depends. But why has this important lineage decision in cardiac
morphogenesis utilized repression of a repressor, or, if Factor X is invoked,
repression of a repressor of a repressor? Superficially, this seems terribly
inefficient, so why has it evolved? The answer to this may lie in how
functional efficiencies in the hearts of mammals and other species were
achieved through the innovation of valves, septa and specialized conduction
components. It is likely that the most expedient evolutionary strategy for
crafting these parts was to modify lineages already existing in the simplest
and most ancient of hearts, namely myocytes and endothelial cells. We propose
that an essential developmental and evolutionary theme in the
chamber/non-chamber lineage split is modulation of the extent of myogenesis
driven by the ancient conserved core transcriptional pathway. In chambers,
ancestral myogenesis is `enhanced', and the neuregulin pathway, essential for
full chamber differentiation, is likely to be involved in this process
(Burden and Yarden, 1997). In
non-chamber myocardium, myogenesis is `repressed', allowing the specialization
of other components, such as conduction cells, which essentially retain the
electrical but not contractile properties of muscle. Tbx2 is the nodal element
that `de-tunes' myogenesis in non-chamber myocardium. It is interesting that
Tbx2 and Tbx3 are also expressed in the second heart lineage mesoderm, so we
can anticipate interesting, perhaps repressive, interactions with Tbx1
function in this region.
Applied to mammalian heart development, this viewpoint would posit that
neither chamber nor non-chamber myocardium is the default evolutionary state.
Importantly, the model also allows us to see how function begets form in heart
development (see Introduction). Pathways responsive to biomechanical stress or
other cues associated with function would somehow exaggerate chamber growth
and differentiation (Sedmera et al.,
2000). The scheme shown in Fig.
4 will therefore serve as a template for expanding our
understanding, not only of chamber formation per se, but of the many other
inputs affecting developmental regulation in this important organ.
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