Cardiovascular Research 2003 58(2):264-277; doi:10.1016/S0008-6363(03)00286-4
© 2003 by European Society of Cardiology
Copyright © 2003, European Society of Cardiology
Molecular pathways in myocardial development: a stem cell perspective
Mark J. Sollowaya and
Richard P. Harveya,b,*
aVictor Chang Cardiac Research Institute, 384 Victoria Street, Darlinghurst 2010, Australia
bFaculties of Medicine and Life Sciences, University of New South Wales, Kensington 2052, Australia
r.harvey{at}victorchang.unsw.edu.au
* Corresponding author. Present address: Victor Chang Cardiac Research Institute, 384 Victoria Street, Darlinghurst 2010, Australia. Tel.: +61-2-9295-8520; fax: +61-2-9295-8528.
Received 9 December 2002; accepted 10 February 2003
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Abstract
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The heart has long been considered to adapt to increased work
or pathology through the cellular growth process of hypertrophy.
However, recent evidence for the existence of endogenous stem
cells and regenerative capacity in the adult heart has given
new impetus to the quest for cell therapies for heart failure,
which remains the number one killer in Western cultures. The
molecular cues driving cardiac development are now being explored
in detail and will come into sharp focus as regimes for stem
cell differentiation and efforts to augment endogenous regeneration
are trialed. This review briefly outlines the current state
of knowledge on the molecular basis of the four modalities of
myogenesis that have been identified in the developing vertebrate
heart. Stem cell-mediated myogenic repair in the heart represents
a fifth modality, and an exciting frontier with basic and practical
implications that remain to be explored.
KEYWORDS Stem cells; Cardiac stem cells; Heart development; Cell therapy; Cardiomyopathy
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1 Introduction
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Stem cells are the essential building blocks of metazoans. Until
recently, it has been assumed that embryonic stem cells become
more and more restricted in their developmental potential during
ontogeny, and that a limited number of stem cell populations
persist in the adult as dedicated servants of single regenerative
organ systems such as blood, skin, gut, liver etc. Findings
that stem cells are much more widespread in adult tissues than
anticipated, and that embryonic and adult stem cells have unexpectedly
broad developmental potential, even able to cross germ-layer
boundaries, has challenged our ingrained beliefs about developmental
processes
[1].
Biologists and clinicians have been quick to grasp the great potential of stem cells or their differentiated progeny for cellular therapies in the treatment of degenerative diseases and diseases of the aged. Differentiation of embryonic or adult stem cells in vitro into organ-specific progenitors or more mature cells has potential to revolutionise therapies for ischaemic heart disease, diabetes, stroke, Parkinson's disease, skeletal myopathies and perhaps a host of other conditions. It may also be possible to re-stimulate organ-specific regenerative processes that are present in lower vertebrates but have become attenuated in mammals [2].
The heart has long been thought to adapt to increased work and compensate for disease exclusively through the cellular growth process of hypertrophy. However, recent evidence for the existence of endogenous stem cells and regenerative capacity in the adult heart has given new impetus to the quest for cell therapies for the diseased or failing heart [3]. A number of cell grafting procedures have already been trialed in animals, including the use of embryonic, foetal and adult cardiomyocytes, cardiomyocyte tumour cells, smooth muscle cells, dermal fibroblasts, skeletal myoblasts, ES-derived cardiocytes and adult stem cells [3]. Practical, biological and ethical issues are associated with some of these approaches. However, the rational guided development of stem cell populations into cardiac muscle lineages in vitro remains a promising way forward for this field. Mobilisation or augmentation of endogenous stem cell populations capable of multi-lineage heart regeneration also appears feasible [4]. Jessell and colleagues have recently reported the orchestrated production of spinal motor neurons from mouse ES cells in vitro [5], an undertaking that relied on a sophisticated knowledge of neurogenic and patterning pathways in the embryo. A similar understanding of cardiac developmental pathways may be of enormous strategic benefit in devising or refining cellular therapies for the heart. The purpose of this review is to briefly outline what is currently known about the genes and developmental pathways that specify the cardiac myocytes of the vertebrate heart. Our understanding of these events is evolving rapidly and we are progressing towards a blueprint for the cardiac muscle program found in the embryo that will be the basis of future work in molecular cardiac bio-engineering.
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2 Overview of cardiac morphogenesis
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Cardiac myocytes are derived from the mesodermal germ layer,
and the position of their progenitor cells can be mapped within
the primitive ectodermal layer prior to gastrulation. The bilaterally-arranged
progenitors migrate through the node and primitive streak at
gastrulation
[6], and move to the anterior and anterior/lateral
aspects of the embryo to form the so-called paired heart fields
[7] which then converge anteriorly over the foregut lip to form
the cardiac crescent (
Fig. 1). Heart field cells are defined
as those that have cardiac developmental potency when explanted
and placed into culture
[8]. In lower vertebrates such as amphibia,
heart fields may include cells that do not ultimately contribute
to the heart, but can do so in a regenerative mode if definitive
precursors are removed or damaged
[9]. In a progression associated
with foregut pocket formation, heart progenitor cells move ventrally
and fuse at the midline to create the linear heart tube. This
consists of an inner endothelial tube shrouded by a layer of
myocardium that remains attached to the ventral aspect of the
foregut. The elongating heart tube then largely loses its connection
to the foregut and adopts a rightward spiral in a process termed
cardiac looping morphogenesis. It is during looping that the
primitive cardiac chambers become evident as a pattern of swellings
and constrictions along the heart tube
[10]. Looping is guided
by an embryonic left/right axial pathway that determines the
rightward direction of ventricular bending and distinct morphological
identities of left and right atria. At the completion of looping
(around E12.5 in the mouse), the heart has assumed a form that
closely resembles that of the adult organ. There is extensive
remodelling of the internal structures of the heart during looping,
with septation and valvulogenesis completing the separation
and connectivity of the chambers.
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Fig. 1 Heart development. Stylised pictures (above) and sections (below) depict the major transitions in early mammalian heart development. Dark shading represents myocardial tissue. AV, atrioventricular; LA, left atrium; LV, left ventricle. Reproduced with permission from Harvey et al., 2003 [149].
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The genetic pathways underlying cardiogenesis are complex and
interconnected (
Fig. 2). There are several modalities of cardiac
myogenesis that may differ in mechanism. Furthermore, inductive
interactions between the endocardium and myocardium, and epicardium
and myocardium, are essential for correct growth and differentiation
of chamber muscle. The morphological and molecular aspects of
these processes will be discussed in further detail below.
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Fig. 2 Lineage map of cardiogenesis. A brief summary of the complex lineage relationships (solid lines) and interactions (dashed lines) that occur during vertebrate heart development. See text for details. AV, atrioventricular; OFT, outflow tract.
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3 Induction of cardiogenesis in the heart fields: the role of bone morphogenetic proteins
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Numerous cardiac-restricted transcription factor genes are induced
in the heart fields
[11]. The earliest known gene encodes the
zinc finger transcription factor, GATA4.
Gata4 is expressed
in anterior endoderm and mesoderm and may help restrict mesoderm
to a cardiac fate
[12]. Later, this gene is vital for activation
of many myocardial and endodermal differentiation genes
[13].
Nkx2-5, encoding a homeodomain factor, is also expressed early
in the cardiogenic program and is often used to delineate cardiac
progenitors
[9,14,15]. Induction of these genes occurs before
the first appearance of endocardium and before imposition of
the intra-embryonic coelom, which separates heart precursors
from pericardial mesoderm (
Fig. 2). Thus, the initial heart
precursor cells are the common progenitors of myocardium, endocardium
and pericardial mesoderm
[14]. A large body of evidence shows
that factors secreted from endoderm immediately juxtaposed to
heart mesoderm play a key role in cardiac induction
[12,16] (
Fig. 3). Several members of the bone morphogenetic protein
(BMP) family of secreted signalling molecules are expressed
in endoderm of the cardiogenic region as well as in ectoderm
and cardiac mesoderm itself
[17–19]. Application of Bmp2
or Bmp4 to explants of cardiac or non-cardiac regions of chick
embryos induces expression of early cardiac markers such as
GATA4,
Nkx2-5,
Tbx2 and
MHC, and a beating phenotype, whereas
inhibition of BMP signalling blocks expression of
Nkx2-5 and
cardiac differentiation
[17,20–22]. The dose of BMP seen
by cardiac progenitors may be critical for correct induction
[12,23]. In contrast to explant studies, ectopic placement of
BMP-soaked beads or BMP-expressing cells into head mesoderm
can induce cardiac markers such as
Nkx2-5, but does not induce
myosin expression or a beating phenotype
[17,20]. Full cardiogenesis
in explants may therefore be due to the interaction of BMP signals
with other components, perhaps fibroblast growth factors (FGFs;
see below), present in serum or embryo extract used in the culture
medium
[23]. Genetic dissection of the role of BMPs in mice
appears complicated by genetic redundancy. However, some embryos
lacking
Bmp2 do not form a heart at all
[24]. Furthermore, the
hearts of most BMP2 mutant embryos, and those lacking its downstream
effector gene
Smad5, are abnormally placed and develop poorly
[22,25,26]. Dominant inhibition of BMP signalling in frog and
chick embryos inhibits cardiogenesis
[27–29].
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Fig. 3 Positive and negative signalling inputs which shape the cardiac progenitor fields. Figure shows a stylised transverse section through the posterior region of the cardiac crescent of a mouse embryo at E7.5–8.0 (Fig. 1). Arrows indicate positive inductive signals from tissue layers neighbouring the myocardium. Bars indicate negative influences. Note that the intraembryonic coelom is shown as formed in this figure. This cavity separates the cardiac mesoderm (ventral) from pericardial mesoderm (dorsal). However, early cardiogenic signals act prior to coelom formation. Adapted with permission from Harvey, 2002 [150].
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Smad proteins are transcription factors that are positively
regulated by phosphorylation as a result of signalling downstream
of TGFβ receptor family members. Smad1, 3 and 5 are regulated
by BMPs and during cardiac induction, BMP-Smads appear to directly
activate early cardiac transcription factor genes, including
Nkx2-5 [30–32]. It is now well established that heart
formation in the fruitfly,
Drosophila, is guided by genetic
pathways homologous to those used in vertebrates
[33–35] and the direct action of BMP-Smads on cardiac transcription
factor genes is one of the features of these pathways that has
been conserved in evolution
[36].
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4 Upstream of bone morphogenetic proteins
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The organiser, or node-equivalent in
Xenopus, has long been
known to be critical for cardiac induction
[37]. Several recent
studies have noted that BMP signalling, while maintaining cardiac
regulatory gene expression, is not involved in the very earliest
phases of induction
[27–29]. Furthermore, in the mouse,
an early phase of
Nkx2-5 expression is lost in embryos lacking
the
Smoothened (
Smo) gene, which encodes a membrane-bound signalling
protein for members of the Hedgehog family of secreted factors
[38]. Expression recovers to normal levels by E9.0. Although
these findings need extending, data suggest that the earliest
stages of
Nkx2-5 expression are initially regulated by a Hedgehog
signal. Cardiac progenitors may be exposed to Hedgehog signals
from the node (Sonic and Indian Hedgehog) and/or visceral endoderm
(Indian Hedgehog)
[39]. Indian Hedgehog can induce expression
of the
Bmp4 gene
[39], possibly connecting early and later inductive
events in the forming heart. The maintenance role played by
BMPs in early cardiac induction is also seen in
Drosophila.
Here, the fly homologues of BMP2 and 4, called Dpp and Screw,
are required to maintain expression of
tinman, the homologue
of
Nkx2-5, in dorsal mesoderm that carries the heart progenitors
[35].
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5 A requirement to block Wnt/β-catenin signalling
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The cardiac crescent is shaped not only by the positive influences
of BMPs and other inducers, but also by repressive signals emanating
from axial mesoderm and the neural plate (
Fig. 3). BMP induction
of cardiac markers in chick anterior mesendodermal explants
is almost completely blocked by juxtaposed explants of neural
tube and notochord
[40]. However,
Nkx2-5 expression is still
induced in these experiments, suggesting that signals from axial
tissues prevent propagation of the cardiac program downstream
of initial
Nkx2-5 expression. Signals from the notochord may
include BMP antagonists such as noggin and chordin
[41]. Furthermore,
two members of the Wnt family of secreted glycoproteins, Wnt-1
and Wnt-3a, are expressed in the dorsal neural plate at the
time of cardiac induction and can mimic its repressive effects
in culture
[42]. Indeed, ectopic expression of a Wnt antagonist
can overcome the repressive effects of the neural plate on cardiogenesis
[42]. Wnt antagonists of the Dickkopf and Frizzled-like families
are expressed in the organizer/node as well as in endoderm neighbouring
the heart mesoderm
[43–45]. Importantly, these antagonists
can activate cardiogenesis in non-cardiac mesoderm of frog and
chick embryos, leading, in the frog, to ectopic beating heart
tubes lined with endothelial cells
[43,44]. Thus, elimination
of Wnt/β-catenin signalling is essential for cardiogenesis
and the balance of Wnts and their antagonists appears to play
a critical role in shaping the cardiac fields.
β-Catenin is one of the best characterised effectors of the Wnt pathway [46]. Stabilisation of cytoplasmic β-catenin within the ‘canonical’ Wnt signalling pathway allows it to enter the nucleus, associate with transcription factors of the TCF/LEF family, and activate transcription of target genes. The serine/threonine kinase, GSK3β, whose normal role is to destabilise β-catenin in the absence of Wnt signalling, can also initiate cardiogenesis in non-cardiac mesodermal explants from Xenopus when over-expressed [43]. Remarkably, targeted inactivation of β-catenin in the node, notochord, and endoderm of the developing mouse results in the formation of ectopic hearts along the anterior/posterior axis [47], consistent with the need to block Wnt/β-catenin signalling for vertebrate cardiogenesis.
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6 The Wnt/JNK pathway
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Recent observations suggest that certain Wnt proteins activate
a non-canonical cytoplasmic signalling pathway
[48]. Wnt-11,
which is expressed in mesoderm, has been implicated in regulating
cell polarity and movement during gastrulation through activation
of c-Jun-N-terminal kinase (JNK) and the small GTPases RhoA
and cdc42
[49]. In contrast to the Wnt/β-catenin pathway
discussed above, which is inhibitory for cardiogenesis, the
non-canonical Wnt/JNK pathway is essential for cardiac induction
in the frog and chick embryo systems
[50,51]. In frogs, ectopic
expression of Wnt-11 or activation of both protein kinase C
(PKC) and JNK induces a cardiogenic phenotype
[50]. Wnt-11 also
induces cardiogenesis in mouse pluripotent P19 teratocarcinoma
cells which normally only form beating cardiac muscle after
treatment with DMSO
[50]. Inhibition of Wnt11, or of PKC or
JNK, inhibits endogenous or induced cardiogenesis. An interesting
twist is that Wnt-11 also inhibits the canonical Wnt/β-catenin
pathway, and that Dickkopf and Frizzled family Wnt inhibitors
actually stimulate the Wnt/JNK pathway at the same time as blocking
Wnt/β-catenin signalling
[50]. Thus, activation of Wnt/JNK
signalling by factors expressed in both endoderm and mesoderm
promotes cardiogenesis, while inhibitory Wnts expressed in the
neural plate help to limit the extent of the cardiac fields
(
Fig. 3).
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7 The role of fibroblast growth factors
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In chick embryos, anterior endoderm can induce cardiogenic mesoderm
in posterior primitive streak explants
[44]. However, this does
not occur with BMPs, Wnt/β-catenin antagonism and Wnt/JNK
activation alone. Other positive factors are clearly required.
The FGF family of signalling molecules is also present in endoderm
implicated in modulating cardiogenesis
[52] and explant experiments
reveal that FGF-4, in the presence of BMP-2, can induce cardiac
differentiation in certain non-cardiac mesodermal explants
[21,53].
Each of these signals, particularly that of FGF, is required
only transiently to induce non-cardiac cells to a cardiac fate
[23]. Both are required to sustain expression of
Nkx2-5 and
the serum response factor gene (
SRF). In a recent study, ectopic
application of FGF-8, expressed in the endoderm of the heart-forming
region of the chick
[12] and mouse
[54], can prevent the loss
of cardiac gene expression in embryos in which inducing endoderm
has been resected
[12]. Furthermore, the expression of cardiac
markers is expanded when FGF-8-soaked beads are placed lateral
to the heart fields
[12]. In keeping with previous explant experiments
[21,53], FGF-8 only has a cardiogenic effect in regions where
BMP signalling is also present. Furthermore,
fgf8 gene expression
appears to be downstream of BMPs
[12].
The above findings strongly suggest that BMP and FGF-specific pathways interact to specify the cardiac lineage. However, support for an essential role for FGFs in cardiac induction is still lacking. Studies in the chick system using inhibitors has thus far failed to show that FGF signalling is essential for inductive events [12,55]. The fgf8 gene is needed for normal cardiac morphogenesis and gene expression in zebrafish [56], although relative roles in mesodermal migration, a clear function for the mouse gene [57], and induction, have not been delineated.
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8 Signalling downstream of BMPs and FGFs
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Cardiogenic signalling pathways downstream of BMPs and FGFs
are now being elucidated. Recent studies in the P19 teratocarcinoma
system as reviewed elsewhere in this issue
[58], in which cardiogenesis
is induced by DMSO, show that myogenesis is dependent upon BMPs,
BMP-Smads and induction of cardiac transcription factors Nkx2-5
and GATA4
[59,60]. However, also required are the mitogen-activated
protein kinase kinase kinase (MAPKKK), TAK1, and the ubiquitously-expressed
ATF/CREB family transcription factor ATF2. TAK1 can be activated
by TGFβ pathways and, in turn, activates p38 MAP kinase,
which phosphorylates ATF2. ATF2, which has intrinsic histone
acetyl-transferase activity, can then associate with Smad hetero-oligomers
and stimulate transcription. Raf1, a MAPKK normally associated
with signalling pathways stimulated by FGFs, cannot substitute
for TAK1 in BMP-mediated cardiogenesis. However, like Raf1,
TAK1 can activate JNK and p38 kinase leading to activation of
transcription factors of the AP-1 family, which can also associate
with TGFβ-Smad proteins and co-activate transcription
[61].
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9 Other cardiogenic inducers
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Numerous other signalling factors have been implicated in cardiogenesis
in vivo or in vitro, although their roles have been less extensively
studied. Cripto-1 belongs to the EGF-CFC family of extracellular
membrane-tethered proteins, members of which serve as obligatory
co-receptors for signalling by the TGFβ-family member Nodal
[62]. Targeted disruption of the mouse Cripto-1 gene causes
embryonic lethality after gastrulation
[63]. Mutant embryos
do form some mesoderm, although no cardiac markers are expressed.
Furthermore, differentiating mutant ES cells can express brachyury
(a marker of mesoderm) and
Nkx2-5, although other markers of
the cardiogenic program are absent.
Activin and platelet-derived growth factor have been shown to promote cardiogenesis in various contexts [21,64]. Activin potentially promotes anterior character in mesendoderm, thereby creating a permissive environment for BMP/FGF-mediated cardiogenesis [21]. Cerberus is a TGFβ-superfamily member that can antagonise both BMP and Nodal signalling by direct ligand binding [65]. Despite its anti-BMP activity, it has been shown to induce ectopic head-like structures sometimes containing hearts in Xenopus embryos. In Xenopus ectodermal explants, it can induce Nkx2-5 expression although not full cardiogenesis. The opioid prodynorphin B has also been found to promote cardiogenesis in P19 cells in the absence of DMSO, possibly through nuclear opioid receptors [66]. Opioid receptor antagonists inhibit cardiogenesis in this system. Interestingly, dynorphin B induces activation and intra-cellular translocation of PKC isoforms [67], perhaps connecting this system to the Wnt/JNK pathway.
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10 The secondary heart field
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Recent papers describe a novel population of cardiac precursor
cells termed the anterior or secondary heart field, that give
rise to myocardium of the outflow tract
[68–70]. Fate
mapping studies using vital dyes and retrovirus lineage tags
in chick and mouse embryos, as well as the expression of a mouse
nlacZ-transgene inserted near the
Fgf10 locus, reveal that cells
dorsal and anterior to the heart migrate into the cranial part
of the formed heart tube to build the outflow tract and possibly
(in the mouse) right ventricular myocardium (
Fig. 4). Although
the extent of the secondary heart field has not been mapped
in detail, it may include pericardial and head mesoderm, and/or
branchial arch mesenchyme of neural crest origin
[68–70].
Non-myogenic cells dorsal to the heart tube express cardiac
transcription factors genes such as
Nkx2-5 and
GATA4, as well
as growth factor genes
fgf8 and
fgf10 [14,69,70]. In vitro analyses
demonstrate that signals from existing outflow tract myocardium
can recruit cells from the secondary heart field to a myocardial
fate
[68,69], an effect that is blocked by addition of the BMP
antagonist Noggin
[69]. Patterns of
fgf10-LacZ expression suggest
that secondary heart field cells arise, at least in part, from
cells that lie medial to the primary heart fields that form
the initial heart tube
[70] (
Fig. 4). These cells are apparently
held in reserve before being deployed to make a major contribution
to heart morphogenesis. The presence of FGFs and dependence
on BMPs suggest that the primary and secondary heart fields
share common mechanisms of myogenesis.
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Fig. 4 Relative contributions of the primary and secondary heart fields to the developing mouse heart. Drawings depict the dynamic relationship between the primary (light-shaded) and secondary (dark-shaded) heart fields from the cardiac crescent through looping stages of heart development (see text). Secondary heart field cells appear to contribute to the right ventricle in mouse [70], although this has not been described in birds [68,69]. Adapted with permission from Kelly et al., 2001 [70].
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11 Notch and early cardiogenesis
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In
Xenopus, the heart field occupies a ventral mesodermal region
that closely corresponds to the expression domain of
Nkx2-5 [9]. The
Xenopus heart field is ‘regulative’ in
the sense that cells located laterally in the field can be recruited
to form a normal heart if definitive progenitors are removed.
Regulating cells normally form the dorsal mesocardium and dorsal
pericardial mesoderm
[9] (
Fig. 1). Over time, heart forming
potential in the field becomes restricted to the definitive
progenitors without a corresponding reduction in the expression
of
Nkx2-5. Thus, restriction in potency occurs downstream of
Nkx2-5.
Recently, Notch-1 and its ligand Serrate have been implicated in the progressive loss of cardiac potency in the Xenopus heart field [71]. Notch signalling mediates numerous cell fate decisions in vertebrates and invertebrates, including roles in repression of differentiation and allocation of alternative cell fates [72]. Initially, Notch-1 and Serrate are co-expressed with Nkx2-5 throughout the cardiac field, although Serrate-1 becomes restricted to cells of the future pericardial roof and dorsal mesocardium, which do not ultimately become myogenic [71]. Activation of the Notch pathway inhibits myogenesis and increases markers of dorsal non-myogenic tissues. Inhibition of the pathway has the converse effect.
The lateral regulative cells of the heart field in Xenopus may be homologous to cells of the secondary heart field in amniotes. Although the extent of secondary heart field cells has not been mapped in detail, it is appealing to believe that a regulative heart field in lower vertebrates has been coopted to drive one of the major morphogenetic innovations that has occurred during evolution of mammals. The implication is that Notch signalling inhibits differentiation of both the primary and secondary heart fields until their staged deployment in the heart.
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12 Specialisation of chamber myocardium
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Anatomical, electrophysiological and gene expression data suggest
that the working muscles of the cardiac chambers are formed
as a specialisation of a more primitive type of muscle found
in the primary heart tube
[10]. Chamber specialisation occurs
in discreet zones at the outer curvature of the looping heart
tube, implicating anterior/posterior and dorsal/ventral patterning
processes in their definition
[10]. These zones are marked by
expression of several genes, such as those encoding transcription
factors Hand1, Cited1 and Irx1/2/3, gap junction proteins connexin
40 and 43, the secreted peptide atrial natriuretic factor (ANF),
and the cytoskeletal protein Chisel
[10,73–75]. Other
genes not strictly chamber-specific in their expression, including
some myofilament genes, become up-regulated in chamber myocardium
as development proceeds
[10]. Thus, formation of definitive
chamber muscles requires a regional myogenic specialisation
leading to unique contractile, conduction and cytoskeletal properties
appropriate for function.
The spatial specificity of chamber formation is guided, in part, by transcriptional repression within the myocardium. The T-box family transcriptional repressor, Tbx2, is expressed in an evolving pattern in the forming heart tube that is mutually exclusive of the zones that become chamber myocardium [76]. Furthermore, Tbx2 acts inter-dependently with Nkx2-5 on a repressive element in the proximal promoter of the ANF gene (a marker of chamber myocardium). Such repression results in down-regulation of ANF gene expression in non-chamber myocardium.
The endocardial layer of the heart also appears to be the source of a key determinant for chamber myocardium. Trabeculae are the spongiform layer of myocytes that form on the inner surface of the developing chambers next to the endocardial layer. Trabeculae are ultrastructurally more differentiated and less proliferative that other layers of the heart [77] and are likely to be the force-generating component of the embryonic ventricle. Trabeculae also have privileged conduction properties and in mice appear to be the precursors of the Purkinje fibers of the conduction system, which are a specialised form of chamber-specific myocyte [78,79]. In mouse, deletion of genes encoding neuregulin-1, an EGF-related signalling molecule expressed in endocardium, or its myocardial receptors ErbB2 or ErbB4, leads to virtually complete elimination of trabecular myocardium [80–82]. Exogenously-delivered neuregulin-1 induces excessive trabeculation and up-regulation of a transgenic marker of the Purkinje system [79,83]. These studies highlight the key role played by endocardium in promotion of chamber muscle and Purkinje fibre differentiation. Recent studies from this laboratory show that neuregulin-1 acts by maintaining expression of a host of cardiac transcription factor genes (unpublished data). The neuregulin-1/ErbB signalling axis is also a homeostatic system for cardiac muscle, its loss in the adult leading to dilated cardiomyopathy and drug susceptibility in myocytes [84,85]. Interestingly, formation of trabeculae in the embryo and their differentiation can be uncoupled. In C3H/HeJ mice lacking the jumonji gene, which encodes a nuclear protein normally expressed in the trabecular layer of the heart, ventricles become filled with trabeculae but their differentiation is inhibited [86].
The endocardium becomes regionally induced by myocardium and contributes profoundly to heart valve and septal morphogenesis [87]. The endocardium associated with chamber myocardium is also a specialised entity, selectively expressing the homeodomain factor gene Irx5 [74]. The cardiac extracellular matrix, termed cardiac jelly, elaborated mainly by myocardium, is essential for correct propagation, integration and/or stabilisation of endocardial signals. This is exemplified in mice lacking the major hyaluronic acid synthase gene, Has2, in which trabeculae and endocardial cushions are completely lacking [88]. ErbB2 signalling in endocardial cushion explants of Has2 mutant embryos is rescued by treatment with neuregulin-1 or hyaluronic acid, suggesting an intimate link between neuregulin signalling and the cardiac matrix [89]. In mice lacking the matrix protein versican, endocardial cushions do not form and the right ventricle and conus are severely under-developed [90].
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13 Control of myocardial growth by epicardium
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The epicardium is the outermost layer of the heart which gives
rise to the coronary circulation, including its smooth muscle
cells, and interstitial fibroblasts
[91]. This cardiac layer
is derived from the proepicardial organ, an out-pocketing of
the septum transversum mesenchyme lying adjacent to the venous
tributaries of the heart. While cardiomyocyte proliferation
in the early phase of chick heart development is dependent on
FGF signalling
[92], later proliferation of chamber myocardium
occurs principally in the sub-epicardial layer
[93], implicating
epicardium in chamber growth. A host of genes have been implicated
in growth and differentiation of chamber myocardium through
analysis of knockout mice, including those encoding retinoic
acid and retinoid-X receptors (RARs and RXRs, respectively),
VCAM-1,
4-integrin, TEF-1, erythropoietin, erythropoietin receptor,
WT-1 and gp130. How most of these genes act is unclear, especially
in the light of evidence that the roles of RXR
, gp130 and erythropoietin
receptor in chamber growth are non-cell autonomous for myocytes
[94–96]. Nevertheless, retinoic acid (RA) signalling may
be a key component of epicardium-mediated chamber growth. Retinaldehyde
dehydrogenase 2 (RALDH2), the rate-limiting enzyme in RA synthesis
in the embryo, is highly expressed in epicardium
[97]. Furthermore,
a RA-responsive-LacZ transgene is expressed during chamber growth
in both epicardium and myocardium. Mouse embryos lacking
Raldh2,
or various RAR and RXRs, display thin-walled ventricles and
precocious differentiation of myocytes
[98].
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14 Myocardialization
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A fourth modality of myogenesis in the heart has been termed
‘myocardialization’ (
Fig. 5). This refers to the
relatively late muscularisation of endocardial cushion tissues
of the atrium, atrioventricular canal and outflow tract, and
of the caval and pulmonary veins
[99,100]. Its degree is species-specific,
more extensive in endocardial cushions of the chick, for example,
compared to mammals. Competence to form cushion myocardial networks
is restricted to the more primitive (primary) myogenic zones
of the heart
[10]. It does not occur adjacent to, nor can it
be directed by, specialised chamber myocardium. Within cushions,
myocardial networks always form in continuity with existing
myocardium (
Fig. 3), implying a migratory mechanism. However,
elegant use of explant assays and conditioned media show that
cushion mesenchyme of endothelial origin can be recruited directly
to the myogenic lineage, suggesting that de novo myogenesis
is at least one mechanism for myocardialisation
[99,100]. The
molecular details of this mode of myogenesis are completely
unknown.
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Fig. 5 Myocardialization of the outflow septum in the developing chicken heart. Brightfield micrographs of sections from chick embryos taken at the level of the outflow tract stained with a monoclonal antibody recognizing myosin heavy chains. (A,B) At Hamburger and Hamilton Stage (H/H) 30, the endocardial ridges are being invaded by cardiomyocytes. (C,D) Muscularization encompasses the entire cushion by H/H 32. Boxes in A,C represent areas shown in B,D. AO, aorta; LA, left atrium; OTC, outflow tract cushion tissue; RA, right atrium; RV, right ventricle. Adapted with permission from van den Hoff et al., 1999 [99].
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15 Transcription factor programs in the myocardium
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Cardiac myogenic and morphogenic programs are regulated by interconnected
networks of transcription factor genes
[11]. Included in these
networks are the ancient factors that play central roles in
all muscle lineages, such as the MADS-box proteins SRF and Mef2A/B/C/D
[101]. More cardiac-restricted factors include Nkx2-5/2-6, GATA4/5/6,
Tbx2/5/20, Hand1/2, Irx1/2/3/4, CITED1/2, COUPTFII, Hey1/2,
Pitx2 and myocardin. Rapid accumulation of data over the past
decade has given us a flavour of the complexity of these circuits
although an integrated model is still lacking. Transcription
factor programs are subject to complex inputs and are interpreted
and integrated at the level of individual target gene enhancers.
The details of individual cardiac transcription factors and
their mutant phenotypes have been recently reviewed
[11,102–104],
and we will restrict comments here to some general features
relevant to myocyte heterogeneity.
15.1 Positive transcription factor circuits in cardiomyogenesis
Cardiac induction in the embryo is accompanied by a positive feed-forward and mutually cross-regulatory circuit [11]. Thus, both Nkx2-5 and Hand2 are regulated by GATA factors [105,106], and gata6 is regulated by Nkx2-5 [107]. Likewise, Nkx2-5 is required for full expression of Hand1 and Irx4 in vivo [108,109], Irx4 is required for expression of Hand1 [110], Hand2 and Tbx5 for Irx4 [108,111], and so on [11]. Furthermore, many cardiac transcription factors interact directly, allowing greater stability of transcriptional complexes on DNA, synergistic activation of target genes and recruitment of individual factors to enhancers that lack their specific binding sites [112–114]. Nkx2-5 has been shown to interact with SRF, GATA4, and Tbx5 [114–116], while GATA4 can also interact with Mef2C, FOG2 and NFATc [112,117,118] and SRF with myocardin and GATA4 [113,119]. Few of these interactions have been tested genetically, although one study characterised mice carrying a point mutation in GATA4 that eliminates its association with FOG2 [120]. These mice have defects similar to those lacking FOG2, and a subset of those seen in mice lacking GATA4 completely. While there does not appear to be a single cardiac master regulatory gene, the combinatorial mode of regulation described above may account for why individual factors, such as Nkx2-5, GATA4 or Mef2C, can activate the cardiogenic program in pluripotent P19 cells without DMSO [121]. Complex post-translational inputs also regulate the activity of cardiac transcription factors. Both Nkx2-5 and Mef2 proteins are positively regulated by phosphorylation [122,123], and GATA4 is phosphorylated by p38 MAP kinase acting downstream of Rho-family GTPases, promoting transcriptional activation of the RhoA gene itself and sarcomere assembly [124].
15.2 Negative regulatory circuits
Cardiac transcriptional programs will also be subject to complex negative regulatory circuits, which may dampen positive pathways and guide region-specific myogenesis and morphogenesis. The importance of such pathways is perhaps highlighted by the exquisite dose-sensitivity of cardiac transcription factors [125]. The inhibitory Smad protein, Smad6, is expressed in the developing heart and may modulate BMP and/or TGFβ-pathways [29,126]. Numerous other transcriptional repressors implicated in heart development have been described [76,110,127–131]. MEF2 proteins may be universally repressed by direct association with histone deacetylases (HDACs), becoming de-repressed by Ca2+-dependent signalling pathways that export HDACs from the nucleus [132]. Nkx2-5 activates two negative feedback cascades in vivo. Acting through a GATA factor-dependent promoter element, it induces the ankyrin-repeat protein CARP, which represses multiple cardiac promoters [127,133]. It also directly induces HOP, a minimal homeodomain protein that associates with and represses SRF-dependent gene expression [129,130].
15.3 Modular gene regulation in the heart
Analysis of cardiac phenotypes in zebrafish and mice, and the patterns of multiple mouse LacZ transgenes carrying cis-regulatory elements of cardiac-expressed genes, suggests a modular basis for gene regulation in the heart [106,133–136]. The implication of these findings is that the complete expression pattern of at least some cardiac genes will be a composite of sub-patterns controlled by different region-specific regulatory modules (reviewed in this issue by Habets et al. [137]). This may reflect the regional diversity of regulatory mechanism and functionality that likely occurred during addition of new regulatory ‘modules’ to the heart in the course of evolution [134]. Stable expression of most transgenes occurs only in the right ventricle and outflow tract, likely reflecting regulation of enhancers active in cells of the secondary heart field. The mechanisms underlying this modular regulation are unknown, although bHLH transcription factors Hand1 and Hand2, and the T-box factor, Tbx5, which show differential expression and function between left and right ventricles, are suggested to be involved [11].
15.4 Atrial and ventricular transcriptional programs
The structural and functional characteristics of the atria and ventricles are distinct. Transcriptional repression and mutually antagonistic pathways appear to be mechanisms utilised to establish chamber-specific identity. There is now considerable evidence that the morphogen, RA, is critical for the initial diversification of the cardiac lineage into atrial and ventricular phenotypes. In the chick and mouse, excess RA causes ventricular chambers to acquire atrial characteristics [138,139]. These teratogenic effects occur only if RA is administered very early in heart development, suggesting a role for RA in the earliest stages of heart tube patterning. Expression of the Raldh2 gene, encoding the enzyme responsible for virtually all RA synthesis in the embryo [97], is initially restricted to the sinuatrial region (primitive atrium and inflow tributaries) of the forming heart, and mouse embryos lacking Raldh2 show unlooped hearts without a distinct atrial chamber [98]. This is also true if RA signalling is inhibited by other means [139,140]. The T-box transcription factor gene Tbx5, mutated in Holt-Oram syndrome in humans, may mediate some of the effects of RA [111]. Tbx5 is initially expressed throughout the cardiac crescent and linear heart tube, but rapidly adopts a graded distribution, high in the atria, low in the left ventricle and largely absent in the right ventricle and outflow tract [141]. Tbx5 is induced by RA and its graded distribution in the heart is flattened in embryos lacking Raldh2 [98]. The hearts of Tbx5-null embryos have a severely hypoplastic sinuatrial region [111], and enforced mis-expression of Tbx5 in the ventricles induces abnormal ventricular morphology and down-regulation of ventricle-specific markers [142]. The orphan nuclear receptor and transcriptional repressor, COUP-TFII, is also important for sinuatrial development [143].
In the ventricles, the homeodomain factor Irx4 appears to be involved in negatively regulating atrial chamber identity. Irx4 is expressed in the right and left ventricles and its deletion in mice leads to inappropriate activation of a transgene carrying an avian slow myosin heavy chain gene promoter, which in mice is atrial-specific [110]. In vitro characterisation of this promoter shows that repression in the ventricles requires an interaction between Irx4 and the RXR component of an RXR/vitamin D receptor dimer. The complex acts on a vitamin D-responsive promoter element to repress transcription [144]. Enforced mis-expression of Irx4 in chick atria induces ventricular gene expression [145], suggesting that mutual antagonism between a RA-directed atrial program, and an Irx4-mediated ventricular program establishes chamber identity.
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16 Summary and conclusions
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Abstract
1 Introduction