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Physiol Rev
83: 1223–1267, 2003; 10.1152/physrev.00006.2003.
Cardiac Chamber Formation:
Development, Genes, and Evolution
ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
Experimental and Molecular Cardiology Group, Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands
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I. Introduction
A. Scope
B. The cardiac cell types: origin and diversity
C. The different manifestations of cardiomyocytes: contraction, conduction, and automaticity
II. Evolutionary Aspects of Chamber Formation
A. Introduction
B. Drosophila
C. Primitive chordates
D. Lower vertebrates
E. The electrical configuration of the chamber heart
F. Tales of the rings: the emerging concept of ballooning chambers
G. Overview of the component parts of the lower vertebrate heart
III. Development of the Parallel-Arranged Four-Chambered Heart
A. Evolutionary aspects and terminological problems
B. Cardiac looping, changing blood flows, and chamber formation
C. The primary ring: development of the right ventricle
D. Completion of septation
IV. Development of Cardiac Function and Conduction System
A. Development of polarity
B. Development of the ECG
V. Lineage Analysis and Chamber Development
A. Formation of the linear heart tube
B. The heart-forming regions include anterior and posterior mesenchyme
C. The inner curvature connection
D. Origin of the nodal tissue
E. Fate of the primary ring
F. Origin of the ventricular conduction system
G. Overview of the component parts of the higher vertebrate heart
VI. Antero-Posterior Patterning and Chamber Formation
A. AP patterning during and after gastrulation
B. Retinoic acid: a cardiac AP patterning molecule
C. Factors involved in AP patterning and morphogenesis
VII. Cardiac Patterning Along the Dorsoventral Axis
VIII. Left-Right Signaling in Chamber Specification and Morphogenesis
IX. Chamber-Specific and Regionalized Transcriptional Programs
A. Chamber-specific patterns of gene expression
B. Regulation of chamber-specific gene expression
C. Gene deficiency reveals pathways involved in chamber formation
X. Conclusions and Perspectives
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Moorman, Antoon F. M., and Vincent M. Christoffels. Cardiac Chamber Formation: Development, Genes, and
Evolution. Physiol Rev 83: 1223–1267, 2003; 10.1152/physrev.00006.2003.—Concepts of cardiac development have
greatly influenced the description of the formation of the four-chambered vertebrate heart. Traditionally, the
embryonic tubular heart is considered to be a composite of serially arranged segments representing adult cardiac
compartments. Conversion of such a serial arrangement into the parallel arrangement of the mammalian heart is
difficult to understand. Logical integration of the development of the cardiac conduction system into the serial
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0031-9333/03 $15.00 Copyright © 2003 the American Physiological Society
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ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
Theory without fact is fantasy, but fact without theory is chaos.
C. O. Whitman (334)
I. INTRODUCTION
A. Scope
In birds and mammals, the four chambers of the
formed heart are arranged in parallel (Fig. 1, A and C).
The right atrium is exclusively connected to the right
ventricle and the left atrium exclusively to the left ventricle. The right half of the heart drives blood from the body
through the lungs to the left half that drives the blood
through the rest of the body. The two halves can only beat
in synchrony, the right half pushing the pulmonary circulation and the left half the systemic circulation. This
parallel-arranged four-chambered heart develops from a
single circuited tubular heart. The current concept is that
this heart tube is composed of a linear array of segments
(21, 56, 254, 330) (Fig. 1, B and D). In this model the
atrium is not connected to the right ventricle and the left
ventricle not to the outflow tract. The conversion of such
a serial arrangement of segments into the proper parallel
arrangement has remained one of the most difficult concepts of heart development (156), for it seems illogical
first to make a serial arrangement of cardiac segments
and then a parallel one. Based on morphological and flow
direction considerations, Steding and Seidl (292) described this concept as “one of the most fatal assumptions.” Recent functional and molecular data also challenged the concept (51, 64, 118, 210), and a new concept
was formulated, “the ballooning model of chamber formation.” In this review we discuss the possibility that
during the evolution and ontogenesis of the heart the
chambers develop from a linear tube by local differentiaPhysiol Rev • VOL
tion and that this local differentiation and expansion sets
the scene for the electrical configuration of the chamber
heart. From the outset, the right and left halves of the
heart are brought into parallel position by looping of the
heart tube. A fundamental question is thus whether indeed all compartments of the formed heart are represented as a linear array in the tubular heart.
In a stimulating review, Fishman and Olson (90) suggested a model of the vertebrate heart as an assembly of
separable genetic modules that were added to the primitive ancestor chordate heart during evolution. The modules are supposed to be component parts or functions of
the heart that can be selectively affected by single gene
mutations. Examples of such modules would be the cardiac chambers and the localized pacemaker activity. Challenging though these ideas are, this concept can only fully
be exploited if the molecular data are integrated morphologically and functionally in a unifying model of chamber
formation. The deep evolutionary conservation of molecular pathways in the hearts from fruitfly to human is
intriguing and instrumental in finding general developmental mechanisms. On the other hand, evaluation of
evolutionary diversity is fundamental to revealing both
conserved and novel modules of the building plan of the
present-day mammalian heart.
De la Cruz and Markwald (66) wrote an exhaustive
review on cardiac development in general. The roles of
endocardium (186), epicardium (191), and neural crest
(85) in heart development, as well as molecular cardiac
genetics (48, 121, 285) were reviewed elsewhere. In this
review, we try to define the basic cardiac building blocks
and examine their developmental and evolutionary origins. This, in turn, requires evaluation of the identity of
the atrial and ventricular chambers and of the conduction
system of the avian and mammalian hearts. We need to
determine whether these structures are indeed basic cardiac building blocks, or are composed of smaller compo-
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concept has remained puzzling as well. Therefore, the current description needed reconsideration, and we decided
to evaluate the essentialities of cardiac design, its evolutionary and embryonic development, and the molecular
pathways recruited to make the four-chambered mammalian heart. The three principal notions taken into consideration are as follows. 1) Both the ancestor chordate heart and the embryonic tubular heart of higher vertebrates
consist of poorly developed and poorly coupled “pacemaker-like” cardiac muscle cells with the highest pacemaker
activity at the venous pole, causing unidirectional peristaltic contraction waves. 2) From this heart tube, ventricular
chambers differentiate ventrally and atrial chambers dorsally. The developing chambers display high proliferative
activity and consist of structurally well-developed and well-coupled muscle cells with low pacemaker activity, which
permits fast conduction of the impulse and efficacious contraction. The forming chambers remain flanked by slowly
proliferating pacemaker-like myocardium that is temporally prevented from differentiating into chamber myocardium. 3) The trabecular myocardium proliferates slowly, consists of structurally poorly developed, but well-coupled,
cells and contributes to the ventricular conduction system. The atrial and ventricular chambers of the formed heart
are activated and interconnected by derivatives of embryonic myocardium. The topographical arrangement of the
distinct cardiac muscle cells in the forming heart explains the embryonic electrocardiogram (ECG), does not require
the invention of nodes, and allows a logical transition from a peristaltic tubular heart to a synchronously contracting
four-chambered heart. This view on the development of cardiac design unfolds fascinating possibilities for future
research.
CARDIAC CHAMBER FORMATION
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nent parts developed during evolution and integrated during embryonic development. It is our goal to set out a
logical model of cardiac chamber formation from these
elementary components. In this review we largely restrict
ourselves to the myocardial component of the heart.
B. The Cardiac Cell Types: Origin and Diversity
The heart essentially has a mesodermal origin (Fig.
2). The minor contribution of neural crest cells is largely
confined to the formation of the aorto-pulmonary septum
at the arterial pole of the heart (86, 157), and the
endoderm only plays a regulatory role (186). Committment of mesodermal cells to the cardiogenic lineage is
established during and shortly after gastrulation (274,
297), upon which the cardiogenic cells are organized in
bilateral epithelial sheets that are part of the visceral
mesoderm lining the developing coelomic cavity. The cardiogenic cells are in close association with the endoderm.
The endocardial cells are formed from the cardiogenic
mesoderm in between the endodermal and mesodermal
Physiol Rev • VOL
FIG. 2. Schematic of the development of endocardium, myocardium, and epicardium from the mesodermal germ layer. See sect. IB for
further explanation.
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FIG. 1. A: cartoon displaying the elementary component parts of the adult mammalian heart in a four-chamber view.
The working myocardium of the formed heart is composed of the right and left atria (RA, LA) and right and left ventricles
(RV, LV). The conduction system is composed of the pacemaking tissues of the sinoatrial node (SAN) and the
atrioventricular node (AVN) and of the fast-conducting components comprising the atrioventricular bundle (AVB), the
left and right bundle branches (BB), and the peripheral ventricular conduction system (PVSC). In chicken hearts, an
additional periarterial conduction system is present that is not present in human and rodent hearts. Note that the
epicardium (EPI), indicated in yellow, is contiguous with the insulating fibrous tissue of the fibrous annulus separating
the atrial from the ventricular myocardium. PV, pulmonary veins; S/I CV, superior/inferior caval vein; CFB, central
fibrous body; MV, mitral valve; TV, tricuspid valve; END, endocardium; MYO, myocardium. B: segmental view of the
embryonic linear heart tube. In the segmental view of the embryonic heart, the heart tube is considered to be composed
of a linear array of segments representing compartments of the adult heart. Rings of “cardiac specialized conduction
tissues” are supposed to be present at the sinoatrial junction (SA-ring), the atrioventricular junction (AV-ring), and at the
so-called interventricular junction. To prevent confusion, we dub the latter the “primary” ring (P-ring), rather than the
commonly used names “interventricular” or “bulboventricular” ring. As is explained in the text, the primary ring is
positioned between two parts of the primary heart tube. The ring is not localized between the left and right ventricles,
which would justify the name interventricular ring. The ring is also not localized between the bulbus (which is the
outflow part of the primary heart tube) and the ventricle, which would justify the name bulboventricular ring. VP, venous
pole; SV, sinus venosus; A, atrium; LV, left ventricle; RV, right ventricle; aos, aortic sac; AP, arterial pole. C: parallel
cardiovascular arrangement in birds and mammals; the left heart drives the systemic circulation that drains to the right
heart, which, in turn, drives the pulmonary circulation that drains to the left heart. D: serial cardiovascular arrangement
in fish and vertebrate embryos; the single circuited heart drives the blood via the pharyngeal arches (gills in fish) through
the body and back to the heart.
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ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
C. The Different Manifestations
of Cardiomyocytes: Contraction,
Conduction, and Automaticity
The vertebrate heart is myogenic, which implies that
all cardiomyocytes are in principle capable of producing
an intrinsic cycle of electrical activity that leads to conPhysiol Rev • VOL
traction (80). This phenomenon is shared with smooth
muscle cells and is called automaticity, intrinsic rhythmicity, or pacemaker activity. Because all cardiomyocytes
are electrically coupled, the cells with the fastest intrinsic
rate, in the mammalian heart usually the cells of the sinus
node, stimulate the whole heart to contract and determine
its rate. The cells with the fastest intrinsic rate are called
the (leading) pacemakers; the others can be categorized
as followers or latent pacemakers. If the pacemakers
were to stop, other cardiomyocytes take over, albeit at a
slower pace. Automaticity requires poor coupling of the
cardiomyocytes, which is achieved by a specific composition and density of gap junctions and ion channels, as
well as by the size and the arrangement of the cardiomyocytes involved. Poor intercellular coupling permits loading of the cells to a threshold value of electrical charge,
resulting in depolarization of the adjacent myocardium
(145). Hence, pacemaker (nodal) cells share automaticity
and slow conduction (Table 1).
Automaticity and slow conduction velocity are also
features of the peristaltically contracting hearts of primitive chordates and vertebrate embryonic tubular hearts,
because slow conduction of the depolarizing impulse is
an absolute prerequisite for peristalsis (see sect. II). The
development of synchronously contracting chambers required higher conduction velocities of the depolarizing
electrical impulse. For this reason, and because unambiguous morphological criteria are lacking, automaticity and
TABLE
1. Basic features of cardiac muscle cells
Type of Myocardium
Characteristic
Primary
Nodal
Bundle and
branches
Working
Automaticity
Conduction velocity
Contractility
SR activity
High
Low
Low
Low
High
Low
Low
Low
High
High
Low
Low
Low
High
High
High
Cardiac muscle cells share a number of characteristic features that
distinguish them from other cell types. All cardiac muscle cells have
sarcomeres and a sarcoplasmic reticulum. The cells are coupled by gap
junctions allowing the depolarizing impulse to travel over the myocardium, and all cells display automaticity. The degree of differentiation of
these features depends on the cardiac muscle cell type considered. Thus
the cells of the atrial and ventricular working myocardium display
virtually no automaticity but are well coupled. They have well-developed
sarcomeres and sarcoplasmic reticular structures. Nodal cells have the
opposite phenotype and are similar to the myocardial cells of the primary heart tube. The atrioventricular bundle, bundle branches, and
peripheral ventricular conduction system have an ambiguous phenotype: the cells of this system are well coupled, allowing fast conduction
of the depolarizing impulse but have otherwise an embryonic phenotype. The division of the myocardium into four groups does not imply
that the cells belonging to one group are identical, but they share a
degree of development of the distinguishing features. For the sake of
simplicity, the characteristics are ranked in “high” or “low” only. This does
not imply that all highs are identical or all lows, but implies that they are in
the same range. The ranking is based on Reference 39 for the morphological
features and on Reference 161 for the electrophysiological features.
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layers. During the subsequent process of folding of the
embryonic disc, the bilateral mesodermal cardiac plates
migrate into the embryo, fuse in the midline, and form the
cardiac tube (64).
The three principal cardiac cell types comprise 1) the
endocardium that forms the endothelial lining of the heart
(see Ref. 186 for a review), 2) the myocardium or cardiac
muscle cells, and 3) the epicardium that is the source of
cardiac fibroblasts and coronary arteries (see Ref. 191 for
a review). The endocardium is formed early in cardiogenesis because, after retroviral labeling, endocardial cells
were shown to share hardly any lineage relationship with
myocardial cells (201). However, the two types of cell
must have separated after recruitment of the mesodermal
cells into the myocardial lineage, because initially the
endocardial cells display a cardiac muscle phenotype as
well (62, 184, 317). Upon signals from the myocardium,
the endocardial cells undergo a transformation from epithelial to mesenchymal cells, by which the mesenchymal
cardiac cushions are formed. These cushions fuse and
form part of the cardiac septa (30, 31, 83, 205, 244). The
mesenchyme of these cardiac cushions, in turn, can be
transformed into cardiac muscle, by which process these
parts of the cardiac septa become muscularized (211, 225,
308, 309, 339).
The epicardium develops from the so-called proepicardial organ that is located caudally from the heart. The
proepicardial organ forms cauliflower-like mesothelial
structures protruding into the coelomic cavity that will
cover the entire heart and form the epicardium. Mesenchymal cells derived from the epicardium contribute to
the cardiac-cushion mesenchyme, to the cardiac fibroblasts, and to the endothelial and smooth muscle cells of
the coronary arteries (72, 203, 238, 242).
Interestingly, endocardium, epicardium, and myocardium share a common lineage as they are all derived from
a group of mesodermal cells that express the basic helixloop-helix transcription factor Mesp1 (266, 267). The role
of this transcription factor in the formation of the cardiovascular system remains to be resolved. It is of great
interest that endothelial cells proved to be capable of
transdifferentiating into cardiac muscle when they were
cocultured with cardiomyocytes (54). Preliminary results
from studies from the author’s lab indicate that proepicardium is able to transdifferentiate into cardiac muscle
in vitro as well.
CARDIAC CHAMBER FORMATION
II. EVOLUTIONARY ASPECTS
OF CHAMBER FORMATION
A. Introduction
The morphologically complex mammalian fourchambered heart has evolved from a simple chordate
tubular heart (Fig. 3) (257). Modification of existing elements and the addition of new elements accomplished
this evolution. This in turn required continuous adaptation of the genomic regulatory programs that control
cardiac development. In general genetic terms, molecular
pathways for regional specification of morphological
structures had to be selected, which resulted in the activation of sets of downstream genes. Subsequently, cycles
of regional specification were required to achieve the final
adult configuration (240).
Many features that seemed unique to vertebrates
were already present in the early chordate ancestors, and
new functions became associated with modified duplicated sets of genes (114). What we thus need to understand both at the morphological and genomic level is what
cardiac elements were added during evolution. It is
equally important to integrate conceptual insights derived
from the cardiac anatomy and physiology of lower vertebrates that underlie mammalian cardiac development. According to the rules of Von Baer (318), the embryos of
different vertebrate groups should be compared rather
Physiol Rev • VOL
FIG. 3. Simplified family tree of the vertebrates, based on Romer
and Parsons (257). In the trunk of the tree, pictographs representing
prototypical hearts of the main groups are shown.
than the adult stages, because the general features of a
vertebrate group appear earlier in development than the
specialized features. Consequently, the mammalian embryo heart is never like the heart of an adult lower vertebrate but only like that of its embryo. A practical problem
is that most experimental data are from extant adult
lower vertebrates and not from embryos. Thus one should
be cautious in formulating generalities and specializations
derived from these adult specimens.
B. Drosophila
Recent molecular data indicate that some essential
aspects of the regulation of heart development in Drosophila resemble those in vertebrate hearts, despite the
long evolutionary distance between both groups (25).
Drosophila has become the paradigm for many developmental studies in vertebrates, so we start our description
with the Drosophila heart.
The heart of Drosophila lies within a dorsal pericardial cavity and is also called the dorsal vessel. It is an
elongated tube that is closed posteriorly and drains into
the head anteriorly. The anterior part is narrowed and
called the aorta. In fact, the Drosophila heart is a blind
sac from which hemolymph is expelled into the body by
rhythmic contractions. During relaxation, hemolymph enters the heart tube via ostia in its wall, which are closed
when the tube contracts. The wave of contractions initiates caudally and moves rapidly in the anterior direction, indicating localized pacemaker activity (253). The
Drosophila heart is myogenic, because the heart continues to beat outside the body for several hours. As yet, no
specialized pacemaker cells have been reported, and it is
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conduction velocity became important characteristics to
distinguish the different areas in the developing heart.
In the formed heart, atrial and ventricular working
myocardium are conventionally distinguished from the
so-called “conduction system” (see Table 1). This notion
is supported by the fact that the atrial and ventricular
muscle cells have well-developed sarcomeric and sarcoplasmic reticular structures (39) but are also well coupled, allowing fast conduction of the depolarizing impulse
and efficient synchronous contractions. On the other
hand, and in contrast to the cells of the working myocardium, the cells of the conduction system all share a poorly
developed contractile and sarcoplasmic reticular apparatus. In this respect, these cells are reminiscent of the
embryonic cells of the tubular heart. In contrast to what
might be suggested by its name, the conduction system is
not just composed of fast-conducting cells, but also of
pacemaking or nodal cells. These cells are slowly conducting and poorly coupled and display high automaticity.
Only the cells of the atrioventricular bundle and bundle
branches are well coupled, permitting fast conduction of
the depolarizing impulse. Because the bulk of the cardiac
muscle cells comprise cells of the working myocardium,
the embryonic-like cells of the developing conduction
system are often called “specialized.”
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ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
C. Primitive Chordates
All chordates have a notochord, a neural tube, and
metameric lateral muscle blocks. In the early representatives, a closed cardiovascular system has developed. The
chordate phylum comprises the subphyla of the urochordates, or tunicates, and related forms, the cephalochordates with Amphioxus as a well-known representative,
and the vertebrates (257).
1. Tunicates
The tunicate heart is a tubular structure that lies in a
pericardial cavity. It consists of a single layer of cardiac
muscle cells that are electrically coupled and are not lined
by endocardial cells (247). These cardiac muscle cells are
also called myoendothelial cells. Although all regions of
the heart possess intrinsic automaticity, the dominant
pacemaker is active at one end of the cardiac tube for a
number of beats, after which a pacemaker at the other
end of the tube takes over (3). The resulting peristaltic
contraction waves travel along the heart tube with velocities of ⬃0.5 cm/s and propel the blood in either of the two
directions (164).
2. Amphioxus
The vascular plan of Amphioxus is basically similar
to that of the vertebrates (207), but a unique feature is the
lack of a true heart. However, several vessels are capable
of contractile movements: the subintestinal vein, the portal (afferent) and hepatic (efferent) veins, and the ventral
aorta (endostylar artery), which acts as the principal
pump. A myoepithelial layer surrounds the vessels (207).
The vessels are not lined by endothelium, although scattered cells called hemocytes are sometimes closely associated with the wall of the vessels (245). These cells might
be comparable to the ancestral cell population that gave
rise to the vertebrate endothelium or vertebrate blood
cells or both (294). The vessels contract in succession,
Physiol Rev • VOL
although coordination is poor and a single pacemaker
area seems to be lacking (247, 319). Very slow, longlasting contraction waves (⬃0.03 cm/s) travel along the
vessels, which results in movement of the blood in these
valveless vessels, indicating some kind of polarity. However, this polarity of pacemaker activity is not well developed because reversal of flow and consequent extinction
of the meeting waves of contraction were observed (319).
D. Lower Vertebrates
1. Fishes
The vertebrates constitute the principal group of the
chordate phylum, and the development of the chambered
heart is a new phenomenon in this group (Fig. 4A). It is
first seen in the jawless fishes the cyclostomes, such as
the hagfish (127, 247, 268). Their hearts consist of a sinus
venosus, atrium, ventricle, and conus arteriosus. The
common atrial chamber bulges out over the right and the
left sides of the ventricle. The conus arteriosus is poorly
developed in hagfish but is clearly present in jawed cartilaginous fishes, such as sharks and rays, and also in
lungfishes. The conus arteriosus is the most distal part of
the primitive fish heart and forms the connection between
the ventricle and the ventral aorta.1 At the sinoatrial, the
atrioventricular, and the ventriculoconal junctions, valves
developed to prevent backflow of blood during relaxation
of the preceding compartment. The cardiac tube is Sshaped with a dorsally positioned atrium and a ventrally
positioned ventricle pointing caudally. This S-shape, by
which the ventricular inlet and outlet become positioned
in almost the same plane, is a general feature of all
vertebrate hearts. It has dynamic advantages because it
redirects the momentum of the bloodstream, which flows
from the atrium into the ventricle, toward the arterial
pole. In a linear chamber heart, the inflowing bloodstream
would be rebounded from the closed semilunar valves
toward the atrium (153).
The sinus venosus became the drainage pool of the
venous system to facilitate atrial filling. However, the wall
of the sinus venosus is poorly muscularized, and its de-
1
The conus arteriosus is considered a component part of the heart
because it has a myocardial wall and lies within the pericardial cavity. It
is a feature of the evolutionary primitive state. In amphibians it is called
the bulbus cordis, a term that is also used for its equivalent in mammalian embryos. The more derived extant bony fish, like the zebrafish, do
not have this cardiac compartment. They have a so-called bulbus arteriosus, which is not enclosed by cardiac muscle, but by elastic tissue and
smooth muscle, and therefore is considered to be a specialization of the
proximal part of the ventral aorta (256). However, similar to the mammalian condition (306, 326, 339), the bulbus arteriosus in zebrafish
embryonic hearts is surrounded by myocardium that disappears with
development (134, 135). The bony fish bulbus arteriosus might thus be
homologous to the shark conus arteriosus and amphibian/mammalian
bulbus cordis.
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unknown whether the localized pacemaker activity is intrinsic or imposed by innervation.
The wall of the Drosophila heart tube consists of two
cell layers. The inner layer lining the lumen is composed
of a single type of myocardial cells forming a contractile
layer (263). The myocardial cells are not fused but interconnected via adherent junctions where the myofilaments
also attach. The outer layer lining the pericardial cavity
consists of pericardial cells called nephrocytes. They do
not have a contractile phenotype but a filtration and secretion function and should not be confused with the
pericardial cells in mammals. The pericardial layer is
attached to the body wall via so-called alary muscle connections at the segmental boundaries.
CARDIAC CHAMBER FORMATION
1229
velopment as a separate cardiac compartment as well as
the development of the sinoatrial valves that physically
separate sinus venosus from atrium during contraction
have allowed the development of the atrium as the principal driving force for ventricular filling (142, 143). This
role has become less important in mammalian hearts
where collection of blood is the principle function of the
atria. In line with this, in mammals the sinus venosus has
become part of the right atrium. Although the lungfish
branch does not give rise to the amphibian group, the
lungfish heart already has an atrial septum comparable to
the amphibian condition. The ventricular septum of the
lungfish is considered to be a derived structure that is not
homologous to the avian and mammalian ventricular septum (130).
cardial wall as well, but has a common chamber because
it has no atrial septum. Hence, rather than the left atrium,
one should consider the atrial septum and possibly additional myocardium that surrounds the pulmonary vein as
the novel components of the amphibian heart. Despite the
presence of a single ventricle, amphibians share a highly
effective separation of oxygenated and deoxygenated
blood with the related lungfishes (143). Most of the left
atrial bloodstream flows to the systemic arteries and most
of the right atrial bloodstream to the pulmo-cutaneous
arteries. The morphofunctional adaptations for this amazing performance are derived features typical for the extant amphibian specimens. However, these are not dealt
with in this review.
E. The Electrical Configuration
of the Chamber Heart
2. Amphibians
A typical amphibian heart has two atria, a single
ventricle, and a bulbus cordis (the amphibian equivalent
of the fish conus arteriosus) (127, 143, 342). The sinus
venosus drains exclusively to the right atrium. The pulmonary vein bypasses the sinus venosus and directly
drains to the left side. The left atrium is often considered
to be the evolutionary novel chamber, because it is a
component of the pulmonary circulation. Obviously, the
atrium of the fish heart has a right and a left atrial myoPhysiol Rev • VOL
The heart tube of tunicates and amphioxus display
peristaltic contraction waves, and all regions of the tube
display intrinsic automaticity. This is so because peristaltic contraction requires slow propagation of the wave of
depolarization, which, in turn, requires poor intercellular
coupling. This characteristic also is a prerequisite of automaticity. However, polarity of pacemaker activity is not
well developed.
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FIG. 4. A: cartoon of the primitive fish heart. SV, sinus venosus; A, atrium; RV/LV, right/left ventricle. [Modified from
Randall (246).] B: scheme of the growth of the cardiac chambers. The atrial chamber develops dorsally at the outer
curvature of the “atrial” part of the S-shaped linear heart tube and the ventricular chamber ventrally at the outer
curvature of the “ventricular” part of the linear heart tube. In this view the in- and outlets of the chambers are flanked
by the myocardium of the original heart tube as described by Benninghoff (21). Because these flanking regions do not
participate in the growth of the chambers, they define the orifices of the chambers as rings. Subsequently these rings
were called rings of cardiac specialized conduction tissue. This representation of chamber differentiation in the fish heart
is also highly reminiscent of that described by Davis for the developing human heart (59) and of that described in this
review for the developing mouse heart; SA-ring, sinoatrial ring; AV-ring, atrioventricular ring; P, primary ring. C: scheme
of the embryonic avian or mammalian heart. Ift, inflow tract; avc, atrioventricular canal; oft, outflow tract.
1230
ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
Physiol Rev • VOL
function, it may not be coincidental that in these areas
nodes have developed, as discussed in section IV.
F. Tales of the Rings: the Emerging Concept
of Ballooning Chambers
In the beginning of the previous century, Benninghoff
(21) described regions in the hearts of fish, amphibians,
reptiles, birds, and mammals that do not participate in the
growth of chambers from the heart tube. Because these
regions hardly grow, they define the openings of the expanding chambers as rings (Fig. 4B). He ascribed a
sphincterlike function to these areas and associated them
with the conduction system. As we discussed in the previous section, these areas indeed have a sphincter function and contribute to the slow components of the cardiac
conduction system (the nodes, or pacemaking tissues).
Later, the rings were retrieved from oblivion in studies on
the development of the conduction system in human embryos, but their sphincter function was denied (330) and
the rings were not explicitly restricted to the slowly conducting components of the conduction system, but to
so-called “cardiac specialized tissue” (7, 330). The latter
term does not distinguish unambiguously between the
slow and the fast components of the cardiac conduction
system. This use of this term is extremely confusing because this term, as well as the term conduction system, is
often linked implicitly to fast conduction. Moreover, what
is “specialized”? In a paper on the embryonic development of cardiac impulse conduction, Lieberman (179)
expresses this ambiguity as follows:
Perhaps, concomitant with an increase in myofibrillar content, a process is triggered in the genetically
determined working myocardial cells which causes
the myocardial cell membrane to lose this “embryonic” property to pacemake. Implicit in this notion is
the idea that only those cells that develop an abundance of contractile proteins (that is, working myocardium) should, in fact, be referred to as the “specialized tissue of the heart.” Or, the corollary may also
be implied: all cells that lose the ability to synthesize
myofibrils and maintain automaticity and conductivity
should be considered specialized.
The studies of Benninghoff unfortunately led to the
abstractive representation of the cardiac tube composed
of a linear array of chambers with rings of conduction
tissue at the transitions, which has become the paradigm
of the development of the mammalian heart and the conduction system (21, 56, 254, 330) (compare Figs. 1B and
4C). However, the atrial chambers balloon out at the
dorsal side and the ventricular chamber at the ventral
side of the heart tube and lead to the typical S-folded
heart of fishes and amphibians as represented in Figure 4
(127, 254).
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The fish heart displays clear polarity of contraction in
a posterior-to-anterior direction. The contraction waves
originate in the sinus venosus and terminate in the conus
arteriosus. The nodal phenotype persists in the inflow
region of the heart, varying from the venosinus to the
sinoatrial junctional areas in different species (246, 268).
Similar to the mammalian situation, pacemaker tissue
with a lower intrinsic rhythmicity is also found at the
atrioventricular junction. Interestingly, conus tissue displays pacemaker activity as well (268). In the fish heart,
the chamber myocardium has acquired a faster conduction of the depolarization wave (268). For instance, in the
ventricular chamber of the shark, the conduction velocities of the depolarization waves are ⬃50 cm/s, whereas
velocities of ⬃3 cm/s were measured in the conus myocardium (300). In ECGs these slow depolarization waves
of the conus are recognizable as a peak between the QRS
complex and the T peaks of the ventricular depolarization
and repolarization, respectively (300). The long contraction that results from this slow conduction in combination
with the length of the conus supports valve function and
prevents backflow of blood from the ventral aorta into the
ventricle during diastole (142, 269).
The electrical configuration of the amphibian heart is
remarkably similar to that of the fish heart (2, 193). The
fastest conduction velocities are observed in the atria and
the slowest in the bulbar region. The atrioventricular
junction propagates the depolarizing impulse slowly as
well. In line with this, ultrastructural analyses of the
atrioventricular region display a phenotype of nodal myocardial cells with the typical absence of a transverse
tubular system, few myofibrils, few mitochondria, the
presence of glycogen particles, small cell diameter, and
scarcity of gap junctions (39). The bulboventricular junction would display the slowest conduction time of the
impulse between ventricle and bulbus. This might be
apparent because of the presence of a sulcus at the bulboventricular junction; the real traveling distance of the
impulse over the sulcus myocardium may be longer than
the direct distance between the measuring points in ventricle and bulbus. Also in amphibians the myocardium of
the bulbus cordis assists valve function (142).
Whereas the peristaltically contracting hearts of the
lower vertebrates do not need valves, the synchronously
contracting chamber hearts of the lower vertebrates do
need one-way valves at both ends of their chambers to
prevent backflow from a downstream compartment during relaxation and to an upstream compartment during
contraction. The electrical configuration described above
shows that the entry and exit of a chamber are flanked by
slowly conducting myocardium guaranteeing a sphincter
function (prolonged contraction) at both orifices, as has
been demonstrated for the myocardium surrounding the
semilunar valves in fish and amphibian hearts (142, 269).
Because slow conduction is also a prerequisite for nodal
CARDIAC CHAMBER FORMATION
This morphological process is typical for the developing mammalian heart as well and has been described in
great detail for the developing human heart (59). It also
explains why the expanding cardiac chambers are flanked
by myocardium of the original heart tube and why this
myocardium displays features of the slow components of
the conduction system.
G. Overview of the Component Parts
of the Lower Vertebrate Heart
travels rapidly from the atrioventricular node toward the
apex and then toward the conal region, achieving activation
from apex to base. However, preferential conduction tracts
have not been identified histologically as yet (268).
The largest changes in the amphibian heart compared with that of fish occurred at the venous pole (Fig.
5). In evolutionary retrospect, the fish atrial wall can be
well described as being composed of a right auricular wall
and a left auricular wall that bulge out from the common
atrial portion of the heart tube. Similar to the mammalian
embryonic condition, a single auricle bulges over the
ventricular component at the right and left side. First, the
sinus venosus became largely incorporated into the right
auricular wall close to the midline. Second, an atrial septum and possibly accompanying pulmonary myocardium
developed, by which the single atrial chamber was divided into a right and a left component. The systemic
venous system drains to the right (systemic) atrium and
the pulmonary system drains to the left (pulmonary)
atrium. Thus the atrial myocardial compartment of the
amphibian heart is composed of the following components: 1) right and left atrial wall or auricular myocardium; 2) sinus venosus myocardium or caval myocardium; 3) septal myocardium, possibly including pulmonary myocardium that surrounds the entrance of the
pulmonary veins; and 4) the myocardium of the atrioventricular canal that forms the so-called atrial vestibulum,
which is the smooth-walled lower atrial wall just above the
atrioventricular junction. Support for the generality of this
1. Novel cardiac components
Three major adaptations, or “novel cardiac components,” that were not present in the ancestor chordate
heart tube can be distinguished in the lower vertebrate
heart: the atrium, ventricle, and possibly the muscular
sinus venosus. Furthermore, within the ventricular component a compact outer myocardial component and an
interiorly localized extensive trabecular component can
be distinguished. The specific activation of the ventricle
adds to its complexity as follows. The depolarizing impulse
Physiol Rev • VOL
FIG. 5. Schematic of the atrial building blocks. The cartoon is
modified from that of the transcriptional domains in the developing
mouse atria (91). The atria are composed of four component parts that
each has a left and right component as identified by the left marker
Pitx-2 (gray rectangle). Av, smooth-walled atrial vestibule, derived from
the embryonic atrioventricular canal; aa, atrial appendage, derived from
the embryonic atrium; pm, pulmonary myocardium, including the atrial
septa; cm, caval myocardium, including the myocardium of the sinus
venosus. [Modified from Franco et al. (91).]
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The hearts of the lower fish and amphibian vertebrates have often been used as prototypes for the development of the avian and mammalian hearts, particularly
to understand the origin of the cardiac conduction system
(21, 56, 89, 139, 227, 254). Yet, it has remained a highly
controversial topic, clearly illustrating the difficulties in
uncovering the essentialities of cardiac design. It is thus
of paramount importance to try to assess the essence of
the design of the fish and amphibian hearts, because these
hearts are considered to be the ancestor avian and mammalian hearts. This basic “prototypic” cardiac building plan will
enable us to evaluate the development of the parallel-arranged hearts of birds and mammals in section III. We will
not discuss the reptilian heart, as the heart of the extant
species is considered to be highly derived. The ventricular
septum is considered to have evolved in the primitive reptilian ancestor that gave rise to the mammalian branch on
the one hand and to the avian/crocodilian branch on the
other hand; its evolution is still controversial (130, 327).
During evolution, the high-volume low-pressure cardiovascular system of the tunicates (blood volume equals
⬃40% of their body weight) developed into a low-volume
high-pressure system in vertebrates (blood volume comprises ⬃6% of their body weight). This change was accompanied by a concurrent transformation of a peristaltically contracting straight tubular heart into the more
efficient and powerful synchronously contracting looped
chamber heart. This type of change allowed reduction of
the circulation time of the blood from ⬃6 min in tunicates
to ⬃1 min in humans. Vessels covered with a contiguous
endothelial sheet replaced the largely leaky vessels in
amphioxus.
1231
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ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
description comes from studies on the developing mouse
heart, in which these component parts have been recognized
on the basis of their unique transcription patterns (91).
2. Conserved cardiac components
III. DEVELOPMENT OF THE PARALLELARRANGED FOUR-CHAMBERED HEART
A. Evolutionary Aspects and
Terminological Problems
The parallel-arranged avian and mammalian hearts
originate from the heart of primitive fish that gave rise to
the lungfishes and the amphibian/reptilian branch (Fig. 3)
(257). By that time, the development of the left atrium had
already been realized in the lungfishes and amphibians
and, therefore, the atrial part of their hearts is already
essentially similar to its avian/mammalian counterparts.
This subject has been dealt with in the previous sections.
The right ventricle is the new component of the fourchambered heart and is therefore the focus of the discussion on the development of the four-chambered heart.
The right ventricle is thought to have developed from
the proximal part of the conus arteriosus, which is the
homolog of the bulbus cordis of amphibians and of avian
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Three components of the lower vertebrate heart
might be considered to be minor adaptations, remnants,
or “conserved components” of the ancestor cardiac tube.
These components are the sinoatrial junction, the atrioventricular junction, and the conus, because these three
areas display nodal activity and phenotype. In the absence
of clear distinguishing features, sinus node and atrioventricular node remain difficult to identify unambiguously in
the lower vertebrates. It is worth noting that nodal cells
are virtually devoid of so-called specific myocardial granules, whereas these granules are abundant in atrial and
ventricular myocardium (342, 343). In mammals, myocardial granules are the storage sites of atrial natriuretic factor
(ANF) precursor (283) and the nodes are devoid of ANF,
similar to the fish nodal tissue (282). Because fish hearts do
contain ANF, it is assumed that these granules are the storage sites of ANF precursor protein in fish as well. It is of
great interest that ANF is not expressed in the early murine
embryonic heart tube (51, 347). For that reason its expression has been used to identify the differentiating chamber
myocardium (51), as discussed in section IXA.
So far, we tried to extract the essentialities of the
body plan of the vertebrate heart from the examination of
adult hearts of lower vertebrates, rather than that of their
embryos. In section III we examine whether these essentialities can indeed be considered to be unifying features
of the vertebrate heart.
and mammalian embryos. Trabecularization of the bulbus
“ventricularized” this region that subsequently became
easily accessible from the right atrioventricular canal by
reduction of the so-called bulbo-auricular ridge at the
right side (127). The use of the term bulbo-auricular
ridge rather than the term ventriculo-auricular ridge may
indicate that the early cardiac embryologists saw no ventricular tissue at the inner curvature. In human embryos,
the proximal part of the bulbus cordis becomes trabeculated and is then called primitive right ventricle (314).
However, trabecules develop at the ventral, outer curvature side of the bulbus only; at the inner curvature
smooth-walled myocardium covered with endocardial
cushions remains (73, 314). In our opinion, the question
can justifiably be asked, whether these authors thought
that the entire proximal bulbus would become the primitive right ventricle or merely the trabeculated part at the
outer curvature? Others dispute the origin of the right
ventricle in the bulbus cordis and argue that it is not a
bulbar structure in its entirety (7). In fact, the latter
authors may have called the proximal part of the bulbus
mentioned by the authors of references (73, 314), right
ventricle. Lack of a clear terminology may have led to
misunderstanding. Again, the same question can be asked,
whether the entire segment of the heart tube was meant
to be the primitive right ventricle or merely the trabeculated ventral part. The conflicting terminology originating
from both phylogenetic and ontogenetic considerations
(241) has undoubtedly contributed to the confusion in the
field, but the severe underexposure of dorsoventral patterning has mystified the field most.
Separate cardiac compartments are clearly recognizable at the outer curvature of the developing four-chambered heart, but one feels uncomfortable when one is
forced to distinguish atrioventricular canal, left ventricle,
right ventricle, and outflow tract at the inner curvature.
Indeed, lumen reconstructions of this area in human embryonic hearts made more than half a century ago do not
lend support to true segments at the inner curvature (Fig.
6) (57, 73, 218, 228). Based on the development of trabeculated myocardium, unanimity exists that the right ventricle develops later on in development, downstream and
visibly separated from the left ventricle as is clear from
Figure 6. These observations indicate that the right ventricle
develops from a part of the heart tube, whatever the name of
that part, that is downstream from the left ventricle, and
argue against development from a common ventricular
chamber by septum formation within a single ventricular
chamber as occurs in the atrial part of the heart. In addition,
the reconstructions clearly showed impressions of the ventricular trabecules at the outer curvature and impressions of
smooth-walled myocardium at the inner curvature.
The significance of the above-mentioned description
is that it unequivocally demonstrates dorsoventral (innerouter curvature) patterning. One way to describe these
CARDIAC CHAMBER FORMATION
1233
factual observations of the cardiac configuration at the
ventricular part of the heart tube is that the looping heart
tube is a single compartment or module, from which two
additional modules have grown, the embryonic ventricles.
The smooth-walled myocardium is covered with endocardial cushions and ventrally where the endocardium approaches the myocardium, trabeculated myocardium develops. In the past, the smooth-walled myocardium was
also called primary myocardium (73, 228), and recently,
this name was reintroduced on the basis of functional and
molecular characteristics (63, 209).
A comparable description could be made of the development at the venous side of the atrioventricular canal (73).
Here, the primary heart tube covered with endocardial cushion tissue also extends toward the venous side. At the dorsolateral sides, where the endocardium approaches the
myocardium, the auricular components develop.
The description given above implicitly considers the
primary heart tube as a single entity, although in certain
aspects the tube is highly patterned, as evidenced by the
local differentiation of the atrial and ventricular chambers
and the localized pacemaker activity at the intake of the
heart (270, 313). Without implying the presence of strict
boundaries (in particular not at the inner curvature) but
to facilitate dialogue, we propose that the primary heart
tube can be divided into an inflow tract part, an atrial part,
an atrioventricular canal, a ventricular part, and an outflow tract (bulbus cordis). The patterning of the tube in
the ventricular and outflow tract parts deserves additional
attention, because it plays a crucial role in the formation
of the right ventricle. After the development of this chamber, an anatomically complete parallel-arranged heart had
been achieved.
Physiol Rev • VOL
B. Cardiac Looping, Changing Blood Flows,
and Chamber Formation
A most conspicuous feature of cardiac looping is that
the ventricular chambers that are positioned along the
antero-posterior axis of the embryo become positioned in
a left-right orientation (32, 190, 291). Looping starts at a
stage in which only the ventricular component and part of
the atrioventricular canal has been formed (60, 190). The
linear heart tube grows at both poles, bends toward the
ventral side, and subsequently turns toward the right side
(190). By this process, the original left lateral wall becomes the ventral ventricular wall, the dorsal side the
inner curvature, and the ventral side the outer curvature
(38, 67, 190). The entire dorsal half including the left and
right portions of the linear heart tube ends up at the inner
curvature of the looped heart in later stages; a label positioned at the ventral midline ends up at the outer curvature
(Fig. 7). This indicates that the original ventral side of the
heart tube expands and bends toward the right. During this
process, the ventricular chambers are formed and the flow
of blood reaches its adult pattern (Fig. 8). All experimental
evidence so far supports the notion that looping of the heart
is intrinsic to the myocardial tube itself (185).
Because the high viscosity of the embryonic blood
results in low Reynolds numbers2 (Re: 0.05–2.0 compared
with an adult value of ⬃1,000), flow in the embryonic
vascular system is laminar (197). This flow can be con2
Reynolds number (Re) is a unitless number representative of the
tendency of a liquid (or gas) to become turbulent. It is proportional to
the velocity of flow and to the density, and inversely proportional to the
viscosity.
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FIG. 6. A: “lumen cast” reconstructed from a human embryonic heart of about 4 wk of development. B: drawing of
a section of a human heart of about the same age. Note that the left and right ventricular cavities are connected via the
primary heart tube. RA/LA, right/left atrium; RV/LV, right/left ventricle; oft, outflow tract; avc, atrioventricular canal.
[From O’Rahilly and Müller (228).]
1234
ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
sidered to consist of numerous parallel streaming layers,
which can be marked and followed by application of
indian ink. Changing flow patterns have been documented
by microcinematography during development (Fig. 8)
(292, 293). Watching a beating embryonic heart during an
early stage of development gives the impression that two
separate bloodstreams flow through the heart tube (344),
because contraction is not circular but asymmetric. Dur-
FIG. 8. Diagrams of the developmental changes in blood flow. Three consecutive embryonic mouse stages are shown
in addition to the adult situation. Note that the left and right flows of blood are parallel in all stages as has been
demonstrated in the chicken heart (292). The primary ring is indicated in purple; at 11.5 days of mouse development, it
is clear that this ring has become the right ventricular inlet and left ventricular outlet. RV, right ventricle; LV, left
ventricle.
Physiol Rev • VOL
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FIG. 7. Lineage analysis experiments of chicken embryonic hearts carried out by De la Cruz and co-workers (67, 68).
Label placed at HH stage 9⫹ at either the left or the right interventricular groove is found at the inner curvature at HH
stage 12. Label placed at the midline, i.e., at the ventral fusion line of the cardiac primordia, is found at the outer
curvature. These experiments demonstrate the enormous growth (ballooning) of the ventral side of the heart tube,
relative to the dorsolateral side, by which the label placed at the left or right interventricular groove becomes positioned
at the inner curvature. They also illustrate the rightward turning of the heart tube by which the ventrally positioned label
becomes positioned at the outer curvature and by which the left part of the heart tube becomes positioned ventrally. The
rightward turning of the cardiac tube also is underscored by the expression of the “left marker” cPitx2 that is almost
exclusively expressed in the ventral ventricular walls and absent from the dorsal ventricular wall from HH 23 chicken
hearts (left panels). [From De la Cruz and Markwald (66) and Campione et al. (38), with permission from Birkhäuser.]
CARDIAC CHAMBER FORMATION
Physiol Rev • VOL
reference points, this notion is not true. Viewed in the
longitudinal (antero-posterior) direction of the primary
heart tube, the right side of the atrioventricular canal is
directly connected to the right ventricle. Similarly, the left
side of the atrioventricular canal is directly connected to
the left ventricle. Both sides expand during further development. In line with this view, the so-called dorsal (inferior or caudal) atrioventricular cushion is not limited to
the classical atrioventricular canal but extends toward
the ventricular septum (Fig. 9) and guides the right blood
flow directly toward the right ventricle and the left blood
flow to the left ventricle.
C. The Primary Ring: Development
of the Right Ventricle
A crucial landmark in the development of the right
ventricle of the four-chambered heart is the so-called
primary ring, also called interventricular or bulboventricular ring (Figs. 8 –10) (43, 212, 333). This ring was
recognized in the beginning of the previous century and
associated with the developing conduction system (see
sect. IIF) (7, 21, 330). Lack of molecular markers had
prevented unambiguous delineation of its developmental
fate and hence full appreciation of its morphological significance. The primary ring comprises the myocardium of
the primary heart tube at the position where the ventricular septum develops. In human embryos, the ring is
characterized by the expression of a protein epitope
called GlN2 (18). In chickens, it exclusively expresses the
Drosophila muscle segment related homeobox transcription factor Msx-2 (43). The complex morphological expression patterns of these factors during further development were described in great detail, and three-dimensional reconstructions were made (43, 154, 173). Both in
humans and in chickens, remarkably similar patterns of
expression appeared. Figure 8 depicts the position of the
primary ring relative to the embryonic blood flow and
clearly shows that the ring demarcates the entrance to the
forming right ventricle and the outlet of the forming left
ventricle. Concordantly, during further development the
primary ring becomes positioned at the lower rim of the
right atrium just above the right atrioventricular junction
and at the left ventricular outlet just below the left ventriculoarterial junction (173, 331). Although this morphological analysis is not strictly speaking a lineage analysis,
the precise description of the changing patterns of expression of Msx-2 and GlN2 in a large developmental series in
two species (chickens and humans) allows assessment of
the contiguity of the changing pattern from stage to stage
and makes this morphological analysis almost similar to a
lineage analysis.
Considering Msx-2 and GlN2 as lineage markers,
their developmental patterns of expression unambigu-
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ing contraction, the lumen becomes ovoid, and transitory
ridges arise at both long-sided walls of the ovoid, as a
consequence of which the central part of the lumen is
emptied earlier than the lateral parts (278, 293). At the
long-sided wall of the lumen the cardiac cushions develop
later. During further development of the ventricles, the
decrease in the lumen during systole gradually becomes
concentric, and thus the pattern of flow becomes adultlike. The blood-flow pattern changes from an initially right
flow at the inner curvature and an initially left flow at the
outer curvature to the adult configuration, with the left
flow directed toward the inner curvature (Fig. 8) (129,
278, 293). These flow patterns have been meticulously
documented during chicken development, and the change
proved to take place between Hamburger and Hamilton
stages (HH stage, Ref. 119) 26 and 28. This is at about
Carnegie stage 16, comparable to about 38 days of human
development or 12 days of mouse development. At this
stage, the peristaltic contraction form disappears from
the ventricle (292).
The change in pattern of flow associated with cardiac
looping and formation of the ventricles, as represented in
Figure 8, makes immediately clear that during development the antero-posterior serial arrangement of the right
and left ventricles at the outer curvature had transformed
into a parallel arrangement. Thus, via the primary heart
tube, the parallel-arranged ventricles are connected with
the atria that were arranged in parallel from the very
outset. Usually this is presented as a problem because the
atrioventricular canal would be entirely positioned above
the left ventricle. However, as we described above, functionally this is not entirely true. Anatomically this is not
entirely true either: when describing developing cardiac
morphology one is confronted with the changing position
of the organ within the body and of the cardiac component parts in relation to each other. This easily results in
confusion: people with a medical background tend to use
the body axes as the points of reference and those with a
biological background use the axes of the organ, while
often terminology is used indifferently (156).
Scanning electron microscopic photographs of the
atrioventricular canal of the embryonic heart disclose a
narrow entrance, not only to the right ventricle but also to
the left ventricle (Fig. 9); the atrioventricular cushions
separating the right and left blood flows occupy most of
the volume of the atrioventricular canal. The scanning
electron microphotograph of the atrioventricular canal
represented in Figure 9 clearly demonstrates that both the
right and the left atrioventricular canal halves have to
expand almost equally to achieve the adult configuration.
Hence, the expansion of the right atrioventricular canal to
the right seems to be overemphasized to match the segmental model in which the common atrium is supposed to
be directly connected to the left ventricle only. When the
axes of the developing heart tube are taken as prime
1235
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ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
ously demonstrate that right atrial myocardium develops
at the inner curvature upstream from the primary ring,
and left ventricular outlet myocardium downstream from
the ring. The simplest explanation of the previous statement is that these areas develop directly from the primary
heart tube rather than from parts of the tube that had
already developed a (left and right) ventricular identity, as
is assumed in the segmental model. This notion is supPhysiol Rev • VOL
ported by molecular data (51, 210) (see sect. IX). The ring
divides the primary heart tube into two parts, an upstream
or atrioventricular part and a downstream or bulboventricular part. For the right blood flow the ring is atrioventricular, demarcating the right ventricular inlet, and for
the left flow of blood the ring is bulboventricular, demarcating the left ventricular outlet (Fig. 8). Similarly, one
could divide the cushions of the primary heart tube into
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FIG. 9. Cardiac septation. A: scanning electron microscopic picture of a human heart of 35 days of development
(Carnegie stage 15). B: scanning electronic microscopic picture of a mouse embryonic heart, of which the embryonic
atria have been removed, showing that the atrioventricular canal is positioned in the middle of the heart. Because the
atrioventricular cushions occupy almost the entire atrioventricular orifices, they leave narrow entrances (marked by the
purple lining) to the ventricles only. Therefore, both the right and the left orifice have to expand to achieve the adult
configuration. [Photograph from Sadler (265).] C: oblique sagittal section of a rat embryonic heart, showing the
continuity between outflow tract and atrioventricular canal at the inner curvature (primary heart tube) and the
expanding atrial and ventricular chambers. The section is hybridized with a probe to sarcoplasmic reticulum ATPase
(SERCA2a), showing high levels of expression in the working myocardium of the atrial and ventricular chambers (210).
Note also the continuity between the dorsal atrioventricular cushion and the dorsal mesocardium. D: cartoon of a heart
of a comparable stage as presented in A to facilitate the understanding of the transition of a looping tubular heart into
a four-chambered heart. The primary heart tube is indicated in purple, the atrial chambers in blue, the ventricular
chambers in red, the cardiac cushions in yellow, and the venous and arterial connections in green. The outflow tract is
hinged to the right to allow visualization of the inner curvature. E: cross section showing the formation of the atrial
septum from the muscular primary interatrial septum (blue), and the mesenchymal atrioventricular cushions (yellow)
and vestibular spine (green). The latter mesenchymal structures become almost entirely muscular with further development. Oft, outflow tract; RV/LV, right/left ventricle; RA/LA, right/left atrium; PAS, primary atrial septum; PAF, primary
atrial foramen; DM, dorsal myocardium; 1, dorsal atrioventricular cushion; 2, ventral atrioventricular cushion; 3, parietal
outflow tract ridge; 4, septal outflow tract ridge. The dashed line in D represents the plane of sectioned represented in
E. [Modified from De Jong et al. (64).]
CARDIAC CHAMBER FORMATION
1237
two types of cushions as well: 1) upstream cushions
rather than atrioventricular cushions, and 2) downstream
cushions rather than truncus cushions (Fig. 9). We will
conform to conventional terminology, but it is important
to appreciate that large parts of the cushions are in fact
localized in parts of the primary heart tube that are conventionally called right or left ventricle. The atrioventricular cushions also extend into the atrial part of the heart
tube upstream from the atrioventricular canal, although
this is less pronounced (Fig. 9).
D. Completion of Septation
Chamber formation in the avian and mammalian
heart is completed with the physical separation of the
blood flows (Fig. 9). For the sake of clarity, we only
consider septation of the three principal components of
the heart: 1) septation of the atrial chamber by formation
of the atrial muscular septum, 2) septation of the ventricular chamber by formation of the ventricular muscular
septum, and 3) septation of the primary heart tube by
formation of the cardiac cushions. For precise descriptions of the changes in these complex morphological
processes we refer to detailed accounts from our laboratory and others (59, 64, 73, 154, 171–173, 228, 292, 314).
Only some very basic principles are given here.
The atrial chamber is divided by a muscular septum
(primary septum) that is formed by local active proliferation of the myocardial cell layer. Its free rim is covered
with a mesenchymal cap (100) that fuses with the atrioventricular cushions and the dorsal mesenchymal protrusion (vestibular spine), as shown in Figure 9 (154, 299,
328, 332). Whether the mesenchymal contribution from
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three distinct origins (atrioventricular cushion, atrial septal cap, and extracardiac mesenchyme) to the formation
of the lower rim of the primary atrial septum is of developmental significance is as yet unknown. Folding of the
atrial wall at the right side of this primary septum forms
the secondary atrial septum (8, 64, 314). Note that the
secondary atrial septum is a unique mammalian structure
and is not present in avian and amphibian hearts.
The muscular ventricular septum is formed by apposition of cardiomyocytes at the outer side concomitantly
with the outgrowth of the ventricles at the outer curvature
(314). In vivo labeling studies have shown that the septum
is derived from ventricular cells located at the ventral
fusion line of the bilateral mesodermal heart epithelia
(65).
The primary heart tube is physically separated into
left and right components by fusion of the so-called cardiac cushions. In the primary heart tube, the endocardium
is separated from the myocardium by an extracellular
matrix layer or cardiac jelly (58, 59). The cardiac jelly
disappears from the chamber-forming regions of the cardiac tube, but remains covering the smooth-walled myocardial tube. Subsequently, upon myocardial signaling, a
subpopulation of endocardial cells overlying the cushions
undergoes a transformation from epithelial to mesenchymal cells, by which the cardiac cushions are formed (31,
83, 264, 336). Neural crest cells also contribute to the
mesenchyme of the outflow tract ridges (55, 243, 320),
while epicardium-derived cells contribute to the atrioventricular cushions (103). However, the specific functions of
these cells are still unclear.
Although the molecular mechanisms of the formation
of cushion mesenchyme are well-described (30, 31, 83,
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FIG. 10. The primary ring displays a unique pattern of expression. The panels show patterns of expression of the
mRNAs encoding Msx-2 (A, overview; B, detail) and atrial natriuretic factor (ANF) (C). Msx-2 mRNA is almost
exclusively expressed in the primary ring, although some expression is visible at the left atrioventricular junction as well.
The photograph shows the right atrioventricular ring bundle (ravrb) and the atrioventricular bundle (avb) at the top of
the ventricular septum. Note that ANF mRNA is not expressed in the Msx-2 positive areas. Its expression identifies the
developing atrial and ventricular chamber myocardium. [Modified from Houweling et al. (133).]
1238
ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
IV. DEVELOPMENT OF CARDIAC FUNCTION
AND CONDUCTION SYSTEM
Some topics in the field of cardiac development have
always attracted special interest, for example, the neural
crest and the cardiac conduction system. The combination is irresistible but has not particularly clarified the
field (for reviews, see Refs. 107, 112, 170, 202, 208, 271).
Controversy often appears to be based on misunderstanding, due to a highly confusing terminology. The pacemaking tissues (sinus node and atrioventricular node) are
well-recognized components of the cardiac conduction
system. As we have seen, pacemaking activity is already
present in the hearts of the chordate ancestor in which
Physiol Rev • VOL
neural crest cells have not yet developed (114). An ECG
(232, 277, 313), indicating coordinated depolarization of
the distinct cardiac compartments, can be recorded concomitantly with chamber formation at a stage in which
neural crest cells (106, 107, 158, 159) as well as cells
derived from the epicardium (103, 191) have not arrived in
the heart. Although a role for these cells in the maturation
of the conduction system can be envisaged, the essential
electrical configuration of the heart has already been laid
down before these cells arrive at the heart and before the
different components of the conduction system can be
morphologically identified. Recently, this notion was confirmed independently by both elegant lineage studies of
Cheng and co-workers using retroviral and adenoviral cell
lineage tracing constructs (49), and clonal analysis of the
fate of the atrioventricular canal (60). A sensible approach to examining how the essential electric configuration is achieved is to relate its development with the
development of the basic components of the heart.
A. Development of Polarity
The first key issue to resolve with regard to the
development of polarity in the heart is the origin of the
sinus node or leading pacemaker activity. Its development
is intimately associated with the development of anteroposterior polarity of the heart. Patten (234) already noticed this and wrote,
One of the most startling and significant facts
about the developing embryonic heart is that its myocardium at different cephalo-caudal levels exhibits
different inherent rates of contraction. Each new part
of the heart that is added behind the first formed
cono-ventricular part has a higher intrinsic contraction rate than the part of the heart tube already
formed. The part of the cardiac tube with the highest
contraction rate at any given phase of development
sets the rate for the entire heart.
Patten referred to experiments in which fragments of
chicken myocardium are cocultured or grafted. As soon
as the fragments have become connected, the faster beating fragment takes the lead and causes the more slowly
beating fragment to beat at a faster rate. He concludes
that the pacemaker is always the part of the heart that
was added most recently. Because electromechanical
coupling is first established in the ventricular region, the
first beats are observed in the ventricle, while the pacemaker is present in the upstream region that does not yet
beat (313). These observations and experiments demonstrate antero-posterior polarity in pacemaker activity and
contraction rate along the linear heart tube. Due to the
slow conduction of the impulse (13, 63, 126), contraction
is peristaltic (234). Taken together, in the early chicken
embryo all regions of the linear heart tube possess intrin-
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205, 244) and the position of cushion formation is clear
from the contraction pattern mentioned above, the molecular cues that determine the precise anatomical positions and extents of cushion formation are as yet unknown. Upstream from the primary ring, the two atrioventricular cushions (upstream cushions) develop (Fig.
9); note that they extend both in the ventricular part and
in the atrial part of the tube. Only the dorsal (inferior or
caudal) atrioventricular cushion extends toward the ventricular septum, guiding the two blood flows separately
into left and right ventricular directions. Two cushions
are also present downstream from the primary ring, usually called ridges. We call the ridge that is contiguous with
the ventricular septum the septal outflow tract ridge (64)
(other authors have called this ridge the left conal swelling, Ref. 314), and the other ridge the parietal outflow
tract ridge (also called the right conal swelling). The
septal ridge guides the blood from the left ventricle toward the aortic outlet. The parietal ridge extends into the
atrioventricular part of the heart tube, and because of
that, it also guides the right flow of blood into the right
ventricle.
Fusion of the cushions affects the physical separation of the right and left blood flows, but the precise
contributions of the individual cushions in the ventricular
part of the heart tube are uncertain. It should be appreciated that the cartoon in Figure 9 overemphasizes the
relative dimensions of the heart tube at the inner curvature. The outflow tract of the heart was hinged to the right
to permit visualization of the cushions at the inner curvature.
Most of the cardiac cushion mesenchyme becomes
muscularized during further development (68, 211, 225,
308, 309, 339), but the extent of muscularization varies
among the different species. For instance, in humans, the
atrioventricular cushion mesenchyme approaching the
top of the ventricular septum remains membranous and
forms the so-called atrioventricular part of the membranous septum (172).
CARDIAC CHAMBER FORMATION
B. Development of the ECG
Already before the onset of overt chamber formation,
an ECG can be recorded in embryonic chicken hearts
from HH stage 11 onwards (232, 277, 289). Initially, the
ECG is sinusoidal in shape, as reflected by the peristaltic
waves of contraction. A delay between the atrial and
ventricular parts of the tube can be recorded, when the
atrioventricular sulcus becomes visible. This development is accompanied by an “adultlike” electrical organization, comprising the typical P wave of atrial depolarization, the QRS complex representing ventricular depolarization, and the T wave representing ventricular
repolarization. Interestingly, a conal wave (C wave) in
between the QRS complex and the T wave is present in
embryonic chicken hearts as well (231). Thus the chicken
embryonic ECG is basically similar to that of the formed
heart of the shark, as discussed in section IIE. Although in
the early embryonic heart (chicken stage HH 14) no specialized/distinct cells of a conduction system can be recognized, it displays coordinated propagation of the impulse, implying a pacemaker at the intake of the heart,
rapid conduction in the atrial region, a delay in the atrioventricular region, rapid conduction in the ventricular
region, and slow conduction in the outflow tract. And,
indeed, slow conduction velocities were measured in the
sinoatrial region (63), atrioventricular region (13, 14, 63,
180, 181, 313), and outflow tract (28, 63, 231), and it was
concluded that the atrioventricular ring tissue is the functional counterpart of the atrioventricular node (181). In
line with this, the primary heart tube consisting of atrioventricular canal, inner curvature, and outflow tract is
virtually devoid of the two main gap junctional proteins
connexin (Cx) 40 and Cx43 (69, 208, 310).
Studies in chicken demonstrated that the linear heart
tube contracts peristaltically and has a matching sinusoiPhysiol Rev • VOL
dal ECG (277). Also, during further development when
chambers have formed, the pattern of contraction
matches the electrical configuration (28, 230). In the linear heart tube the layer of cardiac jelly, in between the
endocardium and the myocardium, plays a crucial role by
supporting the unidirectional propulsion of the blood: the
limited sarcomeric shortening would not permit closure
of the myocardial tube without such a layer (19, 235).
During further development, a progressive lag in contraction duration along the cardiac tube becomes apparent as
follows. The atrium displays short, brisk contractions that
are finished before the ventricle contracts, the ventricle
contracts at a slower pace, and the sluggish, prolonged
contractions of the outflow tract prevent backflow of
blood while the ventricle relaxes (28). This situation is
highly reminiscent of that in the adult shark heart, in
which the prolonged contraction of the conus (outflow
tract) supports the function of the semilunar valves (142,
269). Such a function is also essential for the embryonic
heart, which has no valves; the slow conduction of the
depolarizing impulse in the atrioventricular canal and
outflow tract, and the subsequent prolonged contraction
duration substitute valve function in these areas (63). The
pattern of contraction is in line with the regional differences in calcium-triggered calcium release and with the
mRNA and protein levels of sarco(endo)plasmic calcium
ATPase (210). High levels were observed in the atrium,
intermediate levels in the ventricles, and low levels in the
primary heart tube, that is in the atrioventricular canal,
the inner curvature, and in the outflow tract (Fig. 9C).
In summary, the molecular, functional, and electrical
configurations of the looping heart tube at the onset of
chamber formation fully explain the coordinated contraction as reflected in the ECG.
V. LINEAGE ANALYSIS AND
CHAMBER DEVELOPMENT
Cell-tagging (fate-mapping), grafting, and explantation experiments in several species have yielded a detailed insight into the contribution of cell populations to
the heart at consecutive stages of development (199, 258,
291, 297). Precursor cells of myocardium and endocardium are colocalized in the epiblast. During gastrulation,
the epiblast heart precursors ingress through the primitive streak until late-streak stages [chicken HH3; mouse
embryonic day (E) 6.5–7] to form the bilateral heartforming regions lateral of the anterior gut endoderm
(chicken HH4 – 8; mouse E7–7.5).
A. Formation of the Linear Heart Tube
The tubular heart is formed by migration and fusion
of the two heart-forming regions at the midline [chicken
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sic automaticity and the peristaltic contraction waves
travel along the tube with low conduction velocities. This
condition is reminiscent of the hearts of the primitive
chordates, in which polarity evolved with the dominant
pacemaker at the intake of the heart, but the mechanism
that imposes antero-posterior polarity upon the heart
tube is not settled yet (see sect. VI).
Functional measurements of early mammalian cardiac development are rare, because the mammalian embryo is not easily accessible for experimental manipulation. Furthermore, early mammalian cardiac development
is rapid compared with early chicken cardiac development. For example, in rats the first contractions are already observed before the paired primordia have fused
(108); the myocardial cells are poorly coupled, which
would suggest that also in the early mammalian heart tube
the propagation of the impulse is slow (94, 115, 310).
1239
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ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
B. The Heart-Forming Regions Include Anterior
and Posterior Mesenchyme
In a recent lineage study by Redkar et al. (248), a
precise map of the mesodermal heart-forming region in
chicken embryos was generated by DiI cell labeling. The
stages of labeling were HH4 – 8, which represent the period until just before fusion of the heart-forming region at
the midline to form a tubular heart (from HH9 onwards)
and were followed by the analysis of the fate 20 h later
(until stage HH12). The heart-forming region thus mapped
appeared larger than the heart-forming region that was
defined in a previous study by Ehrman and Yutzey (82), in
which the fate was analyzed at HH10. This discrepancy
may result from the difference in stage of analysis between both studies, because in the latter study cells that
will contribute to the heart tube after HH10 were not
mapped to the heart-forming region. Furthermore, in the
analysis of Ehrman and Yutzey (82) Nkx2–5 and Vmhc
were used as markers for the heart-forming region. At
HH5, however, this gene was not expressed in all cells
fated to become cardiomyocyte at HH12 (248). The posterior border of the Nkx2–5 expression domain within the
heart-forming region was found to represent the posterior
ventricular border of the heart tube at HH12. Because the
entire heart tube of HH12 embryos expresses Nkx2–5, this
finding indicates that between HH5 and HH12 at the posterior side of the heart ongoing recruitment of cells to the
cardiogenic lineage (Nkx2–5 positive) has taken place.
In reality the heart-forming region is even larger,
because at stage HH12 part of the sinoatrial region and
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the outflow tract have not yet been added to the tubular
heart (67) and were thus not mapped to the heart-forming
region. The entire heart-forming region would be found if
the end-stage of analysis is performed at approximately
stage HH18 –22, when all myocardium has been recruited
(67). Indeed, in two independent studies (204, 321) the
outflow tract of the chicken heart at HH18 –22 was shown
to be formed from cells that are localized outside the
“classical” heart-forming region, in a “novel” anterior or
secondary heart field that got unrecognized before. It remains to be defined which cells in the HH4 – 8 embryos will
give rise to the outflow tract. Kelly et al. (150) extended the
observations in chicken to mouse and demonstrated that
between E8.25 and E10.5 pharyngeal and anterior pericardial mesodermal cells are recruited to the heart, forming the
myocardial outflow tract. These myocardial cells originate
medially from the classical heart fields. Whether or not the
right ventricle is part of the anterior heart field is not settled
yet. The pattern of expression of the Fgf10 enhancer trap
LacZ transgenic line suggests it is. However, a small Mlc2v
promoter fragment that is active in the right ventricle and
the outflow tract is expressed already in the anterior region
of the cardiac crescent and linear heart tube (260), which
may indicate that right ventricle precursors may not be part
of the anterior heart field.
C. The Inner Curvature Connection
An important issue to resolve is the lineage relationship between the distinct regions of the linear heart tube
and the component parts of the cardiac chambers. De la
Cruz and Markwald have made relevant contributions as
documented in detail in Reference 66. Some other authors
interpreted these analyses as follows (192): “The segments of the tubular heart, like the somites, form progressively along an antero to posterior axis with each segment
being an undifferentiated, morphogenetic unit separated
by boundary interfaces.”
At present, this segmental view is commonly used,
albeit in general without any conceptual connotation.
Because De la Cruz and Markwald did not explicitly include dorsoventral regionalization in their descriptions, it
seems to be a logical extension of their work, but nevertheless it is fundamentally wrong. De la Cruz and Markwald (66) explicitly state that “In the straight heart tube
there is no region that gives origin either to the right or
the left ventricle in their entirety,” and further that “the
straight heart tube is constituted by two primordia, one
cephalic, i.e., that of the apical trabeculated region of the
anatomical right ventricle, and the other caudal, that of
the apical trabeculated region of the anatomical left ventricle. Consequently there is no single primordium for
each of the definitive cardiac cavities.”
This description is entirely in line with that of the
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HH9 –10 (291); mouse E7.5– 8 (71)]. Labeling experiments
in chicken showed that the cells of the cardiac crescent
fuse in the antero-posterior direction (258, 291). The ventral side of this tubular heart will eventually contribute to
the apical part of the right ventricle of the formed heart
(67). As development proceeds, myocardium is continuously recruited at the posterior side of the elongating
tube. This myocardium will form the apical part of the left
ventricle, the atrioventricular canal, and the sinoatrial
region, respectively (67, 291). At the anterior side of the
tube, myocardium is recruited from the pharyngeal mesoderm that will contribute to the outflow tract (67, 150, 204,
316, 321). From anterior to posterior serially aligned primordia are thus formed. As a result of the continuous
elongation of the heart tube, the morphological inflow
tract and outflow tract of the linear heart tube of HH9 –10
chicken embryos are formed by myocardium fated to
contribute to the left ventricle and right ventricle in the
mature heart, respectively. While recruitment of myocardium at both heart poles takes place, the prospective ventricular portion has already begun to loop and to change its
molecular and morphological phenotype (see below).
CARDIAC CHAMBER FORMATION
D. Origin of the Nodal Tissue
It has been a long-standing issue whether the nodal
components of the cardiac conduction system are rem-
nants of the primary myocardium and escaped differentiation into the chamber lineage (“escape” hypothesis)
(208, 209, 241), or are recruited during later stages of
development from a common precursor that also gives
rise to the working myocardium (“recruitment” hypothesis, Ref. 49). Although placed opposed to each other (49),
the two hypotheses are essentially similar. However, the
escape hypothesis explicitly assumes that the primary
myocardium is the common precursor of nodal and working myocardium. It also assumes that nodal function is
already present in the embryonic heart, as we have discussed in section IV. The recruitment hypothesis, on the
other hand, is indeterminate about the nature of the precursor cells, assumes that recruitment takes place until
late stages of development, and implicitly suggests that
the nodal conduction system is formed de novo in the
developing heart.
Several arguments are in favor of the escape hypothesis, as follows.
The observations described in section IV reveal an
amazing simplicity of cardiac design, in which the slowly
conducting areas of the primary heart tube relatively decline at the expense of the developing fast conducting
working myocardium of the chambers. This description
explains the observed electrical configuration (ECG),
without the need to recruit new “nodal” elements. In line
with the escape hypothesis, the slowly dividing cells of
the primary heart tube map to the conduction system (60,
304, 305). Further evidence comes from Gata-6 gene enhancer studies and analysis of the expression pattern of
minK (60, 169). Burch and co-workers (60) observed that
transgenic mice having the lacZ gene under control of a
chicken Gata-6 gene enhancer expressed the transgene in
the atrioventricular canal. At later stages of development
expression became confined to the atrioventricular node,
atrioventricular bundle, and atrioventricular ring bundle.
By deploying the Cre-lox system, it was shown that these
nodal components are clonally related to the embryonic
atrioventricular canal cells (60). Similarly, when inserted
into the minK gene, the lacZ gene was expressed in
primary myocardium of the atrioventricular canal in the
FIG. 11. Origin of the ventricles. The fate of the
colored segments in the embryonic heart tubes (left) is
depicted in the formed heart (right). Note that in the
tubular heart the atrioventricular canal and the outflow
tract are separated by ventricular primordia, whereas in
the formed heart they are connected. The same holds for
the right ventricle and the atrioventricular canal, and for
the left ventricle and the outflow tract. In reality, the
atrioventricular canal and the outflow tract are directly
connected at the inner curvature (dorsal side) of the
tubular heart, solving this connection problem. [Modified
from De La Cruz and Sanchez-Gomez (67).]
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early cardiac anatomists (57, 73, 218, 228) given in section
III. In the mature four-chambered heart, the atrioventricular junction and the right ventricle are directly connected without intervention by left ventricular myocardium (compare Figs. 1 and 11). Likewise, the left ventricle
is directly connected to the outflow tract, without intervention of right ventricular myocardium. Even the atrioventricular junction and outflow tract are directly connected
without intervention of ventricular cells at the inner curvature. It is clear that these connections cannot be made in a
representation of the tubular heart composed of serially
aligned segments, where atrioventricular canal and outflow
tract are separated by cells fated to become left ventricle
and right ventricle, respectively (Fig. 11).
The discrepancy between the concept of the segmented tubular heart and the adult condition is virtual
and can entirely be solved by appreciating that from the
very outset of heart-tube formation onward a direct connection exists between cells fated to become atrioventricular canal and those fated to become outflow tract. This
connection is found at the dorsal side of the straight heart
tube and at the inner curvature of the looped heart tube.
In line with this assumption is the fact that only the apical
components of the ventricles are formed from the “ventricular segments,” whereas the connected basis and conus parts are generated from the atrioventricular canal
and outflow tract “segments” (Fig. 11) (67). The lineage
analysis studies of De la Cruz and co-workers (67) do not
resolve issues related to the fate of myocardium located
at the cardiac inner curvature, simply because these areas
were not labeled. Hence, the entire concept would be
strengthened if some of the labeling experiments were
repeated in conjunction with analyses of patterns of gene
expression, such as those of the genes encoding Msx2 and
ANF as markers for the primary ring and for the forming
chambers, respectively (Fig. 10).
1241
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ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
E. Fate of the Primary Ring
The fate of the primary ring is of great interest. In
section IIIC we described that this ring can be identified
molecularly by the expression of Msx-2 and GlN2. It is a
part of the primary heart tube that is localized at the crest
of the ventricular septum and that after further development surrounds the right atrioventricular junction and the
left ventricular outlet (43, 212, 333). In adult avian hearts,
these regions were morphologically identified as parts of
the conduction system that still form a ring and comprise
the atrioventricular bundle, septal branch, retroaortic
branch, and right atrioventricular ring bundle (170, 315).
Thompson and co-workers (304, 305) identified these regions as proliferation-quiescent areas derived from the
slowly dividing tubular heart, which was recently confirmed by the above-mentioned lineage studies of Cheng
et al. (49). In the adult mammalian heart, none of these
components can be recognized except the atrioventricular bundle, although in embryonic and neonatal hearts of
various species (including humans) some elements are
still present (4, 5, 9, 11, 208, 333).
Based on its location and origin the ring is expected
to have a “primary heart tube” phenotype. MorphologiPhysiol Rev • VOL
cally this is indeed the case; similar to the nodal tissues
these cells are always described as “more undifferentiated” relative to the working myocardium of the atrial and
ventricular chambers. We mentioned (see sect. IIF) the
paradoxical naming of the conduction system as cardiac
specialized tissue, which is at odds with the poorly developed myofibrillar and t-tubular system (39). However, it
has been argued that the conduction system is specialized
because it has almost withdrawn from the cell cycle (304).
These cells were considered to be terminally differentiated, analogous to skeletal muscle precursor cells that
withdraw from the cell cycle and differentiate into muscle
cells. It is therefore interesting to speculate whether the
derivatives of the ring have retained the slowly conducting feature as well. Arguëllo et al. (14) mapped this area in
great detail in embryonic chicken hearts after 6 days of
incubation. In the atrioventricular bundle, as well as in the
atrioventricular node, atrioventricular canal, and lower
interatrial septum, they measured low conduction velocities compared with the velocities in the atrial auricle and
ventricle. In line with this, the gap junctional proteins
Cx40 and Cx43 are generally found to be absent from the
atrioventricular bundle and proximal part of the bundle
branches in fetal and neonatal mammalian hearts (311). In
adult hearts of both mouse and human, the atrioventricular bundle is fast conducting (128, 161). Thus, although
the cells of the conduction system withdraw from the cell
cycle relatively early in development, they acquire their
mature phenotype relatively late in ontogenesis, indicating that the maturation of the conduction system is an
ongoing process.
An intriguing aspect of the primary ring is that it
constitutes an almost separate compartment. At the right
side of the atrioventricular canal, the insulating fibrous
tissue will interpose the myocardium downstream from
the ring. By this process the ring forms the lower rim of
the right atrium that dorsally penetrates the insulating
fibrous plane as the atrioventricular bundle (331). The
atrioventricular bundle will become surrounded rather
than interrupted by fibrous tissues. In mammalian development, the ventral penetration disappears concomitantly
with the retraction of the myocardium of the outflow tract
by which process the part of the ring that surrounds the
left ventricular outlet disappears as well (326, 339). The
atrioventricular bundle thus connects the cardiac specialized primitive tissue of the atrioventricular node with the
cardiac specialized primitive tissue of the ventricular conduction system (see below), and in itself is also a cardiac
specialized primitive tissue.
We know very little about the development of the
function of the primary ring, its molecular identity, its
interaction with the neighboring compartments, and
about the maturation of the atrioventricular bundle to a
fast-conducting bundle. The role of Msx-2 is still unexplained. We also do not know whether the ring is present
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embryo, and in the region of the atrioventricular node in
the mature heart (169). These observations indicate that
already early in development part of the primary heart
tube (atrioventricular region up to and including the primary ring) is predisposed not to participate in chamber
formation, but fated to atrioventricular node and bundle,
and is not recruited late in development (49).
Birth-dating experiments by label dilution of [3H]thymidine and lineage tracing experiments using replicationincompetent viral lacZ-expressing constructs have been
interpreted by Cheng et al. (49) in terms of the recruitment hypothesis. However, the results of these birthdating experiments also match the escape hypothesis and
confirm the observations of Thompson and co-workers
(304, 305) that the conduction system is recruited from
slowly proliferating myocardium. Lineage tracing studies
that started in primary heart tube stages of necessity
reveal a lineage relationship of cells of the conduction
system with neighboring cells of the working myocardium, as both are formed from this source. Finally, it is
still unknown why some cells of the primary myocardium
in the sinoatrial and atrioventricular region escape differentiation into chamber myocardium and form the nodes,
whereas the primary myocardium of the outflow tract
eventually differentiates in the ventricular direction. It
also is unclear why morphologically distinct sinus and
atrioventricular nodes are present in the formed heart of
mammals only, whereas in other vertebrates the nodes
remain morphologically quite diffuse (39).
CARDIAC CHAMBER FORMATION
in amphibian or fish hearts as a phenotypic distinct entity.
Unraveling the regulatory pathways will add a fascinating
story to the understanding of cardiac morphogenesis.
F. Origin of the Ventricular Conduction System
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the unlooped heart were found in the trabecular regions
of the ventricles (67). The sequence of events implies that
the cells in the trabecules at the endocardial side are the
“oldest” cells directly derived from the embryonic ventricular wall of the heart tube. Indeed, Anf (51, 133, 347),
Cx40 (70, 111, 312) and Irx3 (52) initially are expressed in
the epithelial-like ventricular wall of the embryonic heart.
During chamber expansion their expression becomes restricted to the trabecules and subsequently to the subendocardial ventricular conduction network. A similar process, but less extensive, is observed in the atrial appendages, where the trabecules become the pectinated
muscles.
G. Overview of the Component Parts of the Higher
Vertebrate Heart
The formed heart comprises the right and left atrial
and ventricular chambers and a conduction system encompassing the pacemaking tissues (sinus node and atrioventricular node) and the fast-conducting atrioventricular
bundle and bundle branches. The definitive cardiac chambers are anatomical units. They are built up out of several
embryonic component parts that often constitute distinct
transcriptional domains (88, 91, 93, 151, 152).
1. The atrial and ventricular chambers
Compared with the fish/amphibian ancestor heart,
the essence of the design of the atrial part of the avian and
mammalian heart has not significantly changed (see sect.
IIG, Fig. 5), whereas in the ventricular part a right ventricle has developed and the outflow tract has been incorporated into the ventricular component. This part of the
heart is still a distinct transcriptional domain primarily
within the right ventricle (151). Because the fibrous insulation occurs at the lower boundary of the atrioventricular
canal (331), the atrium can be considered to be composed
of four component parts (see Fig. 5), i.e., 1) sinus venosus, 2) primary atrial septum including the pulmonary
myocardium, 3) auricles (atrial appendages), and 4) atrioventricular canal (atrial vestibule). Each of these component parts has a left and a right domain (91).
Whereas the heart has a left and a right atrium in
terms of identity, this is not so for the ventricles. We
described above that the ventricles develop at the outer
curvature along the antero-posterior axis. After looping,
the ventricular chambers become localized in left-right
orientation; the process of looping positions the left side
of the heart tube ventrally. Thus, in terms of identity, each
ventricle has a left component (located at the ventral
side) and a right component (located at the dorsal side)
(38, 67, 190). Consequently, it might be clearer to call the
left ventricle the systemic ventricle and the right ventricle
the pulmonary ventricle.
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The ventricular conduction system comprises the
right and left bundle branches and the subendocardial
peripheral ventricular conduction system. In rodents, the
peripheral ventricular conduction system is poorly developed, but in chickens and ungulates it is well developed.
In chickens, an additional well-developed periarterial Purkinje system has developed (39, 170, 208, 315). Studies by
Mikawa’s laboratory revealed that in chickens 1) the periarterial Purkinje cells are clonally related to surrounding
working myocardium, 2) these cells develop in close association with and are induced by the developing coronary arteries, and 3) their formation can be induced by
endothelin-1 in vitro and in vivo (49, 113, 136, 296). The
periarterial Purkinje system develops late in chicken development (second half of the incubation time), which is
well after the development of the ECG and of the preferential conduction of the impulse toward the apex of the
ventricle (53, 63, 232, 277, 289). The periarterial Purkinje
system expresses a slow tonic myosin (105) and a distinct
gap junctional protein Cx42 (110), and it displays a unique
transcription factor expression profile (295, 301). The
periarterial Purkinje system has not yet been characterized functionally.
The bundle branches and the peripheral ventricular
conduction system are thought to develop from the trabecular ventricular component (208, 315). The early ventricular wall consists of a compact layer of myocardial
cells and protrusions into the lumen called trabecules.
Before and during looping, the wall of the heart consists
of only a few layers of epithelial-like myocytes and is lined
by a cellular cardiac jelly and endocardium. Just after
looping (chicken HH16/17 and mouse E10), trabecules
become evident at the entire outer curvature between the
atrioventricular and outflow tract cushions (276). The
cells of the ventricular trabecules and of the bundle
branches display a poorly developed contractile apparatus and t-tubular system but well-developed gap junctions
(39). The trabecules display a high abundance of Cx40
and Cx43 protein (94, 111, 310) and the impulse is preferentially conducted via the trabecules (53, 63). Retroviral
lineage analyses revealed that individual myocytes generate a series of myocyte clusters, forming one or a few
trabecules (199, 202). These trabecules fuse and, because
the cells at the epicardial side proliferate faster than those
at the endocardial side (304, 305), cone-shaped clonally
derived structures are formed. Part of the trabecules becomes incorporated in the compact layer of myocardium.
Consistently, label particles placed on the ventral wall of
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ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
2. The conduction system
In the formed heart, three distinct components of the
conduction system can be distinguished morphologically,
functionally, and molecularly (Table 1): 1) the sinus node
and atrioventricular node, consisting of poorly coupled
cells that do not express ANF; 2) the atrioventricular
bundle, consisting of well-coupled cells that do not express ANF; and 3) the bundle branches and the subendocardial extensions, consisting of well-coupled cells that
do express ANF. Other differences exist as well (208), but
those mentioned here are highly distinctive and have been
discussed in the previous chapters.
3. Engrailed-2/lacz transgene expression:
speculations on cardiac design
The distinct components of the cardiac conduction
system have much in common. The sarcomeric and ttubular systems generally are poorly developed (Table 1)
(39, 208), and the entire conduction system originates
from proliferation-quiescent areas (304, 305). In this con-
text the pattern of a recently described cardiac transgene
is of great interest (249). The pattern originates from a
singular transgenic mouse line (engrailed-2/lacZ) harboring a fusion construct of the lacZ reporter gene and the
enhancer region of the homeodomain-containing transcription factor engrailed-2 (Fig. 12). LacZ gene expression was first observed in a widespread pattern in the
(primary myocardial) tubular heart (E8.5–9). During further development, the entire area of the conduction system, including the nodal regions, atrioventricular bundle,
bundle branches, and subendocardial ventricular myocardium expressed the transgene. Interestingly, left and right
atrioventricular junctional myocardium (also called the
smooth-walled atrial vestibular myocardium that is derived from the atrioventricular canal region of the primary
heart tube, Refs. 154, 331) expressed the transgene as
well. In addition, expression was observed in a broad
dorsal region of the right atrium roughly in between the
nodes.
An intriguing possibility is that the transgene discloses the myocardium that has not fully differentiated
into atrial or ventricular working myocardium and identifies the area of slowly proliferating embryonic myocardium (304, 305). The pattern of expression of the transgene is highly reminiscent of that of the proliferationquiescent areas described in the chicken embryonic heart
by Thompson et al. (304), who proposed that the early
conduction system would serve as organizational tissue
for myocardial differentiation. Interestingly, the area of
engrailed-2/lacZ transgene expression can be enlarged by
the addition of neuregulin-1 to whole mouse E8.5–9.5
embryo cultures (250). The transgene is not induced in
the free ventricular wall of mouse E10.5 embryos, when
overt compact myocardium has differentiated. Optical
mapping of the neuregulin-1-treated embryos demonstrated ectopic preferential activation pathways that were
attributed to the recruitment of additional cardiomyocytes to the conduction system. Taken together, the transgene may thus identify the primary myocardium and the
FIG. 12. A: expression of the MC4 Engrailed-2/
lacZ reporter in a mouse embryonic heart of 12.5 days
of development. LacZ expression is seen in the sinoatrial (SAN) and atrioventricular (AVN) nodes as well
as in the internodal myocardium and the atrioventricular junctional myocardium. Expression is also seen in
the ventricular trabecular myocardium. [From Rentschler et al. (249), with permission from The Company
of Biologists Ltd.] B: projection of the pattern of expression of the LacZ transgene in a cartoon of the adult
heart.
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Finally, two components, which we have called “evolutionarily conserved” elements, are now incorporated
into the chambers, namely, the atrioventricular canal into
the atrial chambers (171, 172, 331) and the proximal part
of the outflow tract into the ventricular chambers; the
distal part loses its myocardium to the semilunar valves
(306, 326, 339). The myocardium of the atrioventricular
canal has retained some of its primary features (196), but
the outflow tract has retained hardly any primary feature
(208). It is of great interest that in patients ventricular
tachycardias originating from the right or left ventricular
outflow tract were described that sometimes originated
from the stem of the pulmonary artery (146, 255). These
observations suggest that in these patients the myocardium of the outflow tract has retained some of its primitive characteristics and may even partially persist in the
arterial trunk.
CARDIAC CHAMBER FORMATION
VI. ANTERO-POSTERIOR PATTERNING
AND CHAMBER FORMATION
Cardiac chambers are formed at specific sites of the
heart tube. To understand the underlying mechanisms we
need to know which factors provide the myocardium with
positional information for the site-specific formation of
chambers, and how these factors regulate the gene programs required for chamber formation. To delineate the
complicated process of local chamber formation it is
useful to distinguish antero-posterior (AP), dorso-ventral
(DV), and left-right (LR) patterning and the interpretation
of these patterns by genes that effectuate chamber formation.
Physiol Rev • VOL
A. AP Patterning During and After Gastrulation
The position of cardiac progenitor cells in the primitive streak along the AP axis correlates roughly to the AP
position of their descendants in the tubular heart (97,
258), although this relation is not strict (97). Retroviral
tagging of anterior primitive streak at HH3 resulted in
tagged progenitors mostly in the anterior region of the
embryo including the entire heart tube at HH12 (329),
indicating that the AP position within the streak is largely
conserved after gastrulation. In zebrafish, single cells injected in the midblastula stage formed single colonies in
the atria or ventricles, indicating separation of atrial and
ventricular lineages at that stage (288). Cardiogenic mesodermal cells of HH4 – 6 chicken embryos infected with
recombination incompetent retrovirus formed single colonies in the chambers (201). These observations indicate
lineage separation of the chambers or, alternatively, limited spatial rearrangement and/or a limited number of cell
divisions between the stage of labeling and analysis (288).
Fate-map experiments, however, do not reveal whether
and when cells have interpreted positional information
involved in their phenotypic specification.
Upon transplantation, cells of the anterior streak
(HH3) were able to form posterior heart structures, and
vice versa, indicating that at this stage AP patterning is
not fixed (137). Grafting experiments in chicken embryos
after gastrulation revealed that distinct regions along the
AP axis of the tubular heart of HH12 embryos mapped to
extensively overlapping regions in the precardiac areas of
HH5 embryos, although some AP positional correlation
was observed (291). In line with these observations, recent DiI labeling experiments (248) showed that the position of labeled cells in the tubular heart did not correlate
well with positions on either the AP or the mediolateral
axis in the heart-forming region of HH4 –7 embryos. Only
at HH7– 8 a definitive pattern was formed as the AP
position of progenitors in the cardiac crescent correlated
with the AP position of the cardiomyocytes in the heart
tube. These results indicate that myocytes have not interpreted AP patterning information until HH7. De Haan and
co-workers (270) analyzed developing beat rates in cultured explants of HH5–7 heart-forming regions and in situ,
in “multi-heart embryos” in which the heart-forming region was cut along the AP axis (270). They observed that
at HH12 posterior segments had developed higher beat
rates than anterior segments, indicating that the heartforming region at these stages has interpreted AP positional information. However, when HH5–7 precardiac mesoderm from the anterior heart-forming region was transplanted to more posterior positions, it developed beat
rates appropriate for their new location (270). These results indicate that at these stages the cells seem not to be
determined to their AP fate but that the positional information is imposed by regional cues. Consistently, the
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trabecular ventricular component. Large parts of this
myocardium do indeed develop into components of the
conduction system. However, not all parts will do so, and
these are not called conduction system. For instance, the
myocardium at the left atrioventricular junction is not
considered to be a part of the conduction system, although nodal action potentials and low abundance of gap
junctions have been observed in the lower rim of the left
atrium in the formed heart of various species (196). In
addition, although internodal tracts are not considered to
be significant components of the conduction system, their
existence is an amazingly persistent, but highly controversial issue (16, 56, 138, 182). One of the reasons for this
persistency might again be confusion about terminology.
If this area would be a direct derivative of the primary
myocardium, it would not be surprising to find cardiac
specialized primitive cells in the internodal myocardium,
i.e., in the right dorsal atrial wall (56). Such an observation is not necessarily linked to the existence of internodal tracts, certainly not if these tracts are supposed to
conduct rapidly and to be insulated by fibrous tissue (10).
Linkage with slow (nodal) conduction seems more appropriate but would not have much physiological significance, because the activated atrial cells conduct the depolarization wave much faster to the atrioventricular
node than the internodal myocardium would do.
To fully exploit the challenging data of Rentschler
and co-workers it is of great interest to relate the pattern
of transgene expression with the expression of relevant
molecular markers, such as the genes encoding connexins
(69), Anf (51), chisel (233), and other genes. Definition of
the site of engrailed-2/lacZ transgene integration into the
genome might reveal which endogenous gene regulatory
sequences control the specific lacZ expression pattern,
and thus might provide clues for the regulatory mechanism underlying the restricted pattern of transgene expression.
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ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
B. Retinoic Acid: a Cardiac AP
Patterning Molecule
Retinoic acid (RA) is an important morphogen during
embryogenesis (261). Excess or deficiency in RA has
dramatic effects on cardiogenesis, and RA has been used
extensively to study processes related to AP patterning,
chamber specification, and morphogenesis. Transient exposure of zebrafish to RA specifically truncates the heart
tube, starting with the outflow tract and ventricle at low
doses, and further along the AP axis with higher doses
(287). The effects on cardiac development were already
observed when embryos were treated during and shortly
after gastrulation, indicating that AP polarity can be affected during or shortly after mesoderm has acquired its
cardiogenic fate. In chicken, quail, and mouse, excess RA
causes “posteriorization” of the heart tube (229, 337, 346).
Genes normally expressed at higher levels in the posterior
region expand their domain toward the anterior region.
This suggests an anterior expansion of cells fated to the
posterior phenotype at the expense of anterior myocytes.
RA treatment of HH5– 8 explants of the anterior part of
the heart-forming region comprising meso- and endoderm
results in the induction of Amhc1, suggesting that the
cells are not yet committed to their anterior fate. Conversely, deficiency of RA in quail causes underdevelopment of the posterior structures of the heart, most notably
the sinus venosus and atria (163, 307). RA is not required
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for specification or differentiation of myocardium, because a heart tube expressing Nkx2–5 and sarcomeric
genes including Amhc1 is formed in this model.
Retinaldehyde dehydrogenase type 2 (Raldh2) is a
key enzyme for the synthesis of RA in the embryo (214,
221, 349). In quail and chicken, Raldh2 gene expression
was observed exclusively in posterior mesoderm at HH8,
and in the posterior heart precursors from HH9 onward
(338). In HH9⫺ chicken embryos the most anterior extension of Raldh2 expression within the embryo colocalizes
with the expression of the Amhc1 gene. In mouse, Raldh2
expression is initiated in the posterior mesoderm shortly
after gastrulation (220). In E7.5–7.75 mouse embryos, just
before fusion of the cardiogenic mesoderm, Raldh2 expression and the transcriptional response to RA [using a
retinoic acid response element (RARE) hsplacZ transgene
as a read-out] colocalize and are restricted to the most
posterior cardiac progenitors (214). At E8.5 (4 –5 somite
pairs), when a tubular heart has formed, expression and
response were restricted to the posterior region of the
heart, the sinus venosus. Mouse mutants that lack the
Raldh2 gene, and therefore lack the ability to produce
endogenous RA (221), show severe cardiac malformations, which include severely impaired atria and sinus
venosus (222). Like in the quail RA deficiency model,
expression of cardiac actin and myosin heavy and light
chain genes were not affected.
To further explore the role of RA in AP patterning of
the heart, Xavier-Neto et al. (337) generated transgenic
mice with a 0.8-kbp Smyhc3 promoter, which is derived
from the quail homolog of the chicken Amhc1 gene (325),
coupled to the human alkaline phosphatase (hAP) reporter gene. In transgenic mice, the reporter gene was
active only in the posterior heart precursors of E8.25
mouse embryos, and subsequently in the inflow tract,
atria, and atrioventricular canal of E9 embryos. RA administered to pregnant females at 7.5 days post coitum,
but not after 8.5 days post coitum, induced cardiac dysmorphogenesis including reduced development of the
outflow tract. The ventricles coexpressed Smyhc3-hAP
and Mlc2v, a gene marking the anterior part of the heart
tube. When, conversely, endogenous RA synthesis was
blocked, ventricles were enlarged and tapered off in the
sinus venosus. Smyhc3-hAP and RARE-hsplacZ transgene
expression was reduced. The observed cardiac malformations were similar to the RA deficiency models in quail
and Raldh2 mutant mice. The coexpression of Smyhc3hAP and Mlc2v in the anterior myocytes can be explained
by several mechanisms, which range from the respecification of anterior heart precursors to posterior ones, to
simply the activation of the RA-sensitive Smyhc3 promoter in cells that otherwise have acquired the anterior
phenotype. The coexpression of these two genes also
implies the presence of a distinct pathway, not under
control of RA, which specifies anterior cells.
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expression of Amhc1 could be induced by retinoic acid
treatment in anterior explants before but not after HH8,
the stage at which the expression of differentiation markers in the anterior portion of the heart-forming region is
initiated (345). Therefore, the phenotypic diversification
between anterior and posterior cells is stable after HH8.
Indeed, when rotating the entire “classical” heart-forming
region (comprising mesoderm and endoderm) of HH4 – 6
embryos 180° along the longitudinal axis and placing it
back, judged by their ability to express Amhc1, originally
posterior cells adapted to their new anterior position and
vice versa (236). From stage HH7 onward, however, the
originally posterior cells initiated Amhc1 expression at
their new anterior position.
Knowledge on commitment of cardiac cells in mammals is limited. Gruber et al. (117) demonstrated that in
mice after looping and initiation of chamber formation
(E10.5), the downregulation of Mlc2a and ␣Mhc gene
expression in ventricular cells when grafted into the atria
appeared irreversible. Taken together, from HH7– 8 onward, when anterior cardiac cells start to express differentiation markers, the cells within the heart-forming region are committed to their AP fate. Mechanisms that
pattern the myocardium must therefore act between HH3
(gastrulation) and HH7– 8.
CARDIAC CHAMBER FORMATION
C. Factors Involved in AP Patterning
and Morphogenesis
1. Gata factors
Gata4, -5 and -6 are expressed in, and posterior to,
the heart forming region of HH7 (1 somite) embryos and
in the tubular heart of HH9⫹ (8 somites) embryos. The
three genes, particularly Gata4, are expressed at levels
higher in the posterior inflow tract compared with the
anterior heart tube (141, 163). In E8 mouse embryos,
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Gata4 is expressed in the anterior intestinal portal and in
the inflow tract region of the fusing heart tube just anterior to the intestinal portal (206). In quail, RA deficiency
resulted in specific downregulation of Gata4 expression.
Administration of exogenous RA at the 1– 4 somite stages
(HH7– 8) completely rescues the cardiac phenotype and
the expression of Gata4. RA treatment of early (HH5– 6)
chicken embryos resulted in induction of Gata4 expression in the lateral plate mesoderm including the heartforming region (178). In RA-treated HH7– 8 embryos,
Gata4 expression was expanded and upregulated in the
anterior heart tube and in the lateral plate mesoderm posterior to the heart. Also in RA-treated Xenopus embryos,
Gata4, -5, and -6 are specifically upregulated (140). These
observations suggest that Gata family members, especially
Gata4, are components of the RA signaling pathway for
specification or formation of the posterior heart.
Cardiomyocyte differentiation as defined by expression of Nkx2–5 and other cardiac markers is normal
under RA-deficient conditions, indicating that Gata4 is
not required for specification or differentiation of cardiac
precursors. Indeed, although heart formation is severely
affected in Gata4-deficient mice, the defect does not result from specification of the cardiac cell lineages, but
from affected folding of the embryo and by that failure of
fusion of the heart fields at the midline (167, 206). The
unfused heart regions of Gata4 mutant mice express the
chamber marker Anf (206), indicating that initiation of a
chamber-specific program of gene expression occurs (see
section IXA). Chimeric mice made of Gata4-deficient embryonic stem cells and wild-type blastocysts incorporated
descendants of these cells into the formed heart (167).
Also when Gata4, -5, and -6 were depleted simultaneously with antisense oligomers in chicken, cardiomyocyte differentiation was not affected (141).
Expression of a dominant negative isoform of Gata4
in Xenopus resulted in the expansion of the Nkx2–5 expression domain into posterior direction, whereas in RAtreated embryos the Gata expression domain was increased at the expense of the Nkx2–5 expression domain
(140). These results indicate that Gata factors restrict
Nkx2–5 to a more anterior region of the heart-forming region where it regulates myocardial differentiation. However,
the Nkx2–5 regulatory region is stimulated by Gata factors
(reviewed in Ref. 275) and Nkx2–5 and Gata4 in synergy
activate cardiac promoters in cell culture systems (77, 175).
Taken together, Gata4 may play a role in posterior cardiac
morphogenesis, but its precise role is still unclear.
2. Tbx5
Tbx5 is expressed in a postero-anterior gradient in
the heart-forming region and tubular heart (35, 51, 178;
Fig. 13) and is, like Gata4, upregulated in RA-treated
chicken embryos. Tbx5 gene expression was abnormal
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Rosenthal and Xavier-Neto (259) proposed a model
for the RA mediated specification of the cardiac progenitor cells. In this model posteriorly localized Raldh2 expression is initiated shortly after gastrulation (mouse
E7.5, chicken HH6), resulting in the escape of the most
anterior cells of the migrating cardiac precursors from
contact with RA (259). These anterior cells, which form
the early tubular heart, are specified to the anterior (including ventricular) fate. The posterior cardiac mesodermal precursors will contact an RA-producing region or
express Raldh2 themselves. RA triggers the posterior phenotype (inflow tract, atria) when these cells are recruited
at the posterior side of the heart tube. The model has
several attractive features: it fits with the defined stage
(HH7) at which irreversible programming of cardiogenic
cells along the AP axis takes place; it explains the short
window of RA sensitivity of heart development; it includes cardiac AP patterning in a more general AP patterning mechanism that is also involved in patterning of
the neural tube and axial skeleton, where RA acts as an
important regulator of AP identity (261); it uncouples
formation of the cardiac lineage and patterning; and it is
testable. Several transcription factors have been implicated in mediating AP patterning (see below), but it remains to be established what mechanism effectuates phenotypic AP polarization of the heart tube.
RA may also be involved in the earlier differentiation
of anterior (ventricular) cells compared with posterior
cells during early stages of heart formation (see above).
Vitamin A deficiency in mice mimicked by blocking retinol binding protein expression at E7.5, or by mutation of
retinoic acid receptor genes (Rxr␣, ␤ or Rar␣), causes
early differentiation of subepicardial ventricular myocytes (148). Furthermore, ultrastructural analysis showed
that the differentiation of the ventricular myocytes of E8.5
Raldh2-deficient embryos was more advanced than that
of wild-type embryos (222). Thus RA signaling is required
for a delay in differentiation of these cells. Exposure of
Xenopus embryos to low levels of RA blocks expression
of differentiation markers in the heart (74). It is therefore
tempting to speculate that selective contact of posterior
cells with RA in early cardiogenesis is causal to the AP
gradient in differentiation along the linear heart tube.
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ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
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FIG. 13. Profiles of regionally expressed genes in the developing heart.
Mouse E8.5 is the prototypical linear
heart tube in which chamber-specific
genes are first observed at the ventral
side. Several regulatory genes are expressed in a pattern along either the anteroposterior (AP) or the dorsoventral
(DV) axis. The mouse E9.5 heart is the
prototypical looped chamber-forming
heart. Ventricular and atrial chambers
are visualized by Anf or Irx5 expression.
Both the ventricular and the atrial chamber myocardium are positioned at the
outer curvature (see blue arrows in leftmost panels for the atrial inner curvature, and red arrows in right most panels
for the ventricular inner curvature). Tbx2
expression is restricted to the primary
myocardium in a pattern strictly complementary to Anf. The AP-restricted segmental pattern of Irx4 and Mlc2v results
after looping in expression from the
proximal outflow tract to the atrioventricular canal. The mouse E11.5 heart is
the prototypical septating four-chambered heart. Anf, absent from inflow
tract, atrioventricular canal and outflow
tract is retracting from the compact ventricular myocardium.
CARDIAC CHAMBER FORMATION
3. Other factors
Mice with a targeted deletion of the Coup-TFII gene
have underdeveloped atria and sinus venosus (237).
Coup-TFII gene expression is normally restricted to these
posterior structures. The mechanism for the failure of the
posterior structures to develop is not understood but may
involve affected endothelial-mesenchymal interactions, as
angiopoietin-1, a proangiogenic soluble factor thought to
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mediate this interaction, is downregulated in mutant
mice. Targeted deletion of genes for Nkx2–5, Hand2,
Mef2C, and Bob revealed their importance for heart formation, which in mutants was affected more at the anterior than at the posterior side. These mutants are discussed in section IXC. The homeobox transcription factor
Irx4 has been implicated in the anterior (outflow tract,
ventricles) or posterior (inflow tract, atria) regulation of
myosin genes in chicken and mouse (see sect. IXB). Based
on their expression profiles, a number of genes have been
implicated in specification or differentiation along the AP
axis. Genes for the hairy related basic helix-loop-helix
factors Hey1/HRT1 and Hey2/HRT2 are heterogeneously
expressed along the AP axis throughout cardiac development (176, 216). Their role in AP patterning and morphogenesis remains to be established.
A significant contribution to AP patterning of the
developing heart may come from the continuous recruitment of myocardium at both poles of the tubular heart
during development. The recruited myocardium has been
subject to conditions and signals that change during development, such as RA at the posterior pole of the heart
(see above). At the anterior pole, recruited cells may have
been exposed to signals like fibroblast growth factors
(FGFs) and bone morphogenetic proteins (BMPs) in a
manner different from the myocardium of the tubular
heart (150, 204, 321). The differential exposure may contribute to the distinct phenotype of the anterior cells. The
role of FGFs and BMPs in these patterning events and in
chamber and vessel morphogenesis is not clear yet.
VII. CARDIAC PATTERNING ALONG
THE DORSOVENTRAL AXIS
The dorsal side of the forming linear heart tube is
connected to the body wall by the dorsal mesocardium, a
morphological hallmark for the difference between the
dorsal and ventral side of the heart tube. During looping
the ventral side of the anterior ventricular region and the
posterior dorsal side of the atrial region will become the
outer curvatures from where the chambers will expand
(Figs. 7 and 9). Therefore, chambers form in response to
integrated DV and AP patterning. To date, only a very
limited number of genes are known to discriminate between the ventral and dorsal sides of the heart tube.
Hand1 is specifically expressed at the ventral side of the
linear heart tube at E8 – 8.5 (23, 51, 303; Fig. 13). After
looping, its expression is confined to the left side of the
atrioventricular canal (derived from the ventral side of the
linear heart tube; Refs. 38, 60), the outer curvature of the
ventricles, and the outflow tract. The right ventricle expression diminishes by E8.5–9.5 (88, 303), reflecting AP as
well as DV patterning. The function of Hand1 in heart
formation is not clear. Development of Hand1-deficient
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and downregulated in the posterior part of the tubular
heart of Raldh2-deficient embryos (222). Therefore, Tbx5
may be involved in AP patterning or morphogenesis
linked to RA signaling (178). The function of Tbx5 in
cardiogenesis has been investigated in several species. In
Xenopus, an inducible dominant negative isoform of Tbx5
(Tbx5 coupled to the repressor domain of engrailed)
blocked normal cardiogenesis nearly completely (131),
although interference with the function of other T-box
factors cannot be ruled out in this study. In zebrafish,
Tbx5 deficiency (heartstrings mutation) resulted in failure of cardiac looping and deterioration of both atrium
and ventricle (98). Tbx5 haplo-insufficiency in human and
mouse causes relatively mild cardiac defects, which include defects in the septa and conduction system (20, 36,
177). Tbx5 deficiency in mice caused severe hypoplasia of
posterior heart structures in the linear heart tube from E8
onward, showing that Tbx5 is required for formation of
the posterior heart, compatible with its expression profile
(36). Expression of Nkx2–5 and Gata4, and of the anteriorly expressed genes for Irx4, Mlc2v, and Hey2 and of
the chamber-specific genes for Cx40 and Anf was reduced
in mutant embryos (36).
When Tbx5 was ectopically expressed in the tubular
heart of mice under control of the ␤Mhc promoter, loss of
anterior gene expression (Mlc2v) and retardation of ventricular chamber morphogenesis were observed (178).
These observations are compatible with a role of Tbx5 in
patterning, imposing posterior identity on the heart tube.
Viral expression of Tbx5 in chicken hearts caused inhibition of myocardial growth and of the formation of the
trabecules, consistent with the observed phenotype in the
␤Mhc-Tbx5 transgenic mice (123). The experiments in
chicken indicate that Tbx5 is involved in the downregulation of cell proliferation. Its expression was indeed
found to colocalize with regions of low proliferation in
several tissues. This is not necessarily the case in the
heart where Tbx5 expression is observed in both the
rapidly expanding atria and left ventricle and in the slowly
proliferating inflow tract, atrioventricular canal, and ventricular trabecules. Taken together, Tbx5 is a component
of a pathway controlling AP specification and morphogenesis. However, its position in the AP signaling pathway
and the mechanism by which it imposes its activity on the
forming heart still need to be defined.
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ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
Physiol Rev • VOL
pressed at the outer curvature and, except for Hand1, are
restricted to the forming chambers (see sect. IX), underlining the relation between early DV patterning and chamber formation at the outer curvature.
VIII. LEFT-RIGHT SIGNALING IN CHAMBER
SPECIFICATION AND MORPHOGENESIS
The LR identity of the heart-forming regions and the
direction of looping of the atrial and ventricular regions of
the tubular heart are evolutionary conserved, and both
depend on LR signaling in the embryo (reviewed in Refs.
40, 120). Incorrect decisions regarding laterality may result in serious cardiac malformations (32, 149). LR signaling, however, is not required for chamber formation per
se and is discussed here only briefly. A cascade of signaling molecules that regulate the establishment of LR identity of the embryo (reviewed in Ref. 40) culminate into the
expression of the homeodomain factor Pitx2 in the left
side of the visceral organs, including the heart. Asymmetric Pitx2 expression seems to be sufficient for establishing LR identity of the heart.
During fusion of the left and right heart-forming region, a tubular heart is formed that consists of two halves
with a left and right identity, respectively. The contribution of each heart-forming region to the fused heart tube
is essentially equal. However, the left heart-forming region contributes more to the anterior portion of the tube
and the right heart-forming region more to the posterior
part (248, 290), indicating that LR contributions are influenced by AP patterning. In models of heterotaxy, the
heart is normally patterned and formed in relation to its
AP and DV axis, indicating that AP and DV specification
can act independently from LR specification. In contrast,
RA, involved in AP patterning (see above), is required for
the LR asymmetry pathway, as randomized cardiac situs
occurs under conditions of RA excess or deficiency (45,
350). RA appears to control the level and location of
expression of components of the LR signaling pathway
(lefty, nodal, Pitx2).
The left and right atria are largely derived from the
corresponding side of the tubular heart. This can be
shown by following Pitx2 expression, a bona fide marker
for the fate of the left portion of the heart tube (38), in the
developing heart (Fig. 7). Morphologically, isomerism
leads to two left atrial appendages, which do express
Pitx2, or two right atrial appendages, which do not express Pitx2. Isomerism of the ventricular chambers is less
obvious. The atrioventricular canal, the ventricles, and
the outflow tract are aligned along the AP and DV axis
before looping, and each component is composed of descendants of the left and right heart-forming regions. As
mentioned in section IIIB, the AP arrangement of the
ventricular chambers of the heart tube is converted with
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mice is arrested at E7.5, and embryos die between E8.5
and E9.5 (88, 251). However, a tubular heart is formed
that expresses cardiac markers. Notably, Anf is expressed
in Hand1 null-mice (88), suggesting that a chamber-specific program of gene expression is initiated despite the
failure of the heart to loop and expand. Mlc2v expression
was also observed, indicating that AP patterning was not
severely affected either. In tetraploid rescue experiments,
embryos died at E10.5 due to cardiac failure (251). Development of the hearts of these embryos was arrested at the
onset of looping, and trabecules were not formed, similar
to the Nkx2–5 null mice that lack Hand1 expression in
the heart (see below). When chimeric mice were made of
Hand1 mutant ES cells and wild-type (Rosa26-lacZ) cells,
the outer curvature of the heart tube was generated invariably of descendants of the wild-type cells, demonstrating that Hand1 mutant cells cannot contribute to this
part of the heart in E9.5 embryos (252). It is therefore
likely that Hand1 plays an important role in the morphogenesis of the ventral side/outer curvature of the heart
tube.
In mouse and chicken, Anf gene expression is first
observed at the ventral side of the putative ventricular
region of the fused heart tube, just after the onset of
looping (51, 133, 347). A small region at the dorsal side
does not express Anf. The ventral side of the tubular heart
is derived from the lateral sides of the bilateral heartforming region. In HH9 chicken, just posterior to the site
of fusion of the crescent the Anf gene was expressed
lateral in the cardiac mesoderm (133). This indicates that
mediolateral signaling precedes the DV pattern. Transgenic mice with a chicken Gata6 gene enhancer driving
the lacZ reporter gene showed AP-restricted expression
in heart-forming region (E7.5) and in the inflow tract of
the tubular heart (E8.5; Ref. 60) that was restricted to the
lateral sides. Expression became confined to the dorsal
and ventral myocardial walls of the atrioventricular canal
that are lined by cushions. These results indicate that in
chicken and mouse, mediolateral patterning in the heartforming region is present that precedes DV patterning in
the tubular heart.
Expression of the genes for Chisel and Cited1
(Msg1) confirms the presence of DV patterning in the
tubular heart, as they are expressed selectively at the
ventral side of the future ventricular region of the linear
heart tube of E8.25 embryos (76, 233). After looping,
expression is confined to the chambers. Also, the onset of
Irx3 expression in the myocardium and Irx5 expression
in the endocardium at the ventral side of the E9 mouse
heart (52) indicates DV patterning in the linear heart tube.
In summary, expression patterns of Hand1, Anf,
Chisel, Cited1, Irx3, and Irx5 demonstrate the presence
of DV differences in transcription regulation in the early
tubular heart, in line with the morphological observations
dealt with in section III. These genes are specifically ex-
CARDIAC CHAMBER FORMATION
IX. CHAMBER-SPECIFIC AND REGIONALIZED
TRANSCRIPTIONAL PROGRAMS
A. Chamber-Specific Patterns of Gene Expression
As outlined in previous sections, morphological and
electrophysiological criteria distinguish chamber myocardium from primary myocardium. As a consequence, gene
products that discriminate between these types of myocardium are present as well. The Anf gene is not expressed in the primary linear heart tube before the onset
of looping (E8.5). First expression is detected at the ventral side that will form the trabeculated portion of the
left-ventricular chamber. During and after looping, Anf
gene expression gradually increases in the right ventricular chamber. Atrial expression of Anf is first observed at
the end of looping, E9.25, at the dorsolateral side of the
atrial region. Expression is never observed in the primary
myocardium of the inflow tract, atrioventricular canal,
inner curvatures, and in the outflow tract (Fig. 11) (51,
195, 347), making this gene a suitable marker for the
chambers. The expression in the right ventricle is weak.
Physiol Rev • VOL
From E11 onward, Anf no longer is expressed in the
rapidly proliferating compact myocardium at the epicardial side of the ventricles and becomes restricted to the
trabecules that will form the subendocardial ventricular
conduction system (Purkinje network, see sect. VF and
Ref. 208). Anf expression is not observed in the sinoatrial
node and atrioventricular node that are derived from the
inflow tract and atrioventricular canal, respectively. The
pattern of Cx40 gene expression is highly reminiscent of
that of Anf. In contrast to Anf, Cx40 is expressed in the
atrial septum and in the caval veins (but not in sinoatrial
node), indicating that these component parts form distinct transcriptional domains. Another gap junction gene,
Cx43, shows a cardiac pattern very similar to that of Anf
and Cx40, albeit that atrial chamber myocardial expression is initiated later (69, 312). Furthermore, Cx43 is
expressed at high levels in the compact ventricular myocardium.
Expression of Chisel, encoding a small muscle-specific protein (233), was first detected at the ventral side of
the fusing heart tube at E8.25. It has an expression profile
very similar to that of Anf. However, it is expressed at
comparable levels in left and right ventricles and at higher
levels in the compact layer of the ventricles than in the
trabecules (51, 233). Initiation of Cited1 (Msg1) gene
expression (76) is at the ventral side of the linear heart
tube (E8 – 8.25). Thereafter its expression profile is very
similar to that of Anf, albeit that atrial expression is weak.
Serca2a, which encodes sarcoplasmic reticulum Ca2⫹ATPase, is expressed at moderate levels in the entire
cardiac crescent and linear heart tube in a postero-anterior gradient. The gene is upregulated in the atrial and
ventricular chamber myocardium (colocalized with Anf,
Chisel, and Cx40 expressing myocardium) that form at
the outer curvatures (210), but retains its original pattern
in the remnants of the primary myocardium at the inner
curvatures, atrioventricular canal, and outflow tract. The
homeobox gene Irx5 is expressed in the endocardium
lining the trabecular myocardium of the ventricles and the
atrial myocardium (52). Endocardium lining the cushions,
present in the inflow tract, atrioventricular canal, inner
curvatures, and outflow tract (Fig. 9), does not express
Irx5. Its expression therefore coincides with that of Anf,
Cx40, and Chisel. The gene for Tbx2, a member of the
T-box transcription factor family (44), was found to be
strictly associated with the primary myocardium. It is
expressed both in myocardium and cushions of the inflow
tract, the inner curvature of the atrial loop, the atrioventricular canal, the inner curvature of the ventricular loop,
and the outflow tract (118, 340). Its pattern is strikingly
complementary to that of Anf (Fig. 13). The significance
of this factor in the primary myocardium is discussed in
section IXB. Several genes show regional expression in the
tubular and subsequently looped chamber heart. Mlc2v
gene expression is first observed at E8, in the paired
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looping into a LR arrangement (38, 51, 303). Due to this
process, the ventricular part derived from the left half is
found at the ventral side of the heart, the part derived
from the right side is found at the dorsal side (38) (Fig. 5).
Indeed, in mutant mouse strains that have heterotaxy,
such as the iv/iv mouse, both double-sided absence and
double-sided presence of Pitx2 expression in the ventricles are observed. This phenomenon is associated with
double-outlet right ventricle, a condition of improper
alignment of the ventricles and great arteries. Therefore,
for isomerism of the ventricles, typical features of the
dorsal side or the ventral side of the ventricular region are
expected to be either present or missing at both sides.
These features have not been described, which may indicate that the condition is incompatible with life.
In corrected transposition of the great arteries
(CTGA), the morphologically left and right ventricles are
found at the right and left side, respectively, and each
fulfills the physiological function of the other. This suggests that only the ventricular region has been inverted,
by left-sided looping of the ventricular loop, whereas the
remainder of the heart is correctly specified. Indeed, in
patients with CTGA, the presence of a (rudimentary)
atrioventricular bundle at the ventral (anterior) side of the
ventricles has been reported (6). Furthermore, in the iv/iv
mouse model of cardiac heterotaxy, symmetry in presence or absence of Pitx2 expression was sometimes observed only in atria or in ventricles. Together with the
CTGA, this suggests that regulation of LR identity of
different components of the heart may be modular and
may occur relatively late in development.
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ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
B. Regulation of Chamber-Specific
Gene Expression
To gain insight into the transcriptional program underlying the regionalized chamber formation, two strategies appeared to be informative: 1) transgenic analysis to
Physiol Rev • VOL
study regulatory DNA sequences that provide regional
activity (152) and 2) inactivation or ectopic activation of
genes for cardiac transcription factors. In the next sections, studies that have provided insight into the control
of regionalized gene expression or chamber formation are
reviewed.
1. Regulation of atrial gene expression: Irx4
and the Smyhc3 gene
Bao et al. (17) identified a novel member of the
vertebrate Iroquois family of homeobox genes, Irx4,
which is specifically expressed in the chicken heart from
HH10 onward (17). Expression is confined to an APrestricted segment of the tubular heart (see above). In
later stages Irx4 expression is largely confined to the
ventricles. Vmhc1 and Amhc1 gene expression were detected in the entire tubular heart from HH9⫹ and HH10,
respectively. This Amhc1 pattern is different from the
posterior-restricted pattern found by Yutzey et al. (346).
The probes used in these studies contain regions homologous to other myosin heavy chain genes expressed in the
heart tube. This and differences in the applied sensitivity
of the in situ hybridization account for the discrepancy in
observed patterns (unpublished observations). With further development Amhc1 and Vmhc1 expression became
restricted to posterior and anterior portions of the looping
tubular heart, respectively. The region of Irx4 expression
coincides with the Vmhc1 expression domain and is complementary to the Amhc1 expression domain. Viral expression of Irx4 in the heart caused downregulation of
the Amhc1 gene and upregulation of the Vmhc1 gene in
the atria. Ectopic expression of the homeodomain of Irx4
coupled to the repressor domain of engrailed caused the
opposite effect. Therefore, Irx4 was implicated in chamber-specific gene regulation and maintenance or even determination of cardiac chamber formation in chicken. The
mechanism by which Irx4 activates the Vmhc1 gene in
the ventricle is not clear but may be indirect, since functional studies so far indicated that members of the Iroquois family of factors primarily act as repressors (41,
104, 165). Irx4 expression does not respond to RA signaling (34, 222), indicating that Irx4 is not directly responsible for the RA-inducible expression of Amhc1. However,
Irx4 and Rxr␣ cooperate in the repression of the quail
homolog of Amhc1 (see below). In mouse, Irx4 is expressed in a pattern similar to that of Irx4 in chicken (34,
52). Therefore, Irx4 function may be conserved between
vertebrate species. Irx4 deficiency in mouse caused no
cardiac malformation but resulted in postnatal derepression of Anf and ␣-skeletal actin, and in cardiomyopathy
after several months (33). Before birth, Hand1 was downregulated in the ventricles of Irx4 null mice, whereas the
Smyhc3-hAP transgene, active in the inflow tract, atria,
and atrioventricular canal in mouse (see sect. VIB), was
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lateral regions of the fusing heart-forming regions, just
before the formation of the tubular heart (188, 219). This
pattern provides one of the first indications that the mammalian heart is effectively patterned along the AP axis
before formation of the tubular heart. When the tubular
heart is formed at E8, Mlc2v and Irx4 are expressed in a
large portion of the tube that includes dorsal and ventral
sides but excludes the inflow and outflow tract (Fig. 13)
(223). After looping, the atrioventricular canal, the ventricles, the inner curvature of the ventricular loop, and the
proximal outflow tract express Mlc2v and Irx4 (34, 51, 52,
223). Therefore, neither gene is restricted exclusively to
the ventricular chambers during development, and both
exceed the morphological and phenotypic boundaries of
the ventricular chambers. Because the ventricles rapidly
proliferate and expand, they will form the bulk of the Mlc2vexpressing domain. Therefore, Mlc2v and Irx4 expression
will become more and more “ventricle specific” during development. Using a Cre-lox based method to follow the fate
of myocytes and their progenitors that expressed Mlc2v (Cre
gene under control of the Mlc2v locus; Ref. 47), we found
labeled cells outside the ventricles (e.g., in the lower rim of
the atria). This shows that the Mlc2v-expressing domain in
the embryonic heart is broader than that fated to form the
ventricular chambers (unpublished observations). The patterns of Mlc2v and Irx4 constitute an AP-defined segment
(from atrioventricular canal to proximal outflow tract)
throughout cardiac development (Fig. 13). The ventricular
chambers form at the ventral side and outer curvature
within the Mlc2v- and Irx4-expressing segment (Fig. 13).
A number of genes, including Amhc1, Smyhc3, ␣Mhc,
Mlc2a, and others (51, 92, 187, 325, 346), are selectively
expressed in, or become confined to, the posterior portion
of the tubular and chamber-forming heart. Here, again, the
patterns are restricted along the AP axis only, because also
the inflow tract, inner curvature of the atria, and the atrioventricular canal express these genes. Within this posterior
portion (or segment), atrial chamber myocardium will form
and expand from the dorsolateral sides (Fig. 13).
In conclusion, several chamber-specific genes are restricted to the morphological and physiological chambers at
embryonic stages, whereas others are expressed in a
broader domain along the AP axis, forming segments of
expression within the tubular and looped heart. Positional
information along both the AP and DV axes is required for
the site-specific initiation of expression of the chamberspecific genes.
CARDIAC CHAMBER FORMATION
2. Regulation of ventricular gene expression:
the Mlc2v gene
The mechanism controlling Mlc2v gene expression
has been subject to extensive study (219). Transgenic
mice in which the proximal (250 bp) Mlc2v promoter was
Physiol Rev • VOL
coupled to lacZ showed expression in a steep AP gradient
that after looping results in expression in the outflow tract
and right ventricle, tapering of in the left ventricle (260).
This pattern of transgene expression differs from the
expression profile of the endogenous Mlc2v gene (both
ventricles, the ventricular inner curvature, atrioventricular canal, and proximal outflow tract; see above). A pattern similar to that of the 250-bp promoter was observed
when a dimerized 28-bp HF-1 fragment of the Mlc2v promoter containing HF1a and HF1b/MEF2 elements was
coupled to the minimal Mlc2v promoter fragment. This
core element therefore contains sufficient information to
drive expression in the anterior myocardium of the linear
and looped heart tube. Different lines with the same
250-bp promoter construct and dimerized element constructs showed expression that to a variable degree penetrated into the left ventricle. This indicates that the
promoter senses an AP-graded signal along the heart tube
and that the sensitivity of its response depends on the site
of integration of the transgene construct into the genome.
One of the lines has been used to monitor right ventricular
specification in various mouse-mutant backgrounds with
affected cardiogenesis (122, 341). In light of the pattern of
the Mlc2v -lacZ transgene, it may however be that the
promoter only senses a transcriptional signal in the anterior region of the heart tube within which the outflow
tract and right ventricle will develop.
The patterns of the Mlc2v-lacZ transgene and endogenous gene differ, and the activity of the Mlc2v promoter
is weak compared with the endogenous gene (47) and in
contrast to the endogenous gene is not downregulated in
Nkx2–5 mutant mice (188, 298, 341). Therefore, expression of the endogenous gene depends on sequences outside the proximal promoter (260). Although within the
proximal Mlc2v promoter some specific cardiac activity is
contained, its main function may be to relay transcriptional input from distal regulatory elements to the transcriptional machinery. These distal elements drive very
strong expression and restrict Mlc2v expression within its
boundaries (from atrioventricular canal to proximal outflow tract) along the AP axis. Evidence that supports the
notion that the proximal Mlc2v promoter primarily relays
activity from distal elements was obtained from chimeric
promoter studies in transgenic mice (118 and unpublished
data). When the Anf regulatory sequences (position ⫺638
to ⫺138), which are active in atria and ventricles but not
in atrioventricular canal and outflow tract, were coupled
to the Mlc2v promoter, the Anf pattern was observed,
indicating that the Anf regulatory sequences impose their
activity on this promoter. Similar observations were done
when the Mlc2v promoter was replaced by a small cardiac
troponin I (cTnI) promoter fragment. However, when the
Mlc2v promoter was placed upstream of the cTnI promoter, the pattern typical for the cTnI promoter was
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upregulated. Therefore, Irx4 is involved in some aspects of
the ventricle-specific program of gene expression. The relatively mild phenotype of the Irx4-deficient mice may result
from compensation by other Irx family members. Using the
conserved chicken Irx4 homeodomain as a probe, we isolated five other Irx family members from a mouse cardiac
cDNA library, which all are expressed in highly specific
patterns in the myocardium (Irx1, -2, -3) or endocardium
(Irx5, -6) (52, 215). Their spatial and developmental patterns
are markedly different from Irx4, indicating that they can
compensate Irx4 deficiency only partially. The function of
the other Irx genes in the specification or formation of the
separate cardiac components has yet to be determined.
The Smyhc3 gene, homolog of the chicken Amhc1
gene, is initially expressed in the entire tubular heart and
becomes downregulated in the ventricles during development (325). Wang and co-workers (323, 325) showed that
the first 840 bp of its promoter region are sufficient to
mediate atrial activity and ventricular repression. Functional analysis of the regulatory fragment revealed the
presence of a functional GATA factor binding element and
a vitamin D response element (VDRE). The GATA binding
element is required for cardiac activity of the promoter.
However, mutation of the VDRE completely abolished
ventricular repression. Further studies showed that a heterodimer of retinoid X receptor ␣ (Rxr␣) and vitamin D
receptor (Vdr) binds the VDRE. Because the receptors are
expressed in the entire heart, an unidentified ventriclespecific corepressor involved in the ventricle-specific repression was postulated (325). The observation that the
Smyhc3 transgene is upregulated in the ventricles of Irx4deficient mice suggests that Irx4 could be the corepressor
of Smyhc3. Indeed, Irx4 was found to repress the promoter in a VDRE-dependent manner but did not interact
with the DNA (324). The Rxr␣ receptor within the Vdr/
Rxr␣ heterodimer was found to interact with Irx4. Together these findings have revealed a mechanism for the
ventricle-specific repression of Smyhc3, in which an inhibitory complex composed of Vdr/Rxr␣ receptor and
Irx4 binds to the VDRE and represses transcription. It is
not clear whether the chamber-specific repression mechanism found for the Smyhc3 gene applies to other genes
as well. However, a number of cardiac genes that are
expressed in a chamber-restricted pattern, are initially
expressed in the entire tubular heart (51, 92, 187). It is
therefore tempting to speculate that repression during
chamber formation is a general mechanism by which the
expression domain of genes becomes restricted.
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ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
3. Regulation of chamber-specific gene expression
In section IXA, the Anf and Cx40 genes were introduced as specific markers for the forming chambers. Both
genes are therefore excellent model genes to study mechanisms that control chamber-specific expression. The Anf
gene encodes ANF, a hormone that in the formed heart is
secreted by the atria and involved in blood-pressure regulation. It serves as a marker for cardiac hypertrophy,
during which the gene is strongly upregulated in the ventricles (50). The promoter region of the Anf gene has been
used as a paradigm for regulatory pathways that control
cardiac-specific gene expression (79). The upstream 0.6
kbp of the Anf regulatory region are sufficient to drive
expression in transiently transfected pre- and perinatal
atrial and ventricular cells (12). Among the factors that
interact with the Anf promoter are Gata family members,
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Nkx2–5, Mef2C, Tbx5, Tbx2, Srf, and Myocardin (36, 77,
78, 118, 124, 125, 175, 213, 279, 322). Several of these
factors were shown to interact with each other and with
sites within the promoter. In transient transfection studies, Nkx2–5 was reported to activate the promoter in
synergy with Gata4, Srf, or Tbx5, respectively. From mutant mice it was deduced that Anf requires Nkx2–5,
Mef2C, and Tbx5 directly or indirectly for its expression
(36, 122, 183, 298). Irx4 null mice show higher Anf expression in the ventricles after birth (33), which suggests
that Irx4 may be a component of the pathway responsible
for ventricular downregulation of Anf in the compact
ventricular myocardium after birth (12, 347). Together,
these studies have provided valuable insights into the
mechanism of cardiac-specific expression of Anf, and into
the mechanisms underlying the cooperative action of different classes of transcription factors in the regulation of
cardiac gene regulation. They do not, however, provide a
mechanism for its relatively late onset of expression,
shortly after the onset of looping. Furthermore, the spatial
expression patterns of the factors involved do not explain
the restriction of Anf gene expression to the chamber
myocardium of the embryonic heart and to the atrial
appendages in the postnatal heart.
In transgenic mice, the upstream 0.6-kbp promoter
fragment of the Anf gene appeared to be sufficient to
recapitulate the spatial and developmental pattern of the
endogenous gene (87, 118, 162). Sequences required for
the strong response to hypertrophy in the ventricles, observed for the endogenous Anf gene, are not present in
this fragment in vivo (162). This observation indicates
that the regulatory region of the Anf gene is modular and
that the hypertrophy pathways are different from the
regulatory pathways for the spatial and developmental
regulation. Furthermore, transient transfection analysis
of Anf promoter parts in ventricular, atrial, and noncardiac cells suggest that the promoter is composed of modules (79). The most upstream region was found to be
required for activity in isolated ventricular cells and the
middle portion for activity in atrial cells. Such modularity
has been found for several other genes and seems a
common feature of regulatory sequences (15, 275). Each
regulatory module has a specific contribution to the overall activity of the gene (15). The part most proximal to the
transcription start site of the Anf gene is required to relay
the transcriptional activities from the upstream parts,
reminiscent of the function of the proximal promoters of
the Mlc2v and cTnI genes (see previous section). Indeed,
Mlc2v or cTnI promoters can freely exchange the proximal part of the Anf promoter without loss in activity or
pattern in vivo (118). Whether the ventricular and atrial
modules indeed confer compartment-specific activities in
vivo remains to be established.
In homozygous Tbx5 mutant mice, several genes
were found to be downregulated, including the chamber
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observed, indicating that the Mlc2v promoter itself has
only weak regulatory activity indeed.
The anterior activity of the proximal Mlc2v promoter
has been used as a tool to gain insight into the regulation
of localized gene expression within the heart. The proximal Mlc2v promoter contains binding sites for a number
of cardiac enriched as well as ubiquitous transcription
factors (219). The expression patterns of these factors,
however, do not correlate with the anterior activity of the
Mlc2v promoter. Because the dimerized 28-bp HF-1 regulatory unit confers anterior activity to a reporter gene, the
factors that interact with the sites within this unit have
been studied most extensively in relation to the specific
anterior activity. The ubiquitously expressed factor EFIA,
the rat homolog of human YB-1, was found to bind to the
HF-1a site within the 28-bp HF-1 unit and to activate the
Mlc2v promoter in cardiac cells (351). With the use of
YB-1 as bait in a yeast two-hybrid screening, the factor
Carp (cardiac ankyrin repeat protein) was isolated (352).
Carp, a cofactor for YB-1, functions as a repressor. The
gene is expressed specifically in the heart and at E10 is
reduced in the ventricle compared with atria and outflow
tract, compatible with the hypothesis that Carp negatively
regulates endogenous Mlc2v. The Carp pattern, however,
is not compatible with the pattern observed for the 250-bp
Mlc2v promoter or the dimerized HF-1 elements that Carp
is thought to interact with via YB-1. Further analysis of
Carp regulation (168) showed that the first 0.3 kbp of its
promoter drives reporter gene expression in the outflow
tract and right ventricle from E9 onward, in a pattern
similar to the Mlc2v-lacZ transgenes. When a dimerized
fragment of the upstream 128-bp promoter was used,
expression was restricted to the outflow tract. Therefore,
consecutive restriction of the promoter results in a restriction along the AP axis. In conclusion, both Mlc2v and
Carp regulatory fragments are sensitive to AP patterned
activity, but the underlying mechanism is still enigmatic.
CARDIAC CHAMBER FORMATION
Physiol Rev • VOL
outflow tract, strictly complementary to the expression of
Anf and Cx40 (Fig. 11). Functional studies provided further evidence that Anf and Cx40 are functional targets for
Tbx2 and that Tbx2 forms a stable complex with Nkx2–5
on the combined TBE-NKE of the Anf promoter (118). In
a simple model for chamber-restricted expression, Anf
and Cx40 are activated by Tbx5 in the chamber myocardium and repressed by Tbx2 in primary myocardium (Fig.
14). Nkx2–5 functions as a “cardiac” accessory factor for
both T-box factors, restricting their action to specific
cardiac genes. The T-box factors provide the first clue of
a mechanism underlying the localized differentiation of
chamber myocardium within the tubular heart by repression of a program in the primary myocardium. Results of
transgenic embryos expressing Tbx2 in the heart favor
this hypothesis. Heart development is blocked in the tubular heart stage, and expression of Anf is inhibited (unpublished observations). Preliminary studies have disclosed a similar role for Tbx3. Its expression becomes
confined to the components of the conduction system.
4. Localized activity of other regulatory DNA fragments
A number of cardiac promoters in addition to the
Smyhc3, Mlc2v, Carp, and Anf promoters have localized
activity in the heart (see Ref. 152 for a review). Expression of the gene for Hand2, a factor that is required for
the formation of the anterior region of the tubular heart
(see below), is initially expressed in the entire tubular
heart but becomes restricted to the anterior region during
looping (286). A 1.5-kbp fragment of the regulatory region
of the mouse Hand2 gene drives lacZ reporter gene expression in a pattern similar to that of the endogenous
gene (194). Two conserved Gata factor-binding sites
within the fragment are essential for the regionalized
activity, although Gata factors themselves are not chamber restricted. These findings suggest that positive or
negative coregulators cooperate with Gata factors to control regionalized gene activity. Similar observations were
done for a Desmin gene enhancer fragment. Here, the
right ventricle-outflow tract specific expression was dependent on a binding site for Mef2 factor family members
(166), which are present and active in the entire developing heart (81, 217). Further analysis of the regulatory
regions of Hand2, Desmin, and other genes is required to
understand the mechanisms that are involved in their
regionalized activity.
An intriguing aspect of a number of promoter fragments, most of which have been mentioned above, is their
localized activity either anterior (outflow tract and right
ventricle) or posterior (left ventricle and further posterior) to the interventricular connection. Fragments of
Mlc2v, Carp, Nkx2–5, Gata6, Sm22␣, Desmin, Dystrophin, Hand2, and Hop are active anterior to this connection, whereas fragments of cTnI, Mlc3f (2E), and ␣-car-
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markers Anf and Cx40 (36). Surprisingly, these two genes
were specifically reduced in heterozygous mutants, indicating that Tbx5 is a rate-determining factor in the activity
of both genes. Consistent with the observed requirement
of Anf and Cx40 for Tbx5, careful analysis of expression
patterns revealed that Anf and Cx40 are never observed
outside the Tbx5 expression boundaries. After looping,
expression of Tbx5 is absent from the most anterior components, the outflow tract and most of the right ventricle
(35, 51, 178). Also, in the left ventricle expression disappears gradually from the compact layer. Anf and Cx40 are
not expressed in these parts of the heart. Therefore, Tbx5
represents one of the first examples of a factor that directly determines several aspects of the regionalized patterns of two downstream cardiac genes.
The mechanism by which Tbx5 regulates Anf and
Cx40 was studied in in vitro assays (36, 101, 125). Both
Anf and Cx40 contain T-box binding elements (TBE),
which are homologous to one-half of the palindromic
brachyury (T) binding site (160). Both sites are functional
targets of Tbx5. In a yeast two-hybrid screen, Tbx5 was
found to interact with Nkx2–5 (125). Nkx2–5, which is
implicated in the positive regulation of Anf through NK
factor binding elements (NKE), binds with Tbx5 to a
combined TBE-NKE within the Anf promoter. These factors synergistically activate Anf and Cx40 promoterdriven gene expression in cultured cardiac and noncardiac cells.
To gain insight into the chamber-restricted expression of Anf, we generated a series of chimerical regulatory constructs in which the upstream region of the Anf
gene was placed upstream of different cardiac promoters
(118). A small cTnI gene promoter fragment that is predominantly active in the atrioventricular canal was specifically extinguished in the atrioventricular canal by Anf
sequences, whereas the Mlc2v promoter, active in the
outflow tract and right ventricle, was extinguished in the
outflow tract. However, the typical chamber expression
pattern of the Anf promoter was retained irrespective of
the proximal promoter fragment used. Therefore, upstream Anf sequences are stimulating Anf transcription in
the chambers and are repressing Anf transcription in the
myocardium of the atrioventricular canal and outflow
tract. When we inactivated the TBE or NKE (see above)
within the chimeric Anf-cTnI construct by mutation, the
chamber expression was not abolished. In contrast, the
repression in the atrioventricular canal was relieved.
These observations clearly demonstrate that in vivo these
sites are not essential for activation by either Nkx2–5 or
by Tbx5 but that factors interacting with these sites are
components of an inhibitory pathway. Analysis of the
expression patterns of other T-box genes revealed that
the transcriptional repressor Tbx2 is expressed selectively in the myocardium and cushion mesenchyme of the
inflow tract, atrioventricular canal, inner curvature, and
1255
1256
ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
diac actin are active posterior to this connection (46, 61,
118, 152, 168, 194, 275). The interventricular border coincides with the border of expression of the Fgf10 enhancer
trap LacZ transgene that marks the contribution of cells
to the tubular heart between E8 and E10 (150; the “anterior heart field,” see sect. VB). A possible implication is
that the anterior heart field forms a conserved transcriptional domain, added during evolution of the heart of
higher vertebrates, and that cardiac genes have developed
regulatory fragments to ensure their activation in this
domain. Whether the cells of the anterior heart field form
a distinct population in which the regulatory elements
become active once the cells have differentiated into
cardiac cells, or whether the cells move into an AP patterning field resulting in the activation of the regulatory
elements by anterior signals remains to be established.
5. Regulation of regionalized transcription factors
The role of Tbx2, -3, and -5, but also of Irx4 in the
compartment-specific regulation of genes such as Anf,
invokes the question of what mechanisms are involved in
the regionalized expression of these regulating factors.
Although little is known about their regulation, several
studies indicate that both groups of genes are downstream of BMPs. BMPs are members of the transforming
growth factor-␤ superfamily of extracellular signaling
peptides of which multiple members are expressed in
highly localized patterns in the chicken and mouse heart
during development (1, 75, 144, 189, 284, 340, 348). Functional studies indicate that BMPs are obligatory for early
cardiac development (273, 274). Genetic inactivation of
components of BMP-signaling pathways revealed roles in
Physiol Rev • VOL
multiple steps of heart development, including early heart
morphogenesis and affected cushion formation and outflow tract septation (42, 96, 155, 284, 348).
In the chamber-forming heart (mouse E9.5; chicken
HH16) Bmp4 is expressed in the inflow tract and outflow
tract, whereas Bmp2 is expressed in the atrioventricular
canal (1, 340, 348). The genes for Tbx2 and -3 are expressed in highly specific patterns in the chamber-forming
heart (102, 118, 340, 340; unpublished observations). The
patterns largely coincide with those of Bmp2 and -4 together (inflow tract, atrioventricular canal, inner curvature, outflow tract). Ectopic application of BMP2 via
soaked beads specifically induced Tbx2 and -3 expression
in the cells surrounding the beads (340). Tbx5 was not
upregulated. Moreover, Tbx2 expression was specifically
downregulated in the hearts of Bmp2-deficient mouse
embryos. These observations suggest that BMPs are part
of the regulatory pathway that controls the localized expression of Tbx2 and -3, which in turn repress the activation of chamber-specific genes in the primary myocardium of the inflow tract, atrioventricular canal, inner curvature, and outflow tract (see previous section).
The Irx4 gene is a member of a family of six Iroquois
homeobox genes found in vertebrates (27, 239). The genes
are localized in two paralogous clusters of three genes
each (Irx1, -2, -4 on chromosome 13, Irx3, -5 and -6 on
chromosome 8; Refs. 27, 132, 239). The genes of each
cluster show strong similarity in expression pattern, indicating that they are regulated by shared regulatory elements (132). In Xenopus, the Irx1 homolog Xiro1 was
shown to be activated by Wnt signaling and to be negatively regulated by BMP4, whereas Xiro1 itself was able
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FIG. 14. Scheme showing various
steps in the formation of the cardiac
chambers and conduction system components. The primary myocardium is patterned along the AP axis by retinoic acid
(RA) and along the DV and LR axis.
Within the anterior domain of the primary myocardial tube cells differentiate
into prechamber myocardium that subsequently will form the trabecules, the bundle branches and peripheral ventricular
conduction system, and compact ventricular working myocardium. Within the
posterior domain of the primary myocardial tube cells will differentiate into atrial
chamber myocardium. Part of the primary myocardium is set aside from differentiation and will form the nodal components of the conduction system, the
sinoatrial node, the atrioventricular
node, and the atrioventricular bundle.
CARDIAC CHAMBER FORMATION
to inhibit Bmp4 gene expression (104). Also Xiro3 is
activated by anti-BMP signals and able to repress Bmp
expression (165). Therefore, a role for BMPs in the regulation of the Irx genes in other vertebrates is anticipated.
Although no experimental evidence has been presented
for reciprocal Irx/Bmp regulation in the heart, cardiac
expression of Bmp2 and -4 does not colocalize with any
of the six Irx genes, with the exception of Irx4 that
colocalizes with Bmp2 in the atrioventricular canal.
C. Gene Deficiency Reveals Pathways Involved
in Chamber Formation
1257
diac jelly (37). Mutant mice have no trabecules and atrioventricular cushions. Hyaluronan and other cardiac jelly
components are ligands for CD44, which is associated
functionally with Erbb2 (29), providing a possible link
between hyaluronan and formation of the trabecules. The
endocardial cushions appeared to be reduced in size in
neuregulin- and Erbb3-deficient mice (84), and to a lesser
extent in Erbb2- and Erbb4-deficient mice, indicating that
signaling between neuregulin and Erbb3, which is expressed in cushion mesenchyme (198) and presumably
heterodimerizes with Erbb2, is important for cushion formation.
Physiol Rev • VOL
Drosophila lacking the NK class homeodomain gene
tinman does not form a dorsal vessel (26). Expression of
dominant negative isoforms of XNkx2–3 or XNkx2–5,
Xenopus homologs of tinman, can cause complete elimination of cardiac gene expression and cardiogenesis (95,
116). In contrast, mice deficient in Nkx2–5 do form a
tubular heart that is arrested during looping (188, 298).
Therefore, Nkx2–5 is not required for establishment of
the cardiac lineage, possibly as a result of functional
redundancy. The Nkx2–5-deficient embryos do not show
affected proliferation or enhanced apoptosis, but the
atrial and ventricular chambers fail to expand. The right
ventricle that normally forms later than the left ventricle
does not visibly form, endocardial cushions are not
formed, and the ventricular trabecules do not form either,
suggesting a general inhibition of cardiogenesis after the
establishment of the tubular looping heart. A number of
genes are specifically downregulated. These genes include Anf, Bnf, and Chisel, markers for the forming chamber myocardium; Hand1, required for the formation of the
outer curvature; and homeobox gene Hop, involved in
modulation of Srf activity and required for normal cardiomyocyte proliferation and differentiation (23, 46, 233,
252, 280, 298). The expression of Anf and Bnf is affected
more in the ventricle than in the common atrium, suggesting that Nkx2–5 is more important for anterior than for
posterior gene regulation. Irx4 and Mlc2v, two genes that
are patterned along the AP axis, are expressed in Nkx2–
5-deficient embryos, although their levels fail to reach the
levels that are normal for age-matched wild-type littermates (188, 298). Furthermore, the Mlc2v promoter-lacZ
transgene that is normally active selectively in the anterior part of the linear and tubular heart is expressed
normally in tubular hearts of Nkx2–5-deficient embryos
(122). Taken together, in Nkx2–5 mutant mice a primary
heart tube is formed that is patterned along the AP axis
but fails to further differentiate due to a failure in downstream cardiac gene expression, resulting in arrest during
looping and impaired chamber morphogenesis.
Not only is Nkx2–5 important for early cardiogenesis,
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1. Nkx2–5
Targeted deletion of genes in embryonic stem cells,
and the subsequent generation of mouse lines or chimeric
mice, has revealed the importance of several factors for
cardiac morphogenesis. Overviews of the cardiac defects
in various species that result from gene mutations are
given in References 89, 262, 285. Disruption Rxr and Rar
genes, N-myc, Tef-1, Nf-1, and others results in thin ventricular walls, possibly as a result of proliferation defects
of the myocardium. Disruption of Nf-atc, Smad-6, and
Bmp6/7 leads to defects in septation or valve formation.
In the next paragraphs the consequences of deficiency of
factors involved in chamber formation are discussed.
Signaling between endocard, myocard, and cardiac
jelly, the extracellular matrix in between the endocard
and myocard, is important for the formation of trabecules
at the outer curvature and of cardiac cushions that line
the wall of the nonchamber myocardium. Mice in which
the genes for neuregulin or its tyrosine kinase receptors,
Erbb2 or Erbb4, are inactivated do not form trabecules at
the ventricular outer curvature, whereas the compact
outer layer of the ventricles is thinner or unaffected (84,
99, 174, 198). The lack of trabecules may result from
defects in signaling between neuregulin, expressed in
endocardial epithelium and Erbb2 or -4, expressed in
myocardium. Whether any of these components is responsible for the localization of the formation of the trabecules
is not clear. Expression of the genes for neuregulin or its
receptors in the heart is not restricted to the regions
where trabecules form. Furthermore, expression of the
Erbb2 receptor in the whole heart of Erbb2 mutants did
not result in expansion of the trabecular zone or of the
chamber myocardium, although it rescues the cardiac
phenotype (335). Disruption of the serotonin 2B receptor
resulted in a phenotype similar to that of neuregulin or
Erbb mutant mice, possibly because Erbb2 is downregulated in these mutants or because signaling through heteromeric GTPases G␣q and G␣11 is affected. E10.5 mouse
embryos that lack both GTPase encoding genes have
hypoplastic ventricular wall and trabecules (224). Hyaluronan synthetase-2-deficient mouse embryos are unable
to produce hyaluronan, an important component of car-
1258
ANTOON F. M. MOORMAN AND VINCENT M. CHRISTOFFELS
2. Hand2
Mice with a targeted deletion of Hand2 have severely
hypoplastic anterior heart structures and a malformed
aortic sac (286). Targeted deletion of Bob, a histone
deacetylase-dependent transcriptional repressor, results
in a similar although more severe cardiac phenotype (109).
In Bop mutants Hand2 expression is abolished specifically in the heart, indicating that the cardiac phenotype of
Bob mutant mice is caused by absence of Hand2 in the
heart. Rescue experiments by expression of Hand2 in
Bob-deficient mice may prove this point. In Hand2 mutant
embryos, increased apoptosis was observed in the
branchial arches and the anterior region of the tubular
heart, indicating that the hypoplastic structures are a
result of programmed cell death (302, 341). The regions
that are affected correlate well with the expression pattern in the looped heart of E9.5. Hand2 is initially expressed in the entire cardiac crescent and tubular heart
but is downregulated in the posterior components (atria,
Physiol Rev • VOL
atrioventricular canal, and left ventricle) of the looped
heart. Hand2 deficiency does not affect the anterior expression of the Mlc2v-lacZ transgene in the tubular heart,
indicating that Hand2 is not required for the AP pattern
that regulates this promoter fragment. Endogenous Mlc2v
gene expression is also not affected (34, 341). Hand2
deficiency does however interfere with Irx4 expression
(34). Whether this downregulation is due to specific interference with Irx4 gene regulation has not been established. In double mutants of Nkx2–5 and Hand2, heart
formation is arrested in the tubular stage, and Mlc2v is
strongly downregulated (341). However, some cells at the
ventral side of the small tubular heart that is formed
express Mlc2v. Coup-TFII, Hey1, and Tbx5 expression
was observed in the posterior region of this tube. These
results suggest that a primary heart tube is formed in
compound null mice that is robustly patterned along AP
and DV axes, but that maturation and expansion of the
chamber myocardium, especially the ventricular chambers, is severely affected. Irx4 and Hand1 expression
were undetectable in these mutants. The double mutants
seem to have an additive phenotype of the Nkx2–5 or
Hand2 mutant phenotypes, indicating that the factors act
at least in part independently in the maturation/proliferation of the myocardium of the anterior region.
3. Mef2c
Homozygous disruption of the myocyte enhancer factor (Mef) 2C gene causes severely affected cardiac morphogenesis (183). The heart tube forms but does not loop
and anterior heart structures are malformed and hypoplastic, whereas posterior structures are relatively unaffected. Several cardiac genes, including Anf, are downregulated, but Mlc2v and Hand1 are expressed normally.
This phenotype suggests that a primary heart tube forms
and is patterned along the AP and DV axis, but that due to
a failure in downstream cardiac gene expression the further differentiation of myocardium is blocked, resulting in
arrest during looping and lack of ventricular chamber
morphogenesis and expansion. Mef2C is expressed in the
entire heart (81). Its ventricular-specific requirement
therefore indicates that Mef2C is a necessary cofactor for
ventricle-specific factors. Hand2 is downregulated in
Mef2C mutant mice at the time of looping. As Hand2
null-mice also have hypoplastic/malformed anterior heart
structures and as Mef2 factors cooperates with other
basic helix-loop-helix factors in skeletal muscle development, Hand2 has been implicated in cooperating with
MEF2C in formation of the anterior region of the heart
(226).
X. CONCLUSIONS AND PERSPECTIVES
The descriptions of the developing heart by the early
anatomists and physiologists in the beginning of the pre-
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its expression at the right dose at later stages is also
required for septation of the chambers and formation of a
functional conduction system. In patients with septal defects and atrioventricular conduction defects, mutations
have been found in Nkx2–5 (22, 272). Even though mice
with only one intact Nkx2–5 allele do not show defects as
severe as those in patients (24), haploinsufficiency is
likely to be an important mechanism for the phenotype in
patients (147). In line with these findings, detailed analysis of mice deficient for polycomb-group gene Rae28
showed that Nkx2–5 is required for septation of the cardiac chambers (281). Rae28-deficient mice have septal
defects, and Nkx2–5 expression is not sufficiently sustained during later development. The cardiac phenotype
was rescued by transgenic expression of Nkx2–5 in
Rae28-deficient embryos showing that Nkx2–5 expression later in development is crucial for normal cardiogenesis and septation. Why low levels of Nkx2–5 result in
defects in specific structures is unclear. Although higher
levels of Nkx2–5 mRNA and protein were observed in
components of the conduction system (301), Nkx2–5 is
expressed broadly in the (developing) heart. Nkx2–5 cooperates with Tbx5 and Tbx2. the latter two interacting
through the same sites in the promoter of Anf. Haploinsufficiency for Tbx5 causes septal defects and atrioventricular conduction defects (20, 36, 177), resembling the
phenotype of Nkx2–5 mutants. Tbx2 is expressed in the
atrioventricular and outflow tract myocardium and cushions, which will form the septa and atrioventricular conduction system, and together with Nkx2–5 represses Anf
gene expression in these areas (118). These findings suggest that in vivo the interplay between Nkx2–5, Tbx5, and
Tbx2 is important in the correct septation of the chambers and formation of the atrioventricular canal.
CARDIAC CHAMBER FORMATION
Physiol Rev • VOL
ongoing. Patterning of the heart tube along the AP and DV
axes provides crucial positional information for the sitespecific process of chamber formation in higher vertebrates. Like the site-specific formation of chambers, the
localized conservation of the primary myocardium requires positional information, as is evident from the function of Tbx2 in the repression of chamber-specific genes.
Retinoic acid was shown to play an important role in the
pattern formation along the AP axis of the heart tube, but
other signals and pathways that regulate this patterning
remain to be determined. We hope that our analysis and
attempt to integrate the molecular, morphological, and
physiological data into a unifying concept are a help and
a stimulus for both teaching and research purposes. It is
challenging to examine the nature of the patterning process of the heart tube, which results in the precisely
localized formation of the cardiac chambers and of the
conduction system. Research on these very basic principles may have an important impact on the understanding
of many cardiac malformations and may give inroads into
therapeutic strategies.
We are grateful to our colleagues of the Experimental and
Molecular Cardiology Group and to Drs Jaques de Bakker, Judith Goodship, and Gertien Smits for valuable suggestions that
have much improved the manuscript. We thank Lara Laghetto
and Cees Hersbach for the illustrations and Wendy van Noppen
for linguistic advice.
The authors are supported by the Netherlands Heart Foundation Grant M96.002.
Address for reprint requests and other correspondence:
A. F. M. Moorman, Dept. of Anatomy & Embryology, Academic
Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands (E-mails: a.f.moorman@amc.uva.nl; v.m.christoffels
@amc.uva.nl).
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