REVIEW
193
Development 135, 193-205 (2008) doi:10.1242/dev.001883
Islet1 cardiovascular progenitors: a single source for heart
lineages?
Karl-Ludwig Laugwitz1,2,*, Alessandra Moretti1,2,*, Leslie Caron1, Atsushi Nakano1 and Kenneth R. Chien1,3,*
“Is myogenesis controlled by a single master gene? Possibly,
combinatorial schemes and cell lineage are both used to
generate positional identity in the embryo for each individual
cell.”
Hal Weintraub and colleagues (Davis et al., 1987)
Introduction
Heart is muscle. Or so it would seem to the outside observer. From
this vantage point, the discovery of MyoD almost 20 years ago by
Weintraub, Lassar and colleagues (Davis et al., 1987) represents one
of the seminal advances in our understanding of myogenesis, not
only in skeletal muscle, but also in cardiac muscle. Subsequent
studies by many investigators (for a review, see Srivastava and
Olson, 2000; Olson, 2006) have underscored this point and have
documented the conservation of the pathways and principles of
myogenesis across muscle types, spanning the evolutionary
spectrum from invertebrates to humans. This beautiful body of work
has had major implications for our understanding of both
development and disease, and has uncovered a host of genetic
regulatory circuits that contribute to the control of cardiogenesis. As
such, the molecular paradigm for cardiogenesis has largely been
based on studies of skeletal myogenesis, an approach that has
yielded spectacular advances in the field of cardiovascular science
and medicine.
But is heart more than muscle? And if so, might there be another
major paradigm that accounts for the generation of these diverse cell
types, which include endothelial, smooth muscle and conduction
system cells? Early cell lineage-tracing studies in avian systems
indicated that a common muscle cell precursor exists for both
1
Massachusetts General Hospital – Cardiovascular Research Center, Charles River
Plaza/CPZN 3208, 185 Cambridge Street, Boston, MA 02114, USA. 2Klinikum rechts
der Isar und Deutsches Herzzentrum – Technische Universität München, I.
Medizinische Klinik – Molekulare Kardiologie, Ismaninger Strasse 22, 81675
München, Germany. 3Department of Stem Cell and Regenerative Biology, Harvard
University and the Harvard Stem Cell Institute, Cambridge, MA 02138, USA.
*Authors for correspondence (e-mails: [email protected],
[email protected], [email protected])
working myocardium (atrial and ventricular myocardium) and the
conduction system (Mikawa, 1999). At the same time, other studies
identified a common progenitor, the hemangioblast, for endothelial
and blood cell lineages, a finding which indicated that a common
precursor might also exist for endothelial cells in the heart (Choi et
al., 1998; Fehling et al., 2003; Kouskoff et al., 2005). The recent
discovery of multipotent Islet1-positive (Isl1+) progenitors in several
species, including mouse, rat and human, and in different regions of
the embryonic and adult heart, implies that there might be a stem cell
paradigm for the generation of diverse cell lineages in the heart
(Moretti et al., 2006). Unexpectedly, a growing body of evidence
from multiple independent laboratories now suggests that, with
respect to lineage diversification, the heart could be like blood, an
organ in which a single stem/progenitor cell is able to generate all of
the major cell types of the system (Kattman et al., 2006; Wu et al.,
2006). The concept of progressive lineage restriction is well
accepted for hematopoiesis but has not been established in such
detail in the development of solid organs such as the heart. In this
review, we explore this idea further by focusing on the Isl1
cardiovascular progenitor story, placing it into the context of the
generation of diverse cardiovascular lineages, and discussing its
implications for cardiovascular development and disease. The reader
is also directed to reviews on the potential general role of progenitors
in cardiogenesis and disease (see Buckingham et al., 2005; Chien
and Karsenty, 2005; Srivastava, 2006; Black, 2007).
Cardiovascular cell lineages arise from discrete
embryonic precursors
The heart is composed of diverse muscle and non-muscle cell
lineages: atrial/ventricular cardiac myocytes, conduction system cells,
smooth muscle/endothelial cells of the coronary arteries and veins,
endocardial cells, valvular components and connective tissue (Fig. 1).
During cardiogenesis, the differentiation of these multiple heart
lineages is under tight spatial and temporal control, resulting in the
coordinated formation of the distinct tissue components of the heart,
including the four specialized chambers, diverse structures of the
conduction system, the endocardium, the heart valves, the coronary
arterial tree and the outflow tract (Harvey, 2002; Brand, 2003).
Understanding how this diversity of heart cell lineages arises is a
fundamental question that has major implications for understanding
and treating both congenital and adult heart diseases, a subset of which
have recently been shown to be due to defects in the pathways
involved in heart lineage specification (Schott et al., 1998; Benson et
al., 1999; Garg et al., 2003; Pashmforoush et al., 2004) (Table 1).
In this regard, three major sources of heart cell precursors have
been identified in the embryo: the cardiogenic mesoderm, the
cardiac neural crest and the proepicardial organ (see Fig. 1A,B and
glossary, Box 1). Each of these represents a spatially and temporally
distinct pool of embryonic heart progenitors, which are known to
give rise to distinct cardiac structures and cell components. The
cardiogenic mesoderm forms the linear heart tube (see Box 1) and
ultimately the bulk of the working myocardium in the ventricular
DEVELOPMENT
The creation of regenerative stem cell therapies for heart
disease requires that we understand the molecular mechanisms
that govern the fates and differentiation of the diverse muscle
and non-muscle cell lineages of the heart. Recently, different
cardiac cell types have been reported to arise from a common,
multipotent Islet1 (Isl1)-positive progenitor, suggesting that a
clonal model of heart lineage diversification might occur that is
analogous to hematopoiesis. The ability to isolate, renew and
differentiate Isl1+ precursors from postnatal and embryonic
hearts and from embryonic stem cells provides a powerful
cell-based system for characterizing the signaling pathways
that control cardiovascular progenitor formation, renewal,
lineage specification and conversion to specific differentiated
progeny.
194
REVIEW
A
E7.5
Development 135 (2)
E10.5
E8.5
E8.0
HFs
HFs
Arterial
pole
Heart tube
Cardiogenic
mesoderm
L
RA
Cardiac
neural
crest
Ca
PhA
E14.5
Cr
OFT LA
RV
Ao
AA
LA
RA
RV
ML
Venous
pole
ML
V
L
PRA
Ca
LV
AA arteries
Cr
R
PT
LV
PLA
Coronary
vasculature
IVS
Ca
Aorta smooth muscle cells
B
Cardiogenic
mesoderm
Endocardial cells
Atrial myocytes
Cushion cells
Cardiac
neural
crest
Autonomic nervous system
Conduction cells
Smooth muscle
coronary artery
Ventricular myocytes
?
Proepicardium
Endothelium
coronary arteries
Fibroblasts
and atrial chambers. The cardiac neural crest migrates into the heart
at a later stage of cardiac development, and gives rise to the vascular
smooth muscle of the aortic arch, ductus arteriosus (see Box 1) and
the great vessels; additionally, neural crest contributes to essential
components of the cardiac autonomic nervous system (Kirby et al.,
1983; Epstein and Buck, 2000). The mesenchyme portion of the
developing heart and the majority of epicardial cells are derived
from the proepicardium (Dettman et al., 1998; Moore et al., 1999;
Manner et al., 2001). The epicardial mantle begins to envelop the
myocardium at the same time that coronary precursor cells first
appear in the heart tube (Mikawa and Gourdie, 1996) and retroviral
genetic labeling experiments have shown that single vasculogenic
cells of the proepicardium differentiate into solitary vesselassociated clusters that consist of three cell types: endothelial,
smooth muscle and perivascular connective tissue cells (Mikawa
and Gourdie, 1996). This evidence suggests that the coronary
vasculature might be partially derived from mesodermal cells in the
proepicardium, although the contribution of the proepicardium to
the endothelial lineage is still controversial (Poelmann et al., 2002).
Taken together, these studies on the contribution of the primordial
heart field, cardiac neural crest, and of the proepicardium to
specialized structures during cardiogenesis indicate that lineage
diversification in the heart can arise, in part, via distinct pools of
cardiovascular progenitors that are spatially and temporally
segregated in the developing embryo (see Fig. 1B).
Isl1 identifies the second heart field lineage
One of the earliest steps in cardiogenesis is the formation of the
cardiac crescent (Fig. 2 and see Box 1), which is derived from cells
of the mesoderm that become instructed to adopt a cardiac fate in
response to signals from adjacent tissues (Harvey, 2002; Srivastava
and Olson, 2000). Recent studies have revealed that the cardiogenic
DEVELOPMENT
Fig. 1. Origin and lineage relationship of cardiac cell types. (A) Contribution of the three populations of embryonic heart progenitors,
cardiogenic mesoderm (red), cardiac neural crest (purple) and proepicardial organ (yellow) to different heart compartments during cardiac
morphogenesis in the mouse. Progenitors of the cardiogenic mesoderm are first recognizable under the head folds (HFs) of the embryo at E7.5,
then move ventrally to the midline (ML) and form initially the linear heart tube and ultimately the four chambers of the heart. After the looping of
the heart tube (E8.5), cardiac neural crest progenitors migrate from the dorsal neural tube to engulf the aortic arch arteries and contribute to
vascular smooth muscle cells of the outflow tract (OFT) around E10.5. At the same time in mouse development, the proepicardial organ precursors
contact the surface of the developing heart, give rise to the epicardial mantle (yellow) and contribute later to the coronary vasculature. In the fetal
heart (~E14), the chambers separate due to septation and are connected to the pulmonary trunk (PT) and aorta (Ao). Cranial (Cr)-caudal (Ca), right
(R)-left (L), and dorsal (D)-ventral (V) axes are indicated. (B) Cardiac cell types that arise through the lineage diversification of the three embryonic
precursor pools in the mouse heart. Whereas the contribution of the proepicardium to the smooth muscle cells of the coronary system and to the
mesenchymal cells of the heart is well accepted, the origin of the endothelial lineage in the coronary vasculature is still controversial. AA, aortic
arch; IVS, interventricular septum; LA, left atrium; LV, left ventricle; PhA, pharyngeal arches; PLA, primitive left atrium; PRA, primitive right atrium;
RA, right atrium; RV, right ventricle.
Development 135 (2)
REVIEW
195
Table 1. Gene mutations in human non-syndromic congenital cardiovascular malformations
Gene
Inheritance
Cardiac malformations
References
NKX2-5 (NK2 transcription
factor related, locus 5)
Autosomal dominant
ASD, conduction system defects
GATA4 (GATA binding
protein 4)
TBX20 (T-box 20)
Autosomal dominant
ASD, VSD
Partial penetrance
(Kirk et al., 2007)
JAG1 (jagged 1)
CRELD1 (cysteine-rich with
EGF-like domains 1)
NOTCH1
MYH6 (myosin heavy chain
6)
Partial penetrance
Partial penetrance
ASD, VSD, valve abnormalities,
cardiomyopathy
TOF
AVSD
BAV, calcification
ASD
(Garg et al., 2005)
(Ching et al., 2005)
Autosomal dominant
Autosomal dominant,
partial penetrance
(Schott et al., 1998; Benson
et al., 1999; Goldmuntz
et al., 2001; Elliott et al.,
2003; McElhinney et al.,
2003)
(Garg et al., 2003)
(Krantz et al., 1999)
(Robinson et al., 2003)
ASD, atrial septal defect; AVSD, atrioventricular septal defect; BAV, bicuspid aortic valve; TOF, tetralogy of Fallot; VSD, ventricular septal defect.
mesoderm in fact consists of two populations or fields (see Box 1)
of cardiac precursor cells that contribute to different parts of the
heart. The earliest population of cardiac progenitors, referred to as
the first heart field, originates in the anterior splanchnic mesoderm,
gives rise first to the cardiac crescent, later to the linear heart tube,
and ultimately contributes to parts of the atrial chambers and the left
ventricular region. The second cardiogenic region, known as the
second heart field, lies anterior and dorsal to the linear heart tube and
is derived from the pharyngeal mesoderm medial to the cardiac
crescent (see Fig. 2A and Box 1). Cells from this second heart
lineage are added to the developing heart tube and give rise to the
outflow tract, the right ventricular region and the main parts of the
atrial tissue (reviewed by Kelly and Buckingham, 2002;
Buckingham et al., 2005). The discovery of the second heart field
A
B
E8.5
E8.0
Stage 16
E10.5
E7.5
HFs
Pha
RA
OFT LA
Heart tube
Second heart
field
Cardiac
crescent
LV
RV
ML
R
Cr
Cr
Cr
D
L
R
V
Neural epithelium
L
Ca
Ca
Ca
Second heart
field
Pharyngeal region of
foregut diverticulum
Ca
DOFT
Head mesoderm
V
LV
Cr
Pericardial cavity
Aortic sac
L
RV
Primitive
heart tube
Bulbus cordis region
of the primitive heart
R
L
Stage 22
Ca
Body wall
Fig. 2. First and second heart fields and their contributions to the developing heart. (A) The upper drawings show the relative position,
movement and contribution of the second heart field progenitors (green) relative to the first heart field cells (red) from the cardiac-crescent through
to the looping stages of mouse heart development. The dashed lines indicate the position of the corresponding sections shown in the lower panels.
(B) Location (upper) and contribution (beneath) of the second heart field progenitors (blue) to the outflow tract in the chick embryo. Cranial (Cr)caudal (Ca), right (R)-left (L), and dorsal (D)-ventral (V) axes are indicated. DOFT, distal outflow tract; HFs, head folds; LA, left atrium; LV, left
ventricle; ML, midline; PhA, pharyngeal arches; OFT, outflow tract; POFT, proximal outflow tract; RA, right atrium; RV, right ventricle.
DEVELOPMENT
Truncus arteriosus
Primitive foregut
endoderm
R
LA
POFT
Cardiac
crescent
D
OFT
RA
Foregut
endoderm
REVIEW
Box 1. Glossary of specialized terms
Branchial arches: A series of paired segmental structures composed
of ectoderm, mesoderm and neural crest cells that are positioned on
either side of the developing pharynx. In mammals, the branchial
arches contribute to pharyngeal organs and to the connective,
skeletal, neural and vascular tissues of the head.
Cardiac crescent: Crescent-shaped epithelium that represents the
first recognizable cardiac progenitors at the cranial and cranial-lateral
parts of the embryo. The cardiac crescent fuses at the midline to form
the early heart tube.
Cardiogenic mesoderm: Mesoderm is one of the three layers of
cells in the early embryo that, together with the endoderm and
ectoderm, provides the source of all subsequent cell types that
appear during embryogenesis. The cardiogenic mesoderm gives rise
to the specialized cells of the heart and includes both first and second
heart field precursors.
Ductus arteriosus: A shunt connection between the pulmonary
artery and the aortic arch that allows most of the blood from the
right ventricle to bypass the embryonic lungs.
Heart field: A defined special area of cells in the mesoderm that is
fated to form the cardiac organ.
Linear heart tube: Transient structure of the early developing heart
composed of an inner endothelial tube shrouded by a myocardial
layer.
Myocardial cell lineage: The term lineage emphasizes the history
of a cell and its descendents, and the characterization of their clonal
contribution to the heart. The first and second myocardial lineages
represent the progenitors of the first and second heart fields and
their differentiated progeny.
Neural crest: Progenitor cells present in vertebrates that arise from
the dorsal neural tube and migrate to other sites in the embryo and
differentiate into various cell types depending on location.
Pharyngeal mesoderm: The mesoderm that is located below the
head in the pharynx, which is the part of the embryo where the
developing respiratory and digestive systems are situated.
Primitive streak: Transitory embryonic structure, present as a strip
of cells that prefigures the anterior-posterior axis of the embryo.
Proepicardial organ: A cluster of mesothelial cells located on the
right side of the external surface of the sinus venosus. These cells
contact the dorsal wall of the tubular heart in the region of the
atrioventricular junction and form a monolayer that gradually covers
the heart.
Retrospective clonal analysis: A genetic approach for lineage
analysis that is based on random labeling of precursor cells. The
example cited in this review employed a lacZ carrying a duplication
(laacZ) that renders it non-functional. Only upon a rare, spontaneous
intragenic recombination which removes the duplication do cells
express the lacZ reporter and can be clonally analyzed.
reads like a scientific detective story, in which independent pieces
of evidence were brought together to ultimately identify a prime
suspect.
The initial evidence that the outflow tract was not present in the
linear heart-tube stage came from a series of in vivo lineagetracing experiments performed in chick embryos by de la Cruz
and co-workers in the 1970s, which indicated that the outflow
tract myocardium originates from a progenitor population that is
different from the cardiac crescent and is situated anterior to the
heart tube (reviewed by de la Cruz and Sanchez-Gomez, 2000).
Over the past few years, the source of the outflow tract and the
right ventricle has been addressed by studies from three different
laboratories, two performed in chick embryos and one in mouse
embryos (Waldo et al., 2001; Kelly et al., 2001; Mjaatvedt et al.,
2001). The lineage studies in chick demonstrated that the
Development 135 (2)
splanchnic mesoderm adjacent to the pharyngeal endoderm
migrates in through the aortic sac to contribute cells to the outflow
tract and the right ventricle of the heart, and that the flow of these
progenitors along this path gives rise to distinct outflow tract
regions at different times during cardiac development (Mjaatvedt
et al., 2001; Waldo et al., 2001) (see Fig. 2B). In mouse embryos,
a lacZ reporter gene integrated into the genomic fibroblast growth
factor 10 (Fgf10) locus marked the outflow tract and the right
ventricle of the developing embryonic heart (Kelly et al., 2001).
This transgene was expressed at the cardiac-crescent stage of
heart development at embryonic day (E) 7.5, and ␤-galactosidase
(␤-gal) activity was observed in splanchnic mesoderm that lies
medial to the classical first heart field. As development
progressed, ␤-gal+ cells were found in anterior splanchnic
mesoderm adjacent to pharyngeal endoderm and were
subsequently observed in branchial arch mesoderm proximal to
the heart, in the outflow tract and in the right ventricle. Taken
together, these studies demonstrated that cells comprising the
earliest fusing myocardium do not contain all of the progenitors
of the outflow tract and the right ventricle and that these heart
structures derive, wholly or in part, from precursor cells of a
second heart field, which are added to and supplement the
myocardium that originates from the first heart field progenitors
(Kelly and Buckingham, 2002). Furthermore, an elegant
retrospective clonal analysis (see Box 1) in the mouse embryo
suggested that the first and second lineages (see Box 1) of cardiac
progenitors originate from a common precursor population that
segregates prior to the cardiac-crescent stage (Meilhac et al.,
2004). However, a clear delineation of the existence of the second
myocardial lineage as a separate subset of cardiovascular
precursors, and the ultimate identification of the heart components
that it generates, would await the identification of a suitable
genetic marker.
In this regard, recent studies have revealed that the expression of
the LIM-homeodomain transcription factor Isl1 is a marker of the
second myocardial lineage during mammalian cardiogenesis (Cai et
al., 2003), a finding that ultimately allowed the isolation of the Isl1+
cardiovascular progenitors themselves (Laugwitz et al., 2005).
LIM/HD proteins are a subset of homeodomain (HD)-containing
transcription factors that are defined on the basis of a common LIM
domain, which consists of a conserved cysteine- and histidine-rich
structure of two tandemly repeated zinc fingers. The acronym of LIM
is derived from the first identified members of this family, namely
LIN-11 from Caenorhabditis elegans (Frey et al., 1990), Isl1 from rat
(Karlsson et al., 1990), and MEC-3 from C. elegans (Way and Chalfie,
1988). Subsequent studies of LIM/HD proteins in embryonic
motoneurons have led to the identification of a combinatorial code of
several LIM/HD proteins, including the Isl1 gene, that controls
various aspects of motoneuron identity (Tsuchida et al., 1994; Thor et
al., 1999; Thaler et al., 2004). Aside from its expression in embryonic
motoneurons, Isl1 is expressed in a variety of cell lineages of
pancreatic endocrine origin during embryogenesis, as well as in
normal adult islet cells (Karlsson et al., 1990).
Isl1 knockout mice have been generated and studied for defects
both in motoneuron specification and pancreatic development (Pfaff
et al., 1996; Ahlgren et al., 1997). Homozygous Isl1 mutants exhibit
growth retardation around E9.5-10, and die at approximately E10.511. Histological analysis of mutant hearts between E9.0 and 9.5
revealed that homozygous Isl1 mutants have a severe cardiac
phenotype. Isl1-null hearts fail to undergo looping morphogenesis
and appear to have a common atrium and a uni-ventricular chamber,
whereas the right ventricle and the outflow tract are absent (Cai et
DEVELOPMENT
196
al., 2003). Genetic marker analysis for expression of Tbx5, Hand1
and Fgf10 demonstrated that the remaining ventricular tissue had a
left ventricular identity and confirmed the lack of the right ventricle
and the outflow tract.
These observations suggested that Isl1 is expressed in cells of the
second heart field, which contribute to both the venous and arterial
poles of the heart. Lineage tracing of Isl1-expressing cells using the
Cre-loxP strategy showed that these cells colonize the outflow tract,
the right ventricle, part of the atria and a minor portion of the inner
curvature of the left ventricle, confirming that this transcription
factor marks cardiac progenitors of the second myocardial lineage
(see Box 1). Furthermore, Isl1 seems to be required for the survival,
proliferation and migration of these cells into the cardiac tube, and
its transcription is turned off as the precursor cells differentiate.
Since Isl1 expression delineates undifferentiated and differentiated
progenitor states, it represents an excellent lineage tracer for cardiac
mesodermal cells during embryogenesis.
Is Isl1 expression restricted to the second heart field of cardiac
progenitor cells or is it also transiently expressed in the first? Two
lines of evidence support the latter notion. First, recent lineagetracing experiments that used a highly efficient Isl1-Cre knock-in
mouse line showed that the majority of cells in the left ventricle
express the genetic marker ␤-gal (Park et al., 2006). However, it is
possible that the inefficiency of the excision of the original Isl1IRES-Cre allowed cells in which Isl1 was expressed for a longer
period of time to be preferentially detected (Srinivas et al., 2001; Cai
et al., 2003). The second piece of evidence comes from recent
studies in mouse and Xenopus. Harvey and co-workers report that,
in contrast to Isl1 mRNA, Isl1 protein is expressed at E7.5
throughout the anterior intra-embryonic coelomic walls and
proximal head mesenchyme, regions that encompass both first and
second heart fields in mouse (Prall et al., 2007). Similarly, during
neurula stages in Xenopus, Isl1 is co-expressed with Nkx2-5
throughout the cardiac crescent, which is the first heart field in
amphibians (Brade et al., 2007). These data suggest that Isl1 might
be a pan-cardiac progenitor marker, but additional work is needed
to clarify this issue. In this regard, the identification of specific
markers for the first heart field would be extremely valuable.
However, analyses of the cardiovascular phenotype of Isl1 knockout
mice suggests that Isl1 does not play such an essential role in the first
myocardial precursor lineage as compared with the second. So far,
the earliest molecular pan-cardiac markers for both myocardial cell
lineages are the transcription factors Mesp1/2 (mesoderm posterior
1/2) and Fgf8, which are expressed transiently in cells of the newly
formed mesoderm at the primitive-streak stage (see Box 1), the
descendents of which colonize the whole myocardium (Saga et al.,
2000; Kitajima et al., 2003; Ilagan et al., 2006).
Isl1: an early nodal point in cardiogenesis
A comparison of the cardiovascular defects that arise in Isl1deficient mouse embryos with those of mice with mutations in other
cardiac transcription factors has begun to uncover a genetic
regulatory network that controls the fate of Isl1+ cardiovascular
precursors in the second heart field (Table 2; Fig. 3).
The first and second myocardial lineages seem to be governed
by both shared and distinct genetic programs. Myocardial
regulatory genes that are activated in both lineages include the
zinc-finger-containing transcription factors of the GATA family
(Gata4, 5 and 6) (Charron and Nemer, 1999) and NK2
transcription factor related, locus 5 (Nkx2-5) (Harvey, 1996).
Recent studies have identified Mef2c (myocyte enhancer factor
2C; also known as RSRF, related to serum response factor) as a
REVIEW
197
direct transcriptional target of Isl1 and GATA factors in the second
heart field (Dodou et al., 2004). The forkhead box H1
transcription factor Foxh1 is also expressed in the second lineage
of cardiac precursors, and Foxh1 mutant mouse embryos, like
Isl1- and Mef2c-deficient embryos, display defects in the right
ventricle and in outflow tract formation (von Both et al., 2004).
Isl1 and Foxh1 directly activate transcription of Mef2c by
regulating two independent enhancer regions in collaboration
with GATA factors and Nkx2-5 (Arceci et al., 1993; Dodou et al.,
2004). Thus, these genes appear to act at the top of a cascade of
cardiac transcription factors in the second myocardial lineage.
The SET-domain protein Smyd1 (Bop) is a direct target of Mef2c
and regulates Hand2 (heart and neural crest derivatives expressed
transcript 2) expression during second heart field development,
implying that Smyd1 is an indirect downstream target of
Isl1/GATA factors and Foxh1/Nkx2-5 (Gottlieb et al., 2002; Phan
et al., 2005). Recently, it has been shown that Forkhead factors are
central components of additional regulatory circuits that intersect
and reinforce the Isl1-GATA-Mef2c main pathway in the second
heart lineage transcriptional network. Foxa2, Foxc1 and Foxc2
can bind and activate in vitro a Tbx1 enhancer that is sufficient to
direct expression to the second heart field (Maeda et al., 2006).
Tbx1, in turn, activates Fgf8 (Hu et al., 2004), whose loss-offunction in the second heart field results in a reduction of Isl1
expression in the pharyngeal mesoderm and outflow tract (Park et
al., 2006).
Taken together, the early segregation of the two lineages, the
different time-course of myocytic differentiation and the distinct
regional contributions to the embryonic heart support the idea that
the two progenitor populations may have discrete properties and
distinct transcriptional hierarchies for cardiac development, with
Nkx2-5 as the critical transcription factor in the first and second
lineages, and Isl1, along with Foxh1 and GATA factors, as the key
transcriptional regulators in the second heart progenitor field (Biben
and Harvey, 1997) (see Fig. 3).
Alternatively, one might question the existence of two unbridgeable
progenitor lineages regulated by distinct transcriptional networks
whose descendents form distinct heart compartments, and instead
propose the complex patterning of one primordial precursor field
(Abu-Issa et al., 2004). Such a view carries the implication that cardiac
precursors within this solitary field have the capacity to form different
heart structures, depending on positional cues. Temporal
microenvironmental stimuli and differing concentrations of diffusing
morphogens could create cellular diversity that leads to a progressive
restriction of developmental potency within a field that was initially
homogenous (Moorman et al., 2007).
Isl1+ progenitor cells in the embryonic and
postnatal heart
The purification, renewal and differentiation of native Isl1+ cardiac
progenitors provides a means to unravel the steps for both cardiac
lineage formation and regeneration, and to identify how these steps
are linked to certain forms of congenital and adult cardiac diseases.
Taking advantage of the fact that Isl1 expression is downregulated in
most cardiac precursor cells as they differentiate, recent studies have
utilized inducible Isl1-Cre and knock-in Isl1-nlacZ mice to analyze
the timing of Isl1+ progenitor migration into the looping heart and the
distinct subdomains of the heart that they colonize during embryonic
development (Laugwitz et al., 2005; Sun et al., 2007) (Fig. 4).
Interestingly, a subset of Isl1+ undifferentiated progenitors remains
embedded in the embryonic heart after its formation and a few cells
are still detectable after birth in the compartments that arise from Isl1+
DEVELOPMENT
Development 135 (2)
198
REVIEW
Development 135 (2)
Table 2. Mutant phenotypes of genes involved in second heart field development
Gene
SHF expression
Isl1
SHF progenitors; weak in
distal OFT
Mef2c
Early SHF progenitors
Hand2
Both FHF and SHF
progenitors at cardiac
crescent stage
Yes
Hand1/2
Foxh1
References
No ventricles; only atrial chamber forms
Nkx2-5
Both FHF and SHF
progenitors
Embryonic lethal at E10. OFT reduced or absent; RV does not
develop; disrupted expression of Mef2c, Hand2, Fgf8 and
Tbx5
Embryonic lethal at E10.5. Severe defects in RV and OFT
formation; abnormal coronary vessels; disrupted expression of
Fgf10, Fgf8 and Tbx1
Embryonic lethal. Defective looping and single ventricular
chamber
Single atrial and ventricular compartments; loss of ventricular
tissue; no Hand1 expression
Tbx1
Pharyngeal mesoderm
Perinatal lethal. Defective OFT and ventricular septation
Tbx20
Cardiac crescent stage
Embryonic lethal at E11.5. Failed looping; chambers do not
develop; no Hand1 expression; hypoplastic RV; OFT disrupted
Fgf8
Yes
OFT defects
Fgf10
Yes
No early phenotype detected
Foxc1/c2
Smyd1
SHF progenitors and
pharyngeal mesoderm at
E9
SHF progenitors at E8 and
splanchnic and
pharyngeal mesoderm
Precardiac mesoderm at E7.5
Mutant phenotype in the cardiac tube
Embryonic lethal at E10. Single atrial and ventricular (LV
identity) compartments; no RV or OFT; atria at the venous
pole are abnormal
Embryonic lethal at E10. OFT reduced; RV does not develop
(Hand2 is downregulated); INF abnormalities
Embryonic lethal at E10.5. Defective looping with RV
abnormalities
(Cai et al., 2003)
(Lin et al., 1997;
Dodou et al., 2004)
(Srivastava et al., 1997)
(Yamagishi et al.,
2001; McFadden et
al., 2005)
(von Both et al., 2004)
(Kume et al., 2001; Seo
and Kume, 2006)
(Gottlieb et al., 2002)
(Lyons et al., 1995;
Tanaka et al., 1999;
Yamagishi et al.,
2001)
(Xu et al., 2004; Hu et
al., 2004)
(Takeuchi et al., 2005;
Singh et al., 2005;
Stennard et al.,
2005; Cai et al.,
2005)
(Abu Issa et al., 2002;
Brown et al., 2004)
(Kelly et al., 2001)
FHF, first heart field; INF, inflow region; OFT, outflow tract; RV, right ventricle; SHF, second heart field.
Isl1+ cardiovascular progenitors and heart lineage
diversification
In vivo cell lineage tracing using the Cre-loxP strategy in the mouse
has been an invaluable tool for precisely delineating the specific cell
types that derive from Isl1+ precursors, as well as for defining their
specific locations in the components of the heart, vascular and
conduction systems. To date, several separate Cre mouse lines have
proven to be useful for the in vivo lineage tracing of cardiovascular
Isl1
Foxc1/2
Tbx20
Foxa2
Gata
Tbx1
Nkx2-5
Fgf10
Fgf8
Mef2c
Smyd1
Foxh1
Hand2
Fig. 3. Regulatory networks within the second heart field
lineage. The model presents an Isl1-dependent transcriptional network
for the development of the second heart field lineage. The LIMhomeodomain transcription factor Isl1 (blue) functions as an early
regulator of a core network that determines right ventricle and outflow
tract development. The solid lines indicate that direct in vivo activation
of regulatory sequences has been demonstrated. Dotted lines indicate
genetic data or in vitro activation. Fgf8/10, fibroblast growth factor 8
and 10; Foxa2, forkhead box a2; Foxc1/2, forkhead box c1/2; Foxh1,
forkhead box h1; Gata, GATA-binding proteins; Hand2, heart and
neural crest derivatives expressed transcript 2; Isl1, insulin gene
enhancer protein; Mef2c, myocyte enhancer factor 2C; Nkx2-5, NK2
transcription factor related, locus 5; Smyd1, SET and MYND domain
containing 1; Tbx1/20, T-box 1 and 20.
DEVELOPMENT
second lineage precursors during cardiac development. Tamoxifeninducible Cre-lox technology has enabled this novel postnatal Isl1+
cell population and its progeny to be selectively marked at a defined
time and purified to relative homogeneity (Laugwitz et al., 2005). The
ability of these cells to self-renew in vitro on a cardiac mesenchymal
feeder layer and to be stimulated to differentiate into fully mature
functional cardiomyocytes indicates that these cells represent native
cardiac progenitors, remnants of the embryonic Isl1+ precursors
(Laugwitz et al., 2005). Postnatal Isl1-expressing cells can be detected
in mouse, rat and human myocardium and appear to be distinct from
the previously reported cardiac c-Kit+ and Sca1+ cells (Sca1 is also
known as Ly6a), which were reported to activate cardiomyocytespecific genes in vitro and to differentiate into cardiac muscle cells in
vivo (Beltrami et al., 2003; Oh et al., 2003).
The Isl1 cell-based in vitro system, which can use both mouse
embryos and differentiating ES cells as a source of Isl1-expressing
cells, should facilitate the rapid and direct identification of the
signaling pathways that guide the formation, renewal and
diversification of Isl1+ progenitors into distinct heart cell lineages.
Development 135 (2)
REVIEW
199
progenitors from the first and second heart field. The Isl1-IRES-Cre
line (Srinivas et al., 2001) has the advantage of driving Cre
expression only in undifferentiated cardiac progenitors, but the
disadvantage of being widely expressed later on in embryonic
development. The inducible Isl1-mER-Cre-mER line offers
temporal control over Cre activity (Laugwitz et al., 2005). In this
line, the presence of the two mutated oestrogen-receptors (mERs)
results in Cre being sequestered in the cytoplasm. In the presence of
Tamoxifen, the mER-Cre-mER protein undergoes rapid nuclear
translocation, which allows Cre-mediated recombination to then
occur in those cells that express Isl1. A transgenic Cre mouse line
that allows gene inactivation to be restricted to the second heart field
lineage has been recently described in which Cre is expressed under
a specific Mef2c promoter/enhancer region (Verzi et al., 2005).
Nkx2-5-Cre and Mesp1-Cre deleter lines have also been well
characterized, and can be utilized to trace lineages from both the first
and second heart fields (Moses et al., 2001; Kitajima et al., 2000).
Fate-mapping experiments utilizing these different Cre lines have
demonstrated that Mef2c, Nkx2-5 and Mesp1 can mark cell
populations that contribute to myocardial cells and to subsets of
DEVELOPMENT
Fig. 4. Isl1+ progenitor cells during
cardiovascular development. ␤-gal
expression was analyzed in Isl1-nlacZ
knock-in mouse embryos at the
indicated days of development and in
the postnatal heart at day 3 (PN 3d)
by X-Gal staining. Representative
sections at each developmental stage
from E8.5-14.5 are shown (left), as
are the corresponding heart structures
that express Isl1 (middle) and the
lineage markers used to identify the
different cell types (right). Cranial
(Cr)-caudal (Ca), right (R)-left (L), and
dorsal (D)-ventral (V) axes are
indicated. A, atrial cavity; Ao, aorta;
AS, atrial septum; AVN,
atrioventricular node; CG, cardiac
ganglia; Hcn4, hyperpolarizationactivated cyclic nucleotide-gated K+ 4;
LA, left atrium; LV, left ventricle;
MF20, antibody recognizing
sarcomeric myosin; OFT, outflow
tract; PA, pulmonary artery; RA, right
atrium; RV, right ventricle; SAN,
sinoatrial node; SM, smooth muscle;
sm-actin, smooth muscle actin; V,
ventricle cavity. Parts of the figure are
reproduced with permission (Sun et
al., 2007).
REVIEW
Development 135 (2)
A Adult heart vasculature
Pulmonary artery
Aorta
Proximal aorta
and pulmonary artery
RCA
LCA
Valves
Endothelial cells
Smooth muscle cells
Endothelial cells
Smooth muscle cells
B Adult heart
Ganglia
Fig. 5. Cell lineage tracing of Isl1+ cardiac progenitors and
lineage contribution to the adult heart. Lineage tracing utilizing the
Isl1-IRES-Cre mouse crossed with the indicator R26R line to mark all
cells that once expressed Isl1 during development. The boxes represent
magnifications of different regions of the coronary tree (black boxes)
and of the aorta/pulmonary artery (red boxes), where ␤-gal-positive
cells have been detected. (A) Contribution of Isl1+ progenitors (blue) to
the coronary vasculature, the valves and the pulmonary artery/aorta.
The contribution of Isl1+ cells to the endothelial (purple) and smooth
muscle (yellow) cell layers is limited to the proximal area of the great
vessels and progressively declines from the proximal to the distal parts
of the coronary tree. ␤-gal+ cells were also detected in connective tissue
structures of the aortic and pulmonary leaflets, indicating that
components of the conotruncal cushions, which have an endocardial
origin, are derived from Isl1+ progenitors. (B) Contribution of Isl1+
progenitors (blue) to the atrial/ventricular myocardium (red), the
conduction system (white) and cardiac ganglia (green). In the
conduction system, most genetically marked cells were detected in the
sino-atrial nodal (SAN) region. AS, atrial septum; AVN, atrioventricular
node; CAs, coronary arteries; LA, left atrium; LCA, left coronary artery;
LV, left ventricle; RA, right atrium; RCA, right coronary artery; RV, right
ventricle; VS, ventricular septum.
endocardium (Verzi et al., 2005; Stanley et al., 2002; Kitajima et al.,
2000). Furthermore, the Cre-mediated lineage tracing of cells that
express Flk1 (also known as Kdr), one of the earliest mesodermal
progenitor markers for vascular endothelial and hematopoietic
lineages has, interestingly, shown the potential of Flk1+ precursors
to give rise to both cardiac and smooth muscle during development
(Motoike et al., 2003; Coultas et al., 2005).
To define the contribution of Isl1+ precursors to cardiac cell
lineages in the adult heart, Isl1-IRES-Cre/R26R double heterozygous
mice have been generated and analyzed (Fig. 5) (Laugwitz et al.,
2005; Moretti et al., 2006). In these mice, the Cre-mediated removal
of a stop sequence in the cells that express Isl1 results in the
expression/translation of lacZ, which is itself under the control of the
endogenous Rosa26 (R26) promoter. Thus, this approach marks all
the cells that once expressed Isl1 during development, and its use has
revealed that a high proportion of the right ventricular myocardium
and parts of both atria consist of cells that express lacZ and the
myocytic protein sarcomeric ␣-actinin (Laugwitz et al., 2005). The
histochemical analysis of ␤-gal and acetylcholinesterase expression
has revealed that Isl1+ progenitors make a remarkable contribution to
the cells of the conduction system, primarily to the sino-atrial node,
the pacemaker of the heart. Additionally, ␤-gal staining was observed
throughout the proximal aorta, the trunk of the pulmonary artery and
the stems of the main left and right coronary arteries (see Fig. 5). The
co-expression of lacZ with endothelial and smooth muscle-specific
markers, such as CD31 (Pecam1), VE-cadherin (cadherin 5) and
smooth muscle myosin heavy chain (SM-MHC; also known as
myosin heavy chain 11), revealed that Isl1+ precursors can give rise to
vascular lineages. Additional studies in the mouse embryo have
confirmed that Isl1+ cardiovascular progenitors contribute to over
two-thirds of all the cells in the embryonic heart, and to the major cell
types in all of the cardiovascular compartments and conduction
system, with the exception of the free left ventricular wall (Cai et al.,
2003; Moretti et al., 2006; Sun et al., 2007).
Taken together, these results demonstrate that Isl1 marks cardiac
precursors that give rise to working cardiac muscle, to the
conduction system and to endothelial/smooth muscle cells in
multiple heart tissue compartments during cardiogenesis (see Fig.
5). Furthermore, this evidence raises the question of what role Isl1
plays in the specification of each of these mesodermal lineages. Is
Isl1 marking an early anterior mesodermal progenitor not yet
committed to a cardiac fate? Or is Isl1 defining a multipotent
primordial cardiovascular progenitor, which contributes to distinct
cell lineages within heart components known to originate from the
second heart field? Or do different lineage-restricted precursors
exist, all of which independently express Isl1? The early onset of
Isl1 expression is consistent with the first two possibilities, although
previous retroviral marking experiments have shown, for the
proepicardial progenitors of the coronary vasculature, that clones of
precursors can give rise only to a single phenotype (for example,
only to epicardial cells, endothelial cells, smooth muscle cells or
fibroblasts) (Reese et al., 2002). A clear delineation of the
developmental potency of Isl1+ cells during cardiogenesis awaits in
vivo clonal differentiation analysis.
Multipotent Isl1+ cardiovascular progenitors
Understanding how embryonic precursors generate and control the
formation of distinct endothelial, pacemaker, atrial, ventricular and
vascular smooth muscle lineages, as well as how these cells become
positioned to form the specific chambers, aorta, coronary arteries and
conduction system of the heart, is of fundamental importance for
understanding the developmental logic and molecular cues that
DEVELOPMENT
200
Development 135 (2)
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201
Fig. 6. Cellular hierarchy of cardiac progenitors and their lineage specification. The expression of the lineage markers shown is based on
previous studies (Kattman et al., 2006; Moretti et al., 2006; Wu et al., 2006). The divergence of endothelial and haematopoietic lineages from the
myocardial lineage precedes the expression of Nkx2-5 in the precardiac mesoderm (tissue-specific markers are shown in blue). Bry, brachyury (T);
MLC2a, atrial myosin light chain 2 (also known as Myl7); MLC2v, ventricular myosin light chain 2 (Myl2); cTnT, cardiac troponin T (Tnnt2); Hcn4,
hyperpolarization-activated cyclic nucleotide-gated K+ 4; sm-actin, smooth muscle actin; SM-MHC, smooth muscle myosin heavy chain; VE-Cadh,
VE-cadherin.
concept, a hierarchy of distinct Isl1+ cardiovascular progenitors has
been uncovered (Laugwitz et al., 2005; Moretti et al., 2006) (see Fig.
6), including a rare subset of Isl1+ cardioblasts that persists until
birth and can develop into fully mature cardiac and smooth muscle
cells (Laugwitz et al., 2005; Sun et al., 2007). At the same time,
independent in vivo and in vitro studies from other laboratories have
also identified other multipotent and bi-potent cardiovascular
precursors (Kattman et al., 2006; Moretti et al., 2006; Wu et al.,
2006), which might also arise from the early heart fields. However,
the multi-lineage potential of MICPs still needs to be confirmed by
fate-mapping techniques in vivo. Uncovering the molecular
pathways that control the formation, renewal and differentiation of
cardiovascular progenitors into specific mature cellular progeny will
be crucial for unlocking the potential of stem cell therapy for use in
a myriad of cardiovascular degenerative diseases, such as heart
failure, conduction system disorders and congenital heart disease
(see Table 1).
Wnt/␤-catenin signaling controls renewal of Isl1+
cardiovascular progenitors
The microenvironment provided by a stem cell niche plays a vital role
in stem cell/progenitor maintenance (for a review, see Scadden, 2006).
Previous studies have shown that cardiac mesenchymal cells serve in
vitro as an effective microenvironment that allows embryonic-, ES
cell- and postnatal-derived Isl1+ progenitors to renew in culture
(Laugwitz et al., 2005; Moretti et al., 2006). Utilizing a chemical
screen, recent work from our laboratory has identified the Wnt/␤catenin pathway as being a major component of the cardiac
DEVELOPMENT
underlie both cardiovascular development and disease. As noted
above, the formation of cardiac, smooth muscle and endothelial cell
lineages in the heart has largely been ascribed to a set of nonoverlapping embryonic precursors that have distinct origins. The
discovery of several heart lineage-restricted genes has lead to the
suggestion that the generation of different cardiac cell types could be
driven by a unique combinatorial subset of transcriptional networks
that operate within distinct cardiovascular progenitors (for a review,
see Srivastava and Olson, 2000). An alternative possibility exists that
diverse muscle and non-muscle lineages arise from multipotent,
primordial cardiovascular stem cells, which give rise to a hierarchy of
downstream cellular intermediates that represent the tissue-restricted
precursors of fully differentiated heart cells (Fig. 6). This clonal model
of heart lineage diversification is similar to that of hematopoiesis,
which is initiated by a few multipotent hematopoietic stem cells that
generate large numbers of differentiated progeny by a process of
amplification and progressive lineage restriction (Morrison and
Weissman, 1994; Weissman, 2000).
Recent work from our laboratory has indeed proven the existence
of multipotent Isl1+ cardiovascular progenitors (MICPs), which are
marked by the transcriptional signature of Isl1, Nkx2-5 and Flk1.
These cells can generate the three major cell types of the heart:
cardiac, smooth muscle and endothelial cells (Moretti et al., 2006).
MICPs have been cloned from both mouse ES cells and mouse
embryos, and can make the decision to enter the muscle or
endothelial differentiation pathways at the single-cell level, hinting
at a hematopoietic-like system for how the diverse cardiovascular
lineages can be generated (Moretti et al., 2006). In support of this
REVIEW
mesenchymal microenvironment that controls the pre-specification,
renewal, and subsequent differentiation of a hierarchy of Isl1+
cardiovascular precursors from mouse ES cells, embryos and
postnatal hearts (Qyang et al., 2007). This study reported that the
inhibition of canonical Wnt signaling in the mesenchymal cells of the
feeder layer promotes the pre-specification of mesodermal precursors
into Isl1+ cardiac progenitors in vitro, while reducing the expansion
of already specified Isl1+ precursors. Furthermore, this study and
several others demonstrate that the in vivo activation of ␤-catenin
within the Isl1+ progenitors in the second heart field leads to their
massive accumulation in the pharyngeal mesoderm, the inhibition of
their differentiation, and the onset of outflow tract morphogenetic
defects (Cohen et al., 2007; Kwon et al., 2007). Similarly, loss-offunction studies have confirmed the requirement for ␤-catenin for the
expansion and survival of Isl1+ cardiac precursors in the embryo in
vivo (Lin et al., 2007). Interestingly, chemical agents that inhibit the
activity of glycogen synthase kinase 3 (GSK3), the kinase that
phosphorylates ␤-catenin and promotes its degradation, can markedly
promote the in vitro proliferation of ISL1+ cells from human neonatal
hearts, representing a key advance towards the eventual cloning of
human ISL1+ cardiac progenitors (Qyang et al., 2007).
+
Isl1 progenitors and congenital heart disease
Congenital heart disease (CHD), which is one of the most important
and prevalent forms of human birth defect, is present in nearly 1 in
100 live births and is responsible for the vast majority of prenatal
losses (Hoffman, 1995). Some congenital cardiovascular
malformations occur as syndromes in which multiple organs are
affected. Over the past decade, a number of single-gene mutations
have been correlated with syndromic CHDs, such as the association
of mutations in Tbx1 with DiGeorge syndrome and of Tbx5 with
Holt-Oram syndrome (for reviews, see Gruber and Epstein, 2004;
Ransom and Srivastava, 2007). Although these syndromes have
helped researchers to elucidate some of the mechanisms of CHD,
most CHD occurs in the absence of any other organ malformation.
In humans, 50% of these non-syndromic CHDs manifest as failed
atrial/ventricular septation or outflow tract defects, and necessitate
open-heart surgery to restore normal circulation. A subset of nonsyndromic CHD is familial, and causative genes have been
identified, most of which encode transcription factors that are part
of a conserved regulatory network that controls cardiogenesis (see
Table 1). Mutations in NKX2-5 have been identified in individuals
with atrial/ventricular septal defects and with conduction system
abnormalities, whereas mutations in GATA4 and TBX20 are found
in patients with septation defects, valve abnormalities and
cardiomyopathy (Gruber and Epstein, 2006; Schott et al., 1998;
Garg et al., 2003; Kirk et al., 2007). However, most non-syndromic
CHD cases are sporadic and multifactorial, with no single gene
being wholly responsible. Therefore, a major challenge is to dissect
the transcriptional and signaling pathways that regulate
cardiogenesis and to better understand at a genetic and mechanistic
level how a cardiac progenitor cell can develop into each of the
specific cell lineages that form the heart.
The identification of two distinct populations of cardiac
precursors, one that exclusively forms the left ventricle and the other
that mainly forms the outflow tract, the right ventricle and most of
the atria, hints at a new approach to understanding CHDs, not as a
defect in a specific gene or transcription factor, but rather as a defect
in the lineage decisions of a defined subset of cardiac precursors. In
this manner, CHDs might be associated with alterations in the
formation, expansion and differentiation of embryonic cardiac
progenitor cells, which in turn form essential components of the
Development 135 (2)
heart, such as the atria, ventricles, coronary arteries and conduction
system (Goldmuntz et al., 2001; Pashmforoush et al., 2004; Prall et
al., 2007). The discovery of a unique Isl1+ cardiovascular progenitor
that contributes to all of these structures in the heart has important
implications for understanding the mechanistic origins of CHD. In
this regard, a series of studies of the localization of Isl1+ progenitors
in patients with diverse forms of CHD (atresia of the aortic and
mitral valve, transposition of the great arteries, ventricular septal
defects and an interrupted aortic arch) have added important new
insights into the potential role of Isl1+ progenitors in the closure of
the atrial septum, in remodeling events that occur in the latest stages
of cardiac morphogenesis or in the immediate postnatal window
when the right ventricle starts to pump blood into the pulmonary
circulation (Laugwitz et al., 2005). Moreover, the recent discovery
of the role of ␤-catenin pathways in regulating the renewal and
differentiation of Isl1+ cardiovascular precursors in vivo (Cohen et
al., 2007; Kwon et al., 2007; Lin et al., 2007; Qyang et al., 2007) is
likely to have a significant impact on our understanding of several
forms of CHDs that involve outflow tract defects, especially those
implicating mal-rotations or outflow tract dysplasia.
Isl1+ progenitors and cardiovascular regenerative
medicine
Whereas the pivotal role of Isl1+ cardiovascular progenitor cells in the
formation of the major heart lineages is clear, the importance of these
cells in endogenous programs of cardiovascular regeneration is still
unknown. Cardiomyocytes rarely seem to enter the cell cycle after
birth and, consequently, the heart has a very limited regenerative
capacity following injury. Given that the number of Isl1+ cells within
the heart is vanishingly small after the postnatal window, it is unlikely
that they play a role in the regeneration of the adult working
myocardium. However, there is a persistence of the postnatal Isl1+
cells within the cardiac autonomic nervous system in regions that
intersect with the cardiac conduction system, raising the question as
to their role in the maintenance of normal cardiac conduction (Moretti
et al., 2006; Sun et al., 2007). The location of Isl1+ cells in the
embryonic/fetal structures that are associated with CHDs (outflow
tract, inter-atrial septum, etc) suggests that these cells might participate
in regenerative pathways in these tissues in response to genetic or
environmentally induced injury in the hearts of newborns. Uncovering
the stimuli that might lead to the in vivo mobilization of Isl1+
cardiovascular progenitor cells will be of importance for
understanding their contribution to endogenous regenerative
pathways.
The ability to isolate and clonally expand multipotent Isl1+
cardiovascular progenitors from mouse ES cells encourages the
hope that the same could be achieved in human ES cells. If so, it
could form the basis for the generation of models of human CHD
and of adult forms of heart disease. The technology to genetically
manipulate human ES cells is moving forward rapidly, as are
technologies that will allow the generation of ES cells via somatic
cell nuclear transfer (SCNT) without requiring the use of eggs
(Hochedlinger and Jaenisch, 2002). Moreover, recent work from
several independent groups has demonstrated the possibility of
reprogramming in vitro, differentiated mouse adult fibroblasts into
a pluripotent ES cell-like state through the ectopic expression of
only a few defined factors: Oct3/4 (also known as Pou5f1), Sox2, cMyc and Klf4 (Maherali et al., 2007; Okita et al., 2007; Takahashi
and Yamanaka, 2006; Wernig et al., 2007). This finding represents
an important achievement in controlling pluripotency and might
allow patient-specific pluripotent ES-like cells to be created directly
from somatic cells, which could be a powerful tool for studying
DEVELOPMENT
202
human disease in a ‘culture dish’. The human ES cell-based ISL1+
cardiovascular progenitor model system could prove to be extremely
valuable in designing new human-based assay systems for screening
the toxicity of new drugs during the early stages of cardiac
development, as well as for identifying and validating new
therapeutic targets in specific human cardiovascular cell types, such
as in coronary vascular smooth muscle, pulmonary arterial smooth
muscle, coronary endothelial cells and conduction system cells.
Conclusion
In the search for master cardiovascular genes, it has become
increasingly apparent that the generation of the diverse cell lineages
of the heart is likely to be due to the employment of combinatorial
codes for specific cell types, akin to the theory proposed by
Weintraub and colleagues almost 20 years ago (Davis et al., 1987).
At the same time, recent studies of Isl1+ cardiovascular progenitors
suggest that a key element of lineage diversification in the heart
relates to the presence of a renewable, rare subset of multipotent
cells, perhaps a master heart progenitor, that ultimately gives rise to
over two-thirds of the heart and to the heart’s three major cell types:
cardiac muscle, smooth muscle and endothelium. The generation of
specific cardiovascular cell types might ultimately relate more to a
series of decisions that result in a sequential increase in the
restriction of multipotent cardiovascular progenitors to specific
cellular intermediates and their differentiated derivatives, than to the
functioning of dominantly acting genes that enforce lineage
specification. As such, understanding cardiogenesis at the level of
specific decisions that are made by discrete heart cell lineages
requires that we identify the pathways that govern critical steps in
the formation, renewal, specification and differentiation of the
hierarchy of Isl1+ progenitors and their derivatives. The
identification of specific markers that enable FACS purification of
these intermediates, and genetic approaches to reveal key steps in
their cell fate decisions, will be essential. In short, if heart is like
blood, then approaching cardiogenesis as ‘cardiopoiesis’ might
represent the road ahead.
The authors especially thank Sylvia Evans for her continuous support. We also
thank members of the laboratories of Karl-Ludwig Laugwitz and Kenneth R.
Chien for their helpful discussions and comments. We apologize to colleagues
whose work is not mentioned here owing to space limitations. The authors are
supported by the Massachusetts General Hospital and the Cardiovascular
Disease Program of the Harvard Stem Cell Institute, a Marie Curie Excellence
Team Grant from the European Research Council (MEXT-23208), the German
Research Foundation (La 1238 3-1/4-1), the National Heart, Lung and Blood
Institute, and the Jean Le Ducq Foundation.
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