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First published online February 8, 2008
doi: 10.1242/10.1242/dev.016865
Review |
Cardiovascular Institute, Institute for Regenerative Medicine, Departments of Medicine and Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.
* Author for correspondence (e-mail: emorrise{at}mail.med.upenn.edu)
SUMMARY
Emerging evidence indicates that Wnt signaling regulates crucial aspects of cardiovascular biology (including cardiac morphogenesis, and the self-renewal and differentiation of cardiac progenitor cells). The ability of Wnt signaling to regulate such diverse aspects of cardiovascular development rests on the multifarious downstream and tangential targets affected by this pathway. Here, we discuss the roles for Wnt signaling in cardiac and vascular development, and focus on the emerging role of Wnt signaling in cardiovascular morphogenesis and progenitor cell self-renewal.
Introduction
Although its role in the differentiation of many cell types has been explored in detail, the role of Wnt signaling in cardiovascular morphogenesis and differentiation is less well understood. Previous studies have suggested a model in which β-catenin-dependent canonical Wnt signaling inhibits cardiac specification while Jun-N-terminal kinase (JNK)-dependent, non-canonical Wnt signaling promotes cardiovascular development. However, recent findings have indicated that canonical Wnt signaling has a more complex temporal role during cardiac differentiation than previously thought, with an activating role in specification and an inhibitory role in later cardiomyocyte differentiation (see Table 1).
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Cardiovascular development
The mouse heart develops from two domains of anterolateral mesoderm that
fuse together at the anterior midline to form a crescent-shaped swathe of
mesoderm that underlies the head folds and is known as the cardiac crescent
(Fig. 1). The cells located at
the lateral edges of the cardiac crescent migrate medially towards the ventral
midline, where the opposing edges of the cardiac crescent meet and fuse
together to form the initial heart tube. As the heart tube forms, cells within
this primary or first heart field (FHF) differentiate into functional
cardiomyocytes and the heart begins to beat. Meanwhile, a second field of
cardiogenic mesoderm, or second heart field (SHF), initially located medial to
the edges of the cardiac crescent, is displaced dorsally into the pharyngeal
mesoderm by the heart tube. As the heart tube undergoes rightward looping and
forms distinct chambers, cells from SHF migrate into both the inflow and
outflow tracts of the heart, and contribute most of the cells within the
outflow tract and right ventricle, a substantial proportion of cells within
the atria and some cells to the left ventricle
(Kelly et al., 2001
;
Kelly and Buckingham,
2002).
The expression of several transcription factors, including GATA binding
protein 4 (Gata4), NK2 transcription factor related 5
(Nkx2.5) and myocte enhancer factor 2c (Mef2c), mark both
the FHF and SHF (Kuo et al.,
1997; Lin et al.,
1997; Lyons et al.,
1995; Molkentin et al.,
1997). Additionally, the LIM-homeodomain gene islet 1
(Isl1) is expressed in the SHF and marks this group of cardiac
progenitors as a distinct cell population
(Cai et al., 2003). Although
mutations in these genes cause severe heart defects, none of these factors
appears to be solely required for the specification of cardiogenic mesoderm
(Cai et al., 2003;
Kuo et al., 1997;
Lin et al., 1997;
Lyons et al., 1995;
Molkentin et al., 1997).
Cardiac specification must therefore rely on a more complex network of
interactions between these factors. These interactions are likely to be
mediated by several families of secreted signaling molecules and their
associated signaling pathways implicated in heart development. These include
bone morphogenic proteins (BMPs), transforming growth factor β
(TGFβ) family members, fibroblast growth factors (FGFs) and, more
recently, Wnt proteins.
Wnt signaling: many components, multiple pathways
Wnt proteins are a large family of secreted signaling molecules that
regulate crucial aspects of development, including cell-fate specification,
proliferation, survival, migration and adhesion (for a review, see
Nusse, 2005). Many of these
effects are mediated by a canonical Wnt signaling pathway
(Fig. 2A), which begins with
Wnt proteins binding to a co-receptor complex that consists of frizzled (Fzd)
family, seven-pass transmembrane proteins and the lipoprotein receptor related
5/6 (Lrp5/6) proteins. Wnt receptor binding activates the intracellular
effector protein dishevelled (Dvl), which in turn inactivates a protein
complex that includes the constitutively active serine-threonine kinase
glycogen synthase kinase 3β (Gsk3β), as well as the scaffolding
proteins axin and adenomatosis polyposis coli (APC). This complex normally
phosphorylates β-catenin and targets it for degradation. Upon Wnt
stimulation, however, the inhibition of the degradation complex allows high
levels of β-catenin to accumulate in the nucleus, where it complexes with
LEF/TCF family DNA binding proteins to activate the transcription of Wnt
target genes.
While the canonical Wnt pathway mediates many Wnt effects, some Wnt
regulated processes are β-catenin independent. This non-canonical Wnt
signaling (Fig. 2B) is often
attributed to one of two pathways termed the Ca2+/protein kinase C
(PKC) and RhoA/JNK pathways (for a review, see
Veeman et al., 2003). In
Ca2+/PKC signaling, Wnt binding activates the heterotrimeric
G-protein-dependent activity of Fzd receptors, which in turn activate
intracellular Ca2+ signaling, as well as Ca2+-dependent
protein kinases, such as protein kinase C (PKC) and calmodulin-dependent
protein kinase II. In RhoA/JNK signaling, Wnt proteins activate Rho family
GTPases, such as RhoA and Rac, as well as their downstream effectors,
including Rho associated kinase (ROCK) and JNK through the Dvl protein.
Although the Ca2+/PKC and RhoA/JNK pathways mediate most
β-catenin-independent, non-canonical Wnt signaling, it is still unclear
whether these pathways are distinct from one another, and the combinations of
effectors that mediate this signaling often vary. Furthermore, activating
non-canonical Wnt signaling attenuates canonical Wnt signaling in some systems
(Pandur et al., 2002;
Schneider and Mercola, 2001).
This raises the intriguing possibility that these pathways are actually part
of a larger Wnt signaling network in which unique combinations of effectors
are activated in a cell type-dependent manner.
There are 19 Wnt proteins, 10 Fzd receptors and two Lrp co-receptors in
mammals, suggesting that Wnt signaling specificity may be immensely complex.
Wnt proteins have traditionally been separated into two classes based on their
ability to induce secondary axis formation in frog embryos and to transform
mammary epithelial cells, both of which rely on canonical Wnt signaling. For
example, expression of Wnt1, Wnt3 and Wnt8 induce a
secondary axis in Xenopus and transform mammary epithelial cells and
are thought to signal through the canonical Wnt pathway, while expression of
Wnt5a and Wnt11 does not, and these ligands are thought to
act primarily through non-canonical Wnt pathways
(Shimizu et al., 1997;
Wong et al., 1994). However,
more-recent studies suggest that individual Wnt proteins can activate either
canonical or non-canonical Wnt signaling, depending on cellular context
(Mikels and Nusse, 2006;
Tu et al., 2007). Thus, the
decision to signal through the canonical versus non-canonical pathway is
likely to depend on the specific Wnt-Fzd combinations that are present at the
cell surface, as well as on the intracellular factors that shunt signaling
between the two pathways. This issue remains one of the most intriguing, but
poorly understood, aspects of Wnt signaling, and has an enormous impact on our
understanding of Wnt signaling in cardiovascular development, as discussed
below.
Wnt expression and function during cardiac specification and early differentiation
Studies on chick and frog embryos indicate that the initial specification
of cardiac tissue is governed by the balanced expression of canonical Wnt
activators and repressors (Fig.
3A). Activating canonical Wnt signaling in the anterior mesoderm
inhibits the expression of early cardiac genes, including Nkx2.5 and
Gata4, in the cardiac crescent
(Marvin et al., 2001;
Schneider and Mercola, 2001).
Canonical Wnts such as Wnt1 and Wnt3a, which are expressed
in the neural plate and dorsal neural tube, are therefore thought to inhibit
cardiac specification in the posterior-medial mesoderm. Conversely, several
secreted Wnt antagonists including crescent, which competes with Fzd
for Wnt binding via its homology to the Fzd extracellular domain, and also
Dikkopf (Dkk), which inhibits canonical Wnt signaling by
causing Lrp5/6 receptor internalization, are expressed in the endoderm that
underlies the cardiac mesoderm (Schneider
and Mercola, 2001). The expression of crescent or
Dkk in the non-cardiac posterior mesoderm induces cardiac gene
expression and the appearance of beating cardiomyocytes
(Marvin et al., 2001;
Schneider and Mercola, 2001).
The inhibition of canonical Wnt signaling by crescent and
Dkk is therefore thought to promote the specification of cardiac
precursors in the anterolateral mesoderm that forms the cardiac crescent.
|
;
Ueno et al., 2007). Blocking
canonical Wnt signaling during this early period of differentiation inhibits
the expression of early cardiac markers and the appearance of beating
cardiomyocytes (Naito et al.,
2006). These data suggest that canonical Wnt signaling acts early
to potentiate cardiac specification. Interestingly, the activation of
canonical Wnt signaling slightly later during ES cell differentiation blocks
cardiac induction and differentiation
(Naito et al., 2006).
Consistent with these data, the deletion of β-catenin in the definitive
endoderm during mouse development leads to ectopic heart induction along the
body axis (Lickert et al.,
2002). These data indicate that the role for canonical Wnt
signaling in early cardiogenesis is, at least in part, due to a cell
autonomous role for Wnt signaling in the early endoderm. Although this early
positive role for Wnt signaling in cardiac induction may be specific to mouse
embryos, it is more likely that it was masked in previous studies in chick and
frog embryos by the inability to temporally control the over-activation of
canonical Wnt signaling in these experiments.
The possibility that canonical Wnt signaling plays an early positive role
in cardiac induction is especially interesting in light of recent data that
Wnt2a is required for cardiac differentiation in ES cells. Wnt2a has
been shown to activate canonical Wnt signaling in several contexts and is
expressed in the cardiac crescent (Monkley
et al., 1996). Wnt2a-deficient ES cells exhibit enhanced
hematopoietic differentiation at the expense of cardiac and endothelial cell
types (Wang et al., 2007).
Thus, Wnt2a, and possibly its homologue Wnt2b, may play an
important role in the specification of cardiac cell types from the early
mesoderm. Although defects in heart development have not been reported in
Wnt2a knockout mice, redundancy with Wnt2b or with other
canonical Wnts, such as Wnt8a, which is expressed throughout the
early heart tube (see Fig. 4A),
may mask such effects in vivo (Jaspard et
al., 2000; Monkley et al.,
1996; Zakin et al.,
1998). Taken together, these data show that canonical Wnt
signaling plays a biphasic role in mouse cardiac induction, positively
regulating cardiac gene expression early and then inhibiting cardiac
differentiation at a later stage.
Non-canonical Wnt signaling by Wnt11 is also required for heart
specification. Wnt11 is expressed in the anterolateral mesoderm that
is fated to become the cardiac crescent
(Christiansen et al., 1996;
Eisenberg and Eisenberg, 1999;
Garriock et al., 2005;
Pandur et al., 2002). Blocking
Wnt11 signaling in the anterior mesoderm of Xenopus embryos blocks
the expression of early cardiac genes, including Nkx2.5, Gata4 and
Tbx5, while expressing Wnt11 in the posterior mesoderm of
frog and chick embryos induces ectopic expression of these markers, as well as
the appearance of beating cardiomyocytes
(Eisenberg and Eisenberg,
1999; Schneider and Mercola,
2001). In Xenopus animal pole explants, which normally
take on a neuro-ectodermal fate, Wnt11 induces cardiac tissues without
inducing the expression of pan-mesodermal markers, suggesting that the effect
of Wnt11 on cardiac specification is direct and not the result of increased
mesoderm induction (Pandur et al.,
2002). Wnt11 expression similarly coincides with the
onset of cardiac gene expression in differentiating mouse ES cells and
treating these cells with Wnt11 increases the specification of cardiac
progenitors, indicating that Wnt11 also plays an essential role in
murine heart induction (Ueno et al.,
2007). Inhibiting either JNK or PKC signaling blocks the ability
of Wnt11 to induce cardiac specification, while co-activating JNK and PKC
induces cardiac specification, indicating that both the RhoA/JNK and Ca2+/PKC
pathways mediate Wnt11 signaling (Pandur
et al., 2002). Taken together, these data indicate that the
activation of non-canonical Wnt signaling by Wnt11 is required for the
induction of cardiac tissues through JNK and PKC signaling.
Wnt signaling in the specification and expansion of secondary heart field progenitors
Several well-characterized mouse strains, TOPGAL, BAT-GAL and TCF/Lef-lacZ,
contain transgenic reporters that express β-galactosidase from
multimerized LEF/TCF DNA-binding sites and allow canonical Wnt signaling to be
visualized in vivo (DasGupta and Fuchs,
1999; Maretto et al.,
2003; Mohamed et al.,
2004). These reporters are active during the development of
multiple cardiac structures, including the pericardium, which surrounds the
heart, the endocardial cushions, which differentiate into the
atrio-ventricular (AV) valves, and the outflow tract (OFT)
(Gitler et al., 2003;
Maretto et al., 2003).
Remarkably, none of these reporters displays high levels of activity in the
developing myocardium, despite the expression of Wnt8a, a strong
activator of the canonical Wnt pathway, and β-catenin throughout the
developing heart tube (Cohen et al.,
2007; DasGupta and Fuchs,
1999; Lin et al.,
2007; Maretto et al.,
2003).
|
).
Consistent with these data, Isl1 positive (Isl1+) cardiac
progenitor cells from the SHF display high levels of BAT-GAL activity in both
the pharyngeal mesenchyme and OFT as they migrate into the heart tube
(Cohen et al., 2007).
Moreover, loss of β-catenin expression in the SHF leads to a dramatic
reduction in the numbers of Isl1+ cardiac progenitor cells and the
subsequent loss of SHF derived structures, while activating β-catenin
signaling in the SHF leads to an increase in the numbers of Isl1+
cells (Ai et al., 2007;
Cohen et al., 2007;
Kwon et al., 2007;
Lin et al., 2007). Taken
together, these data indicate that canonical Wnt/β-catenin signaling in
the SHF and OFT plays an essential role in Isl1+ cardiac progenitor
cell expansion (Fig. 5).
Canonical Wnt/β-catenin signaling appears to regulate both the
specification and the proliferation of Isl1+ cardiac progenitor
cells (reviewed by Laugwitz et al.,
2008). The deletion of β-catenin in Isl1-cre mice, in which
cre-recombinase is inserted into the Isl1 locus, causes a dramatic
reduction both in the levels of Isl1 expression and the numbers of
cells that express Isl1 (Cohen et al.,
2007; Lin et al.,
2007). Consistent with this result, chromatin immunoprecipitation
and in vitro reporter assays indicate that β-catenin directly binds to
and regulates the Isl1 promoter
(Lin et al., 2007). These data
strongly suggest that canonical Wnt/β-catenin signaling plays a role in
the initiation of Isl1 expression and in the specification of
Isl1+ cardiac progenitors. Additionally, deleting β-catenin in
the heart with either the SM22
-cre mouse line, in which cre is
expressed in both myocardial and vascular smooth muscle cells, or the
Nkx2.5-cre line, in which cre is expressed in myocardial cells, causes a
reduction in the overall numbers of Isl1+ cells without affecting
the levels of Isl1 expression
(Cohen et al., 2007;
Kwon et al., 2007). This
effect appears to result from a dramatic reduction in the proliferation of
Isl1+ cells in the OFT, preventing the expansion of these
progenitors (Ai et al., 2007;
Cohen et al., 2007;
Kwon et al., 2007;
Lin et al., 2007). Canonical
Wnt/β-catenin signaling therefore regulates both the specification and
the proliferation of the Isl1+ cardiac progenitors of the SHF.
The effect of β-catenin on the proliferation of Isl1+ SHF
cells is mediated, at least in part, by a reduction in FGF signaling. SHF
cells express high levels of Fgf10 when in the pharyngeal mesenchyme,
as well as when in the OFT and right ventricle
(Cohen et al., 2007). Although
mutations in Fgf10 do not cause defects in right heart structures,
mutations in Fgfr2, the Fgf10 receptor, cause severe OFT and
right ventricle phenotypes, consistent with FGF10 acting redundantly with
other FGF ligands (Marguerie et al.,
2006). In support of this hypothesis, Fgf3, Fgf16 and
Fgf20 are also induced by Wnt signaling in SHF explants
(Cohen et al., 2007).
Moreover, treating SHF explants with an inhibitor of FGF signaling blocks the
expansion of Isl1+ cells caused by the addition of purified Wnt3a
to the culture media, suggesting that increased FGF signaling mediates the
effects of Wnt signaling on SHF cells
(Cohen et al., 2007). Thus,
Wnt signaling acts upstream of a complex FGF-mediated pathway that includes
the Fgf3, Fgf10, Fgf16 and Fgf20 genes. Moreover, several
observations point to both Fgf10 and Fgf20 as being direct
targets of Wnt/β-catenin signaling. In the case of Fgf10, its
expression is strongly reduced in β-catenin mutant hearts, as are levels
of ERK1/2 phosphorylation. Reporter constructs that contain the Fgf10
promoter are responsive to β-catenin signaling, and chromatin
immunoprecipitation assays indicate that β-catenin binds to the
Fgf10 promoter in vivo (Cohen et
al., 2007). In the case of Fgf20, it has been shown to be
a direct target of Wnt/β-catenin signaling by similar criteria, including
its induction by exogenous Wnts, reduction in its expression through
inhibition of Wnt signaling, and by the fact that β-catenin forms a
complex on the Fgf20 promoter
(Chamorro et al., 2005).
Wnt-FGF signaling is also required for zebrafish fin regeneration, indicating
a wider use of such a signaling axis beyond that observed in the heart, in
tissue regeneration and in the activation of resident stem/progenitor cells
(Stoick-Cooper et al.,
2007).
|
) (reviewed by Laugwitz
et al., 2008). A small molecule screen was performed to identify
compounds that could drive the expansion of Isl1+ progenitors in
vitro, which resulted in the identification of
6-bromoindirubin-3'-oxime, also known as BIO, a known Gsk3β
inhibitor. Addition of BIO or of recombinant Wnt3a to the medium of purified
Isl1+ cardiac progenitors leads to their dramatic expansion. This
finding suggests that it may be possible to harness canonical Wnt signaling to
expand such progenitor cells either in vivo or ex vivo for tissue replacement,
although the ability of expanded progenitors to fully differentiate into
cardiomyocytes has not been tested. Along with a recent report in zebrafish
that show that TOPGAL activity is activated at high levels in zebrafish heart
regeneration (Stoick-Cooper et al.,
2007), these studies provide some of the first clues as to how Wnt
signaling may be harnessed as a potent regeneration pathway for the heart.
However, whether Wnt signaling can activate cardiac regeneration in higher
vertebrates is unknown and will require direct testing, most importantly in
mouse models, as mammals do not exhibit the same ability for cardiac
regeneration after injury as lower vertebrates. Non-canonical Wnt signaling in cardiac morphogenesis
In addition to its potential role in cardiac specification and progenitor
expansion, non-canonical signaling by Wnt11 plays a crucial role in
cardiac morphogenesis. During Xenopus development Wnt11R, a
second Wnt11 gene found in lower vertebrates, is expressed in the
precardiac mesoderm just prior to the fusion of the cardiac primordia at the
ventral midline (Garriock et al.,
2005). Inhibiting Wnt11R signaling by morpholino injection
disrupts the fusion of the cardiac primordia at ventral midline resulting in
cardia bifida. The overexpression of Wnt11R activates JNK in
Xenopus embryos and the pharmacological inhibition of JNK results in
a phenotype that is similar to the loss of Wnt11R. These data suggest that
Wnt11R regulates cell movements that are important for the proper
migration of early cardiac precursors to fuse and form the primitive heart
tube through a JNK-mediated pathway.
Two non-canonical Wnt genes, Wnt5a and Wnt11, are
expressed in the OFT of the developing mouse heart, and mutations in these
genes cause OFT defects, including double outlet-right ventricle (DORV) and
persistent truncus arteriosus (PTA), which are identical to some of the most
common forms of human CHD (Schleiffarth et
al., 2007; Zhou et al.,
2007). The Wnt5a and Wnt11 mutant mouse
phenotypes are associated with disrupted cytoskeletal architecture in both the
myocardial and smooth muscle components of the OFT, as well as reductions in
matrix deposition and in the expression of matrix adhesion receptors.
Interestingly, Wnt11 positively regulates TGFβ2 expression in the OFT
through the JNK-dependent activation of activating transcription factor/cAMP
responsive element binding protein (ATF/CREB) family transcription factors
(Zhou et al., 2007). Mutations
in Tgfb2 cause OFT phenotypes similar to those caused by the loss of
Wnt5a and Wnt11 function, suggesting that the effects of
non-canonical Wnt signaling on cardiac morphogenesis are mediated, at least in
part, by Tgfβ2 signaling (Bartram et
al., 2001).
Other components of non-canonical Wnt signaling have demonstrated roles in
cardiovascular morphogenesis. Van Gogh/strabismus is a cell surface
transmembrane protein that has a key role in the planar cell polarity (PCP)
pathway in Drosophila (Wolff and
Rubin, 1998). Mice have two Van Gogh/strabismus genes
(Vangl1 and Vangl2), and a naturally occurring
Vangl2 mutant, called the loop-tail (Lp) mouse,
exhibits defects in OFT development that leads to DORV
(Henderson et al., 2006;
Phillips et al., 2005). In
Lp mice, this defect is associated with aortic arch abnormalities,
including persistent right-sided aorta. Phillips et al. showed that the
myocardial cells in the OFT of Lp embryos have less extensive
lamellipodia, indicating that these cells have defective cell-cell and
cell-ECM interactions (Phillips et al.,
2005). Given the importance of the PCP pathway in cell migration
and polarity, these data suggest that Vangl2 plays an important role in
cardiac OFT development through the regulation of cell migration and cell-ECM
interactions. Whether the OFT defects observed in Wnt5a, Wnt11 and
Vangl2 mutants are due to the expression of these genes in the neural
crest or in the SHF-derived myocardium is unknown and will require the
conditional inactivation of these genes in mice for clarification. However,
the defects observed in mice that carry mutations in these genes strongly
implicate non-canonical Wnt signaling, and possibly the PCP pathway, in OFT
development.
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In addition to the myocardium, Wnt signaling has been implicated in the
development of cardiac neural crest cells (CNCCs). CNCCs delaminate from the
dorsal neural tube and migrate first into the pharyngeal arches and, second,
along the arch arteries into the OFT. These cells form the initial OFT septum,
become the smooth muscle layer of the ascending aortic arch and contribute to
the OFT cushion mesenchyme. Two canonical Wnt genes, Wnt1 and
Wnt3a, are expressed in the dorsal region of the neural tube from
which the CNCCs arise and have been implicated in CNCC specification and
delamination. Furthermore, mutations in Dvl2 cause OFT defects that
resemble those caused by the ablation of the CNCCs, such as DORV and PTA, and
the loss of CNCC-specific markers (Hamblet
et al., 2002). Consistent with these results, the tissue-specific
deletion of β-catenin from CNCCs results in OFT defects similar to those
seen in Dvl2 mouse mutants, as well as a severe decrease in CNCC
proliferation (Kioussi et al.,
2002).
The effects of canonical Wnt/β-catenin signaling on CNCCs may be
mediated by both the transcriptional upregulation and functional activation of
paired-like homeodomain 2 (Pitx2). Pitx2 expression is lost
in both Dvl2 mutant mouse embryos and in embryos in which
β-catenin has been conditionally deleted in the CNCCs with
Wnt1-cre. The complete knockout of Pitx2 results in OFT defects that
are similar to those seen in these mutants
(Kioussi et al., 2002).
Furthermore, β-catenin complexes with Pitx2 and activates Pitx2
target gene transcription in a manner that is analogous to its activation of
LEF/TCF. Deleting a floxed-allele of Pitx2 with Wnt1-cre does not
result in OFT phenotypes, whereas deleting Pitx2 in the SHF with
either Isl1-cre or Mef2c-cre, both of which drive the expression of cre in the
SHF, results in OFT defects that are identical to those reported in the
Dvl2 knockout mouse or following the CNCC-specific deletion of
β-catenin (Ai et al.,
2006). These data indicate that Pitx2 has an autonomous
role in SHF development and that its role in the CNCCs is mediated by
paracrine signaling between these two closely apposed cell populations. How
this signaling occurs is unclear but recent evidence shows that Pitx2 is
required for Wnt11 expression in the OFT, indicating that the effects
of Pitx2 deletion are mediated by paracrine Wnt11 signaling from the
SHF cells to the CNCCs (Zhou et al.,
2007). Alternatively, the cell-cell adhesion function of
β-catenin might be responsible for some aspect of these defects
(Luo et al., 2006). This is
supported by the phenotype of mice in which N-cadherin has been deleted in the
CNCCs, which results in similar OFT defects.
Wnt and β-catenin in adult myocardium
As mentioned above, there is little evidence that active canonical Wnt
signaling occurs in the primary developing or adult myocardium. This view is
based primarily on studies that have used the TOPGAL and BAT-GAL reporter
mice, and it contrasts with several reports of Wnt ligand expression being
present in the adult mammalian heart
(Garriock et al., 2005;
Jaspard et al., 2000;
Monkley et al., 1996;
Zakin et al., 1998). Other
studies have suggested an important role for β-catenin and Gsk3β in
postnatal myocardial growth (Hardt and
Sadoshima, 2002; Masuelli et
al., 2003; Tseng et al.,
2006). These studies have, for the most part, not invoked a Wnt
ligand and are based on the role of Gsk3β in stabilizing β-catenin,
leading to increased LEF/TCF activation. Stabilized β-catenin in isolated
cardiac myocytes or in vivo results in increased myocyte growth with or
without the presence of hypertrophic stimuli
(Haq et al., 2003;
Tseng et al., 2006). This
stabilization of β-catenin is thought to occur in the heart through a
protein kinase B/Akt-dependent pathway and not through Wnt activation.
Additional studies have demonstrated that the inhibition of LEF/TCF-dependent
transcription through the expression of a dominant-negative LEF protein or
through the postnatal deletion of β-catenin results in decreased cardiac
growth and in the loss of the hypertrophic response that usually occurs after
the surgical constriction of the aorta (a technique called aortic banding)
(Chen et al., 2006). These
studies are supported by recent findings that the treatment of mammalian
cardiomyocytes with the Gsk3β inhibitor BIO leads to their increased
proliferation in culture (Tseng et al.,
2006). From these studies, Gsk3β is implicated as being an
integration point for cardiomyocyte growth and hypertrophy in response to
stress. The question remains: do any of the other pathways known to regulate
Gsk3β activity stabilize β-catenin in the heart? Recent evidence
suggests that histone deacetylase 2 (Hdac2) can inhibit Gsk3β through the
regulation of an inositol polyphosphate-5-phosphatase and Akt dependent
pathway (Trivedi et al.,
2007). Although this study did not look at β-catenin
stabilization, it would be worth determining whether changes in cardiac growth
and whether stress-induced hypertrophy can be correlated with altered
β-catenin signaling. Together, these studies indicate that the activation
of Gsk3β/β-catenin signaling is important for normal cardiomyocyte
growth and proliferation, but they do not directly implicate Wnt signaling per
se. The failure to implicate a Wnt ligand directly in this physiological
process could be the result of the significant redundancy that exists between
the Wnt ligands that are expressed in both the developing and postnatal heart,
the limitations of the TOPGAL and BAT-GAL Wnt reporter mice, or the fact that
β-catenin/LEF-TCF signaling is affected by other non-Wnt signals, such as
Akt. More extensive genetic experiments using Wnt-ligand or frizzled-receptor
loss-of-function models, particularly in mice, will be required to determine
whether Wnt signaling is directly involved in these processes.
|
Wnt signaling in endothelial and vascular smooth muscle cells
Evidence for an in vivo role for Wnt/β-catenin signaling in vascular
development and remodeling has been reported. Deletion of β-catenin in
developing endothelial cells (EC) leads to embryonic death and loss of ECs
integrity (Cattelino et al.,
2003). Although the authors of this report implicated
β-catenin in cell-cell adhesion, rather than in Wnt signaling in
developing EC in the peripheral vasculature, part of the phenotype could be
attributed to a loss of canonical Wnt signaling, even though TOPGAL activity
is not apparent in these cells. EC-specific loss of β-catenin also leads
to defective endocardial cushion/cardiac valve development through defective
endothelial-mesenchymal transformation, and this process coincides with a
decrease in TOPGAL reporter activity in the developing endocardial cushions
(Liebner et al., 2004).
Several Wnt ligands have been implicated in regulating EC development. In vivo
loss of Wnt2a in mice leads to defective placental vascular
development, with a reduced number of fetal-derived placental capillaries
(Monkley et al., 1996).
Moreover, Wnt2a mutant ES cell lines show impaired endothelial
maturation and vascular plexus formation
(Wang et al., 2007).
Interestingly, loss of Fzd5 also leads to defective placental
vascular development and embryonic lethality
(Ishikawa et al., 2001).
Synergy between Wnt2a and Fzd5 in ectopic axis induction assays in
Xenopus embryos supports the notion that this ligand-receptor pair
cooperatively regulate EC development
(Ishikawa et al., 2001).
Wnt7b has been implicated in apoptosis-mediated retinal EC regression
associated with postnatal development
(Lobov et al., 2005).
Interestingly, the source of Wnt7b in this system is circulating macrophages,
indicating the existence of a precise short-range paracrine mechanism for
Wnt-mediated regulation of retinal EC development. Wnt5a has also been shown
to regulate angiogenesis and to induce endothelial cell proliferation via the
non-canonical pathway (Masckauchan et al.,
2006). In the same report, the authors showed that inhibition of
Wnt5a expression leads to reduced capillary branching in matrigel
assays. Together, these data support an important role for Wnt signaling in
endothelial cell development with the functional role dictated by the
endothelial bed and by the Wnt ligands that are expressed.
A role for Wnt signaling in the developing epicardium, where progenitors
for the coronary vasculature reside, has also been demonstrated. The
contribution of the epicardium to the developing coronaries has been
substantiated using fate-mapping analysis with a promoter fragment from the
chicken GATA5 gene driving cre expression
(Merki et al., 2005). Merki et
al. showed that loss of retinoid X receptor specifically in the
developing epicardium resulted in decreased β-catenin protein stability
and correlates with decreased Wnt9b expression
(Merki et al., 2005). This
leads to a failure in epicardial development and to the loss of ventricular
compaction and to defective coronary vascular branching. Whether Wnt9a is the
critical ligand in this process or acts redundantly with other Wnts awaits in
vivo confirmation. This study reiterates the importance of Wnt signaling in
epithelial-mesenchymal transformation, as in the developing heart valves
mentioned above, and supports the notion that epicardial-derived paracrine
signals, including Wnt, are of crucial importance for myocardial
development.
As mentioned above, active Wnt signaling can be observed in VSMCs of the
OFT during early development. However, what the roles, if any, of Wnt
signaling are in VSMC development in other tissues remains relatively unknown.
Some clues come from a study that shows that loss of paracrine Wnt7b signaling
leads to defective VSMC development in the lung
(Shu et al., 2002).
Wnt7b is expressed at high levels in the developing airway epithelium
during development but is absent from mesenchymal cell types in the lung,
including the vasculature. Wnt7blacZ-/- mutants develop
abnormally large blood vessels during embryogenesis, and at birth, when
pulmonary vascular pressure increases dramatically, these mutants display
profound vessel rupture and hemorrhage around these abnormal vessels
(Shu et al., 2002). Subsequent
studies to determine the mechanism of Wnt7b signaling revealed that
Wnt7b signals via the canonical pathway preferentially through Fzd1 and Fzd10
and the co-receptor Lrp5 (Wang et al.,
2005). Wnt7b did not activate non-canonical JNK signaling in these
experiments, although this ligand has been reported to activate other aspects
of non-canoncial Wnt signaling, including protein kinase C 
(PKC)-dependent pathways (Tu et
al., 2007). This again reiterates the importance of experimentally
determining the mechanism and downstream effectors of a given Wnt ligand in
the specific cell type of interest.
In VSMCs, Wnt signaling appears to regulate cell proliferation. Quasnichka
et al. found that inhibition of canonical Wnt signaling resulted in decreased
VSMC proliferation and cyclin D1 expression, while increasing the
expression of the cyclin dependent kinase inhibitor p21
(Quasnichka et al., 2006).
Myc, a proliferation-associated Wnt target gene, is overexpressed in
proliferating medial and intimal smooth muscle cells in human stenotic bypass
grafts (Hilker et al., 2001).
Given that cyclin D1 and Myc are well known targets of
canonical Wnt signaling, these studies suggest that Wnt signaling acts to
regulate cell proliferation in VSMCs during development and possibly after
injury. Other studies that show canonical Wnt signaling is activated after rat
carotid vessels are injured support this hypothesis and suggests that Wnt
signaling could become a target of anti-angiogenic or anti-restinotic
therapies (Wang et al.,
2002).
Conclusion
Wnt signaling has been known for many years to be one of the primary pathways that is involved in directing embryonic development. Given its crucial role in controlling proliferation and differentiation in other tissue systems, the finding that Wnt signaling regulates similar processes in the various cell types of the vertebrate cardiovascular system is at first glance not surprising. However, it is becoming increasingly clear that Wnts do not simply promote these processes but rather regulate and fine tune them in a cell-type- and temporally-specific manner.
One of the most important unanswered questions that remain concerning Wnt signaling in cardiac development is what function does canonical Wnt signaling serve in the primary myocardium during development? Given the high-level expression of Wnt2a, Wnt2b and Wnt8a in the developing mouse heart, it will be important to determine through genetic loss-of-function techniques whether any of these individually or in combination regulate cardiac differentiation or morphogenesis. Much has been gained from the genetic ablation of β-catenin but interpreting the findings that arise from this approach is complicated by the multiple roles that β-catenin has in cell-cell adhesion and Wnt signaling. The role of non-canonical versus canonical Wnt signaling and whether non-canonical signaling antagonizes canonical signaling in the developing myocardium are also concepts that need to be addressed directly using in vivo loss-of-function mouse models. Given the expression of multiple components of non-canonical Wnt signaling in the myocardium (such as RhoA, Daam1, etc.), one reason why active canonical Wnt signaling is not observed in the developing myocardium could be the presence of high levels of non-canonical Wnt signaling.
Recent evidence that Wnt/β-catenin signaling drives the expansion of
Isl1+ cardiac progenitors and is activated during zebrafish heart
regeneration also underline the importance of this pathway in stem cell
biology and tissue regeneration
(Stoick-Cooper et al., 2007).
It is no longer speculative to assert that Wnt signaling is a key pathway in
the regenerative process in many tissues, including the heart. An important
challenge in the future will be to determine when the activation of Wnt
signaling could promote cardiac regeneration and which cardiac stem/progenitor
cells of the many types reported in the literature are responsive to Wnt
signaling. To advance the field significantly, it is essential that such
studies include the further analysis of potential cardiac stem/progenitor cell
populations using in vivo fate-mapping techniques to determine whether these
cells can indeed generate new myocardium.
The re-activation of β-catenin signaling after cardiac stress and
vascular injury also suggests that this pathway may provide an important new
therapeutic area for intervention in human cardiovascular diseases as diverse
as heart failure and re-stenosis after vascular injury. Wnt signaling is
clearly a major cause of certain colon and intestinal tumors and probably
plays a role in tumorigenesis in other tissues, including the skin
(Chan et al., 1999;
Korinek et al., 1997;
Morin et al., 1997). Along
with this attention has come increasing interest in identifying
pharmacological antagonists and agonists of Wnt signaling. These compounds,
which are being developed as anti-cancer therapies, could provide unique
opportunities to address the enormous clinical impact of cardiovascular
disease.
Studies into the paradigm that the response to injury in adult tissues leads to the reactivation of embryonic gene expression programs required for tissue repair, has revealed that some of these important signaling pathways, including the Wnt pathway, are valuable targets for future medicines. It should come as no surprise if in the next decade we see novel therapies directed towards both cancer and cardiovascular disease based on modulating Wnt signaling. However, these studies need to include the best of in vivo genetic work in the mouse coupled with exacting biochemical analysis of how Wnt signaling promotes important developmental processes required for tissue repair. Only then will the fruits of basic research be translated into the discovery of treatments for human ailments.
ACKNOWLEDGMENTS
The authors thank members of the Morrisey laboratory for discussion and input during the process of writing this review. E.E.M. is supported by funding from the National Institutes for Health and the American Heart Association. E.D.C. is supported by a postdoctoral fellowship grant from the American Heart Association.
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)-dependent Ca2+ release.
This activates PKC and calmodulin-dependent protein kinase II (CaMKII). In
RhoA/JNK signaling (yellow), Wnt proteins activate Rho signaling, including
Rho/Rac, and JNK through Dvl, leading to ATF/CREB activation. Non-canonical
Wnt signaling often antagonizes β-catenin-dependent canonical signaling
through mechanisms that remain poorly understood. PM, plasma membrane.
