DEVELOPMENT AND STEM CELLSRESEARCH ARTICLE 1931
Development 139, 1931-1940 (2012) doi:10.1242/dev.069377
© 2012. Published by The Company of Biologists Ltd
Wnt5a and Wnt11 are essential for second heart field
The heart arises from two domains of lateral mesoderm that join at
the anterior midline to form a crescent-shaped swathe of
cardiogenic mesoderm under the head folds known as the cardiac
crescent (Dyer and Kirby, 2009; Watanabe and Buckingham,
2010). The cardiac crescent contains two distinct fields of cardiac
progenitors. The first heart field (FHF) occupies the medial region
of the crescent and will coalesce at the ventral midline to form the
initial linear heart tube, which is fated to become the myocardium
of the left ventricle (LV). The second heart field (SHF) occupies
more lateral regions of the crescent that are shifted dorsally into the
pharyngeal mesenchyme as the heart tube fuses. SHF progenitors
remain undifferentiated in the pharyngeal mesenchyme until later
in development, when they migrate into the heart and contribute
the majority of cardiomyocytes in the right ventricle (RV) and atria.
How SHF progenitors and their derivatives differ from those of the
FHF is poorly understood, but this knowledge will be crucial in our
understanding of congenital heart defects and the potential
differences in populations of cardiomyocytes derived from
pluripotent stem cells.
Wnt proteins are secreted ligands that regulate diverse cellular
behaviors and play pivotal roles in the development of cardiac
progenitors (Tzahor, 2007; Cohen et al., 2008; Tian et al., 2010a).
The canonical Wnt pathway stabilizes -catenin and allows it to
enter the nucleus, where it complexes with TCF family DNA-
binding proteins to activate gene transcription (Molenaar et al.,
1996; Brunner et al., 1997; Korinek et al., 1998; Young et al.,
1998). Experiments performed on embryonic stem (ES) cell-
derived embryoid bodies (EBs) and zebrafish embryos have
revealed at least two temporally distinct roles for Wnt/-catenin
signaling during cardiogenesis (Naito et al., 2006; Ueno et al.,
2007). The first phase promotes the induction of cardiac
progenitors, whereas the second phase inhibits the maintenance of
cardiac progenitor fate. SHF-specific -catenin deletion reduces the
numbers of SHF progenitors and their expression of the crucial
SHF transcription factor Islet1 (Isl1) in mouse embryos, indicating
that canonical Wnt signaling is required in SHF cells (Ai et al.,
2007; Cohen et al., 2007; Klaus et al., 2007; Kwon et al., 2007).
However, forced activation of -catenin signaling in Isl1+cells also
disrupts SHF development, confirming that canonical Wnt
signaling plays both positive and negative roles in Isl1+SHF
progenitors (Kwon et al., 2009).
Wnt proteins can also activate Rho family GTPases and
intracellular Ca2+release to affect cytoskeletal organization,
adhesion and motility (Malbon, 2004; James et al., 2008;
Schlessinger et al., 2009). This non-canonical Wnt signaling also
inhibits Wnt/-catenin signaling in many contexts and the
balance between these activities may fine-tune the effects of Wnt
proteins on responding cells (Torres et al., 1996; Ishitani et al.,
1999; Ishitani et al., 2003; Mikels and Nusse, 2006; Yuan et al.,
2011). Wnt5a and Wnt11 promote cardiogenesis in frog and
chick embryos as well as in differentiating stem cells through the
non-canonical Wnt pathways (Eisenberg and Eisenberg, 1999;
Schneider and Mercola, 2001; Pandur et al., 2002; Terami et al.,
2004; Koyanagi et al., 2005; Koyanagi et al., 2009). Although
these data suggest that Wnt5a and Wnt11 act in early cardiac
progenitors, Wnt5a and Wnt11 mutations cause mild heart
defects due to problems in cell-cell adhesion and cytoskeleton
organization in differentiating cardiomyocytes (Schleiffarth et
al., 2007; Zhou et al., 2007; Nagy et al., 2010). Here we show
1Department of Medicine, Division of Endocrinology, University of Rochester,
Rochester, NY 14642, USA. 3Howard Hughes Medical Institute, Institute for Stem
Cell and Regenerative Medicine, Department of Pharmacology, University of
Washington School of Medicine, Seattle, WA 98195, USA. 2Department of Cell and
Developmental Biology, 4Department of Medicine, 5Institute for Regenerative
Medicine and 6Cardiovascular Institute, University of Pennsylvania, Philadelphia,
PA 19104, USA.
*Authors for correspondence (firstname.lastname@example.org;
Accepted 20 March 2012
Wnt/-catenin has a biphasic effect on cardiogenesis, promoting the induction of cardiac progenitors but later inhibiting their
differentiation. Second heart field progenitors and expression of the second heart field transcription factor Islet1 are inhibited by
the loss of -catenin, indicating that Wnt/-catenin signaling is necessary for second heart field development. However,
expressing a constitutively active -catenin with Islet1-Cre also inhibits endogenous Islet1 expression, reflecting the inhibitory
effect of prolonged Wnt/-catenin signaling on second heart field development. We show that two non-canonical Wnt ligands,
Wnt5a and Wnt11, are co-required to regulate second heart field development in mice. Loss of Wnt5a and Wnt11 leads to a
dramatic loss of second heart field progenitors in the developing heart. Importantly, this loss of Wnt5a and Wnt11 is
accompanied by an increase in Wnt/-catenin signaling, and ectopic Wnt5a/Wnt11 inhibits -catenin signaling and promotes
cardiac progenitor development in differentiating embryonic stem cells. These data show that Wnt5a and Wnt11 are essential
regulators of the response of second heart field progenitors to Wnt/-catenin signaling and that they act by restraining Wnt/-
catenin signaling during cardiac development.
KEY WORDS: Wnt, Cardiac development, Progenitor, Mouse
Ethan David Cohen1,*, Mayumi F. Miller2, Zichao Wang1, Randall T. Moon3and Edward E. Morrisey2,4,5,6,*
that Wnt5a and Wnt11 are co-required to restrain the inhibitory
effects of prolonged canonical Wnt signaling on Isl1+SHF
MATERIALS AND METHODS
Generation of Wnt5a; Wnt11 double-mutant mice
Mice carrying previously published alleles of Wnt5a and Wnt11
(Yamaguchi et al., 1999; Majumdar et al., 2003) were intercrossed to
produce double-mutant, single-mutant and wild-type embryos. To examine
Wnt/-catenin signaling in vivo, Wnt5a–/+; Wnt11–/+mice were crossed into
the BAT-GAL strain, which carries a TCF-responsive lacZ reporter
(Maretto et al., 2003). Genotyping for Wnt5a, Wnt11 and BAT-GAL was
performed as described (Yamaguchi et al., 1999; Majumdar et al., 2003;
Maretto et al., 2003). All protocols were approved by the University of
Pennsylvania Institutional Animal Care and Use Committee.
Embryos were fixed in 4% PFA overnight before being embedded and
sectioned. Staining with Isl1 (University of Iowa Hybridoma Bank,
40.2D6), Nkx2.5 (Santa Cruz, sc-8697) and Ki67 (DAKO, m7249)
antibodies as well as TUNEL staining were performed as described (Cohen
et al., 2007; Zhang et al., 2008) and examined with a confocal microscope.
Labeling indexes for sections stained for Isl1, Nkx2.5, Ki67 and TUNEL
were obtained by dividing the number of labeled cells by the total number
of cells within the fame. Graphs represent the average value of indexes
from images of three embryos of each genotype. Error bars represent s.d.
P<0.05 by Student’s t-test indicated statistical significance.
In situ hybridization was performed as described (Cohen et al., 2007;
Zhang et al., 2008). Probes were amplified from Wnt5a and Wnt11 cDNAs
(Open Biosystems) using the primers listed in supplementary material
Generation and culture of EBs
TL1 mouse ES cells were grown on inactivated mouse embryonic
fibroblasts in DMEM with 15% FBS (Hyclone) and LIF (Millipore
ESGRO). To initiate differentiation, cells were dissociated, suspended at
5?104cells/ml in LIF-free media containing 100 g/ml ascorbic acid and
used to make 20 ml droplets on Petri dish lids. Cells were cultured as
hanging drops for 2 days to form EBs, which were then cultured in poly-
HEMA (Sigma-Aldrich)-treated dishes for another 2 days. On day 4, EBs
were transferred to gelatin (Sigma-Aldrich)-coated dishes containing
control media, media with 200 ng/ml mouse Wnt5a and/or human WNT11
(R&D Systems), or media with 200 ng/ml human WNT3A (R&D
Systems). EBs were cultured with meBIO (50 nM, Millipore), BIO (50
nM, Millipore) or XAV-939 (25 nM, Cayman Chemical) between days 4
and 6 of differentiation. The phosphorylation mutant form of active -
catenin (pCMV-act--catenin) has been reported previously (Provost et al.,
2003). Additional groups of EBs were lysed on days 8, 10 and 12 for RNA
isolation to detect Wnt and cardiac gene expression. ES cells were
transfected with either pCMV-lacZ or pCMV-act--catenin using Effectine
(Qiagen) as previously described (Ko et al., 2009; Liou et al., 2010).
Measuring TCF activity in EBs
EBs were made from ES cells transduced with pBarls, a TCF-responsive
firefly luciferase virus (Fuerer and Nusse, 2010), and with a constitutive
Renilla luciferase virus. On day 4, EBs were placed in gelatin-coated dishes
containing either control media or media with 250 ng/ml Wnt5a and/or
WNT11. After 24 hours, luciferase activity was assayed with the Dual
Luciferase Assay System (Promega). Renilla luciferase activity was used
to normalize for transduction efficiency. Reported values are the mean ±
s.e.m. for the normalized pBarls activity from at least three assays. P<0.05
by two-tailed Student’s t-test indicated statistical significance.
Quantitative PCR (Q-PCR) and western blotting
Relative gene expression was calculated by the CT method using Gapdh
as an endogenous control. For Q-PCR analysis of embryonic hearts, graphs
represent the average results from three independent pools of RNA isolated
from the heart-containing regions, defined as the first pharyngeal arch to
the septum transversum, of at least five embryos. For Q-PCR analysis of
EBs, graphs represent the average values from three pools of RNA isolated
from independent experiments. RNA was reverse transcribed using
Superscript III reverse transcriptase (Invitrogen) and Q-PCR performed on
an ABI 7900 Cycler with SYBR Green (Applied Biosystems) and the
primers listed in supplementary material Table S1. The CT values of all
samples were normalized to the average CT of control samples, thus
allowing the standard deviation to be calculated from the CT values of
individual controls. Error bars represent s.d. P<0.05 by Student’s t-test
indicated statistical significance.
For western blotting, cells were collected 48 hours following
transfection, lysed, and nuclear and cytoplasmic fractions were generated
as described (Wang et al., 2005). Proteins were resolved on SDS-PAGE
gels and transferred to PVDF membranes. Antibodies used in western
blotting include: -catenin (BD Transduction Laboratories), Gapdh
(Abcam) and hnRNP (Santa Cruz).
Wnt5a and Wnt11 are co-expressed at the anterior
pole of the heart tube
In situ hybridization was performed to determine whether Wnt5a
and Wnt11 expression domains overlap in the embryonic heart,
which could suggest a shared role in early cardiac development.
We performed in situ hybridization for Wnt5a and Wnt11 on
sections of E7.5 embryos. Expression of Wnt5a was observed in
the allantois, as has previously been reported, but expression of
Wnt5a or Wnt11 was not detected in the cardiac mesoderm at this
stage (Fig. 1A,E) (Yamaguchi et al., 1999). By E8.5, Wnt5a and
Wnt11 were both expressed in the presumptive outflow tract (OFT)
and RV and at lower levels in the LV and atria, similar to what has
been reported previously (Fig. 1B-D,F-H) (Kispert et al., 1996;
Yamaguchi et al., 1999; Terami et al., 2004). Co-expression of
Wnt5a and Wnt11 was maintained at the anterior pole of the heart
until E10.5, at which time the patterns of Wnt5a and Wnt11
expression began to diverge. Wnt5a became enriched in the
mesoderm surrounding the branchial arch arteries and the
laryngeal-tracheal grove as well as the medial layer of the OFT
wall (Fig. 1I). Wnt5a expression continued in the compact
myocardium of the RV, LV and atria (Fig. 1J). By contrast, Wnt11
was expressed in both the inner and outer layers of the OFT wall
and inner lining of the OFT cushion (Fig. 1K). Within the
developing RV, LV and atria, Wnt11 was expressed in the
epicardium and endocardium, with the highest levels of expression
observed in the epicardial cells surrounding the atria and inner
lining of the endocardial cushions (Fig. 1L). Wnt11 was also
expressed in the endothelium of the arch arteries (Fig. 1K,
arrowhead) and in the mesenchyme surrounding the trachea (Fig.
Wnt5a and Wnt11 are co-required for cardiac
The overlap between Wnt5a and Wnt11 expression in the OFT and
RV of the embryonic heart suggested that these ligands play
redundant roles in cardiac development. To test this hypothesis,
mice carrying null alleles of Wnt5a and Wnt11 (Yamaguchi et al.,
1999; Majumdar et al., 2003) were intercrossed to generate
Wnt5a–/–; Wnt11–/–embryos, as well as wild-type and single-
mutant controls. Intriguingly, whereas Wnt5a–/–and Wnt11–/–
embryos survived until birth, no Wnt5a–/–; Wnt11–/–embryos were
found after E10.5. Wnt5a–/–; Wnt11–/–embryos were found at the
expected Mendelian ratios in litters harvested at E9.5 and had
beating hearts, indicating that Wnt5a–/–; Wnt11–/–embryos were
viable at this stage.
Development 139 (11)
To assess the extent of disruption to cardiac development by loss
of Wnt5a and Wnt11 expression, wild-type, Wnt5a–/–, Wnt11–/–and
Wnt5a–/–; Wnt11–/–double-mutant embryos were harvested at E9.5
and processed for histological sectioning. Examining sections from
anterior to posterior reveals severe cardiovascular defects in
Wnt5a–/–; Wnt11–/–double-mutant embryos. Wnt5a–/–; Wnt11–/–
embryos had a centrally located OFT leading to a single sac-like
heart chamber that connects directly to the dorsal mesocardium at
the posterior pole of the heart (Fig. 2Da-e). Although
morphological abnormalities were apparent in Wnt5a–/–and
Wnt11–/–hearts, these single-mutant hearts displayed evidence of
chamber septation and development similar to wild-type hearts
(Fig. 2Aa-d,Ba-e,Ca-e). By contrast, no chamber septation was
apparent in Wnt5a–/–; Wnt11–/–hearts (Fig. 2Da-e). The OFT and
RV of Wnt5a–/–hearts appeared dilated and dysmorphic relative to
those of wild-type hearts (Fig. 2Ba-c), whereas the OFT and RV of
Wnt11–/–hearts were small relative to those of controls (Fig. 2Ca-
d). Although atrioventricular (AV) canal development was apparent
in Wnt5a–/–and Wnt11–/–single mutants (Fig. 2Bc,d,Cc,d), no
obvious AV canal development occurred in Wnt5a–/–; Wnt11–/–
hearts (Fig. 2Da-e).
Development of SHF cardiac progenitors requires
Wnt5a and Wnt11
Wnt5a and Wnt11 are co-expressed in the presumptive OFT and
LV, and the single-chambered phenotype of Wnt5a–/–; Wnt11–/–
hearts was highly reminiscent of the cardiac defects found in Isl1
mutants, suggesting that Wnt5a and Wnt11 act together to
promote the development of SHF cardiac progenitors (Cai et al.,
2003). To determine whether the numbers of Isl1+SHF
progenitors were reduced in Wnt5a–/–; Wnt11–/–embryos,
sections of E9.5 double-mutant, single-mutant and wild-type
embryos were stained for Isl1 protein expression. Although
nearly 40% of cells in the OFT and pharyngeal mesenchyme of
wild-type and single-mutant control embryos were Isl1+, less
than 10% of these cells stained for Isl1 in Wnt5a–/–; Wnt11–/–
embryos (Fig. 3A-D,I). Furthermore, the Isl1+cells in Wnt5a–/–;
Wnt11–/–embryos stained much less intensely than those in wild-
type and single-mutant controls (Fig. 3A-D). By contrast,
staining for Nkx2.5, which marks both FHF and SHF cardiac
progenitors, was similar in all genotypes tested (Fig. 3E-I).
To further quantify FHF and SHF gene expression, Q-PCR for
Isl1 and Nkx2.5 mRNA was performed on cardiac tissue dissected
from E9.5 embryos. Consistent with the results for Isl1 and Nkx2.5
immunostaining, Isl1 mRNA levels were more than 80% lower in
Wnt5a–/–; Wnt11–/–embryos than in wild-type and single-mutant
controls, whereas the levels of Nkx2.5 mRNA were unaffected (Fig.
3J). Like Isl1, the basic helix-loop-helix transcription factor Hand2
is expressed in the SHF and is crucial for its development
(Tsuchihashi et al., 2011), whereas expression of the highly related
Hand1 is restricted to the FHF and its derivatives (Riley et al.,
1998; Thomas et al., 1998). Consistent with the loss of Isl1
expression in Wnt5a–/–; Wnt11–/–embryos, Hand2 expression was
significantly reduced in double-mutant embryos, whereas Hand1
expression was unaffected (Fig. 3J). These data indicate that Wnt5a
and Wnt11 are required for the development of SHF cardiac
Cardiac development was also assessed at E8.5 to determine
whether there were any early defects associated with cardiac
progenitor development, proliferation or apoptosis in Wnt5a–/–;
Wnt11–/–embryos. Although the Wnt5a–/–; Wnt11–/–embryos were
smaller than single-mutant or wild-type controls at E9.5, Wnt5a–/–;
Wnt11–/–embryos collected at E8.5 were similar in size and gross
morphology to their wild-type and single-mutant littermates (Fig.
3K-N). Wnt5a–/–; Wnt11–/–hearts at E8.5 had a centrally aligned
OFT that connected to a single cardiac chamber (Fig. 3M,N).
Moreover, there was little AV canal development in Wnt5a–/–;
Wnt11–/–embryos at E8.5 (Fig. 3M,N). Similar to the E9.5 embryos,
Isl1 expression was reduced but Nkx2.5 expression was unaffected
in E8.5 Wnt5a–/–; Wnt11–/–embryos, consistent with abnormal OFT
and ventricular development observed at this stage (Fig. 3O-R,W).
To assess proliferation in Wnt5a–/–; Wnt11–/–hearts at E8.5, Ki67
immunostaining was performed. These data show increased
proliferation in Wnt5a–/–; Wnt11–/–embryos in areas corresponding
to the SHF (Fig. 3S,T,W). By contrast, TUNEL staining did not
reveal any changes in apoptosis in Wnt5a–/–; Wnt11–/–embryos at
Wnt5a/Wnt11 in heart development
Fig. 1. Expression of Wnt5a and Wnt11 during
mouse early cardiac development. (A)In situ
hybridization showing Wnt5a expression in the allantois
(asterisk) at E7.5 but not in the precardiac mesoderm
(arrow). (B-D)Expression of Wnt5a is observed in the
developing OFT and right ventricle myocardium at E8.5.
(E-H)Expression of Wnt11 is not observed in the early
precardiac mesoderm at E7.5 (arrow) and only at low
levels in the allantois. Expression of Wnt11 is observed
in the developing OFT and right ventricle myocardium at
E8.5. (I,J)Wnt5a expression is observed in OFT
mesenchyme (I, arrows) and the branchial arch region at
E10.5 (I, arrowhead) and also in the ventricular and
atrial myocardium (J, arrows) at E10.5. (K,L)Wnt11
expression is observed in the myocardium of the OFT (K,
arrows) and at lower levels in the atrioventricular region
and atrial myocardium (L, arrows) at E10.5. Expression
of Wnt11 is observed diffusely and at low levels in
ventricular myocardium at E10.5 (L). OFT, outflow tract;
LV, left ventricle; RV, right ventricle. Scale bars: 50m in
A-H; 100m in I-L.
E8.5 (Fig. 3U-W). Taken together, these data suggested that the
loss of Isl1+cells in Wnt5a–/–; Wnt11–/–embryos reflected a co-
requirement of Wnt5a/Wnt11 signaling in SHF development and
is not the result of either a broad developmental delay or changes
in the survival of cells within the SHF.
Since Isl1+progenitors contribute to all of the major cell types
within the heart, markers of myocardial and non-myocardial cell
populations within the heart were examined to determine whether
these cell types are affected in Wnt5a–/–; Wnt11–/–embryos.
Immunostaining for the endothelial marker Flk1 (Kdr – Mouse
Genome Informatics) was unaffected in E8.5 Wnt5a–/–; Wnt11–/–
embryos relative to controls (Fig. 3X,Y). Q-PCR revealed that
mRNA levels for the endothelial markers Flk1 and Cd31 (Pecam1
– Mouse Genome Informatics) and smooth muscle markers smooth
muscle -actin (Acta2) and Sm22a (Tagln – Mouse Genome
Informatics) were unaffected in the hearts of Wnt5a–/–; Wnt11–/–
embryos (Fig. 3Z). By contrast, expression of the myocardial-
specific markers cTnI (Tnni3 – Mouse Genome Informatics) and
Mlc2v (Myl2 – Mouse Genome Informatics) was significantly
reduced in Wnt5a–/–; Wnt11–/–embryos (Fig. 3Z).
Taken together, these data suggest that the myocardial
differentiation of Isl1+SHF progenitors is specifically affected by
the combined loss of Wnt5a and Wnt11.
Wnt5a and Wnt11 are co-required to induce
cardiac progenitor gene expression in
differentiating ES cells
To examine the temporal relationship between Wnt5a and Wnt11
expression and cardiac differentiation, EBs were formed from
mouse ES cells using the hanging drop method and used to
assess expression of various Wnt proteins. Expression of Wnt3a
and Wnt5a, which are both expressed in the primitive streak, first
began to rise on day 4 of EB differentiation and increased
steadily, hitting a peak on days 8 and 10, respectively, and
declining thereafter (Fig. 4A,B) (Yamaguchi et al., 1999;
Baranski et al., 2000). By contrast, Wnt11 expression increased
slowly between days 0 and 8 before it increased abruptly on day
10 and remained high through day 14 of EB differentiation (Fig.
4C). To compare the timing of Wnt5a and Wnt11 expression with
the appearance of cardiac progenitor gene expression, the
percentage maximum expression for Wnt5a, Wnt11, Isl1, Nkx2.5
and the precardiac mesoderm marker Mesp1 was plotted over the
course of EB differentiation (Fig. 4D). This comparison shows
that expression of Wnt5a roughly coincided with markers of
early cardiac mesoderm including Isl1, Nkx2.5 and Mesp1,
whereas Wnt11 expression increased somewhat later.
To determine whether Wnt5a and Wnt11 could induce markers
of early cardiac mesoderm progenitors, EBs were formed as
hanging drops and allowed to develop until day 4, the point at
which Mesp1+cardiac competent mesoderm first appears
(Bondue et al., 2008). EBs were then cultured in either control
media, media containing recombinant Wnt5a and/or WNT11
proteins or media containing recombinant WNT3A protein and
Q-PCR used to measure gene expression. Since Wnt/-catenin
signaling was previously shown to promote the formation of
cardiac competent mesoderm, we first examined the expression
of Mesp1, a helix-loop-helix transcription factor expressed in
early precardiac mesoderm and the cardiac crescent but later
extinguished as the heart tube develops (Saga et al., 1999;
Kitajima et al., 2000; Saga et al., 2000; Lindsley et al., 2006;
Development 139 (11)
Fig. 2. Wnt5a–/–; Wnt11–/–embryos
develop a single-chamber heart.
Hematoxylin and Eosin (H+E) staining of
histological sections at E9.5 of (Aa-e)
Wnt5a+/+; Wnt11+/+, (Ba-e) Wnt5a–/–;
Wnt11+/+, (Ca-e) Wnt5a+/+; Wnt11–/–and
(Da-e) Wnt5a–/–; Wnt11–/–mouse embryos.
Wild type as well as Wnt5a–/–and Wnt11–/–
mutants display proper chamber septation.
However, Wnt5a–/–; Wnt11–/–mutants lack
chamber septation and form a single-
chamber heart (question mark in D). Arrows
indicate where branchial arch arteries flow
into the OFT of the heart. AT, atrium. Scale
Lindsley et al., 2008). Treatment with Wnt5a, WNT11 or
WNT3A individually caused a ~50% increase in peak Mesp1
expression relative to controls, and treatment with Wnt5a and
WNT11 together caused only a slight increase in Mesp1
expression relative to treatment with either Wnt alone (Fig. 4E).
By contrast, expression of Tbx5, which is expressed in the
precardiac mesoderm and cardiac crescent before being restricted
to the venous pole of the heart (Bruneau et al., 1999), is
increased in EBs treated with Wnt5a and WNT11 together as
compared with those treated with Wnt5a, WNT11 or WNT3A
alone (Fig. 4F). Most importantly, combined Wnt5a WNT11
treatment also caused cooperative increases in the levels of both
Nkx2.5 and Isl1 expression over the levels of expression found
in either control EBs or those treated with Wnt5a, WNT11 or
WNT3A alone (Fig. 4G,H). This increase in Nkx2.5 could be the
result of an expansion of early precardiac mesoderm by Wnt5a
and WNT11 that leads to an overall increase in cardiogenesis.
Taken together, these data suggest that Wnt5a and Wnt11 are co-
required to increase both FHF and SHF progenitor markers during
Wnt5a/Wnt11 in heart development
Fig. 3. Wnt5a and Wnt11 are necessary for SHF progenitor development and myocardial differentiation. (A-D)Immunostaining shows
that Isl1 protein expression (arrows) is dramatically reduced in Wnt5a–/–; Wnt11–/–mouse embryos compared with wild type or single mutants.
(E-H)By contrast, Nkx2.5 protein expression (arrows) is not significantly altered in Wnt5a–/–; Wnt11–/–mutants compared with wild type and single
mutants. (I)Quantitation of Isl1+cells in single and double mutants. (J)Q-PCR shows that expression of Isl1 and Hand2 is reduced in Wnt5a–/–;
Wnt11–/–mutants, whereas Nkx2.5 and Hand1 expression is not altered. (K-N)H+E-stained sections of wild type and Wnt5a–/–; Wnt11–/–mutants
at E8.5 showing lack of chamber septation in the Wnt5a–/–; Wnt11–/–heart. Question mark indicates single-chamber heart. (O-R)Isl1 (O,P) and
Nkx2.5 (Q,R) immunostaining at E8.5 in wild type and Wnt5a–/–; Wnt11–/–mutants. (S-V)Ki67 (S,T) and Nkx2.5 (U,V) immunostaining at E8.5 in
wild type and Wnt5a–/–; Wnt11–/–mutants. (W)Quantitation of Isl1+, Nkx2.5+, Ki67+and TUNEL+cells in wild type and Wnt5a–/–; Wnt11–/–mutants.
(X,Y)Flk1 immunostaining in wild type (X) and Wnt5a–/–; Wnt11–/–mutants (Y). (Z)Q-PCR for Mlc2v, cTnI, smooth muscle -actin, Flk1 and Cd31 in
wild type and Wnt5a–/–; Wnt11–/–mutants. P<0.05 by Student’s t-test indicates statistical significance. DKO, double knockout. Error bars indicate
s.e.m. Scale bars: 50m in A-H; 150m in K-N; 100m in O-V,X,Y.
Wnt5a and Wnt11 are co-required to repress
canonical Wnt signaling in SHF progenitors
Although Wnt5a and Wnt11 are thought to signal via non-
canonical pathways, they can also co-activate Wnt/-catenin
signaling during early Xenopus development in a manner that is
reminiscent of their cooperative regulation of Isl1 and SHF
development (Cha et al., 2008; Cha et al., 2009). To assess
canonical Wnt signaling activity in the SHF of Wnt5a–/–; Wnt11–/–
embryos, we crossed the BAT-GAL strain Wnt reporter line into
the Wnt5a+/–; Wnt11+/–mutants (Maretto et al., 2003). Sections
through the hearts of E9.5 embryos stained with X-gal reveal that
the number of stained cells within the walls of the OFT and
surrounding mesenchyme are increased in Wnt5a–/–; Wnt11–/–
mutants relative to controls, suggesting that Wnt5a and Wnt11 are
co-required to repress canonical Wnt signaling in the SHF (Fig.
A previous study identified several genes, including Bhlhb2
(Bhlhe40), Ndrg1, Fgf11 and Rora, as being upregulated by -
catenin signaling in Isl1+SHF progenitors (Kwon et al., 2009). To
confirm that -catenin signaling is increased in the SHF
progenitors of Wnt5a–/–; Wnt11–/–embryos, the expression levels
of these genes were examined by Q-PCR. These data show that
levels of Bhlhb2, Ndrg1, Fgf11 and Rora mRNA were all
dramatically increased in Wnt5a–/–; Wnt11–/–hearts relative to
either single-mutant or wild-type controls, further indicating that
-catenin signaling is upregulated in double-mutant SHF
progenitors (Fig. 5C-F).
Forced activation of -catenin signaling blocks the
effects of Wnt5a and Wnt11 on cardiac
The increased BAT-GAL activity in Wnt5a–/–; Wnt11–/–embryos
suggested that Wnt5a and Wnt11 are normally co-required to
attenuate canonical Wnt signaling in the SHF. To determine
whether Wnt5a and Wnt11 are sufficient to inhibit canonical
Wnt signaling, EBs were produced from ES cells transduced
with the TCF-responsive luciferase reporter virus pBarls
(Biechele et al., 2009). On day 4 of differentiation, these EBs
were placed in control media as well as media containing
recombinant Wnt5a, WNT11, or Wnt5a plus WNT11. After 24
hours, EBs were lysed and luciferase activities determined.
Concordant with the increased levels of -catenin signaling in
Wnt5a–/–; Wnt11–/–embryos, treating EBs with media containing
both Wnt5a and WNT11 caused a significant decrease in pBarls
activity (Fig. 6A).
To further test the effects of activated Wnt/-catenin signaling
on cardiac progenitor development in differentiating ES cells using
the pBarls Wnt reporter, day-4 EBs were transferred to media
containing combinations of the Gsk3 inhibitor BIO, its inactive
form meBIO, and Wnt5a and/or WNT11 proteins, for 48 hours.
The combined action of Wnt5a and WNT11 inhibited pBarls Wnt
reporter activity as expected in the presence of the inactive Gsk3
inhibitor meBIO (Fig. 6B). However, In the presence of BIO,
Wnt5a and WNT11 were unable to repress pBarls Wnt reporter
activity (Fig. 6B). Q-PCR showed that activation of Wnt signaling
Development 139 (11)
Fig. 4. Wnt5a and Wnt11 cooperatively promote
early cardiac progenitor development in
differentiating ES cells. (A-D)Temporal expression of
Wnt3a (A), Wnt5a (B) and Wnt11 (C) in mouse ES cell-
derived embryoid body (EB) cultures at the days indicated.
Overlay of temporal expression patterns of Wnt5a,
Wnt11, Mesp1, Isl1 and Nkx2.5 (D). (E-H)Q-PCR analysis
showing effects of recombinant WNT3A, Wnt5a, WNT11,
or Wnt5a plus WNT11 on expression of Mesp1 (E), Tbx5
(F), Isl1 (G) and Nkx2.5 (H) in ES cell-derived EB cultures at
the days indicated. Error bars indicate s.e.m.
by BIO inhibited the increase of Isl1 and Nkx2.5 gene expression
caused by treatment of differentiating EBs with Wnt5a and WNT11
(Fig. 6C,D). Moreover, expression of an activated form of -
catenin containing mutations in the Gsk3 phosphorylation sites
during EB differentiation also inhibited the increase in Isl1 and
Nkx2.5 cardiac gene expression in comparison to control lacZ
expression (Fig. 6E,F).
To assess whether the combined action of Wnt5a and WNT11
affected additional readouts of canonical Wnt signaling, we
performed western blots to assess the levels of nuclear -catenin
upon treatment of differentiating EBs. These data show that the
combination of Wnt5a and WNT11 causes a decrease in nuclear -
catenin levels (Fig. 6G). Moreover, this decrease in -catenin was
not observed in the presence of BIO, consistent with the observed
effects on the pBarls Wnt reporter above (Fig. 6H). Finally, to
assess whether direct inhibition of Wnt/-catenin signaling could
affect cardiac gene expression, we treated differentiating EBs with
the tankyrase inhibitor XAV-939, which promotes axin-Gsk3
interactions and thereby represses canonical Wnt signaling (Huang
et al., 2009). Using this method, we observed a significant increase
in Isl1 and Nkx2.5 gene expression (Fig. 6I). Thus, the ability of
Wnt5a and Wnt11 to cooperatively repress -catenin signaling in
differentiating EBs is essential for their activation of cardiac gene
expression and promotion of cardiac progenitor development.
The data presented here support a novel role for Wnt5a and Wnt11
in SHF development that is mediated by blocking the later
inhibitory effect of Wnt/-catenin signaling on Isl1+SHF
progenitors. We show that Wnt5a–/–; Wnt11–/–embryos die early in
embryogenesis and have single-chamber sac-like hearts reminiscent
of those found in Isl1–/–embryos. Moreover, expression of the SHF
markers Isl1 and Hand2 is significantly reduced in Wnt5a–/–;
Wnt11–/–embryos, whereas Nkx2.5, which is expressed in both
FHF and SHF, is unaffected. Interestingly, treating EBs with Wnt5a
and Wnt11 causes a strong cooperative increase in the expression
of Isl1, as well as other early cardiac progenitor markers including
Nkx2.5, that far exceeds those caused by either Wnt alone, and this
requires inhibition of -catenin signaling. These data suggest that
Wnt5a and Wnt11 are co-required to promote cardiogenesis, in
part, through inhibition of -catenin signaling.
Experiments in ES cells and zebrafish embryos indicate that
canonical Wnt signaling plays two temporally distinct roles in
cardiogenesis, first promoting the appearance of early precardiac
mesoderm and then later inhibiting cardiac differentiation.
Consistent with these data, the canonical Wnt ligand Wnt3a is
sufficient to induce Mesp1 expression in differentiating ES cells,
but overexpressing Mesp1 in ES cells promotes cardiac
differentiation only if canonical Wnt signaling is simultaneously
inhibited (Bondue et al., 2008). We were unable to detect either
Wnt5a or Wnt11 in the cardiogenic mesoderm of E7.5 head-fold
stage mouse embryos, which is consistent with the results of
previous studies indicating that expression of Wnt11 in the heart
begins at E8.0 (Kispert et al., 1996; Terami et al., 2004). These data
suggest that reports of Wnt11 expression in the cardiac crescent of
the chick embryo might reflect species-specific differences between
avian and mammalian development or that Wnt11 expression is
present in early precardiac mesoderm prior to heart tube formation
but is dramatically upregulated upon fusion of the lateral cardiac
primordia (Eisenberg and Eisenberg, 1999; Hardy et al., 2008). Our
expression studies on Wnt5a and Wnt11 in E8.5 embryos revealed
a previously unappreciated degree of overlap between the
expression of these Wnt ligands in the presumptive OFT and RV at
the anterior pole of the heart tube. Furthermore, although Wnt5a
and Wnt11 expression no longer overlap in the mouse heart at
E10.5, they are expressed in a complementary pattern that allows
them to signal cooperatively in a paracrine fashion at this stage.
Thus, the in vivo developmental role of Wnt5a and Wnt11 appears
to be restricted to regulation of SHF progenitor development.
How Wnt5a and Wnt11 co-regulate early cardiac development
is still unclear. Our data show that the combination of Wnt5a and
Wnt11 mediates a decrease in nuclear -catenin levels, which
corresponds to a decrease in canonical Wnt activity. This function
of Wnt5a and Wnt11 appears to act after the time period when
Wnt3a induction of canonical Wnt signaling occurs in
cardiogenesis in differentiating EBs. Previous studies have shown
that Wnt5a and Wnt11 can dimerize to promote canonical Wnt
Wnt5a/Wnt11 in heart development
Fig. 5. Combined loss of Wnt5a and Wnt11 during mouse development increases canonical Wnt/ -catenin signaling activity and SHF
target gene expression. (A,B)BAT-GAL lacZ expression (arrows) is increased in Wnt5a–/–; Wnt11–/–mutants compared with wild-type controls.
(C-F)Expression of Bhlhb2 (C), Rora (D), Ndrg1 (E) and Fgf11 (F) is increased in Wnt5a–/–; Wnt11–/–hearts but not single-mutant hearts. For all four
genes, only the increased expression in the Wnt5a–/–; Wnt11–/–mutants was considered significant (P<0.01). Error bars indicate s.e.m. Scale bars:
signaling in Xenopus (Cha et al., 2008). In cardiac development
and in cardiogenesis in differentiating EBs, we observe an
inhibition of nuclear -catenin levels and canonical Wnt signaling
activity. This discrepancy might simply reflect differences in the
activity of these Wnt ligands in different developmental contexts.
Interestingly, previous reports have shown that Wnt11 can promote
cardiogenesis through caspase 3-mediated degradation of -catenin
that appears to be independent of the cytoplasmic destruction
complex (Abdul-Ghani et al., 2011). Although we did not observe
decreased nuclear -catenin with treatment of EBs with Wnt11
alone, such a mechanism could still be active during the
cardiogenesis promoted by the combination of Wnt5a and Wnt11.
Although Isl1+SHF progenitors are specifically affected in
Wnt5a–/–; Wnt11–/–embryos, levels of Tbx5, Nkx2.5 and Isl1
mRNAs are all dramatically increased in EBs treated with the
combination of Wnt5a and Wnt11. Whereas Nkx2.5 is expressed
in SHF progenitors, Tbx5 is not expressed in the SHF or its
derivatives but rather in the LV and atria and is therefore
considered a specific marker of the FHF (Bruneau et al., 1999).
Given these data, the increased Tbx5 expression in EBs treated
with Wnt5a and Wnt11 suggests that cooperative signaling by these
Wnt proteins might be sufficient to promote both FHF and SHF
fates if present during early precardiac mesoderm development.
These data are consistent with the results of studies of heart
development in Xenopus, which indicate that inhibition of
canonical Wnt signaling is both necessary and sufficient for FHF
formation (Schneider and Mercola, 2001). The ability of Wnt5a
and Wnt11 to induce FHF markers including Mesp1 and Tbx5
occurs earlier than induction of SHF markers, suggesting that
Wnt5a and Wnt11 might act via different mechanisms in a
temporal-specific fashion during cardiogenesis. In the EB culture
system, FHF and SHF progenitors are likely to co-develop at
similar times and might be affected in the same fashion in gain-of-
function experiments using exogenous Wnt ligands. Also, such
differences are often observed between gain-of-function and loss-
of-function experiments and might not be reflective of the
physiological role for the specific Wnt ligands in question.
The endogenous factors responsible for inhibiting the negative
effects of prolonged Wnt signaling during cardiac progenitor
development have yet to be identified, and the mechanisms by
which the activities of Wnt agonists and repressors are balanced in
both FHF and SHF cardiac progenitors remain poorly understood.
Additional research will be needed to determine how pathways that
inhibit canonical signaling, such as the suppression of -catenin
signaling by Wnt5a/Wnt11, are balanced with the active promotion
of Wnt/-catenin signaling in the heart by ligands such as Wnt3a,
Wnt8a and Wnt2 (Tian et al., 2010b).
Development 139 (11)
Fig. 6. Wnt5a and Wnt11 cooperatively
inhibit Wnt/ -catenin signaling activity in
differentiating ES cells. (A)Recombinant
Wnt5a and WNT11 in combination can repress
Wnt/-catenin signaling in mouse EBs as shown
using the Wnt/-catenin reporter pBarls. (B)The
Gsk3 inhibitor BIO inhibits the repressive
effects on Wnt/-catenin signaling caused by
the combined addition of Wnt5a and WNT11
to EBs, whereas the inactive form meBIO does
not. (C,D)Q-PCR analysis showing that addition
of BIO inhibits the activation of Isl1 (C) and
Nkx2.5 (D) caused by co-treatment with Wnt5a
and WNT11, whereas DMSO and meBIO have
little effect. (E,F)Expression of an activated
form of -catenin inhibits the activation of Isl1
(E) and Nkx2.5 (F) caused by co-treatment with
Wnt5a and WNT11. (G)Western blot for
expression of nuclear -catenin showing that
the combined action of Wnt5a and WNT11
decreases nuclear -catenin levels in
differentiating EBs. Sequestered protein
expression of hnRNP and Gapdh show that the
nuclear and cytoplasmic fractionation worked
and that these fractions are relatively pure.
(H)Western blot showing that nuclear levels of
-catenin could not be decreased by the
combined action of Wnt5a and WNT11 in the
presence of BIO. (I)Treatment of differentiating
EBs with XAV-939 increases Isl1 and Nkx2.5
expression. Decreases in pBarls activity in A and
B (P<0.05), increases in Isl1 and Nkx2.5 gene
expression in C-F (P<0.005) and the increase in
Isl1 and Nkx2.5 expression in I (P<0.001) are
significant. Error bars indicate s.e.m.
These studies were supported by funding from the National Institutes of
Health [HL100405 and HL087825 to E.E.M.] and the American Heart
Association Jon Holden DeHaan Myogenesis Center Grant (E.E.M.). E.D.C. is a
recipient of an American Heart Association Scientist Development Grant.
R.T.M. is an Investigator of the Howard Hughes Medical Institute. Deposited in
PMC for release after 6 months.
Competing interests statement
The authors declare no competing financial interests.
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Development 139 (11)