A Wnt- and ?-catenin-dependent pathway for
mammalian cardiac myogenesis
Teruya Nakamura*†, Motoaki Sano*†, Zhou Songyang‡, and Michael D. Schneider*†§¶?
*Center for Cardiovascular Development and Departments of‡Biochemistry and Molecular Biology,†Medicine,§Molecular and Cellular Biology, and
¶Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030
Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved February 28, 2003 (received for review
September 17, 2002)
Acquisition of a cardiac fate by embryonic mesodermal cells is a
avians requires positive signals from adjacent endoderm, including
signal, Wnt proteins, from neural tube. By contrast, mechanisms of
mesodermal commitment to create heart muscle in mammals are
largely unknown. In addition, Wnt-dependent signals can involve
either a canonical ?-catenin pathway or other, alternative mediators.
Here, we tested the involvement of Wnts and ?-catenin in mamma-
lian cardiac myogenesis by using a pluripotent mouse cell line
system, early and late cardiac genes are up-regulated by 1% DMSO,
and spontaneous beating occurs. Notably, Wnt3A and Wnt8A were
induced days before even the earliest cardiogenic transcription fac-
tors. DMSO induced biochemical mediators of Wnt signaling (de-
creased phosphorylation and increased levels of ?-catenin), which
were suppressed by Frizzled-8?Fc, a soluble Wnt antagonist. DMSO
provoked T cell factor-dependent transcriptional activity; thus, induc-
inhibited the induction of cardiogenic transcription factors, cardio-
genic growth factors, and sarcomeric myosin heavy chains. Likewise,
differentiation was blocked by constitutively active glycogen syn-
thase kinase 3?, an intracellular inhibitor of the Wnt??-catenin path-
way. Conversely, lithium chloride, which inhibits glycogen synthase
kinase 3?, and Wnt3A-conditioned medium up-regulated early car-
diac markers and the proportion of differentiated cells. Thus, Wnt?
this pluripotent model system.
anterolateral regions of the embryo during late gastrulation (1, 2).
In this process, morphogenic movements and cardiac fate deter-
mination are believed to be regulated by multiple extracellular cues
factor-?, and fibroblast growth factors (FGFs) secreted from un-
derlying endodermal cells (3). This general model is based on a
as early heart formation is most easily observed in embryos that
develop outside the mother, becoming accessible to explant exper-
can differentiate into beating muscle cells in the absence of
endoderm (9, 10), implying that several discrete steps initiate heart
development: a specification step before gastrulation, which leads
to the appearance of myocardial precursor cells, and a subsequent
step during gastrulation, in which endoderm serves to enhance the
rate of myocyte differentiation and degree of heart tube morpho-
genesis. Therefore, it is important to elucidate when, where, and
how mesodermal cells are instructed to assume the cardiac fate for
understanding the entire body of mechanisms that operate later in
initial instructive events are largely unproven.
he earliest event in heart formation is commitment of meso-
dermal cells to a cardiogenic ‘‘fate’’ and their migration into
Wnt?Wg genes, related to wingless in Drosophila, encode a
number of secreted proteins that play critical roles in the develop-
ment of many organisms, especially in cell fate and patterning
(11–13). Notably, the prototype wingless itself collaborates with the
tube in flies (14–17). In the absence of Wnt proteins, cells under-
take active measures to maintain low levels of the Wnt signaling
protein, ?-catenin. Under baseline circumstances, ?-catenin is
phosphorylated at its N terminus by glycogen synthase kinase 3?
(GSK-3?), targeting ?-catenin for destruction by the ubiquitin–
proteosome pathway (18). Wnt binding to the Frizzled (Fz) family
of serpentine receptors activates an associated downstream com-
stabilizing ?-catenin. Accumulation of ?-catenin in the cytosol
results in its translocation to the nucleus (no specific control is
known at this step), its interaction with T cell factor (TCF)?
Wnt-responsive genes (19). Thus, beyond the role of membrane-
associated ?-catenin in adherens junctions, soluble ?-catenin has a
pivotal function in the canonical Wnt signal transduction pathway.
In addition, Wnt proteins also activate an alternative cascade,
involving protein kinase C and Jun N-terminal kinase (18, 20, 21).
Recent provocative studies using Xenopus and chick embryos
indicate that Wnt proteins are potent negative regulators of
heart muscle specification in those species (6–8, 22). Apart from
the a priori concern that such findings are not necessarily
predictive of mammalian biology, isoform and pathway differ-
ences both likely exist. For instance, in Xenopus, Wnt3A and -8
were inhibitors acting via GSK-3 (6), whereas Wnt11 was an
inducer of heart formation through the ?-catenin-independent
A genetic analysis of the Wnt family in mammals, even
from straightforward, given extraordinary diversity and overlap-
ping expression of the ligands and receptors. Besides Fz proteins,
lipoprotein-receptor-related proteins also couple Wnts to
?-catenin (24). In addition, no known promoter has sufficient
early, potent, and specific expression to undertake a conditional
deletion of Wnt receptors or conditional expression of Wnt
inhibitors in the presumptive heart-forming region. For these
reasons, multipotential cells that can be made to adopt the
as an interim step, for understanding the earliest mechanisms of
cardiac determination. By using one such system, mouse P19CL6
cells (25–31), we demonstrate an essential role for the Wnt??-
catenin pathway in mammalian cardiac myogenesis.
Materials and Methods
Materials and Reagents. P19CL6 mouse embryonic carcinoma cells
were kindly provided by I. Komuro (Chiba University, Chiba,
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: BMP, bone morphogenetic protein; GSK-3?, glycogen synthase kinase 3?;
MHC, myosin heavy chain; QRT-PCR, quantitative RT-PCR; TCF, T cell factor; CM, condi-
tioned media; CMV, cytomegalovirus; HA, hemagglutinin; LiCl, lithium chloride; FGF,
fibroblast growth factor.
?To whom correspondence should be addressed. E-mail: email@example.com.
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vol. 100 ?
hypophosphorylation of ?-catenin, accumulation of ?-catenin,
and TCF?lymphoid enhancer factor-dependent transcription.
Blocking Wnt–receptor interactions with soluble Fz proteins and
inhibition of ?-catenin with GSK-3? evoked equivalent effects,
largely or completely blocking the cardiogenic pathway, includ-
ing induction of Tbx5 and GATA4, the earliest marker of
differentiation here and in microarray profiles of this system
(30). Intriguingly, lymphoid enhancer factor 1 itself was an early
response to DMSO in the study cited (30). Thus, endogenous
Wnts mediate cardiogenesis in P19CL6 cells and do so via the
later responses of the cells to DMSO (BMP2 and BMP4, Fig. 1;
BMP5, ref. 30), required but insufficient to trigger the cardiac
fate (26, 27). Although other endogenous signals might coexist,
our data support the conclusion that Wnts, coupled to ?-catenin,
are the long-sought essential early targets of DMSO for cardiac
specification. We emphasize that, in Drosophila, the cardiogenic
signal from Wg itself depends on armadillo, the ?-catenin
augmented by extracellular and intracellular interventions that
stimulate this pathway, Wnt3A CM and LiCl, respectively. Our
conclusions differ, obviously, from inhibitory roles found for
Wnt3A and -8 in Xenopus and chicks (6–8). Differences from
mammalian cardiac specification exist in both species, e.g., in the
apparent involvement of activins (23, 39, 40). Apart from potential
phylogenetic dissimilarities, the studies also differ inherently (cul-
tured cells vs. explants and embryos) in the stage of maturation,
perturbations, and mix of cell types present. Indirect inductive
FGF8 was induced by DMSO and suppressed by the Wnt inhibitor
(Figs. 1D and 3A). Although no direct extrapolation to mammals is
yet available from the inductive function of this gene in avian
cardiogenesis (42, 43), we speculate that Wnt3A and -8 act at least
in part indirectly by controlling the creation of endoderm or
endoderm-like cells as the source of these cardiogenic factors.
Our results do not contradict the stimulation of cardiogenesis by
Wnt11 via the noncanonical pathway in Xenopus and in P19 cells
(the parent line for the clone we used). Only sufficiency, not a
requirement for Wnt11, was tested (23), and perhaps both classes
is lethal at gastrulation (44), obscuring any role of the gene in
mice after conditional deletion of ?-catenin (45) is discordant with
our findings only at first glance. In this case, ?-catenin was deleted
address the function of ?-catenin in normal cardiac progenitors
from inhibitor studies than from stimulation of the pathway, that
Wnt CM and LiCl promote the formation of heart muscle cells has
auspicious translational implications, in addition to posing ques-
tions for stem cell biology and mammalian cardiac development
more broadly. Endogenous stem cells from bone marrow and
commit to a cardiac fate, albeit in numbers too small to be effective
for cardiac myocyte formation in adults.
We thank R. J. Schwartz, T. Miura, H. Oh, Y. Hamamori, M. Xie, and
D. Zhang for helpful suggestions. This work was supported in part by
National Institutes of Health grants (to M.D.S.) and the M. D. Anderson
Foundation Professorship (to M.D.S.).
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