Epicardium-derived progenitor cells require ?-catenin
for coronary artery formation
Mo ´nica Zamora*, Jo ¨rg Ma ¨nner†, and Pilar Ruiz-Lozano*‡
*Development and Aging Program, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037; and†Department of
Anatomy and Embryology, Georg-August-University of Go ¨ttingen, Kreuzbergring 36, D-37075 Go ¨ttingen, Germany
Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved September 25, 2007 (received for review March 14, 2007)
We have previously identified several members of the Wnt/?-
catenin pathway that are differentially expressed in a mouse
model with deficient coronary vessel formation. Systemic ablation
with failure of both mesoderm development and axis formation.
To circumvent this early embryonic lethality and study the specific
role of ?-catenin in coronary arteriogenesis, we have generated
conditional ?-catenin-deletion mutant animals in the proepicar-
dium by interbreeding with a Cre-expressing mouse that targets
coronary progenitor cells in the proepicardium and its derivatives.
Ablation of ?-catenin in the proepicardium results in lethality
between embryonic day 15 and birth. Mutant mice display im-
paired coronary artery formation, whereas the venous system and
microvasculature are normal. Analysis of proepicardial ?-catenin
mutant cells in the context of an epicardial tracer mouse reveals
that the formation of the proepicardium, the migration of proepi-
cardial cells to the heart, and the formation of the primitive
epicardium are unaffected. However, subsequent processes of
epicardial development are dramatically impaired in epicardial-?-
catenin mutant mice, including failed expansion of the subepicar-
dial space, blunted invasion of the myocardium, and impaired
differentiation of epicardium-derived mesenchymal cells into cor-
onary smooth muscle cells. Our data demonstrate a functional role
of the epicardial ?-catenin pathway in coronary arteriogenesis.
cardiovascular development ? smooth muscle ? proepicardium ?
cells that are in direct contact with the pericardial fluid. Cells
that migrate from the epicardium give rise to the cellular
elements of the coronary blood vessels (reviewed in ref. 1).
The embryonic epicardium originates from a primarily extracar-
diac primordium, the proepicardum, which is located at the septum
transversum near the venous pole of the heart (2, 3). In mouse
embryos, proepicardial cells reach the heart predominantly in the
form of free-floating vesicles that traverse the pericardial cavity,
adhere to the initially naked myocardial surface, and subsequently
form the epicardial covering of the heart (reviewed in ref. 1). The
recruitment of coronary vessel progenitor cells from the proepi-
cardium and embryonic epicardium involves several steps of epi-
thelial-mesenchymal transition (EMT). As a result of proepicardial
becomes populated with mesenchymal cells (4–8). Subsequently,
the primitive epicardium gives rise to the subepicardial mesen-
chyme, also by means of EMT.
Recent data suggest that the primitive epicardium and epicardi-
um-derived cells (EPDCs) modulate the maturation of other car-
diac components, including the embryonic myocardium and the
cardiac conduction system (9–14). We have previously shown that
the embryonic epicardium is a key signaling tissue responsible for
the transmission of the morphogenic signal derived from retinoic
acid and identified several components of the Wnt/?-catenin sig-
naling pathway that are down-regulated upon retinoid signaling
deficiency, in particular, ?-catenin and its activator Wnt9b (15).
he epicardium is the outermost cell layer of the postlooped
heart. It consists of a single layer of flattened mesothelial
However, it remains to be shown whether Wnt/?-catenin signaling
?-Catenin is an important signaling molecule throughout devel-
opment and organogenesis, because it is the final effector of the
canonical Wnt ligands (16, 17). Wnts are secreted, cysteine-rich
glycoproteins that are highly conserved among species and bind to
frizzled (Fz) receptors. In the absence of a receptor-bound canon-
ical Wnt, cytosolic ?-catenin is serine- and threonine-phosphory-
lated, rapidly ubiquitinated, and degraded. Binding of Wnts to Fz
proteins in the presence of the coreceptor low-density lipoprotein
receptor-related protein 5 or 6 results in activation of Dishevelled.
Dishevelled inhibits the glycogen synthase kinase 3?-containing
phosphorylation complex, thereby promoting accumulation of cy-
tosolic ?-catenin, which then translocates to the nucleus, and
activates the transcription of numerous genes implicated in prolif-
eration, differentiation, and other cellular processes (reviewed in
The development of numerous organ systems depends on Wnt
signaling (reviewed in ref. 19). In the heart, Wnt signaling plays a
pivotal role in cardiomyocyte specification, and both activation and
inhibition of Wnt signaling affect the formation of the cardiomy-
ocyte compartment (20–27), suggesting that the muscular compo-
nent of the heart depends on the cellular context in which Wnt
stimulation occurs (27).
To explore the role of Wnt/?-catenin in the development of
coronary progenitor cells and its implication on cardiac mor-
phogenesis, we have generated a conditional ?-catenin mutant
mouse lacking ?-catenin expression in the proepicardium and its
Inactivation of the ?-Catenin Gene by G5-Cre-Mediated Deletion
Leads to Embryonic Lethality. We generated a conditional knockout
(KO) mouse lacking ?-catenin expression in the proepicardium
(epiBC-KO) by interbreeding a floxed ?-catenin mouse (28) with
the proepicardium-expressed Gata5-Cre (G5-Cre) mouse (15).
Double heterozygous mice for the ?-catenin floxed allele and
G5-Cre heterozygous (BCf/f:G5cre/?) were found at birth. We
subsequently determined that lethality of epiBC-KO mice occurs
between embryonic stage 15.5 (E15.5) and birth [supporting infor-
mation (SI) Table 1]).
We tested the ability of Cre to ablate ?-catenin in the G5-Cre
Author contributions: P.R.-L. designed research; M.Z. performed research; M.Z., J.M., and
P.R.-L. analyzed data; and M.Z. and P.R.-L. wrote the paper;.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: EMT, epithelial-mesenchymal transition; EPDC, epicardium-derived cell; Fz,
frizzled; KO, knockout; G5-Cre, Gata5-Cre; E(n), embryonic stage n; SMC, smooth muscle
cell; PECAM-1, platelet/endothelial cell adhesion molecule-1; SMA, smooth muscle actin.
‡To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
November 13, 2007 ?
vol. 104 ?
no. 46 ?
expressing cells by direct examination of the ?-catenin protein
content by using immunohistochemistry with a ?-catenin-specific
antibody. At day 9.5, ?-catenin is already efficiently removed in the
?-catenin expression (Fig. 1 A and B). In the hearts of older
with no detectable modification of ?-catenin expression in the
myocardium (Fig. 1 C–D?), thus demonstrating that G5-Cre effi-
ciently removes ?-catenin expression from the proepicardium and
its derivatives without substantially affecting myocardial cells.
Impaired Cardiac Growth in Epicardium-Restricted ?-Catenin Mutant
Embryos. Despite their normal overall embryonic growth (data not
shown), epiBC-KO mice display a pronounced reduction in cardiac
size (Fig. 2 A–C) that clearly manifest after E13.5. Other defects
include rotation of the heart axis toward the left hemi-thorax (Fig.
2 D and E), thin compact-zone myocardium (Fig. 2 H and I), and
expansion (ballooning) of the ventricles (Fig. 2 F and G, arrow-
heads). The thin myocardium defect correlates with a decrease in
myocardial cell proliferation, which has been verified by BrdUra
incorporation analysis (Fig. 2 J–N). Quantification of myocardial
BrdUra incorporation indicates a trend reduction (15.7% reduc-
tion) of BrdUra-positive cells in E12.5 mutant hearts. At E13.5,
reduction in BrdUra incorporation reaches statistical significance
with an 18% decrease at E13.5 and an additional 39.7% reduction
at E15.5 as compared with their WT littermate controls (Fig. 2N).
Similar data have been gathered upon analysis of phosphorylated
histone-3 immunohistochemistry in the epiBC-KO hearts (SI Fig.
6), thus indicating that the ablation of ?-catenin in the epicardium
causes a deficiency in the proliferative capacity of the myocardium.
No increased apoptosis was observed in epiBC-KO hearts (SI
Epicardial ?-Catenin Expression Is Essential for Subepicardial Layer
Formation and Proper Epicardial EMT. To analyze the fate of
epicardial cells in epiBC-KO mice, we interbred the epiBC
mouse with a transgenic mouse line in which epicardial cells are
genetically labeled with ?-galactosidase (Wilms’ tumor1-LacZ,
LacZWT1) (29). Backcross of BCf/?:G5cre/?:LacZWT1with BCf/f
yield epicardial-LacZ?cells deficient in ?-catenin (BCf/f:
G5cre/?:LacZWT1; named epiBC-KOLacZ) and epicardial-LacZ?
cells expressing ?-catenin (BCf/f: LacZWT1; named wtLacZ).
In both wtLacZand in epiBC-KOLacZmice, the outer layer of the
myocardium becomes enveloped with a continuous epithelial sheet
of epicardial cells (Fig. 3 A and B, blue), indicating that migration
from the proepicardium is largely unaffected by proepicardial
mutation of ?-catenin. The epicardium of wtLacZmouse is con-
nected to the myocardium by a subepicardial tissue layer (Fig. 3A,
arrow) that is rich in extracellular-matrix proteins and that contains
mesenchymal cells of epicardial origin (1). In contrast, the subepi-
physical contiguity between the epicardium and the myocardial
layers (Fig. 3B, arrow). Also, nascent subepicardial vascularization
is absent in epiBC-KOLacZhearts (Fig. 3A, arrowhead).
Epicardial cells that transform to subepicardial mesenchymal
Immunohistochemical staining of ?-catenin protein in sections from embry-
onic hearts. (A–B?) WT E9.5 (A and A?) and epicardial ?-catenin mutants E9.5
(epiBC-KO) (B and B?) show the specific ablation of ?-catenin in the proepi-
cardial mutant cells, compared with the WT control littermate. Dotted circles
A and B are magnifications of the proepicardia in WT (A) and mutant (B). (C
epicardial cells (C), whereas its expression is ablated in the mutant epicardial
cells (D). (C? and D?) Zoom image that visualizes details of epicardial ?-catenin
expression. ?-Catenin signal is detected by peroxide staining (brown) mainly
in the cell membrane of WT embryos and is absent in the mutant epicardium
(arrows). Nuclei are counterstained with hematoxylin staining (blue). V, ven-
tricle; Atr, atrium; OFT, outflow track; PE, proepicardium; epi, epicardium;
D, ?400; C? and D?, ?630.)
Ablation of ?-catenin protein in proepicardial and epicardial cells.
mice. (A–C) Gross morphological comparison of cardiac size between E12.5
(A), E15.5 (B), and E18.5 (C) WT hearts (left hearts) and epiBC-KO hearts (right
hearts). (D–I) H&E staining of E13.5 WT mice (D, F, and H) and epiBC-KO mice
(E, G, and I). As indicated by the boxes, F–I are magnifications of images in D
and E15.5 (L and M) hearts. BrdUra staining is brown, and nuclear counter-
staining with hematoxylin is blue. Atr, atrium; RV, right ventricle; LV, left
ventricle; ivs, interventricular septum; cz, compact zone myocardium; epi,
epicardium. Arrowheads point to the interventricular sulcus. (N) Quantifica-
tion of BrdUra-positive nuclei in cardiac samples at different ages of devel-
opment shows hypoproliferation of epiBC-KO hearts after E13.5. Data are
expressed as percentage of the mean ? SE relative to control and compared
by using two-tailed Student’s t analysis. Significant differences were defined
as P ? 0.05. (Magnifications: A–C, ?20; D and E, ?100; F–I, ?200; J–M, ?400.)
Cardiac growth defects in epicardium-restricted ?-catenin mutant
www.pnas.org?cgi?doi?10.1073?pnas.0702415104Zamora et al.
cells express the marker vimentin (30). Costaining of EPDCs
LacZ?/vimentin demonstrates a dramatic reduction of vimen-
tin?cells in the mutant subepicardium (Fig. 3 C and D). In
contrast to wtLacZ, coexpression of LacZ (Fig. 3 C and D, blue)
and vimentin (Fig. 3 C and D, brown) is rarely observed within
the epicardium of epiBC-KOLacZ(Fig. 3 C and D, arrowheads).
Using a combination of LacZWT1and the cell motility marker
tenascin (31), we monitored the evolution of EPDCs during their
invasion of the myocardium. At E13.5, WT EPDCs undergoing
migration (LacZ?/tenascin?) are detected in the interventricu-
lar septum as clusters arranged subepicardially and migrating
inward to the myocardium (Fig. 3E). WT EPDCs, at E14.5, are
located in a medial position of the compact zone myocardium
(Fig. 3G), whereas LacZ expression is down-regulated, indicat-
ing differentiation of EPDCs (29). In contrast, epiBC-KOLacZ
hearts show sparse LacZ-tenascin costaining that remains sub-
epicardial without down-regulation of LacZ staining (Fig. 3H).
of migrating EPDCs in the interventricular septum and compact
zone of 68.8% and 53.3%, respectively (SI Fig. 8) in the
epiBC-KO hearts. These data indicate that EPDCs mutant for
?-catenin have a reduced capacity to invade the myocardium.
Impaired Coronary Artery Formation in Epicardium-Restricted ?-
Catenin Mutant Hearts. Despite a well preserved microvasculature,
the main arterial vessels that run within the myocardial layer from
the base toward the apex of the ventricle (see Fig. 4 A and A?, red
arrows) are completely absent in the epiBC-KO hearts (five
epiBC-KO and five WT mice analyzed, 100% penetrance of the
defect), as shown by platelet/endothelial cell adhesion molecule-1
(PECAM-1) staining (Fig. 4 B and B?). In addition, the aorta of
epiBC-KO mice is not connected to the coronary arteries but
instead is connected to a primitive vascular plexus via two capillary
structures that are located at the sides where the two coronary
arteries normally take their origin (SI Figs. 9 and 10).
The coronary veins are less severely affected compared with the
coronary arteries of the epiBC-KO mice. In normal E18.5 mouse
hearts, three veins, their main branches, and a network of small
veins are found within the subepicardial tissue layer of the dorsal
In the E18.5 epiBC-KO mice recovered alive, a well developed
layer of the ventral and left-lateral aspects of the free ventricular
line in Fig. 4B?).
The normal remodeling of the arterial portions of the coronary
vascular plexus starts at the aorta from where it proceeds in a
proximo-distal direction toward the ventricular apex. We do not
11). The developing coronary arteries, thereby, become invested by
parietal supporting cells that form the tunica media of smooth
muscle cells (SMCs) (32). Remarkably, no parietal supporting cells
are observed in the epiBC-KO mice, as indicated by ?-smooth
by Van Gieson staining (Fig. 4H). These data suggest that epicar-
differentiation into coronary SMCs.
?-Catenin Plays a Role in EPDCs’ Differentiation to Smooth Muscle
Lineage. In the previous section we described that ?-catenin
mutant EPDCs have impaired the potential in vivo to express
mesenchymal markers, decreased migration, and failed to form
the arterial tunica media. To further test the mechanism of
?-catenin action in EPDCs, we tested in vitro the ability of
EPDCs to undergo EMT and smooth muscle differentiation.
transgenic mouse shows deficient expansion of the subepicardial space and
by coexpression of Wilm’s tumor and vimentin in the epicardium. Epithelial cells
activated to EMT coexpress both epithelial (Wilm’s tumor, blue) and mesenchy-
mal markers (vimentin, brown). Coexpression (arrowheads) is not detected in
performed in hearts of E13.5 WTLacZ(E), E13.5 epiBC-KOLacZ(F), E14.5 WTLacZ(G),
and E14.5 epiBC-KOLacZ(H) mice. Red arrows point to migrating cells. (Magnifi-
cations: A and B, ?200; E–H, ?400; C and D, ?630; Insets A, B, G, and H, ?100.)
coronary veins vasculature in WT (C and C?) and epiBC-KO (D and D?) embryos.
Note that the coronary veins are well developed in epiBC-KO mice (blue in
filled cysts are detected in the subepicardial space (B?, blue circles). (E and F)
WT (E) and mutant (F) mice to analyze the smooth muscle component of the
coronary vessels (arrows). (G and H) H&E stain (G) or Van Gieson stain (H) were
used for histological analysis of the smooth muscle layer on the vessels. White
arrowheads point to elastic fibers. (Magnifications: A–D?, ?20; E and F, ?200; G,
H, and Insets E and F, ?630.)
Zamora et al.
November 13, 2007 ?
vol. 104 ?
no. 46 ?
Epicardial cultures dissected from E12.5 hearts were used to
cells. In a set of three independent experiments, WT EPDCs
treated with EMT-inducing media (FBS and FGF2) lose their
epithelial nature by reduction of cell-to-cell contact and adopt a
motile morphology (mesenchymal transformation) (Fig. 5A Left).
Phalloidin staining shows redistribution of F-actin in the WT cells.
A similar mesenchymal phenotype was found in explanted EPDCs
isolated from epiBC-KO mice (Fig. 5A Right and SI Fig. 8 for
During cardiac development, part of the EPDCs that migrate
into subepicardial space and through the myocardium differentiate
into SMCs. Differentiation conditions can be recapitulated in vitro
upon addition of specific factors, including TGF-?1 (33). As shown
in Fig. 5B, treatment with TGF-?1 induces the expression of the
SMC marker ?-smooth muscle actin (SMA) in WT EPDCs after 8
days in culture. In contrast, ?-catenin mutant EPDCs fail to
of Wnt canonical pathway in EPDCs by treatment with Wnt3a
induces differentiation to SMC as demonstrated by the up-
12), smooth muscle lineage markers (34). Three independent
experiments in duplicate were analyzed.
?-Catenin in the Epicardium.Duringtheinitialphaseofformationof
to the myocardium. Subsequently, a space filled with extracellular
matrix forms between the primitive epicardium and the myocar-
dium. The formation of the subepicardial space is probably caused
by a combination of local down-regulation of cell adhesion mole-
cules and enhanced secretion of extracellular matrix proteins (re-
viewed in ref. 35). Using a double epiBC-KOLacZtransgenic mouse
model, we show that proepicardial cells lacking ?-catenin colonize
the heart and establish the primitive epicardium. This finding
of the proepicardium and primitive epicardium. However, epicar-
dial cells devoid of ?-catenin expression fail to trigger the normal
expansion of the subepicardial space. They, furthermore, fail to
suggesting that active ?-catenin expression in the embryonic epi-
cardium is required to trigger transcriptional modifications neces-
sary for epicardial development.
Mutations that alter epicardial EMT impair coronary vessel
formation (36, 37). We demonstrate that, in contrast to WTLacZ,
coexpression of LacZ (Wilms’ tumor, epicardial marker) and
vimentin (mesenchymal cells) is rarely observed in epiBC-KOLacZ,
indicating that the loss of ?-catenin compromises the epicardial
EMT normally required in vivo for the proper development of the
subepicardial space. Furthermore, in mouse embryonic heart,
with cell motility during tissue remodeling, and it labels the cells
invading the ventricular myocardium (31). We show a significant
reduction of LacZ/tenascin costaining in epiBC-KO hearts, thus
indicating impairment of EPDCs to migrate through the myocar-
dium. Importantly, although in vivo EMT and cell migration are
affected, they are not totally obliterated, demonstrating some
in vitro studies show that both WT and epiBC-KO epicardium-
explanted monolayers have a similar capacity to reorganize F-actin
?-catenin is not essential for EMT stimulation. The apparent
discrepancy between in vitro and in vivo data may result from
differences in the cellular environment. EpiBC-KO hearts lack a
alternative hypothesis can explain a reduced number of epicardial
cells within the myocardial wall, including increased cell death,
lower cell proliferation, and defective cell migration. In this regard,
we show here that, whereas apoptosis is unchanged in epiBC-KO
hearts, cell proliferation and cell migration are consistently de-
creased upon epicardial ?-catenin ablation. This result is consistent
with the described role of Wnt9a in the promotion of cell prolif-
eration during cardiac cushion mesenchyme development (39).
We report here a failure of remodeling of the primitive coronary
plexus into coronary arteries, although some degree of perfusion
from the aorta into the vasculature occurs (Fig. 4). The mature
coronary vascular system arises from the remodeling of a primitive
endothelial vascular plexus (40). The primitive coronary vascular
and remains unaffected in epiBC-KO hearts (SI Fig. 11), further
supporting a specific defect in the vascular smooth muscle lineage.
remodeling process during which two of the connections to the
aorta are transformed into the proximal stems of the two coronary
arteries and the adjacent portions of the coronary vascular plexus
are transformed into the distal stems and branches of the two
coronary arteries (40). In the epiBC-KO mice, the aorta is not
connected to coronary arteries but is connected to a primitive
vascular plexus via two small vascular tubes that are located at the
sides where the two coronary arteries normally take their origin.
This finding suggests that in the epiBC-KO mice, the ingrowth of
the coronary plexus-derived endothelial sprouts into the aortic
sinuses took place but the remodeling of the coronary vascular
of a vascular media layer. It is important to note that although the
main alteration of the epiBC-KO is observed at the arterial pole,
some venous structures are affected as well, with nonvascularized
activation upon treatment with FBS and FGF2 in epicardium explanted cells
from WT (Left) or epiBC-KO (Right) E12.5 embryo hearts. (B) SMC differenti-
ation of WT epicardium cultured cells upon stimulation with vehicle (Upper
Left), Wnt3A (Upper Right), or TGF-?1 (Lower Left). Stimulation with TGF-?1
was also tested in epicardium cells from epiBC-KO (Lower Right). Phalloidin
(red) shows cell morphology, DAPI (blue) indicates nuclear staining, and
?-SMA expression by immunostaining is green. (Magnifications: ?200.)
EMT and SMC differentiation of epicardium cultured cells. (A) EMT
www.pnas.org?cgi?doi?10.1073?pnas.0702415104Zamora et al.
tissue, preferentially in the ventral and left lateral walls of the
ventricles. Whether arterially derived signals are involved in the
proper development of the venous pole is currently unknown.
Impaired Development of EPDCs and Myocardial Growth Defects.
Several data suggest that the growth of the embryonic and fetal
myocardium depends on proper development of the epicardium
deficient formation of the epicardium, deficient coronary vascula-
ture, and a severe growth defect of the compact layer myocardium
called ‘‘thin myocardium syndrome’’ (41). Genetic ablation of
epicardium and EPDCs but also causes remarkable growth defects
of the compact layer myocardium (13, 41, 42). In the present study,
we have found that epicardium-specific deficiency in ?-catenin
expression does not only disturb the development of EPDCs but
additionally leads to hypoplasia of the compact layer myocardium
and reduced expansion (ballooning) of the ventricles. This result is
explained by the significant reduction of myocardium proliferation
in epiBC-KO mice. We demonstrate here that ?-catenin is specif-
?-catenin protein in the myocardium is not modified by the
for ?-catenin in the myocardium and the epicardial compartments.
At the present time, we do not know whether the myocardial
hypoplasia of epiBC-KO mice results from nutritional defects
the growth defects before vascular remodeling suggest that epicar-
dial ?-catenin mutation, at least in part, impairs the release of
growth-promoting signals normally generated by EPDCs.
Role of Wnts in Epicardial Development. Several canonical Wnt
ligands and Fz receptors are expressed in a spatially and temporally
regulated manner consistent with a function during early heart
development. At gastrula stages of mouse embryos, Wnt2a is
expressed in the heart-forming fields and it is later restricted to the
pericardium (43). Within or lateral to the primitive streak, Wnt3a,
Wnt8 is detected in the myocardium and four additional members
of the Wnt family (Wnt-2b, Wnt5a, Wnt7a and Wnt9a) are ex-
pressed in the tubular heart of chicken embryos (reviewed in ref.
49). In addition to the diversity of Wnt ligands, many different Wnt
receptors of the Fz family have been detected in the mesoderm of
the heart-forming fields, cardiac neural crest cells, and the adult
Fz-10b, or human Fz-1, Fz-2, Fz-7, Fz-8, Fz-9 (reviewed in ref. 49).
This large number of different ligands and receptors suggests that
Wnt proteins influence diverse aspects of cardiogenesis.
It is important to note that ?-catenin is part of the E-cadherin/
?-catenin complex that links this complex to the cytoskeleton and
plays a role in epithelial polarity (50). ?-catenin is a bifunctional
protein involved in both cell–cell adhesion through its participation
in the adherens junctions and the regulation of gene transcription
potentially important in epicardial development and may be re-
sponsible for the defective coronary vasculature observed in the
epiBC-KO mouse. Our previous observations in which lithium
chloride (glycogen synthase kinase ? inhibitor) treatment enhances
vascular tube formation (15) and differentiation of epicardial-
derived SMCs (data not shown) suggest a direct involvement of the
Two recent reports support this hypothesis. Missense mutation of
and multiple cardiovascular risk factors (51). In addition, a TCF/
Lef-LacZ reporter of ?-catenin transcriptional activity has identi-
fied both the proepicardium and the epicardium as active targets of
Wnt-mediated transcriptional activity (27). Here, we have demon-
strated progenitor differentiation into the smooth muscle lineage is
largely impaired in the epiBC-KO epicardium. Conversely, stimu-
further supporting a model in which the actions of ?-catenin on
coronary remodeling are, at least in part, directly mediated by
canonical Wnt stimulation. The source of Wnt that activate coro-
nary differentiation is currently unclear.
Generation of Epicardium-Restricted ?-Catenin Mutants. Epicardi-
um-restricted ?-catenin mutant mice (epiBC-KO) were generated
upon crossing the G5-Cre line (15) with the ?-catenin floxed mice
gous for the floxed ?-catenin (BCf/?) were crossed with BCf/?mice
that are heterozygous for the epicardial Cre transgene (Cre/?).
Primers for genotyping were: G5cre, G5cre1, 5?-ATC TTC CAG
CAG GCG CAC CAT TGC CCC TGT-3?, G5cre2, 5?-TGA CGG
TGG GAG AAT GTT AAT CCA TAT TGG-3?; BCfl,RM41,
CAT GTC CTC TGT CTA TTC-3?. RM41/RM42 results in the
amplification of a WT band of 221 bp and a floxed ?-catenin band
of 324 bp. The presence of the Cre gene is determined by the
amplification of a 450-bp band by using Cre1/Cre2 primers. Em-
bryos were obtained from time pregnancies, with plug date defined
Generation of Epicardium-Restricted ?-Catenin Mutants in the Con-
generated upon crossing epiBC line with the line expressing
?-galactosidase under Wilms’ tumor 1 promotor (LacZWT1)
(29). For breeding purposes, BCf/?: G5cre/?: LacZWT1mice were
crossed with BCf/fmice, to generate BCf/f: G5cre/?: LacZWT1
(epiBC-KOLacZ) mice. Primers for genotyping were: LacZ1,
5?-CGG GTT GTT ACT CGC TCA CAT-3?, LacZ2, 5?-ATG
CAG AGG ATG ATG CTC GTG-3?. Primers used for G5cre
and BC floxed allele are described above.
Histology, Immunohistochemistry, and ?-Galactosidase Staining. Dis-
sected mouse embryos were fixed in 4% paraformaldehyde over-
or cryopreserved in 30% sucrose and embedded in OCT. Sections
For immunostaining, paraffin sections were processed for heat-
induced epitope retrieval by using citrate buffer at pH 6.0 and
heated for 45 s in the microwave. Antibodies used were as follows:
1:5,000 diluted ?-catenin (Abcam, Cambridge, MA), 1:200 diluted
vimentin (Sigma, St. Louis, MO), 1:200 diluted tenascin (Chemi-
con, Temecula, CA), undiluted ?-SMA-HRP (DAKO, Glostrup,
Elastin fibers were labeled with Van Gieson stain. To visualize the
epicardial cells in the epiBC-KOLacZmouse, embryos were fixed in
2% paraformaldehyde and washed with PBS. ?-Galactosidase
staining was performed on whole mount with X-Gal solution at
37°C until color developed. Nuclear red fast (Vector, Burlingame,
CA) was used as a counterstain.
BrdU Labeling Analysis. Pregnant, WT, and epiBC mice were i.p.-
injected with BrdU (50 mg/kg) 1 h before death. Detection of
BrdUra-positive cells was performed by immunohistochemistry
with the BrdU Staining Kit from Zymed Laboratories (Carlsbad,
CA). Results were obtained after scoring the percentage of BrdU-
positive nuclei/total nuclei in five different areas of five histological
sections per embryo in a total of three embryos per stage. Three
independent WT embryos were compared with three independent
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vol. 104 ?
no. 46 ?
Whole-Mount PECAM-1 Immunohistochemistry. Hearts were har-
vested from WT and epiBC-KO embryos and fixed in 4% para-
overnight in 100% methanol at ?20°C. Whole-mount immuno-
used was rat anti-mouse PECAM-1 (1:200; R&D, Minneapolis,
Vector) was followed by Vectastain ABC-peroxidase reagent and
diaminobenzidine visualization (Vector). After hearts were pho-
tographed and analyzed, they were embedded in paraffin and
sectioned. Paraffin sections were then stained with eosin and
mounted, and histological sections were analyzed.
Arterial/Vein Morphological Identification. In the prenatal and post-
intramyocardial course, whereas the coronary veins have a subepi-
cardial course (53, 54). Both types of coronary vessels have typical
branching patterns and are connected either to the aorta (coronary
arteries) or the sinus venosus (coronary veins). In PECAM-stained
whole-mount hearts, coronary arteries thus were identified on the
basis of (i) their intramyocardial course, which was indicated by the
fact that these vessels were superficially crossed by myocardial
capillaries, and (ii) their branching pattern. Coronary veins were
identified on the basis of (i) their subepicardial course, which was
indicated by the fact that these vessels run superficially from the
myocardial capillaries, and (ii) their branching patterns and con-
in the mature heart).
Isolation and Culture of Embryonic Epicarial Cells. HeartsfromE12.5
were dissected, and ventricular chambers were placed epicardial
cultured in 1:1 mixture of DMEM and medium 199 containing 100
units/ml penicillin, 100 ?g/ml streptomycin, 10% FBS, and 2 ng/ml
basic fibroblast growth factor (B&D, Franklin Lakes, NJ) as
described (33). After 48 h, epicardial cell monolayers were formed
and the explanted hearts were removed. For EMT activation
experiments, the epicardial explants were cultured for 6 days and
the media were changed every 2 days. Phalloidin staining was used
to identify motile phenotype. To study SMCs differentiation, after
removal of the hearts, the culture medium was replaced with
DMEM-PS without growth factors or with recombinant human
TGF-?1 (PeproTech, Rocky Hill, NJ) or Wnt3a (B&D) at a final
concentration of 50 and 10 ng/ml, respectively. The cells received
immunostaining at 8 days.
M.Z. was the recipient of an award from the Spanish Ministerio de
Educacio ´n y Ciencia and is currently a trainee of the California Institute
for Regenerative Medicine. This work was supported by National
Institutes of Health Grant HL065484 (to P.R.-L.). Work in J.M.’s
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www.pnas.org?cgi?doi?10.1073?pnas.0702415104Zamora et al.