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Contribution of the Primitive Epicardium to the
Subepicardial Mesenchyme in Hamster and Chick Embryos
Department of Animal Biology, Faculty of Science, University of Málaga, Málaga, Spain
A study about the hypothetical
contribution of the epicardial cells to the subepicardial mesenchyme was carried out in Syrian
hamster embryos of 9–12 days post coitum (dpc)
and chick embryos of 3–5 days of incubation. In
the epicardium and subepicardium of these embryos we have immunolocated the proteins cytokeratin (CK), vimentin (VIM), fibronectin (FN),
and two antigens related to the transformation of
endocardial cells into valvuloseptal mesenchyme,
ES/130 and JB3. In the hamster embryos, CK1
subepicardial mesenchymal cells (SEMC) were
apparently migrating from the primitive epicardium from 9.5 dpc at the atrioventricular (AV)
groove and proximal outflow tract (OFT). The
morphological signs of delamination extended by
11 dpc to the epicardium of the interventricular
groove and the dorsal part of the ventricle. The
relative abundance of the CK1 SEMC decreased
in embryos of 12 dpc. VIM colocalized with CK in
most SEMC, and in some epicardial mesothelial
cells, mainly at the areas of delamination. CK
immunoreactivity was also found in some early
subepicardial capillaries. Similar observations
were made in the chick embryos studied. The
immunoreactive patterns obtained at the subepicardium with anti-FN, ES/130, and JB3 antibodies were similar to those reported in the areas of
endothelial transformation of the endocardial
cushions. We suggest that these observations are
compatible with an epithelial-mesenchymal transformation involving the epicardial mesothelium
and originating at least a part of the SEMC. Dev.
Dyn. 1997;210:96–105. r 1997 Wiley-Liss, Inc.
well known (Choy et al., 1993). Some of them contribute
to the cellular component of the atrioventricular valves
(Wenink, 1992), others secrete the extracellular matrix
elements of the subepicardium (Tidball, 1992), and
others might coalesce to form the primary cardiac
capillary plexus (Viragh and Challice, 1981; Tokuyasu,
1985; Icardo et al., 1990; Bolender et al., 1990; Viragh et
al., 1990).
Our previous morphological studies in a primitive
vertebrate, the dogfish (Scyliorhinus canicula), have
suggested that, in this species, the early subepicardial
mesenchymal cells (SEMC) originate from the epicardial mesothelium. In the dogfish, we could discard the
migration of extrinsic mesenchymal cells since the
subepicardium is not connected with extracardiac areas until many SEMC and a part of the capillary plexus
have already appeared (Muñoz-Chápuli et al., 1996).
We wanted to know if a similar process to that
described in the dogfish (i.e., delamination of SEMC
from the epicardial mesothelium) actually occurs in the
embryos of higher vertebrates. In fact, this possibility
has been suggested by some authors on a morphological
basis (Viragh et al., 1990, 1993; Icardo et al., 1990).
Our approach, conducted on Syrian hamster and
chick embryos, was based on the immunohistochemical
localization in the epicardium and SEMC of the proteins cytokeratin (CK) and vimentin (VIM), which
constitute the intermediate filaments characteristic of
epithelial and mesenchymal cells, respectively. The
expression of VIM prior to the transformation of an
epithelial cell is probably involved in the premigratory
shape changes (Hay, 1990). On the other hand, CK
immunoreactivity persists for some time in the mesenchymal cells originated from CK1 epithelial cells (Fitchett and Hay, 1989; Hay, 1990). Thus, if epicardial
mesothelial cells detach and migrate into the subepicardium, colocalization of CK and VIM should be expected
at the migratory as well as at the premigratory stages.
We have also assessed the presence in the subepicardium of three molecules involved in the transformation
of endocardial cells into mesenchymal cells, which
occurs in the embryonic heart, i.e., fibronectin (FN), the
In the vertebrate embryos, the epicardial investment
of the heart is closely followed by the establishment of a
space between the primitive epicardium and the myocardium, a space that is initially acellular and filled with
an amorphous extracellular matrix. This space, the
subepicardium, is rapidly populated by mesenchymal
cells whose origin and developmental fate is not yet
Grant sponsor: Ministerio de Educación y Ciencia, Spain; Grant
number: PB95-0475; Grant sponsor: Junta de Andalucı́a.
*Correspondence to: R. Muñoz-Chápuli, Dept. of Animal Biology,
Faculty of Science, University of Málaga, 29071 Málaga, Spain.
Received 10 March 1997; Accepted 11 June 1997
Key words: epicardium; mesothelium; subepicardial mesenchyme; cytokeratin; vimentin; fibronectin; coronary vessels; chick embryo; Syrian hamster
fibrillin-like protein recognized by the monoclonal antibody JB3, and the antigen ES/130. Fibronectin shows a
characteristic distribution in the subendocardium during the formation of the atrioventricular (AV) and
outflow tract (OFT) endocardial cushions (Icardo and
Manasek, 1984; Mjaadvedt et al., 1987). On the other
hand, only the endocardial cells expressing the JB3
antigen are susceptible to be transformed in mesenchyme cells (Wunsch et al., 1994; Barton et al., 1995;
Bouchey et al., 1996). Finally, the ES/130 antigen
seems to be involved at a critical step in the initiation of
the epithelial-mesenchymal transformation of the cardiac endothelium (Rezaee et al., 1993; Markwald et al.,
These immunohistochemical markers do not provide
a direct proof of the SEMC lineage, but they can afford
evidence that mesothelial cells from the primitive epicardium are able to transform, migrate, and contribute
to the subepicardial mesenchyme.
The animals used in our research program were
handled in compliance with the international guidelines for animal care and welfare. The Syrian hamsters
(Mesocricetus auratus) were housed in polypropylene
cages in a room with controlled temperature and photoperiod. Commercial mouse food (UAR/Panlab, Barcelona, Spain) and water were given ad libitum, starting
at weaning. Eggs from chick (Gallus gallus) were
obtained from commercial suppliers and placed in a
rocking incubator at 38°C.
The Syrian hamster sample consisted of 39 embryos.
Mature male and female specimens of hamster were
mated, and the end of the coitus was considered as the
day 0 of the embryonic development. The embryos were
collected at the stages 9 (3), 9.5 (5), 10 (9), 10.3 (4), 11
(13), and 12 (5) days post coitum (dpc). Pregnant
females were killed by chloroform overdose, and the
embryos obtained by laparotomy and uterotomy, freed
from fetal membranes and fixed.
The sample of chick embryos consisted of seven
specimens of the stages HH17–HH27 of Hamburger
and Hamilton (1951) (3–5 days of incubation).
The embryos were fixed in 40% methanol, 40% acetone, 20% distilled water for 8–12 hr. A few embryos
were fixed either in the same fixative including 16% of
saturated picric acid solution or in Bouin. After fixation,
the embryos were dehydrated in an ethanolic series
finishing in butanol, and paraffin-embedded. Serial
sections (5 and 10 µm) were obtained with a Leitz
(Wetzlar, Germany) microtome and collected on poly-llysine coated slides. The sections were dewaxed in
xylene, hydrated in an ethanolic series, and washed in
Tris-phosphate buffered saline (TPBS, pH 5 7.8). The
ulterior treatment of the slides varied according to the
antibody used, as described below.
For the histological study of the subepicardium, two
embryos (11 dpc) were fixed by perfusion through the
left ventricle with 2% glutaraldehyde and 1% parafor-
maldehyde in 50 mM cacodylate buffer. The embryos
were then immersed in the same fixative for 45 min,
washed in cacodylate buffer for 30 min, and postfixed in
1% OsO4 for 90 min. After washing in distilled water
(308) the embryos were dehydrated in an ethanolic
series finishing in acetone and embedded in Araldite 502.
Semithin sections were obtained in a Reichert UMO-2
ultramicrotome and stained with toluidine blue.
Peroxidase Immunohistochemistry
Endogenous peroxidase activity was quenched by
incubation for 30 min with 3% hydrogen peroxide in
TPBS. After washing with TPBS, non-specific binding
sites were saturated for 30 min with 10% sheep serum,
1% bovine serum albumin, and 0.5% Triton X-100 in
TPBS (SBT). The slides were then incubated overnight
at 4°C in the primary antibody diluted in SBT. Control
slides were incubated in SBT only or with non-immune
rabbit serum diluted 1:200.
After incubation, the slides were washed in TPBS,
incubated for 1 hr at room temperature in biotinconjugated anti-mouse or anti-rabbit goat IgG (Sigma,
Dorset, UK) diluted 1:100 in SBT, washed again and
incubated for 1 hr in avidin-peroxidase complex (Sigma)
diluted 1:150 in TPBS. Peroxidase activity was developed with Sigma Fastt 3,38-diaminobenzidine tablets
according to the indications of the supplier. In some
cases, the staining was intensified with metallic cations
or the slide was counterstained with hematoxylin.
Two anti-cytokeratin antibodies were used. Monoclonal anti-pan cytokeratin (C2562, Sigma) is a mix of six
monoclonal antibodies, which stains most types of
keratin in epithelial cells of several vertebrates, but it
shows no cross-reaction with non-epithelial normal
human tissues. It was used at 1:200 dilution. Polyclonal
anti-cytokeratin (Z622, Dakopatts, Glostrop, Denmark)
is used for wide screening of keratins in several tissues.
This antibody was diluted at 1:500. No significant
differences were found between the results obtained
with both antibodies, but the monoclonal one gave less
background staining.
Polyclonal rabbit anti-human fibronectin antibody
(ICN Pharmaceuticals, Irvine, CA) was used only for
hamster embryos at a 1:500 dilution. The polyclonal
anti-ES/130 (Rezaee et al., 1993) and the monoclonal
JB3 (Wunsch et al., 1994) were a generous gift from Dr.
Edward Krug (Medical University of South Carolina).
The anti-ES/130 antiserum was diluted 1:500 and used
only with hamster embryos. The anti-JB3 supernatant
was diluted 1:100 and used only with chick embryos.
Fluorescence Immunohistochemistry
and Double Labellings
For VIM immunofluorescence, the dewaxed sections
were saturated for 30 min with 10% rabbit serum, 1%
bovine serum albumin, and 0.5% Triton X-100 in TPBS
(RBT). The slides were then washed and incubated as
described above. Polyclonal goat anti-human vimentin
(ICN Pharmaceuticals) was used as the primary anti-
body diluted 1:60 in RBT. After washing, the slides were
incubated in FITC-conjugated rabbit anti-goat IgG
diluted 1:75 in RBT for 1 hr, washed, and mounted in
The CK/VIM double immunolabelling was performed
with a mix of monoclonal and polyclonal primary
antibodies. For the hamster embryos, the slides were
blocked with SBT for 30 min and incubated for 1 hr
with both antibodies, monoclonal anti-human VIM
(Boehringer, Mannheim, Germany) diluted 1:50, and
polyclonal anti-CK diluted 1:50 in SBT. After washing
in TPBS, the slides were first incubated for 45 min in
TRITC-conjugated goat anti-mouse IgG (Sigma) diluted 1:50. This solution had been preadsorbed for 1 hr
with 10% rabbit serum. After washing, the slides were
blocked again in SBT, incubated for 1 hr in biotinconjugated goat anti-rabbit IgG (Sigma) diluted 1:100
in SBT, washed and incubated in extravidin-FITC
conjugate (Sigma) diluted 1:100. For the chick embryos,
the procedure was similar, but a monoclonal anti-chick
VIM (Developmental Studies Hybridoma Bank, University of Iowa, clone AMF-17b) was used as primary
antibody. Controls were incubated only with one primary antibody and then with both secondary antibodies, in order to detect any cross-reaction between the
secondary and the primary antibodies.
The sections were observed in a Nikon Microphot
FXA equipped with epifluorescence and in a laser
confocal microscope Leica TCS-NT (Heidelberg, Germany), using filters specific for the FITC and TRITC
fluorochromes. Selected images were captured and
printed in a Sony digital color printer.
tract (OFT). By 10 dpc only the distal OFT and some
areas of the dorsocephalic part of the atrium were not
covered by the epicardium. The epicardial investment
was complete in the embryos of 11 dpc, when the
mesothelial villi of the transverse septum had already
The subepicardial space first appeared around the AV
canal and proximal OFT, and it was at its maximum
width by 10 dpc (Fig. 1). A subepicardial space subsequently appeared in the sinoatrial (SA) groove, dorsal
surface of the ventricle, interventricular (IV) groove,
and in the ventral part of the atrium. The cephalic
areas of the atrium usually lacked a subepicardium in
the embryos studied. In the embryos of 12 dpc, the
relative volume of the subepicardium decreased markedly.
Only a few SEMC could be seen in embryos of 9.5 dpc,
but they were already abundant by 10 dpc in those
areas where the subepicardium was the widest, i.e., the
SA, AV, and conoventricular grooves (Fig. 1). SEMC
were also present in the ventral part of the sinus
venosus and dorsal part of the ventricle, and they
appeared by 11 dpc in the IV groove. These cells were
frequently in contact with the primitive epicardial cells.
A number of epicardial cells, specially those of the
intercameral grooves, were large and spheric, they
showed rounded nuclei and long basal cytoplasmic
processes. Cell overriding also occurred in these areas
(Fig. 2). By 11 dpc, the morphological signs of delamination of SEMC from the primitive epicardium had extended to the dorsal part of the ventricle and the IV
groove. However, they only persisted at the AV, conoventricular, and IV grooves in the embryos of 12 dpc.
Development of the Epicardium, Subepicardium,
and SEMC in the Syrian Hamster
Cytokeratin Immunohistochemistry
There are no available data in the literature on the
development of the epicardium, subepicardial mesenchyme, and subepicardial capillary vessels in embryos
of Syrian hamsters. For this reason, we will start with a
brief morphological description of these processes. The
chronology of these developmental events was very
similar to that of the mouse embryo.
In embryos of 9 dpc, the myocardium was not covered
by epicardial cells. Just a few cells, probably precursors
of the primitive epicardium, could occasionally be seen
adhered to the cardiac wall, mainly in the neighbourhood of the AV junction. Round cells and small
vesicles seemed to be released in the coelom from the
mesothelial villi of the transverse septum. The vesicles,
composed of epithelial cells, occasionally contained
mesenchymal cells inside.
The epicardial lining of the heart was relatively fast,
as observed in embryos of 9.5 and 10 dpc. The first areas
of the heart to be covered by the epicardium, about 9.5
dpc, were the AV canal, the ventral part of the atrium,
the ventral part of the sinus venosus, the dorsal part of
the ventricle, and a ring around the proximal outflow
In all the embryos studied, the anti-CK antibodies
labelled the cells of the primitive epicardium as well as
those from the splanchnic mesothelium, the epidermis,
and the endodermal epithelium. We will describe now
the distribution of the CK1 SEMC in the heart of the
embryos studied.
As described above, SEMC were already abundant by
10 dpc in the sinoatrial and AV grooves and also around
the proximal OFT. Most of these SEMC were CK1 (Figs.
3,4). CK1 mesenchymal cells were also abundant in the
limit between the liver and the sinus venosus, but a
clear discontinuity was usually present between this
population and the SEMC of the AV groove. CK immunoreactivity also labelled cells that were forming ringlike structures, sometimes involving several cells. The
blood island-like vessels that could be observed in the
subepicardium occasionally showed CK1 cells in their
endothelial lining (Fig. 3).
In the hamster embryos of 11 dpc, CK1 SEMC have
appeared in the dorsal part of the ventricle and in the
interventricular (IV) groove. Some of them could be
seen infiltrated in inner areas of the myocardial wall,
specially at the AV canal and IV septum. Ring-like
structures were abundant, and they were usually formed
Fig. 1. Sagittal section of the heart of a Syrian hamster embryo (10
dpc). Hematoxylin-eosin staining. A wide subepicardium (labelled with
asterisks) is present around the atrioventricular junction and proximal
outflow tract, the areas where the atrioventricular (AVC) and outflow tract
(OTC) cushions are present. Some epicardial cells of these areas are
large and rounded (arrowheads) and show basal cytoplasmic processes
(arrow). A, atrium; V, ventricle. Scale bar 5 25 µm.
Fig. 2. Morphological signs of epithelial-mesenchymal transition at
the proximal outflow tract (conoventricular groove) of a hamster embryo
(11 dpc). Semithin section stained with toluidine blue. There is morphological evidence of cell delamination (arrows) from the epicardial mesothelium (E) to the subepicardium. Some mesenchymal cells are forming
ring-like structures (stars), which sometimes involve cells from the epicardium
itself. OTC, outflow tract cushion; V, ventricle. Scale bar 5 12 µm.
by CK1 cells (Fig. 5). By 12 dpc the proportion of CK1
SEMC had decreased, as well as the intensity of their
immunoreactivity. The mesenchymal cells more intensely labelled with the anti-CK antibody were restricted to the AV and IV grooves, the only places where
the morphological signs of epicardial delamination
remained. Some CK1 mesenchymal cells were located
in deep areas of the myocardium, as described above.
CK1 immunoreactivity was detected in the endothelial
lining of some subepicardial capillaries (Fig. 6).
In the chick embryos of 3–5 days of incubation (HH
stages 17–27), the CK immunoreactive pattern was
similar to that described above for the Syrian hamster
embryos (Figs. 9, 10). The primitive epicardium of the
AV groove and OFT showed signs of delamination,
which extended, in the embryos of stages HH21–23, to
all the ventricles. CK1 cells were very abundant in the
subepicardial space as well as inside the proepicardial
protrusions located in the sinus venosus wall and in the
liver-cardiac limit. There was not a discontinuity be-
tween the CK1 cells of the subsplanchnic and the
subepicardial spaces (Fig. 10). Ring-like structures and
blood-containing vessels showed CK1 cells in their
walls (Fig. 9a,c). Furthermore, some cells lining the
hepatic sinusoids were also CK1 in these stages (Fig.
Cytokeratin/Vimentin Colocalization
Vimentin antibodies labelled all the endothelial cells,
including the endocardial cells, the subendocardial
mesenchyme, and most SEMC. In the 10 and 11 dpc
hamster embryos, as well as in the chick embryos
studied, colocalization of CK and VIM was relatively
frequent in SEMC, either isolated or forming cord-like
or ring-like structures (Figs. 7, 8). In both animal
models studied, the epicardial mesothelial cells from
the areas where delamination was presumably occurring were frequently also CK1/VIM1. However, the
squamous epicardial cells covering other parts of the
heart devoid of subepicardial space were CK1/VIM2.
Fibronectin Immunohistochemistry
In the subepicardium of the Syrian hamster embryos,
the anti-fibronectin antibody stained the basal surface
of the epicardial cells as well as the SEMC. The
mesenchymal cells were stained in a pattern of irregular patches associated to the cell membranes. Filopodial processes were always distinctly stained (Fig. 11).
In the areas where the squamous epicardial cells are
directly adhered to the myocardial wall, without a
subepicardial space, a distinct FN immunostaining was
present between the myocardium and the epicardium,
probably related to the presence of a basal lamina.
JB3 Immunohistochemistry
In the chick embryos, the fibrillin-like protein detected by the JB3 antibody was present at the subepicar-
Figs. 3–6. Cytokeratin (CK) immunoreactivity in the heart of the
Syrian hamster embryos.
Fig. 3. Embryo of 10 dpc, sagittal section of the dorsal part of the
atrium and atrioventricular groove. CK1 mesenchymal cells are abundant
in the liver (L)-cardiac limit, but other CK1 cells (M) seem to be
delaminating from the epicardial mesothelium (E) of the atrioventricular
groove. There is a clear separation between these populations of CK1
mesenchymal cells. Some cells of the blood island-like structures (B) also
are CK1 (arrowheads). AM, atrial myocardium; AVC, dorsal atrioventricular cushion; S, splanchnic mesothelium; V, ventricle. Scale bar 5 12 µm.
Fig. 4. Embryo of 10 dpc, sagittal sections. Signs of delamination of
CK1 cells from the epicardial mesothelium (E) in the proximity of the
atrioventricular (AVC) and outflow tract (OTC) cushions (a, b and c,
respectively). Some CK1 cells are apparently migrating into the myocardial wall (arrowheads). A, atrium; B, blood island-like structure; V,
ventricle. Scale bars: a, b 5 20 µm; c 5 24 µm.
Fig. 5. Embryo of 11 dpc, transverse section, laser confocal microscopy. CK1 cells are present at the atrioventricular subepicardium and into
the myocardial wall (arrows). An epicardial cell shows a large basal
cytoplasmic process (star). Note the CK1 ring-like structure (arrowhead).
A, atrium; V, ventricle. Scale bar 5 10 µm.
dium of the areas where SEMC were presumably
delaminating from the primitive epicardium (Figs. 12,
13). In these areas, JB3 stained the cytoplasm of some
epicardial cells, their basal cytoplasmic projections, the
surface of a number of SEMC, and the subepicardial
capillaries. This pattern of immunostaining was similar to that observed in the endocardial cushions (Fig.
ES/130 Immunohistochemistry
In the hamster embryos, the ES/130 antigen labelled
the endocardium as well as a part of the epicardial cells
that covered the areas of wide subepicardium (Fig. 14),
especially those large and with round nuclei. However,
the squamous epicardial cells adhered to the myocardium usually were ES/1302.
We have carried out a study on a poorly known aspect
of the cardiac development, the origin of the subepicardial mesenchyme. The hypothesis to be tested was the
existence of a contribution of epicardial mesothelial
cells to the subepicardial mesenchyme, a process already shown by us in a fish model (Muñoz-Chápuli et
al., 1996).
The first evidence that supports our hypothesis is the
existence of morphological signs of delamination in
specific areas of the epicardium, mainly in those that
are not directly adhered to the myocardium and cover
the AV and IV grooves as well as the proximal OFT.
These evidences, which include cell hypertrophy, basal
cytoplasmic processes, and cell overriding, are the same
that appear in the endocardium during the formation of
the cushion mesenchyme (Bolender and Markwald,
1979; Markwald et al., 1985). The earliest SEMC
appear in these areas of wider subepicardium, frequently in contact with the epicardial cells.
Fig. 6. Embryo of 12 dpc, transverse section of the dorsal part of the
ventricle (V). The proportion of CK1 cells has decreased. Some of them
can be seen into the myocardial wall (arrowhead) and others in the wall of
blood-containing capillaries (arrows). Note the lack of CK1 mesenchymal
cells in the subepicardium of the atrium (A). Scale bar 5 9 µm.
Fig. 7. Colocalization of cytokeratin (a) and vimentin (b) in a Syrian
hamster embryo (11 dpc). Transverse section of the atrioventricular
groove, laser confocal microscopy. The epicardial mesothelium (E) as
well as most subepicardial mesenchymal cells (M) contain both types of
intermediate filaments. However, the mesenchyme cells of the atrioventricular cushion are VIM1/CK2 (arrow in b). Other cells of this embryo,
such as those from the gut and the epidermis were VIM2/CK1, demonstrating that the double labelling was not due to cross-reactions. Scale bar 5
9 µm.
Fig. 8. Colocalization of cytokeratin (a) and vimentin (b) in a chick
embryo (4.5 days of incubation, stage HH24). Transverse section of the
atrioventricular groove, laser confocal microscopy. There is a clear
colocalization of CK and VIM in part of the epicardial mesothelium (E) as
well as in a number of subepicardial mesenchymal cells (M). A presumable capillary containing a red blood cell is also double-labelled (arrowhead). The tube-like structure shown by the arrow is formed by two cells
coexpressing CK and VIM. Scale bar 5 10 µm.
Figs. 3–8.
The immunolocation of CK and VIM in the primitive
epicardium and SEMC constituted a second test of our
hypothesis. CK immunoreactivity is strong in the primitive epicardial cells, and it has been applied to describe
the epicardial lining of the heart (Vrancken Peeters et
al., 1995). The persistence of the original epithelialtype intermediate filaments, after the transdifferentiation of an epithelium, has been reported both in in vivo
and in vitro systems (Fitchett and Hay, 1989; Hay,
1990). On the other hand, the expression of VIM prior
to the transformation of an epithelial cell is probably
involved in the premigratory shape changes (Hay, 1990)
and it has been demonstrated in the primitive streak
and neural tube (Franke et al., 1982). For these reasons, we expected colocalization of CK and VIM in the
mesenchymal cells presumably derived from the epicardial mesothelial cells, as well as in the epicardial cells
The presence of CK1 mesenchymal cells had been
described in the proepicardium and subepicardium of
the quail embryo, forming a network of tubular structures morphologically identic to the true capillaries but
not expressing the vascular marker QH-1 (Viragh et al.,
1993). These authors suggest that the tubular structures form by invagination of the surface mesothelium.
In spite of this interesting feature, the significance of
the CK1 SEMC has not been hitherto established, and
there is no data in the literature about their presence in
the mammalian embryos.
In the hamster and chick embryos of the stages
studied, CK immunoreactivity was present throughout
the epicardial mesothelium, but it also labelled most
SEMC cells. The persistence of the epithelial-type
intermediate filaments is probably transient. This is
suggested by the marked decrease in the relative
abundance of CK1 SEMC recorded in the hamster
embryo between 10 and 12 dpc. On the other hand, CK
colocalized with VIM in the SEMC and in a number of
epicardial mesothelial cells, mainly those covering the
cardiac grooves. VIM immunoreactivity was scarce or
absent in the squamous epicardial cells directly adhered to the myocardium.
The third evidence of an epicardial contribution to
the subepicardial mesenchyme comes from the immunolocalization of the antigens JB3 and ES/130 in a
number of epicardial mesothelial cells, the same which
were showing the signs of a delamination to the subepicardium. The labelling of these cells was identic to that
reported in the endothelial cells which originate mesenchymal cells in the endocardial cushions (Rezaee et al.,
1993; Wunsch et al., 1994; Bouchey et al., 1996). The
JB3 antigen seems to be expressed only in the subset of
endocardial cells which are responsive to the inductive
signal for a transformation in a migratory mesenchymal cell (Markwald et al., 1995). According to these
authors, the JB31 endocardial cells secrete ES/130
upon induction from the myocardium, before their
transformation in mesenchymal cells. We think that
the presence of these antigens in a subset of epicardial
cells can be significant.
The distribution of FN at the subepicardium was
very similar to that reported in the endocardial cushions (Icardo and Manasek, 1984; Mjaadvedt et al.,
1987), with a patchy staining of the surface of the
mesenchymal cells and the basal filopodial processes of
the epithelial cells. Distinctive staining of a continuous
basal lamina was only observed beneath the epicardial
cells adhered to the myocardium. The presence of FN at
the subepicardium had been reported (Choy et al.,
1991; Tidball, 1992) but its relation with the migration
of epicardial cells to the subepicardium was not suggested.
The mechanism of differentiation of mesenchymal
cells from the primitive epicardium, which we herein
suggest, seems to be analogous to that occurring at the
endocardial cushions and, in fact, there is a relative
spatiotemporal colocalization of both phenomena of
mesenchyme formation. The epicardium of the AV
groove, dorsal part of the ventricle, and proximal OFT
showed the larger abundance and persistence of the
morphological signs of delamination. Their cells were
frequently stained with the anti-VIM, JB3, and anti-ES/
130 antibodies. It will be interesting to check if a
common inductive mechanism originating from the
myocardium might be involved in both processes of
mesenchyme formation. The presence of adheron-like
particles in the subepicardial space (Bolender et al.,
1990), analogous to that reported as playing an inductive role in the endocardial cushions, might be regarded
in this context. It is also interesting to quote the report
of molecules in the epicardium and subepicardium,
which probably play a role in the transformation of
epithelial cells, for example, the extracellular matrix
proteins tenascin-X (Burch et al., 1995), fibulin-1 and
fibulin-2 (Zhang et al., 1995), and the transforming
growth factor b-1 (Choy et al., 1991). Furthermore, the
gene Msx-1 is expressed specifically in the epicardium
and subepicardium of the AV canal during the development of the SEMC (Chan-Thomas et al., 1993). These
observations, together with those reported in this paper, are consistent with an event of epithelial-mesenchymal transformation involving the epicardial mesothelium.
Sources other than the primitive epicardium probably contribute to the subepicardial mesenchyme. The
migration of cells from the liver/transverse septum area
has been demonstrated in the avian embryos (Poelmann
et al., 1993; Viragh et al., 1993). This contribution
might be less significant in the hamster embryos. We
have found that the CK immunostaining of many
SEMC, specially by 10 dpc, is as intense as that of the
neighbour epicardial cells. If CK is progressively disappearing from the SEMC, it is conceivable that mesenchymal cells migrating from extracardiac areas should lose
a significant part of their CK immunoreactivity during
Figs. 9–10. Cytokeratin (CK) immunoreactivity in the heart of the
chick embryos.
Fig. 9. a: Chick embryo of 4 days of incubation (HH21), sagittal
section. Many subepicardial mesenchymal cells are CK1 in the wide
subepicardium between the sinus venosus (SV), atrium (A), and ventricle
(V). In the epicardial mesothelium (E), there are signs of delamination,
such as basal cytoplasmic processes (arrowhead, shown at higher
magnification in b). Some vascular structures also involve CK1 cells,
either forming channels devoid of blood cells (star) or true capillaries (C).
This capillary is shown at higher magnification in c. Note the blood cells
(arrowheads) and the CK1 immunoreactivity of some cells of its wall
the time of their migration. On the other hand, the first
appearance of the SEMC in the hamster embryo is
virtually simultaneous at the subepicardium of the
sinus venosus, AV canal, and OFT, but these cells are
scarce or absent in the intermediary areas (see Figs. 1
and 3, and compare with Fig. 10).
Another possibility that has also been suggested is
the migration of mesenchymal cells from the transverse
septum through free-floating mesothelial vesicles (Van
den Eijnde et al., 1995). We regard this possibility as
feasible, but it is difficult to consider it as the main
source for the subepicardial mesenchyme. Most of the
free mesothelial vesicles observed by us contained no
mesenchymal cells inside, and there are CK1 SEMC in
the AV groove of the hamster embryo by 12 dpc, when
the epicardial investment of the heart is complete and
(arrows). CK1 cells (arrow in a) were always seen at these stages in the
wall of the liver (L) sinusoids. S: splanchnic mesothelium. Scale bars: a 5
26 µm; b 5 14 µm. c 5 15 µm.
Fig. 10. Subepicardium of a chick embryo of 4 days of incubation
(HH21). There is a wide communication between the liver (L) mesenchyme and the subepicardium (large arrow), which could be a potential
way for the migration of mesenchymal cells. In fact, CK1 mesenchymal
cells can be seen in all these areas. However, there also are basal
cytoplasmic processes (small arrows) in the epicardial mesothelial cells
(E). There is a CK1 channel (star) similar to that shown in 9a. P:
proepicardial villi. Other abbreviations as in Figure 9. Scale bar 5 15 µm.
the mesothelial villi of the transverse septum have
disappeared. In conclusion, we think that the primitive
epicardium significantly contributes to the subepicardial mesenchyme, this contribution probably being more
substantial in the mammalian than in the avian models.
Two questions have been raised by our findings and
they remain open. First, is it possible that the CK1
mesenchymal cells of the liver, transverse septum, and
mesothelial villi originate from the splanchnic mesothelium in the same way as the SEMC do from the
epicardial mesothelium? Second, what is the mechanism involved in the subsequent differentiation of the
SEMC either as fibroblasts or as cardiac vessel precursors? Is this mechanism related with a differential
origin of the SEMC? Although we have not further
investigated these questions at this time, we have
Figs. 11–14. Immunolocation in the subepicardium of antigens involved in the epithelial-mesenchymal transition.
Fig. 11. Syrian hamster embryo (10 dpc), posterior part of the
atrioventricular groove, fibronectin (FN) immunostaining. The FN antibody
labelled the surface of the mesenchymal cells (M) in the subepicardium
(SE) and the basal area of the epicardial mesothelial cells (E), especially
the cytoplasmic processes (arrow). The basal surface of the endocardium
(EN) is also labelled. Scale bar 5 8 µm.
elements of the extracellular matrix, and capillaries (arrows). E: epicardial
mesothelium. Scale bar 5 12 µm.
Fig. 13. Chick embryo (5 days of incubation, HH27), JB3 immunostaining, counterstained with hematoxylin. This view of the dorsal part of the
ventricle shows that the JB3 antigen is present in some epicardial
mesothelial cells, presumably in the cytoplasm (arrows). Other abbreviations as in Figure 12. Scale bar 5 9 µm.
Fig. 12. Chick embryo (4 days of incubation, HH21), JB3 immunostaining, counterstained with hematoxylin. The JB3 antigen was present in the
endocardial cells (EN) of the atrioventricular cushion as well as in the
subendocardium. The subepicardium (SE) showed a similar immunoreactive pattern, including staining of the epicardial cells (arrowhead), fibrous
Fig. 14. Syrian hamster embryo (10 dpc), transverse section of the AV
region, ES/130 immunostaining. The ES/130 antigen is present in the
endocardial cells (EN), the mesenchyme of the atrioventricular cushion
(AVC) as well as in some epicardial mesothelial cells (E), particularly
those that are large and rounded. Other squamous epicardial cells,
directly adhered to the myocardial wall, were ES/1302. A, atrium; V,
shown that many ring-like and tube-like structures as
well as some early cardiac vessels contain CK1 cells,
although the intensity of the immunoreactivity was
always inversely proportional to the degree of differentiation of the structure. It is also important to remark
that a number of cells in the wall of the liver sinusoids
in the chick embryos were CK1, although we cannot
precise if they were endothelial. An interesting, although highly speculative, hypothesis would be that
the liver endothelial cells and the endothelial cells of
the primary cardiac plexus share a common mesothelial origin, from the splanchnic mesothelium covering
the liver primordium and from the epicardial mesothelium. This hypothesis would be consistent with the
experimental approaches to the study of coronary vessel development. Mikawa and Fischman (1992) demonstrated, through retroviral cell labelling, that the precursors of the chick coronary arteries enter the heart
during the epicardial morphogenesis, and that the
coronary arteries form by coalescence of discontinuous
colonies. Poelmann et al. (1993) obtained quail coronary endothelial cells after transplantation of quail
liver tissue to chick. Furthermore, the formation of
coronary vessels is severely disturbed or impeded when
the epicardium does not develop properly, either by
mechanical (Manner, 1993) or genetical (Kwee et al.,
1993) manipulations. In order to test this hypothesis,
the possibility of a transient colocalization of CK with
specific vascular markers in the SEMC will be the
subject of our further studies.
The authors sincerely thank Drs. Roger R. Markwald
and Edward Krug (Medical University of South Carolina) for the kind gift of the anti-ES/130 and JB3
antibodies, Borja Fernández for their valuable suggestions, and Jesús Santamarı́a for his help with laser
confocal microscopy. This work was supported by grant
PB95-0475 (Ministerio de Educación y Ciencia, Spain).
David Macı́as and J.M. Pérez are the recipients of
fellowships from the Junta de Andalucı́a and the Ministerio de Educación y Ciencia, Spain, respectively.
Barton PR, Boheler KR, Brand NJ, Thomas PS. Molecular Biology of
Cardiac Development and Growth. Austin: R.G. Landes/Springer,
Bolender D, Markwald RR. Epithelial-mesenchymal transformation
in chick atrioventricular cushion morphogenesis. Scan. Electr. Micr.
Bolender D, Olson LM, Markwald RR. Coronary vessel vasculogenesis. In: Bockman DE, Kirby ML, eds. Embryonic Origins of
Defective Heart Development. Ann. N.Y. Acad. Sci. 1990;588:340–
Bouchey D, Drake CJ, Wunsch AM, Little CD. Distribution of connective tissue proteins during development and neovascularization of
the epicardium. Cardiovasc. Res. 1996;31:E104–E115.
Burch GH, Bedolli MA, McDonough S, Rosenthal SM, Bristow, J.
Embryonic expression of tenascin-X suggests a role in limb, muscle
and heart development. Dev. Dyn. 1995;203:491–504.
Chan-Thomas, PS, Thompson RP, Robert B, Yacoub MH, Barton PJR.
Expression of homeobox genes Msx-1 (Hox-7) and Msx-2 (Hox-8) during
cardiac development in the chick. Dev. Dyn. 1993;197:203–216.
Choy M, Armstrong MT, Armstrong PB. Transforming growth factor-b1 localized within the heart of the chick embryo. Anat. Embryol. 1991;183:345–352.
Choy M, Oltjen S, Ratcliff D, Armstrong MT, Armstrong PB. Fibroblast
behavior in the embryonic chick heart. Dev. Dyn. 1993;198:97–107.
Fitchett JE, Hay ED. Medial edge epithelium transforms to mesenchyme after embryonic palatal shelves fuse. Dev. Biol. 1989;131:455–
Franke WW, Grund C, Kuhn C, Jackson BW, Illmensee K. Formation
of cytoskeletal elements during mouse embryogenesis. III. Primary
mesenchymal cells and the first appearance of vimentin filaments.
Differentiation 1982;23:43–59.
Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J. Morphol. 1951;88:49–92.
Hay ED. Epithelial-mesenchymal transitions. Semin. Dev. Biol. 1990;
Icardo JM, Manasek FJ. An indirect immunofluorescence study of the
distribution of fibronectin during the formation of the cushion tissue
mesenchyme in the embryonic heart. Dev. Biol. 1984;101:336–345.
Icardo JM, Fernández-Terán MA, Ojeda JL. Late heart embryology.
The making of an organ. In: Meisami E, Timiras PS, eds. Handbook
of Human Growth and Developmental Biology. Boca Raton: CRC
Press, 1990:25–49.
Kwee L, Baldwin HS, Shen HM, Stewart CL, Buck C, Buck CA, Labow
MA. Defective development of the embryonic and extraembryonic
circulatory system in vascular cell adhesion molecule (VCAM-1)
deficient mice. Development 1993;121:489–503.
Manner J. Experimental study on the formation of the epicardium in
chick embryos. Anat. Embryol. 1993;187:281–289.
Markwald RR, Krug EL, Runyan RB, Kitten GT. Proteins in cardiac
jelly which induce mesenchyme formation. In: Ferrans VJ, Rosenquist G, Weinstein C, eds. Cardiac Morphogenesis. New York:
Elsevier, 1985:60–69.
Markwald RR, Rezaee M, Nakajima Y, Wunsch A, Isokawa K, Litke L,
Krug E. Molecular basis for the segmental pattern of cardiac
cushion mesenchyme formation: Role of ES/130 in the embryonic
chick heart. In: Clark EB, Markwald RR, Takao A, eds. Developmental Mechanisms of Heart Disease. Armonk, NY: Futura Publishing,
Mikawa T, Fischman DA. Retroviral analysis of cardiac morphogenesis: Discontinuous formation of coronary vessels. Proc. Natl. Acad.
Sci. U.S.A. 1992;89:9504–9508.
Mjaadvedt CH, Lepera RC, Markwald RR. Myocardial specificity for
initiating endothelial-mesenchymal cell transition in embryonic
chick heart correlates with a particulate distribution of fibronectin.
Dev. Biol. 1987;119:59–67.
Muñoz-Chápuli R, Macı́as D, Ramos C, Gallego A, Andrés V. Development of the subepicardial mesenchyme and the early cardiac vessels
in the dogfish (Scyliorhinus canicula). J. Exp. Zool. 1996;275:95–
Poelmann RE, Gittenberger-de Groot AC, Mentink MMT, Bökenkamp
R, Hogers B. Development of the cardiac coronary vascular endothelium, studied with antiendothelial antibodies, in chicken-quail
chimeras. Circ. Res. 1993;73:559–568.
Rezaee M, Isokawa K, Halligan N, Markwald RR, Krug E. Identification of an extracellular 130 kDa protein involved in early cardiac
morphogenesis. J. Biol. Chem. 1993;268:14404–14411.
Tidball JG. Distribution of collagens and fibronectin in the subepicardium during avian cardiac development. Anat. Embryol. 1992;185:
Tokuyasu KT. Development of myocardial circulation. In: Ferrans VJ,
Rosenquist G, Weinstein C, eds. Cardiac Morphogenesis. New York:
Elsevier, 1985:226–237.
Van den Eijnde SM, Wenink ACG, Vermeij-Keers C. Origin of subepicardial cells in rat embryos. Anat. Rec. 1995;242:96–102.
Viragh S, Challice CE. The origin of the epicardium and the embryonic
myocardial circulation in the mouse. Anat. Rec. 1981;201:157–168.
Viragh S, Kalman F, Gittenberger-De Groot AC, Poelmann RE,
Moorman AFM. Angiogenesis and hematopoiesis in the epicardium
of the vertebrate embryo heart. In: Bockman DE, Kirby ML, eds.
Embryonic Origins of Defective Heart Development. Ann. N.Y. Acad.
Sci. 1990;588:455–458.
Viragh S, Gittenberger-De Groot AC, Poelmann RE, Kalman F. Early
development of quail heart epicardium and associated vascular and
glandular structures. Anat. Embryol. 1993;188:381–393.
Vrancken Peeters M-PFM, Mentink MMT, Poelmann RE, Gittenberger-de Groot AC. Cytokeratins as a marker for epicardial formation in the quail embryo. Anat. Embryol. 1995;191:503–508.
Wenink A. Quantitative morphology of the embryonic heart: An
approach to development of the atrioventricular valves. Anat. Rec.
Wunsch AM, Little CD, Markwald RR. Cardiac endothelial heterogeneity defines valvular development as demonstrated by the diverse
expression of JB3, an antigen of the endocardial cushion tissue. Dev.
Biol. 1994;165:585–601.
Zhang H-Y, Timpl R, Sasaki T, Chu M-L, Ekblom P. Fibulin-1 and
fibulin-2 expression during organogenesis in the developing mouse
embryo. Dev. Dyn. 1995;205:348– 364.
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