Download Epicardial-like cells on the distal arterial end of the cardiac outflow

DEVELOPMENTAL DYNAMICS 227:56 – 68, 2003
ARTICLE
Epicardial-like Cells on the Distal Arterial End of
the Cardiac Outflow Tract Do not Derive From
the Proepicardium but Are Derivatives of
the Cephalic Pericardium
José M. Pérez-Pomares,1,2 Aimée Phelps,1 Martina Sedmerova,1 and Andy Wessels1*
A series of recent studies strongly suggests that the myocardium of the cardiac distal outflow tract (d-OFT) does not
derive from the original precardiac mesoderm but, instead, differentiates from a so-called anterior heart field. Similar
findings were also reported for the endocardium of the d-OFT. However, very little information is available on the
origin of the epicardium of the OFT. To address this issue, we have performed a study in which we have combined
experimental in vivo and in vitro techniques (construction of proepicardial chimeras, proepicardial ablation, OFT
insertion of eggshell membrane pieces, and culture on collagen gels) with molecular characterization techniques to
determine this origin and define the properties of d-OFT epicardium compared with proepicardially derived
epicardium. Our results demonstrate that the coelomic/pericardial epithelium in the vicinity of the aortic sac (and not
the proepicardium) is the origin of d-OFT epicardium. This “pericardially” derived epicardium and the proepicardially
derived epicardial tissues differ in their morphologic appearance, gene-expression profile, and in their ability to
undergo epithelial-to-mesenchymal transformation. We conclude that the heterogeneity in the epicardial cell
population of the OFT could be a factor in the complex developmental remodeling events at the arterial pole of the
heart. Developmental Dynamics 227:56 – 68, 2003. © 2003 Wiley-Liss, Inc.
Key words: heart development; outflow tract; epicardium; pericardium
Received 25 October 2002; Accepted 24 January 2003
INTRODUCTION
The fusion of two bilateral heart fields
of precardiac mesoderm results in
the formation of a single tubular
heart consisting of an outer myocardial sleeve and an internal lining of
endocardial cells. Both myocardium
and endocardium classically are regarded as primary derivatives of the
embryonic heart fields (reviewed in
Mikawa, 1999; Tam and Schoenwolf,
1999). These two tissue-layers are
separated by a well-hydrated extracellular matrix commonly referred to
as the cardiac jelly (reviewed in
Mjaatvedt et al., 1999). The remnants of cardiac jelly in the atrioventricular (AV) junction and outflow
tract (OFT) will eventually be invaded by a mesenchyme derived
from epithelial-to-mesenchymal transformation (EMT) of the endocardium
(Wessels and Markwald, 2000). These
mesenchymalized endocardial cush-
ion tissues will contribute to the formation of the cardiac valves and (mesenchymal) septal structures. Once
primitive cardiac segments are formed
and cushion morphogenesis has
been initiated, an additional population of cells arrives at the surface of
the heart. The development of this
layer, the epicardium, is a relatively
late event (Vrancken Peeters et al.,
1995; Männer et al., 2001).
It is now commonly accepted that
1
Department of Cell Biology and Anatomy, Cardiovascular Developmental Biology Center, Medical University of South Carolina, Charleston,
South Carolina
2
Department of Animal Biology, Faculty of Science, University of Málaga, Málaga, Spain
Grant sponsor: NIH/NHLBI; Grant number: 1PO1 HL52813-06; Grant sponsor: AHA; Grant number: GIA 005099U.
*Correspondence to Andy Wessels, Ph.D., Department of Cell Biology and Anatomy and Cardiovascular Developmental Biology Center,
Medical University of South Carolina, 173 Ashley Avenue, Basic Science Building, Room 648, P.O. Box 250508, Charleston, SC 29425.
E-mail: [email protected]
DOI 10.1002/dvdy.10284
© 2003 Wiley-Liss, Inc.
d-OFT CELLS DERIVE FROM CEPHALIC PERICARDIUM 57
the epicardium derives from the proepicardium (Viragh et al., 1993), a
cluster of coelomic/splanchnic mesothelial cells located between the
sinus venosus and the liver primordium in avians (Hiruma and Hirakow,
1989; Männer, 1992, 1993; Viragh et
al., 1993), and analogous regions in
different vertebrates (Viragh and
Challice, 1981; Komiyama et al.,
1987; Kuhn and Liebherr, 1988;
Fransen and Lemanski, 1990; Van
den Eijnde et al., 1995; Muñoz-Chápuli et al., 1997). The proliferating proepicardium generates epicardial progenitor cells, which attach to the
myocardium and spread over the
heart in a well-defined spatiotemporal pattern, a process that was nicely
demonstrated in a series of different
morphologic and experimental studies (for a review, see Männer et al.,
2001). After forming an outer epithelial
layer, separated from the myocardial
surface by a subepicardial extracellular matrix-rich space, a subpopulation
of the epicardial cells transforms into
mesenchymal cells through an EMT.
This transformation generates a population of epicardially derived cells
(EPDCs; Gittenberger-de Groot et al.,
1998), which, in turn, differentiates into
multiple cell lineages (Mikawa and
Gourdie, 1996; Pérez-Pomares et al.,
1998, 2002; Gittenberger-de Groot et
al., 1998; Dettman et al., 1998; Männer, 1999; Vrancken Peeters et al.,
1999).
As already indicated, several experimental studies (Argüello et al.,
1975; De la Cruz et al., 1977, 1983,
1998) have shown that the atrial and
ventricular components of the heart
derive from the tissues found in the
straight heart tube. In addition, these
studies also suggest that the OFT does
not directly derive from the primitive
splanchnic precardiac epithelium
(i.e., the lateral heart fields), a hypothesis that has gained considerable support from recent experimental and
genetic studies that indicate an anterior/noncardiac origin for the OFT in
avians (Noden, 1991; Mjaatvedt et al.,
2001; Waldo et al., 2001) and mammals (Kelly et al., 2001). The embryonic
OFT is one of the most controversial
segments of the heart. Controversies
exist about its origin, its fate, and
about the descriptive terminology of
its components during remodeling
(Pexieder, 1995; Ya et al., 1998;
Qayyum et al., 2001). The notion that
myocardial and endocardial cells in
the OFT derive from an “anterior” or
“secondary” heart field, combined
with the observation that the distal
OFT (d-OFT) of experimentally manipulated avian hearts is populated with
a subpopulation of epicardial-like
cells that are of nonproepicardial origin (Männer, 1999; Gittenberger-de
Groot et al., 2000), prompted us to
investigate the origin, phenotypical
characteristics, and fate of OFT epicardium in more detail.
The results presented and discussed in this study strongly suggest
that d-OFT epicardium is not derived
from the proepicardium but rather is
a derivative of the cephalic coelomic/pericardial epithelium covering
the base of the aortic sac. Our morphologic and experimental data
show that this tissue resembles that
of other mesothelial tissues. The phenotypical characteristics of the
d-OFT epicardial-like cells, is different
from that of the epicardium found in
the rest of the heart. This includes a
difference in the level of expression
of the retinoic acid converting enzyme RALDH2 in the OFT epicardiallike cells compared with the epicardium found in the other heart
chambers (see Moss et al., 1998,
Xavier-Neto et al., 2000; Pérez-Pomares et al., 2002), and differences
in the expression levels of the intermediate filament proteins cytokeratin (CK) and vimentin (VIM). We also
studied the in vitro behavior of these
cells and found that, in contrast to
proepicardially derived epicardial
cells, under standard conditions the
pericardially derived epicardial cells
do not readily undergo epithelial-tomesenchymal transformation. Finally, our results indicate that the
two epicardial populations on the
cardiac OFT might differentially contribute to the development of the
segment.
RESULTS
Anatomical Description of the
Outflow Tract During
Remodeling
The terminology used in the literature
to describe the different regions of
the developing OFT (e.g., conus,
truncus, and aortic sac) varies considerably, and consensus seems
hard to reach (Pexieder, 1995,
Qayyum et al., 2001). The events involved in the remodeling of the
elongated tubular outflow tract
(Thompson et al., 1985, 1987; Ya et
al., 1998), as well as the clinical relevance of congenital defects of the
cardiac outlet (Clark, 1986), are areas of open debate.
In this study, the subdivision of the
OFT into different components will
follow the dynamics of OFT morphogenesis, and will be based on histologic criteria (e.g., myocardial vs.
nonmyocardial) as well as on external anatomic features (e.g., flexure
or bending). Thus, we consider the
outflow tract as the segment that
connects the ventricular (i.e., trabeculated) portion of the heart to the
aortic sac (i.e., the peribranchial,
mesenchyme-embedded, extrapericardial cavity in which the OFT
drains; see also Los, 1978; Orts-Llorca
et al., 1982); therefore, the OFT extends from the trabeculated portion
of the right ventricle to the pericardial reflections. As the epicardium
will not spread over the myocardium
of the OFT until Hamburger and
Hamilton (1951) stage 18 (Vrancken
Peeters et al., 1995), we will distinguish two stages in OFT development.
H/H16 –18.
Starting at H/H16, the OFT can be
subdivided into a proximal segment
(p-OFT, analogous to the conus; for
an extensive review, see Pexieder,
1995) and a distal segment (d-OFT).
The lower boundary of the p-OFT is
internally defined by the transition
between the trabeculated right
ventricle to the untrabeculated
myocardial OFT, and the upper
boundary is internally demarcated
by the upper boundary of the proximal (or conal) cushions and externally by a characteristic “dog-leg”
bend (see Qayyum et al., 2001). In
the d-OFT, a lower and an upper
part can be distinguished. The lower
part has a myocardial component,
which is internally lined with endocardium and endocardially derived
tissues and, therefore, can be
termed myocardial d-OFT. The up-
58 PÉREZ-POMARES ET AL.
per part of the d-OFT (toward the
aortic sac), which is quite a short
segment, consists of a mesenchymal
(nonmyocardial) belt, externally
covered by an epithelium and internally lined by endocardium. We refer to this segment as the mesenchymal (nonmyocardial) d-OFT.
H/H19 –27.
At these stages, the p-OFT is formed
by epicardium, myocardium, endocardium, and endocardial-derived
mesenchyme. For the d-OFT, the
wall of the lower part of the segment
remains myocardial and epicardial,
whereas the wall of the upper part
of the d-OFT, which is now a long
segment connecting to the aortic
sac, is a cylinder of mesenchyme externally covered by an epicardiallike epithelium. This upper d-OFT is
analogous to the “arterial segment”
as described by Qayyum et al.
(2001). Due to the developmental
complexity of this area, and following the example of others (Thompson and Fitzharris, 1985), we include
this cardiovascular segment, which
extends to the base of the aortic
sac–pharyngeal arches, in the description of the OFT.
Histologic and
Immunohistochemical Analysis
of the Developing Outflow
Tract
Stage H/H19.
At this stage, the OFT is a long cylinder consisting of an outer layer of
myocardium and an inner layer of
endocardium. The OFT connects the
prospective right ventricle to the
aortic sac and shows a “dog-leg”
bend (described as a “change in
the directionality in the sagittal and
parasagittal planes” by Pexieder,
1995). This bend roughly divides the
OFT in two halves, proximal and distal. The aortic sac is an extrapericardiac cavity located ventrally to the
pharynx, connecting the lumen of
the OFT with the developing aortic
arches. The OFT myocardium extends from the ventricle to the pericardial reflections that mark the
level of the aortic sac region. Only
the very upper part of the d-OFT is
devoid of myocardium (Fig. 1A). This
area consists of a collar of cuboidal
epithelium reminiscent of the adjacent pericardial epithelium. This epithelium covers a population of mesenchymal cells (Fig. 1A,B).
At this point, the lower part of the
p-OFT is partially covered by a thin
flattened epicardium. The myocardium of the upper part of the proximal OFT, as well as the myocardium
of the entire d-OFT, is not yet covered by epicardium (Fig. 1C,D).
The epicardial cells and the epithelial component of the upper part
of the d-OFT and aortic sac express
cytokeratin (CK). The myocardium
expresses myosin heavy chain, as indicated by the staining with MF-20.
Coexpression of CK and MF-20 is frequently observed in a population of
cells of the upper rim of the d-OFT
myocardium (Fig. 1A–D).
Stage H/H21.
The overall morphology of the OFT at
stage H/H21 is reminiscent of that
described for stage H/H19. The major difference is the increase in
length of the mesenchymal (upper)
part of the d-OFT.
The p-OFT is now completely covered by epicardium. Large areas of
the myocardial distal OFT, however,
remain uncovered by epicardium.
The epicardium shows a flattened
morphology that makes it clearly distinguishable from the cuboidal epithelium covering the upper part of
the d-OFT. An interesting feature at
this stage is that the epithelium that
covers the upper part (nonmyocardial portion) of the d-OFT at the junction with the pericardial reflections
forms protrusions that give the region
a ruffled aspect. These protrusions
have the appearance of small “proepicardia” (Fig. 1E,F).
The epicardium and all mesothelial cells (upper d-OFT epithelial
cells, coelomic epithelium, and
pericardium) are CK-positive. Double-labeling with CK and vimentin
(VIM) antibodies delineates cushion mesenchyme from epicardial/
mesothelial tissue (Fig. 1E,F). The
epicardium covering the atria,
atrioventricular junction, ventricles,
and the p-OFT coexpress both CK
and VIM. This epicardium also expresses RALDH2 (see also Pérez-Pomares et al., 2002). The expression
of vimentin in the “epicardial-like”
cell layer of the d-OFT is relatively
low compared with the epicardium of other heart chambers (Fig.
1E,F). The level of expression of
RALDH2 in the epicardial-like cells is
considerably lower than that of the
epicardial cells covering the rest of
the heart (Fig. 1G).
Stages H/H26 –29.
At stage H/H26, the OFT is still an
elongated structure. The characteristic bend that indicates the boundary between distal and proximal OFT
now approximates 90 degrees (Fig.
1H).
From stage H/H26 –27 onward, the
entire heart is covered by epicardium, mostly with a flattened appearance, as well as by an “epicardial-like tissue,” located at the upper
(mesenchymal) distal OFT. This latter
epithelial tissue consists of relatively
large cuboidal cells (Fig. 1J,K).
Immunohistochemistry shows that
the distal end of MF-20 –stained tissue (the junction between myocardial and nonmyocardial components of the d-OFT) approximately
determines the distal-most margin of
the flattened epicardium (CK-positive) with its related subepicardial
space containing isolated CK-expressing mesenchymal cells (Fig. 1I).
The upper (mesenchymal) d-OFT
consists of a densely compact mesenchyme. This mesenchyme is
strongly CK-positive and, generally,
does not express MF-20. Some isolated mesenchymal cells close to
the myocardium, however, express
little MF-20 (Figs. 1I, 2C). The entire
nonmyocardial d-OFT is covered by
a cuboidal CK-positive epithelium.
The proepicardial-like structures
close to the pericardial reflections
are especially prominent between
stages H/H26 –29 and are expressing
CK (Fig. 1F) and RALDH2 (not
shown). It is important to note that
the “normal” epicardium of the
lower portion of the OFT and the
“epicardial-like” cells at the upper
part of the d-OFT are in continuity at
these stages (Fig. 1I).
Proepicardial chimeras.
Quail-to-chick proepicardial chimeras were constructed to obtain in-
d-OFT CELLS DERIVE FROM CEPHALIC PERICARDIUM 59
Fig. 1. Normal distal OFT development (Hamburger and Hamilton stage [H/H] 19; H/H21; H/H26). A–D: Different developmental aspects
of the OFT in an H/H19 chick embryo. The sections in A–D are immunostained for myosin heavy chain with MF-20 (green) and for
cytokeratin (CK; blue) and counterstained with propidium iodide to reveal nuclei (red). The boxed area in A is magnified in B. The most
proximal portion of the distal OFT myocardium is not yet covered by epicardium. The more distal (cephalic) region of the OFT lacks
myocardial tissue and consists of a compact mesenchyme covered by a thick cuboidal epithelium (arrowheads in B–D). The left box in
B is magnified in C; the right box is magnified in D. Arrows in C and D indicate the distal limit of the myocardium (MF-20 –positive), and
double arrowheads show the absence of epicardium over this myocardium. E,F: Demonstrate vimentin (VIM; blue) and CK (green)
expression in the OFT of a stage H/H21 chick embryo. In E, VIM is strongly expressed in the OFT endocardium and the endocardial cushion
mesenchyme (asterisks in E). VIM expression is very low in the OFT myocardium (compare with atrial myocardium, which is clearly
VIM-positive). CK expression, however, is noticeable in the myocardial tissue of the OFT (double arrowheads). Epicardial tissue is
CK-positive as shown by the green staining (arrow). Proliferations of the epithelium covering the distal part of the OFT are indicated with
arrowheads. Higher magnification in F shows the strong CK/VIM coexpression in the epicardium that covers the distal OFT myocardium
(indicated by a dashed line). This coexpression (arrow, light blue staining) is similar to that found in the epicardium of other heart
chambers (compare with atrial epicardium). Epicardium of the distal regions does not cover myocardial tissue and has a lower VIM
expression level and high CK expression level (greenish staining, arrowheads). Proliferations of the epicardial-like tissue of the distal OFT
are indicated by a double arrowhead). G: Expression of the retinoic acid converting enzyme RALDH2 in the OFT of the same embryo. In
the more distally located OFT, patches of epicardium are seen with a significantly lower level of RALDH2 expression (arrowheads) as
compared with the level of RALDH2 expression in the epicardium of the proximal OFT (arrow) and the rest of the heart. H–K: The OFT in a
stage H/H26 chick embryo. The section in H (enlarged in I) is stained for myosin heavy chain with MF-20 (green) and cytokeratin (blue).
In H, the distal rim of the OFT myocardium is indicated by an arrow. The epicardium covers the myocardium of all the heart chambers.
In the OFT, flattened epicardium (arrowheads) is seen in association with the underlying myocardium. More distally, the OFT consists of
mesenchymal cells (MF-20 negative) covered by a cuboidal epithelium (double arrowheads). In I, the boundary between the flattened
epicardium and the cuboidal epicardium is indicated with an arrow. Some faint MF-20 labeling can be found in the loose tissue distal to
the cephalic border of the OFT myocardium (arrowheads). J and K show H&E stainings of the more distal OFT epicardium and proximal
OFT epicardium respectively. Note the characteristic cuboidal appearance of the epicardial cover directly lying over a mass of compact
mesenchyme in J (arrowheads). In K, the epicardium is basically flattened (arrows) and covers a subepicardial space with dispersed
mesenchymal cells (asterisk). A, atrium; AEP, atrial epicardium; AM, atrial myocardium; CVC, conoventricular canal; AoS, aortic sac; EN,
endocardium; EP, epicardium; OFT, outflow tract; PE, pericardium; Ph, pharynx. Scale bars ⫽ 100 ␮m in A,E,H, 50 ␮m in B,F,G, 25 ␮m in C,D,
30 ␮m in J,K, 15 ␮m in I.
60 PÉREZ-POMARES ET AL.
sight into the contribution of proepicardially derived cells to the OFT. In
the chimeras, epicardial development is complete around stage
H/H26 –27 (Pérez-Pomares et al.,
2001), which coincides with the normal end point for the same process
in control embryos (Vrancken
Peeters et al., 1995). Thus, at stage
H/H29, the atria (with the exception
of small patches of host tissue in the
atrial roof and walls), the atrioventricular region, the ventricles, and
the proximal part of the OFT are all
covered by QCPN-positive, quailderived, epicardium. The upper distal-OFT, however, is covered by host
(i.e., chick), QCPN-negative epicardial-like cells. Almost all of the subepicardial mesenchyme in the atrioventricular junction, ventricles, and
p-OFT is of donor (quail) origin. No
subepicardial mesenchyme is found
in the atria. In addition, subsets of
EPDCs have started to invade the
myocardial layers of the ventricle at
this stage (H/H29). EPDCs are
scarce, however, in the atrioventricular myocardium and atrioventricular cushions at this stage and are
completely absent from atrial and
OFT myocardium and from the conal cushions (Fig. 2D–F).
Immunolabeling with QCPN and
RALDH2 reveals differences in
RALDH2 expression between the
quail proepicardially derived epicardium and the chick-derived epicardial-like cells of the d-OFT. The proepicardially derived epicardium
and associated subepicardial mesenchyme show a strong and equally
distributed expression. The OFT epicardial-like cells show a more diffuse
expression of RALDH2, with a few
patches of cells expressing higher
levels of RALDH2. These latter cells
correspond to the areas where coelomic proliferations/proepicardiallike structures are found and are
found on the mesenchymal (upper)
portion of the d-OFT close to the roof
of the pericardial cavity (base and
walls of the aortic sac) and on the
ventral aspect of the d-OFT (Fig. 2D).
This pattern of expression is similar to
the one described in the control embryos (see above).
The border between quail- and
chick-derived cells in the chimeras is
demarcated by QCPN immunostaining and is consistently found at
the junction between myocardial
and nonmyocardial tissues of the dOFT (approximately from H/H29 onward). The donor and host-derived
epithelial cells seem to form a continuous sheet of cells (Fig. 2F).
Fig. 2. OFT mapping (MF-20 whole-mounts and quail-to-chick proepicardial chimeras). A–C: MF-20 –immunostained whole-mount hearts
of Hamburger Hamilton stage (H/H) 30 chick embryos (green). A and B illustrate ventral and right lateral aspects, respectively. Note the
absence of MF-20 immunoreactivity in the upper (mesenchymal) part of the d-OFT. C shows a detail of the border between the
myocardial (lower) and mesenchymal (upper) portions of the d-OFT. Arrowheads show the intricate architecture of the distal-most
myocardial rim (arrowheads) as well as the presence of isolated groups of myocardial cells that are not directly in contact with the lower
d-OFT myocardial mass (arrow). D,E: Expression of QCPN (green) and retinoic acid converting enzyme RALDH2 (red) in a stage H/H29
quail-to-chick proepicardial chimera. In D, almost all the epicardial cover is formed by donor (quail-derived) cells, as indicated by the
QCPN-positive nuclei (green dots) found in the chimeric epicardial mesothelium (arrowheads) and subepicardial epicardially derived
cells (EPDCs; arrows). RALDH2 expression (red), characteristic of epicardial tissue, is also conspicuous in the epicardial epithelium as well
as in part of the subepicardial space of the ventricle and atrioventricular and conoventricular grooves. Double arrowheads in D indicate
the upper boundary of the donor-derived (QCPN-positive) epicardium on the OFT. The more distal/cranial epicardium is not of quail origin
and contains groups of cells with low RALDH2 expression (boxed areas). In E, a different section of the same chimera is presented to show
some details of the chimerization process. EPDCs immigrate into ventricular myocardium (arrow) but not into the atrial and OFT
myocardium. Arrowheads point to the distal border of the proepicardially derived (QCPN-positive) epicardium. RALDH2 expression is low
in the more cephalic (non-proepicardially derived, QCPN-negative) OFT epicardium (double arrowhead). The asterisk indicates the
presence of a non-quail– derived mesenchyme underlying this epithelial tissue. QCPN staining in F illustrates several aspects of the
donor-derived/host-derived epicardial interface. The distal rim of myocardium is indicated with a dashed line. Host (QCPN-negative) and
donor (QCPN-positive) epicardium integrate without discontinuities (arrow). Host-derived epicardial cells have a characteristic cuboidal
phenotype (arrowheads) that can be compared with the flattened aspect of donor-derived epicardial cells. Scale bars ⫽ 400 ␮m in A,
300 ␮m in B, 100 ␮m in C–E, 25 ␮m in F. A, atrium; AoS, aortic sac, AVC, atrioventricular canal; AVE, atrioventricular epicardium; CV,
conoventricular groove; d-OFT, distal OFT; EP, epicardium; LA, left atrium; ld-OFT, lower distal OFT; LV, left ventricle; OFT, outflow tract; OFTE,
outflow tract epicardium; PE, pericardium; p-OFT, proximal OFT; RA, right atrium; RV, right ventricle; SE, subepicardium; ud-OFT, upper distal
OFT; V, ventricle.
Fig. 3. Epicardial-like tissue at the distal outflow tract of proepicardially ablated embryos. A: Expression of the retinoic acid converting
enzyme RALDH2 (red) and MF-20 (green) in a Hamburger and Hamilton stage (H/H) 27 ablated chick embryo. RALDH2 expression is only
detected in the few epicardial cells (arrows) found on the heart after the ablation procedure. In the distal OFT, strong RALDH2 expression
is observed in the epicardial-like tissue covering the upper rim (arrowheads). Coelomic epithelial proliferations are indicated with an
asterisk. B: A hematoxylin and eosin (H&E) -stained section of a proepicardial ablated H/H27 chick embryo. Arrowheads point to the lower
boundary of the “collar” of epicardial-like tissue around the most distal part of the OFT, the arrows point to the upper boundary of OFT
myocardial tissue. C: Detail of the distal OFT of an ablated embryo (same as shown in A). RALDH2 expression is shown in red, and MF-20
expression in green. The panel shows that, even after proepicardial ablation, the distal OFT epicardium still forms and strongly expresses
RALDH2. The arrowheads mark the lower limits of the collar of distal OFT epicardium, the asterisk the presence of coelomic epithelial
proliferations in the cephalic pericardium (both RALDH2-positive), and the arrows point to the upper boundary of the myocardium in the
OFT (MF-20 staining). D: A different section of the same specimen is shown after MF-20 (green) and cytokeratin (CK; red) staining. Again,
the arrowheads indicate the lower boundary of the collar of epicardial-like tissue. The arrows in this panel point to CK-positive cells in the
compacting layers of the developing great arteries. E: The H&E staining shows the proliferations in the cephalic coelomic (pericardial)
epithelium in a stage H/H27 ablated embryo. The arrowheads indicate the mesothelial cover and the arrows the associated mesenchyme. F: Detail of one of these proliferations is presented. Mesothelial (arrowheads) and underlying mesenchymal cells (double
arrowheads) express CK (red). Note the formation of finger-like protrusions (arrows). A, atrium; AVC, atrioventricular canal; d-OFTE, distal
outflow tract epicardium; MYO, myocardium; OFT, outflow tract; OFTC, outflow tract cushions; V, ventricle. Scale bars ⫽ 200 ␮m in A, 120
␮m in B, 100 ␮m in C,D, 50 ␮m in E, 25 ␮m in F.
Fig. 2.
Fig. 3.
62 PÉREZ-POMARES ET AL.
Characterization of
proepicardially ablated
embryos.
Proepicardial ablations were performed to obtain additional information about the origin of the “epicardial-like” tissues at the upper aspect
of the d-OFT. Proepicardial ablations
result in a clear delay in epicardial
development. Of interest, as reported before (Pérez-Pomares et al.,
2002), the procedure does not completely prevent epicardial spreading
over the heart. Ablation of proepicardium actually seems to induce a
secondary outgrowth of coelomic
mesothelium in the area from which
the proepicardium has been removed. This compensatory tissue
eventually contacts the myocardium in the sinus venosus region and
subsequently migrates over the cardiac surface/generating a late epicardial epithelial-like tissue that
strongly stains with CK and RALDH2
(Fig. 3A). Although normal epicardial covering of the heart is completed between stages H/H26 –27,
stage-matched experimental (ablated) embryos still show numerous
bare areas (not covered by epicardium) in the ventral regions of the
ventricles and OFT (Fig. 3A). Discontinuities in epicardial development
are also seen in the more proximal
segment of the OFT, where the delayed epicardium, growing over the
proximal region, has not made contact with the epicardial-like cell population of the d-OFT. This finding is
especially evident in H/H28 –29 embryos after CK or RALDH2 staining.
The distal part of the OFT in the ablated embryos is completely covered by an epicardial-like tissue that
forms a “collar” covering the upper
area of the d-OFT (Fig. 3A,C,D).
These cells closely resemble the pericardial epithelium surrounding the
basal aortic sac with a characteristic cuboidal morphology. Proliferations of coelomic epithelium are often found in this area both in normal
as well as in ablated embryos. It appears, however, that these proliferations are more prominent in the ablated specimens (Fig. 3E). These
structures, that are CK/RALDH2-positive, are characteristically found
close to the pericardial reflections
around the base of the aortic sac.
They have a heterogeneous morphology, with finger-like protrusions,
and are reminiscent of the normal
proepicardium located between
the sinus venosus region and the liver
primordium. This d-OFT epicardiallike tissue is associated with an underlying, relatively compact, mesenchymal cell population (Fig. 3F).
Eggshell membrane insertion.
To further characterize the nature of
the “proepicardial-like” tissues at the
uppermost aspect of the d-OFT, we
designed an experimental strategy
to isolate these cells. A piece of eggshell membrane was inserted between the inner curvature of the
heart and the junction between OFT
and aortic sac (pericardial reflections) in normal and proepicardially
ablated hearts at H/H17 (Fig. 4A).
The eggs were then reincubated for
approximately 12 hr. In both sets of
experiments, this strategy resulted in
the growth of an “epicardial-like” tissue on the distal (cephalic) tip of the
membrane. Hematoxylin and eosin
(H&E) staining of the embryos shows
how the epicardial-like tissue
spreads over the surface of the
membrane, acquiring an epithelial
morphology. Importantly, this procedure does not interfere with the development of epicardial-like cells on
the d-OFT. The more proximal part of
the membrane (i.e., the part inserted into the inner curvature) becomes covered by proepicardially
derived epithelial cells. The central
part of the membrane remains devoid of any epithelial overgrowth.
The epithelial cells at both ends of
the membrane express CK (Fig.
4B,C) and RALDH2. At the distal end,
the RALDH2 expression is most intense close to the site of insertion at
the pericardial reflection, the expression being weaker in the epicardial-like cells that have migrated
away from the point of insertion (Fig.
4D).
Collagen cultures and
immunohistochemistry.
To obtain more insight into the differences in migration and transformation properties between true proepi-
cardially derived cells, pericardially
derived cells, and cells derived from
the “proepicardial-like” proliferations at the junction of OFT and aortic sac, we performed in vitro collagen
gel
assays.
The
d-OFT
epicardial-like tissue spreads over
the surface of the gels, forming an
epithelial monolayer (Fig. 5A). These
cells are CK- and RALDH2-positive
and have an elongated shape (Fig.
5B). Their expression patterns as well
as their morphology resemble that
of the cells derived from pericardial
explants (Fig. 5D). The CK and
RALDH2 expression found in the proepicardially derived cells is comparable to that of OFT epicardial-like
cells and pericardial cells. The proepicardially derived cells, however,
show a characteristic rounded-polygonal shape, which distinguishes
them from the elongated-shaped
pericardial and epicardial-like OFT
cells (Fig. 5E,F). Distal OFT epicardiallike cells on the surface of the gel
typically loose their epithelial context after 24 hr of incubation and
acquire a spindle shape morphology. Only a few cells, however, invade the collagen matrix (Fig. 5B,C).
The pericardially derived epithelium
basically shows the same behavior
(Fig. 5D).
In the proepicardial (quail)/d-OFT
(chick) cocultures, the epithelial
sheets derived from both tissues
grow over the surface of the gel often making contact with each
other. Many quail epicardially derived cells (QCPN-positive) undergo
epithelial-to-mesenchymal transformation and migrate into the collagen matrix (Fig. 5G,H).
DISCUSSION
Our results demonstrate for the first
time the presence of two distinct
epicardial populations in the normal developing avian heart. Our
experimental studies show that, although the majority of the epicardial OFT cells (the proximal population) is proepicardially derived, the
most distal epicardial population is
derived from coelomic pericardial
proliferations at the base of the
aortic sac. We show that the difference in the origin of these two
populations is reflected in morpho-
d-OFT CELLS DERIVE FROM CEPHALIC PERICARDIUM 63
Fig. 4. Collecting epicardial-like tissues using an eggshell membrane insertion technique. A: The insertion of a piece of the eggshell
membrane (arrow) between the inner curvature of the heart and the base of the aortic sac in a Hamburger and Hamilton stage (H/H)
19 chick embryo. B: Cytokeratin (CK) staining in an immunolabeled sister section of the same embryo shown in the boxed area in A. Note
the insertion of the tip of the eggshell membrane into the coelomic surface in the roof of the pericardial cavity (arrow). C: The same area
(CK staining) at higher magnification. The upper edge of the membrane is highlighted with a dashed line. The epicardial-like tissue
growing over the membrane is indicated with arrowheads. D: Membrane insertion in a stage H/H20 chick embryo is shown after staining
with the retinoic acid converting enzyme RALDH2. Note the strong staining in the pericardial reflection (asterisk) and the staining of the
epicardial-like tissue on the eggshell membrane (arrowheads). The level of RALDH2 expression is highest in the distal part of the growing
epicardial tissue, i.e., the regions closer to the coelomic/pericardial epithelium. Coelomic mesothelial cells display a strong RALDH2
expression (arrow), compared with the tissue spreading over the more caudal areas (lower part) of the membrane. Scale bars ⫽ 300 ␮m
in A, 50 ␮m in B,D, 25 ␮m in C. A, atrium; AVC, atrioventricular canal; EM, eggshell membrane; OFT, outflow tract; Ph, pharynx; V, ventricle.
logic, molecular, and functional
characteristics.
The pericardial proliferations, in
the vicinity of the so-called pericardial reflections, have a characteristic ruffled appearance and closely
resemble the coelomic epithelium
at the caudal end of the heart in the
early stages of proepicardial development. Unlike the proepicardium,
however, the pericardial proliferations never develop into a true cauliflower-shaped cell cluster. The epicardium at the d-OFT that derives
from these proliferations is morphologically different from the proximal,
proepicardially derived, epicardium. At the d-OFT, the cells have a
cuboidal morphology, closely resembling that of the coelomic epi-
thelium, whereas the epicardium on
the p-OFT is flat and has a thin lamellar or squamous phenotype. A similar
difference in phenotypical characteristics was also observed in the epicardial population on the OFT of
quail-to-chick chimeras. Compared
with the epicardium in the rest of the
heart, the d-OFT epicardium is characterized by a relatively low expres-
64 PÉREZ-POMARES ET AL.
Fig. 5. In vitro culture of distal outflow tract (d-OFT) epicardium. A: A d-OFT explant in a collagen gel assay (imaged using Hoffman
Modulation Contrast optics). The tissue grows as an epithelial monolayer from the eggshell membrane (EM; arrows). In the borders of the
culture, cells tend to detach and to migrate as isolated cells over the surface of the collagen gel (arrowheads). B,C: An epicardial d-OFT
explant on collagen gel, immunofluorescently stained for cytokeratin (CK) expression (green). All the cells on the surface of the gel are
CK-positive. Below the surface of the gel, no mesenchymal cells can be detected (C). D: A pericardial explant on collagen gel. The
pericardial epithelium of this explant also expresses CK and is phenotypically very similar to that observed in the d-OFT explants (compare
with A,B). It differs, however, from the epithelium seen in proepicardial explants (compare with E,F). E–H: The results from proepicardial/
d-OFT epicardium cocultures on collagen gels. Quail proepicardial cell nuclei are QCPN-positive (green) in E–H. In E, both proepicardial
(QCPN-positive) and d-OFT tissue (QCPN-negative) express the retinoic acid converting enzyme RALDH2 (red). The boxed area presents
an area of the border between the two explants and is magnified in F. Both proepicardial and OFT tissues integrate normally but do not
seem to mingle (arrows). G and H show details of another coculture of quail proepicardium with chick distal OFT epicardium. Both tissues
have been stained with a CK antibody (red). Quail (pro)epicardial cells (QCPN-positive, green nuclei) remain in the surface of the
collagen gel (arrow in G). Almost all the mesenchymal cells found in the depth of the collagen gel (H) are from quail proepicardial origin
(QCPN-positive, arrowheads). In both cases, the area covered by the eggshell membrane is indicated with a dashed line. EM, eggshell
membrane; PEe, proepicardial explant; OFTe, distal outflow tract explant. Scale bars ⫽ 100 ␮m A–E, 50 ␮m F–H.
sion of RALDH2 and vimentin. The
pericardial proliferations themselves,
however, express a relatively high
level of RALDH2.
To obtain more information on the
characteristics of the pericardial
proliferations, we performed in ovo
microsurgical experiments where
pieces of eggshell-membrane were
inserted between the floor of the
aortic sac and the inner curvature of
the heart in normal and proepicardially ablated hearts. It was found
that, after 12 hr of reincubation, the
cephalic portion of the membrane
was covered by epithelial tissue expressing moderate levels of CK and
relatively high levels of RALDH2. This
high RALDH2 expression in the cephalic portion of the membrane illustrates that the epithelium has developed in close proximity of the
pericardial proliferations (“proepicardial-like structures”). Cells that
have migrated away from this site
express considerably lower levels of
RALDH2.
The dual origin of OFT epicardium
explains the characteristic patchy
expression of RALDH2 seen in the dOFT epicardium of control and chimeric embryos. It is important to emphasize that, in the case of the
chimeras, the heterogeneity in
RALDH2 expression does not reflect
species-specific differences, but
rather reflects the tissue origin of the
cells (proepicardium vs. pericardial
proliferations) as shown by control
(nonchimeric) chick embryos. It is
likely that the strong CK/VIM coexpression in the flattened p-OFT epicardial tissue (but not in the d-OFT
epicardium) is related to the ability
of this population to transform into
epicardially derived cells (Pérez-Pomares et al., 1997, Morabito et al.,
2001) as already shown in other systems (Fitchett and Hay, 1989). This
ability to generate a mesenchymal
cell population (composed of epicardially derived cells, EPDCs)
through EMT has been reported by
different authors (Dettman et al.,
1998; Gittenberger-de Groot et al.,
1998; Pérez-Pomares et al., 1998;
Männer, 1999).
Because the migration of EPDCs
into myocardial layers and their
subsequent differentiation into a
variety of cell types has been suggested to be essential in the proper
development of the myocardium
and coronary vessels (Pérez-Pomares et al., 1998, 2002; Dettman
et al., 1998; Gittenberger de Groot
et al., 1998; Vrancken Peeters et
al., 1999), we determined the potential of the nonproepicardially
derived d-OFT epicardium to transform into mesenchyme in a collagen gel assay. The in vitro experiments showed that proepicardial
tissue initially forms an epicardial
monolayer over the collagen gel.
d-OFT CELLS DERIVE FROM CEPHALIC PERICARDIUM 65
As a result of an EMT, the monolayer subsequently produces a
population of invasive mesenchyme. Cells isolated from the dOFT also spread over the surface of
the gel to form an epithelial monolayer, but, in contrast to the proepicardially derived epithelium, they
only generate very few invasive
mesenchymal cells. The epithelial
cells in isolated d-OFT cultures usually arrange in parallel rows. The
integrity is generally lost after 24 hr,
a phenomenon also seen in most
of the pericardial control explants.
Thus, this supports the histologic observations that the d-OFT epicardium is a derivative of coelomicpericardial tissue. It also indicates
that the two epicardial populations
on the developing OFT have different potentials to undergo EMT, similar to what has been demonstrated before for the endocardial
populations in the respective segments of the developing heart
(Mjaatvedt and Markwald, 1989).
It was demonstrated recently that
proepicardial ablation results in the
formation of a “compensatory mesothelial collar” in the truncal (distal)
OFT region (Gittenberger-de Groot
et al., 2000). Our current study supports this finding. Moreover, we
demonstrate that, in the normal as
well as in the quail-to-chick proepicardial explant-model, the epicardial population on the OFT also has a
dual origin. The epicardium on the
surface of the proximal OFT is proepicardially derived, the cephalic pericardium is the tissue source for the
“epicardial-like” cells on the distal
OFT. Inhibition of proepicardial development apparently creates a
“permissive” environment for the
pericardially derived cell population, resulting in an expansion of this
population toward the proximal end
of the OFT. The extent of this expansion in our ablation experiments and
in those described by Gittenberger-de Groot et al. (2000), however, are not identical. This finding is
not surprising as the methods used to
delay epicardial growth were different (eggshell membrane block and
direct ablation of the proepicardium
respectively, see also Pérez-Pomares
et al., 2002). Therefore, we conclude
that the prominent presence of epi-
cardial-like tissue on the distal OFT in
proepicardium ablated specimens
reflects an expansion of a pericardially derived tissue, which in normal
development can also been found
on the d-OFT, rather than a compensatory growth under abnormal
conditions.
Recently, we showed that, in the
ventricles, proper development of
the epicardium is crucial for the differentiation and maturation of the
underlying myocardium (Pérez-Pomares et al., 2002). Here, we show
the presence of two “epicardial”
cell populations associated with the
OFT. Although it remains to be established whether—and if so, to what
extent—these cell populations contribute to the regulation of OFT morphogenesis, the spatiotemporal relationship between the different
tissues of the OFT suggests that the
interface between the two epicardial populations might be playing an
important role in the developing
OFT. The mechanisms involved in
OFT morphogenesis are contentious.
Specifically, controversies exist regarding two pivotal events, i.e., arterialization of the distal part and the
apparent torsion and retraction of
the conal myocardium (Thompson
et al., 1987). Although during early
development a part of the d-OFT
myocardium is covered by pericardially derived epicardium, eventually the OFT myocardium is completely covered by proepicardially
derived epicardium, the pericardially derived cells ending up covering
the roots of the great vessels. This
apparent shift of the “proepicardial/
pericardial” interface with respect
to the distal myocardial rim of the
OFT leads to a few possible models:
(1) the mature epicardium moves
freely over the myocardial surface;
(2) the nonmyocardial (upper) portion of the d-OFT grows in a distal-toproximal manner (cephalocaudal
direction), displacing the distal myocardial edge proximally as a result of
mechanical forces with a synchronic local regulation of cell division
(Thompson et al., 1995); or (3) the
myocardium in the d-OFT disappears as a results of transdifferentiation of myocardial cells (Argüello et
al., 1978; Ya et al., 1998) and/or
myocardial death (Watanabe et al.,
1998), possibly involving EPDC-regulated apoptosis (Rothenberg et al.,
2002). At this point, we do not have
any data to determine the exact
relationship between the formation
of the interface between the epicardially derived and pericardially
derived epithelial cells on the surface of the OFT and the spatiotemporal changes that occur at the
myocardial/mesenchymal junction
in the OFT. The observation that, in
the setting of proepicardial ablations, the pericardially derived epicardium can occupy a relatively
large segment of the myocardial
OFT (see also Gittenberger-de
Groot et al., 2000), resulting in a
change of the relationship between the above-mentioned tissue
interfaces, indicates that the myocardial/mesenchymal boundary itself is not the determining factor in
establishing the extent of the migration of the pericardially derived
cells over the OFT. Future studies on
the development of the myocardial OFT in animal models with perturbed epicardialization (e.g., microsurgical intervention, knockouts) will undoubtedly provide
more insight in this matter.
EXPERIMENTAL PROCEDURES
Animals
Chick and quail eggs were kept in
an incubator at 37°C. The embryos
were staged according to Hamburger and Hamilton (1951).
Normal embryos for
immunohistochemical
characterization.
Chick eggs were incubated until
stages H/H19 –29. The embryos were
fixed in Amsterdam fixative, embedded in Paraplast plus, sectioned in a
microtome (5 ␮m), and guide series
were stained with H&E. Selected
slides were immunostained using antibodies against VIM, the retinoic acidsynthetic enzyme RALDH2, or CK.
RALDH2 and CK were used as markers
for epicardium (Pérez-Pomares et al.,
2002). The MF-20 antibody was used
to specifically label the myocardium
(Van den Hoff et al., 1999). Cell nuclei
were counterstained with propidium
iodide.
66 PÉREZ-POMARES ET AL.
Fig. 6. Microsurgical procedures to alter/study epicardial development. A: The normal process of proepicardial attachment to the
myocardial wall. B–D: Three microsurgical procedures that provide different and independent information about outflow tract (OFT)
epicardium development. In A, the arrow indicates the attachment of the proepicardium (asterisk) to the inner curvature of the heart.
B presents the proepicardial quail-to-chick transplantation as described by Männer (1999, see their materials and methods for a
careful description). In C, ablation of the proepicardium is illustrated. The proepicardial cell mass (arrowhead) is carefully removed
from the coelomic surface using tungsten needles and iridectomy scissors and then extracted with thin forceps. In D, a piece of
eggshell membrane (depicted as a double dashed line, see arrow) is inserted between the inner curvature of the heart and the base
of the aortic sac. When used to explant the distal OFT epicardial tissue onto collagen gels, only the more distal portion of the
membrane is dissected (arrowhead). A, atrium; AoS, aortic sac; OFT, outflow tract, d-OFT, distal outflow tract; p-OFT, proximal outflow
tract; V, ventricle.
Proepicardial chimeras.
Quail-to-chick proepicardial chimeras were prepared as described by
Männer (1999) with the following
modifications. Host chick embryos
were incubated until stages H/H16 –
17. The eggs were windowed, and
sharp tungsten needles were used
to create small openings through
the vitelline and chorionic membranes exposing the pericardial
cavity. For each embryo, a small
piece of the eggshell membrane
was cut with iridectomy scissors and
made to fit exactly between the sinoatrial sulcus and the caudal
vitelline veins. Then, stage H/H16 –17
quail embryo donors were excised
and perfused with Earles Balanced
Salt Solution (EBSS; GIBCO). The
heart was removed by cutting it
through the outflow tract and the
sinoatrial sulcus. The previously prepared eggshell membrane was introduced through the omphalomesenteric vein of the quail embryo and
pushed until it reached the cardiac
lumen, so that the sinus venosus
formed a cuff around the membrane, holding the donor proepicardium in its surface. The membrane
carrying the quail (donor) proepi-
cardium was inserted facing the
ventricular heart surface (Fig. 6B). After the operation, the eggs were
sealed with Scotch tape and reincubated for 96 –108 hr (H/H25–26), 132–
144 hr (H/H28 –29), or 180 hr (H/H32),
then fixed in modified Amsterdam’s
fixative (methanol:acetone:water ⫽
2:2:1) and embedded in Paraplast
(Paraplast Plus, OXFORD Labware,
St. Louis, MO). Finally, 5-␮m serial
sections were mounted on microscope slides (Superfrost/Plus, Fisherbrand, Fisher Scientific, Pittsburgh,
PA) and immunostained by using
the QCPN antibody to localize the
donor (quail) cells. The anti-RALDH2
and anti-CK antibodies were basically used to characterize mesothelial tissues.
Proepicardial ablation.
For proepicardial ablations, chick
embryos were incubated until
stages H/H16 –17. Openings in the
vitelline and chorionic membranes
were used to enter the pericardial
cavity. The proepicardium was carefully ablated by using fine dissecting
tungsten needles (Fig. 6C). The embryos were fixed in modified Amsterdam’s fixative (see above), embed-
ded in Paraplast, sectioned (5 ␮m),
and either stained with H&E or immunostained with RALDH2, CK, and/or
MF-20 as described below.
Immunohistochemistry in
normal and experimental
embryos.
For both single antigen localization
(RALDH2,
CK)
or
colabeling
(RALDH2/QCPN; RALDH2/MF-20; CK/
MF-20), paraffin sections (5 ␮m) were
used. The sections were dewaxed
and hydrated in a graded series of
ethanol, and nonspecific bindings
sites were blocked with 5% normal
goat serum, 1% bovine serum albumin(BSA), and 0.5% Triton-X 100 for 1
hr at room temperature (RT). The incubation with polyclonal antibodies
(CK, Dakopatts, diluted 1:100 in
phosphate buffered saline [PBS];
RALDH2, gift from Drs. P. McCaffery
and U. Drägger, diluted 1:5,000 in
PBS, both incubated overnight at RT)
was followed by an AlexaFluor-568
goat anti-rabbit immunoglobulin G
antibody incubation (Molecular
Probes), 1:400 in PBS, 2 hr at RT. The
sections incubated with monoclonal
antibodies (QCPN [undiluted], MF-20
diluted 1:20 in PBS, and anti-vimentin
d-OFT CELLS DERIVE FROM CEPHALIC PERICARDIUM 67
[Amf-17b] diluted 1:75 in PBS [all from
DSHB]) were incubated overnight at
RT, followed by incubation with AlexaFluor-488 rabbit anti-mouse antibody (Molecular Probes, 1:400 in
PBS, 2 hr at RT). In the case of colabeling, the method for localizing the
polyclonal antibodies was followed
by the labeling procedure for monoclonal antibodies. When this method
was applied in combination with a
propidium iodide counterstaining
(red fluorescence), AlexaFluor-568
was substituted by Cy5-conjugated
donkey anti-rabbit secondary antibody (Jackson Laboratories). In a
subset of experiments Cy5-conjugated donkey anti-mouse secondary antibody (Jackson Laboratories,
1:100 dilution in PBS, 4 hr of incubation at RT) in combination with AlexaFluor-488 goat anti-rabbit secondary antibody were applied. Some
sections were also counterstained
with propidium iodide. After staining,
slides were cover-slipped by using a
1:1 PBS/glycerol solution and analyzed under a Bio-Rad MRC 1024 laser scanning confocal microscope.
Whole-mount MF-20 stainings were
performed as described elsewhere
(Van den Hoff et al., 1999).
In ovo and in vitro
characterization of epicardiallike tissue of the distal OFT.
To isolate precursor tissue of the epicardial-like cells of the distal OFT,
H/H16 –17 chick embryos were used.
A small piece of eggshell membrane
was cut with iridectomy scissors to fit
exactly between the inner curvature
of the heart and the base of the
aortic sac (Fig. 6D). After insertion of
the membrane, the embryos were
sealed and reincubated for 12 or 24
hr. A subset of these embryos was
excised, fixed in Amsterdam’s fixative, embedded in Paraplast, sectioned, and processed for H&E staining or immunohistochemistry (CK or
RALDH2). The inserted eggshell
membranes from other experimental specimens were isolated, and the
segments closest to the aortic sac
were used for culture on 1.5 mg/ml
drained collagen gels (rat tail type I
collagen, Collaborative Research,
500 ␮l/well in four-well NUNC plates)
in a CO2 incubator at 37°C. In some
cases, these “explants” were cocultured with small pieces of chick pericardium (stages H/H16 –17) and/or
with quail proepicardia (H/H16 –17).
In these coculture experiments, explants were typically placed 2–3 mm
apart. The explants were allowed to
attach to the collagen for 5 hr. Then,
M199 medium (GIBCO) supplemented with 1% chick serum (SIGMA)
and insulin/transferrin/selenium (ITS,
Collaborative Research) was added.
All the explants were cultured for 1–2
days at 37°C and 5% CO2 and inspected daily by using an inverted microscope with Hoffman Modulation
Contrast optics or an Olympus microscope coupled to a SPOT digital camera and photographed. At the end of
the culture period, the explants were
fixed in 4% paraformaldehyde, extensively washed in PBS, blocked in a 1%
BSA-PBS solution and incubated overnight in a 1:100 dilution of the CK antibody or a 1:2,500 of the RALDH2,
both in PBS. The cultures were washed
at least three times (1 hr each) in PBSBSA and finally incubated overnight
again in 1:400 dilution in PBS secondary Alexa-conjugated anti-rabbit antibody (AlexaFluor-568 or AlexaFluor488, Molecular Probes). In quail– chick
tissue cocultures CK or RALDH2 antibodies were used to characterize the
mesothelial tissue (see above), and
QCPN was used to localize quail cells.
In brief, the gels were incubated in
undiluted QCPN supernatant, extensively washed and incubated in a
1:400 dilution in PBS of the secondary
Alexa-conjugated anti-mouse antibody (AlexaFluor-488 or AlexaFluor568, Molecular Probes), washed, and
mounted. The cultures were analyzed
in a Bio-Rad MRC 1024 scanning laser
confocal microscope.
ACKNOWLEDGMENTS
We thank Tanya Rittmann for creative assistance in preparing cartoon illustrations. The authors also
thank Dr. Peter McCaffery and Dr.
Ursula Dräger for their kind gift of the
RALDH2 antibody. The MF-20, vimentin (Amf-17b), QCPN, and QH1
monoclonal antibodies were obtained from the Developmental
Studies Hybridoma Bank maintained
by the Department of Pharmacology and Molecular Sciences, Johns
Hopkins University School of Medicine, Baltimore, MD, and the Department of Biological Sciences, University of Iowa, Iowa City, IA, under
contract NO1-HD-2-3144 from the
NICHD. A.W. and J.M.P.-P received
funding from the NIH and the AHA.
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