1 Stahlman Cardiovascular Laboratories, Department of Medicine and Program for
Developmental Biology, Vanderbilt University Medical Center, Nashville, TN
37232, USA
2 MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4
2XU, UK
3 Department of Cell and Developmental Biology, Fox Chase Cancer Center,
Philadelphia, PA 19111, USA
* Author for correspondence (e-mail: david.bader{at}vanderbilt.edu)
Accepted 4 October 2005
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SUMMARY |
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Key words: Blood vessel development, Gut, Mesothelia
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Introduction |
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Studies of blood vessel development to coelomic organs, such as those
encased in the pericardial and peritoneal cavities, are limited and have
focused primarily on the heart. These studies have shown that the mesothelial
covering of the embryonic heart [the proepicardium (PE) and its derivative the
epicardium] is a major source of cells to the coronary system
(Dettman et al., 1998;
Manner, 1993
;
Manner et al., 2001
;
Mikawa and Fischman, 1992
;
Reese et al., 2002
). Although
debate remains about whether all coronary vasculogenic cells are derived from
the PE/epicardium (Cox et al.,
2000
; Drake et al.,
1997
; Munoz-Chapuli et al.,
2002
), it is well established that cells of this mesothelium
undergo EMT, migration and subsequent differentiation into coronary vessels
(Dettman et al., 1998
;
Mikawa and Gourdie, 1996
;
Perez-Pomares et al., 2002
;
Vrancken Peeters et al.,
1999
). This form of blood vessel development is thought to be a
unique mechanism, as its progenitors are derived from an epithelial
mesothelium that subsequently produces vasculogenic mesenchyme
(Dettman et al., 1998
;
Gittenberger-de Groot et al.,
1998
; Wada et al.,
2003b
).
The origin of vasculogenic cells to the alimentary canal, which is encased
in the peritoneal coelom, is unknown. The structure of the gut is conserved
among vertebrates and consists of the epithelial mucosa, submucosa, muscularis
externa and serosa (serosal mesothelium and underlying mesenchyme)
(Netter, 1997;
Roberts et al., 1996
). The gut
is formed by simple embryonic structures: endoderm gives rise to the
epithelial mucosa while the other layers are thought to arise from the lateral
splanchnic mesoderm (Kiefer,
2003
). Additionally, neural crest cells migrate into the gut and
differentiate into neurons of the enteric plexus
(Young et al., 2000
;
Young and Newgreen, 2001
).
Still, the origin of the major vessels to the gut is not understood.
Vascular systems of the heart and gut have several striking similarities.
Most significantly, the major vessels to heart and gut run on the surface of
the organ and are intimately associated with their mesothelial covering
(Netter, 1997). These
characteristics have led us to test whether a conserved developmental
mechanism, similar to coronary vasculogenesis, accounts for blood vessel
formation in the gut. In the current study, we provide molecular genetic and
experimental data demonstrating that the serosal mesothelium is the major
source of vasculogenic cells of the gut. Our data show that the gut is
initially devoid of its mesothelial covering and its surface blood vessels.
Soon after formation of the tubular gut, non-resident cells migrate to and
over the gut to form the serosal mesothelium. Subsequently, a sub-population
of these cells undergoes epithelial-mesenchymal transition (EMT), migrates
within the gut and gives rise to vascular smooth muscle cells that populate
all major vessels of the gut. Our data indicate that the formation of coelomic
mesothelium, whether it be epicardial or serosal, is coupled to
vasculogenesis, and suggest that elements of a common developmental mechanism
regulate the generation of blood vessels to the heart and gut.
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Materials and methods |
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Immunohistochemistry and lacZ staining
Embryos were either embedded in OCT and snap frozen directly after
dissection, or fixed in 4% paraformaldehyde (PFA), protected in 30% sucrose
overnight, and then OCT embedded and snap frozen. Frozen sections were
generated at 7 µm on a Leica Cryostat. PFA-fixed tissue was used for the
detection of ß-galactosidase (ß-Gal) protein. Immunohistochemistry
was performed according to standard protocols
(Bader et al., 1982;
Reese et al., 1999
;
Wada et al., 2003a
). The
following primary antibodies were used: polyclonal Wt1 (C-19) at a dilution of
1:200 to 1:500 (sc-192, Santa Cruz); monoclonal Wt1 (6F-H2) at 1:50 (M3561,
Dako); polyclonal ß-Gal at 1:5000 (55976, Cappel/ICN); polyclonal
cytokeratin at 1:500 to 1:2000 (Z0622, Dako); monoclonal
-SMA (1A4) at
1:100 to 1:200 (A2547, Sigma); monoclonal Pecam at 1:50 (550274, Pharmingen).
Mouse monoclonal antibodies to be incubated on PFA-fixed tissue were directly
labeled using the Zenon labeling kit (Molecular Probes), according to
manufacturer's instructions. Secondary antibodies were Alexa
fluorophore-coupled (Molecular Probes) and were used at a dilution of
1:2000.
Whole-mount lacZ staining was performed according to standard
protocols (Hogan et al.,
1994). Staining was usually allowed to continue overnight,
especially in younger embryonic stages. In larger embryos and mice, the gut
and other tissues of interested were either dissected out, or the body wall
was opened to fully expose the intestinal organs. For histological analysis,
lacZ-stained specimens were dehydrated in Isopropanol and
subsequently embedded in paraffin. Serial sections (7 µm) were collected
and counterstained with Eosin. Hematoxylin and Eosin staining of sections from
unstained tissues and embryos was performed following standard protocols.
In vivo labeling of embryonic guts and their culture
E12.5 embryos were freed from extraembryonic tissues, but remained attached
to the placenta. CCFSE
[5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate,
succinimidyl ester `mixed isomers', Molecular Probes] was diluted to 24 µM
in sterile PBS, and, through a small opening in the ventral body wall covering
the herniated intestines, injected with a mouth pipet into the cavity
surrounding the guts. After CCFSE application, embryos were incubated for 1
hour at 37°C and 5% CO2 in DMEM/10% FCS under sterile
conditions. Subsequently, embryonic guts were isolated and cultured in 4-well
dishes (Nunc) in Optimem (Life Technologies), supplemented with 1 mM
L-Glutamine (Life Technologies) and a Penicillin/Streptomycin antibiotic
mixture at 37°C and 5% CO2
(Natarajan et al., 1999).
Embryonic gut explants were fed every 2 days. At least two embryonic guts were
fixed at the same time point, day 0 to day 3, with fresh 4% PFA for 30 minutes
on ice, washed in PBS, protected overnight in 30% sucrose, OCT embedded and
snap frozen. Control explants without CCFSE treatment showed no difference in
tissue integrity and viability (not shown).
Quantification of labeled cells
Images of sections labeled with CCFSE and ß-Gal, or ß-Gal and
Pecam/SMA were used for quantification. Images were viewed in Photoshop, where
the color channels for blue (DAPI), red and green were separated for the
counting of cell populations. Percentages were collected for each vessel, and
an average percentage calculated for the number of vessels counted.
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Results |
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At early stages of mouse embryogenesis [embryonic day (E) 8.5], the gut
consists of two epithelial layers: endoderm and lateral splanchnic mesoderm.
At E9.5-E10.5, the mesoderm of the gut has thickened into several cell layers
by cell proliferation. Antibodies for Wt1 detected strong reactivity in cells
of the nephrogenic mesoderm at E9.5 (Fig.
1C), as previously reported
(Armstrong et al., 1993;
Carmona et al., 2001
;
Moore et al., 1998
). Still, at
E9.5, the simple squamous serosal mesothelium that also expresses Wt1 protein
was completely absent from the mesenteries and gut tube
(Fig. 1C)
(Armstrong et al., 1993
;
Carmona et al., 2001
;
Moore et al., 1998
). At E10.5,
Wt1-positive mesothelial cells were first detectable at the proximal base of
the dorsal mesentery, whereas the gut tube was still devoid of a mesothelium,
as evidenced by the lack of Wt1 and cytokeratin surface staining
(Fig. 1D,E). The absence of a
mesothelium at this stage was also confirmed by histology, as cells on the
embryonic gut surface were irregularly shaped and arranged, and did not have a
squamous phenotype (see Fig. S1 in the supplementary material). Residual
cytokeratin staining in splanchnic lateral mesoderm of the mesentery
(Fig. 1E) was observed at
E10.5, as it loses its epithelial nature. One day later (E11.5), Wt1-,
cytokeratin-positive cells were present on the gut surface and the peritoneal
wall along the entire anteroposterior axis
(Fig. 1F,G). Staining of these
two markers was confined to the mesothelial lining of the gut and to the
mesentery, indicating that, by E11.5, the entire gut was enclosed by
mesothelium (Fig. 1F,G). Thus,
serosal mesothelial cells are the only cell type to express Wt1 in the gut at
this stage, as has been previously reported
(Armstrong et al., 1993
;
Carmona et al., 2001
;
Moore et al., 1998
).
Histological analysis at this stage revealed that cells on the gut surface
were arranged in a regular manner, although not yet with a squamous phenotype
(see Fig. S1 in the supplementary material). By E13.5, however, these cells
comprise a thin layer of simple squamous epithelium that is typical for the
serosal mesothelium (see Fig. S1). Thus, Wt1- and cytokeratin-positive serosal
mesothelial cells appear in a proximal to distal manner from E10.5 onwards; by
E11.5 they have completely enclosed the embryonic gut, and by E13.5 they have
fully differentiated into a simple squamous epithelium. Genetic lineage
marking (see below) confirmed this pattern of differentiation. Once
established, the serosal mesothelium was present over the gut surface
throughout prenatal and postnatal life (see Fig. S1).
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The presence of ß-Gal-positive cells in the submesothelial space of
the gut suggests that these cells are the progeny of mesothelial cells that
have undergone EMT. To experimentally confirm that surface serosal cells
undergo EMT, we combined an explant culture system of the embryonic gut with
genetic and in vivo labeling of serosal mesothelial cells
(Natarajan et al., 1999).
Embryonic guts from crosses between Wt1-Cre and Rosa26R were exposed to the
lipophilic dye CCFSE, which has been well established to mark only surface
cells (Morabito et al., 2001
;
Perez-Pomares et al., 2002
;
Perez-Pomares et al., 2004
).
CCFSE was placed locally on the herniated gut by injecting under the ventral
body wall of E12.5 embryos, and, after one hour of incubation, the embryonic
gut tubes, including the mesentery, were dissected out for subsequent culture
in CCFSE-free medium. This approach should initially mark only surface serosal
mesothelial cells (in localized areas), while cells undergoing EMT would be
subsequently labeled in the delaminated underlying mesenchyme. In order to
quantitatively delineate this process, we counted the number of surface and
submesothelial CCFSE-labeled cells that were positive and negative for
ß-Gal protein (Table 1).
As expected, at the time of CCFSE application, only mesothelial cells in
labeled spots on the surface of the gut were positive for CCFSE
(Fig. 4G and
Table 1; arrowheads define the
area of CCFSE labeling). Note that the overwhelming majority (96.1%) of these
cells were also ß-Gal positive (Table
1). No sub-serosal mesenchymal cells were labeled, indicating the
effectiveness of surface labeling and the lack of immediate EMT. After 24 to
48 hours of culture, CCFSE-marked cells in labeled patches were still found in
the serosal mesothelium, but were also present in significant numbers in the
submesothelial mesenchyme of labeled areas
(Fig. 4H,J). Indeed, when
CCFSE-labeled patches were analyzed, on average 25.5% of all CCFSE-labeled
cells were found in the submesothelial space
(Table 1). Of this group of
CCFSE-labeled submesothelial cells, on average 74.4% were also positive for
the ß-Gal marker (Fig.
4J,K and Table 1).
Two important findings arise from these results. First, CCFSE-labeled cells in
the submesothelium reveal that a subset of mesothelial cells undergoes EMT in
this in vitro system (Fig.
4H,J). This result was similar to our observations with serosal
EMT in vivo (Fig. 4A-F). Second, a large majority (74.4%) of CCFSE-labeled cells in the submesothelial
space are co-labeled for ß-Gal (Fig.
4J,K and Table 1),
confirming that these marked cells are descendants of serosal mesothelial
cells. By contrast, Wt1 expression was largely confined to the mesothelial
surface of CCFSE-labeled cultured guts, whereas CCFSE-positive cells were
readily apparent in the connective tissue space
(Fig. 4H,I), reiterating the in
vivo situation (Fig. 4A-C).
This indicates that Wt1 expression is downregulated as cells become
mesenchymal, as has been previously reported
(Carmona et al., 2001
;
Moore et al., 1999
), whereas
ß-Gal protein continues to be expressed. As expected, we also found
ß-Gal-positive cells that do not carry CCFSE, owing to the locally
restricted uptake of the compound. In addition, we find a limited number of
CCFSE-marked cells on the serosal surface (11.4%) and in the submesothelial
space (25.6%) that are not reactive for ß-Gal
(Fig. 4J; Table 1). The implications of
this finding are discussed below (see Discussion). Our analysis of
CCFSE-labeled domains at random positions along the anteroposterior axis of
the embryonic gut did not reveal any qualitative or quantitative differences
in EMT. This indicates that EMT occurs in the same fashion independently from
the position along the anteroposterior axis of the gut tube. Taken together,
these data indicate that a subset of serosal mesothelial cells, like
epicardial mesothelial cells, undergoes EMT.
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Descendants of the serosa differentiate into vascular smooth muscle
To determine the identity of the lacZ-expressing cells of
developing blood vessels in the gut, we performed immunohistochemistry for
ß-Gal, coupled with markers for vascular smooth muscle and endothelium.
Immunostaining for ß-Gal also serves to corroborate the
lacZ-staining pattern, as recent reports have suggested that
lacZ analysis of ß-Gal activity in tissues does not always fully
recapitulate the ß-Gal expression pattern
(Couffinhal et al., 1997;
Mahony et al., 2002
). Using
standard fluorescence microscopy, ß-Gal protein was found in smooth
muscle cells that were co-stained for SMA and desmin adjacent to the vessel
lumen (Fig. 6A-D, see also Fig.
S2 in the supplementary material). However, colocalization of Pecam- with
ß-Gal-antibody was not observed to any significant degree
(Fig. 6E-H). Of particular
interest are three additional findings. First, Pecam-positive endothelial
cells of the vessels are oriented perpendicularly with respect to the vascular
smooth muscle cells (Fig.
6F-H), which in these images are labeled for ß-Gal protein.
Second, it should be noted that ß-Gal protein is not expressed in the
longitudinal and circumferential visceral smooth muscle layers, as seen in
Fig. 6A-D. This indicates that
the visceral smooth muscle is of different origin than the vascular smooth
muscle of the gut. Third, we find cells that express ß-Gal protein but
are not associated with the vascular system of the gut
(Fig. 6), suggesting that the
serosal mesothelium contributes to other cell populations in the intestinal
tract.
To further delineate the colocalization of ß-Gal protein with endothelial and smooth muscle markers, we followed two approaches. First, we performed confocal microscopy on sections from adult intestines. Colocalization of ß-Gal protein with SMA was readily apparent (Fig. 7A-C, see also Fig. S2 in the supplementary material), whereas overlap of ß-Gal with the endothelial cell marker Pecam in blood vessels was not detected to any significant degree (Fig. 7D-F). Next, in order to quantify these results, we determined the percentage of ß-Gal-positive cells in sections of SMA- or Pecam-stained blood vessels of the gut (Table 2). Our quantification revealed that, on average, 77.7% of SMA-positive cells of the gut vasculature co-label with ß-Gal (from a total of 392 cells counted). By contrast, on average, 6.8% of Pecam-marked cells in the gut are also positive for ß-Gal (from a total of 259 cells counted; Table 2). This result indicates that in the gut, a vast majority of vascular smooth muscle cells are descendants of the serosal mesothelium. The presence of ß-Gal-negative vascular smooth muscle may indicate an additional source of this cell type.
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|
Once again, confocal experiments revealed that all major arteries and veins were populated by ß-Gal-positive cells. Taken together, the results indicate that the smooth muscle cells of blood vessels in the gut are the progeny of serosal mesothelium.
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Discussion |
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On the basis of these data, we propose the following mechanism concerning blood vessel development to the gut (Fig. 8). At early embryonic stages (E9.5-E10.5), the gut tube consists of endoderm and splanchnic mesoderm without mesothelial covering. A vascular plexus associated with the endoderm is present before arrival of the mesothelium, whereas no blood vessels are found on the surface of the gut. From E11, the serosal mesothelium can be visualized on the surface of the gut and the mesenteries, as well as covering the peritoneal cavity. At E12.5, mesothelial cells on the surface of the gut tube undergo EMT and are seen in the submesothelial space of the gut. From E13.5, endothelial tubes extending from the vascular plexus are seen at the surface of the gut. From E16.5, a subset of the descendants of mesothelial cells has differentiated into vascular smooth muscle cells that are found in all arteries and veins of the mesentery and gut. Therefore, we show that progeny of the serosal mesothelium differentiate predominantly into vascular smooth muscle and other non-vascular cells of the gut.
|
The Wt1-driven Cre recombinase marks descendants of serosal mesothelium
The intriguing similarities between the epicardium and the serosal
mesothelium, and recent findings that the epicardium is the source of the
coronary vessels, led us to analyze the fate of the serosal mesothelium. We
used a two-component genetic system in the mouse consisting of a Cre
recombinase driven by the Wt1 promoter and the Rosa26R reporter mouse line,
and have shown that serosal mesothelial cells are specifically and faithfully
marked by the reporter (i.e. ß-Gal expression). Also, we have combined
this genetic system with the use of CCFSE as a surface marker
(Morabito et al., 2001;
Perez-Pomares et al., 2002
;
Perez-Pomares et al., 2004
)
and have revealed that mesothelial cells undergo EMT, as a large majority of
CCFSE-positive submesothelial cells co-label for the ß-Gal protein.
Finally, our quantitative data indicate that the majority of vascular smooth
muscle cells in the mesentery and the gut are ß-Gal-positive and thus, we
postulate, are descendants of the serosal mesothelium. Although the origin of
the small, but significant, number of non-ß-Gal-labeled vascular smooth
muscle cells is unclear, we have no way to determine their origin at present.
Possibly these non-labeled vascular smooth muscle cells originate directly
from the splanchnic lateral plate mesoderm that overlies the endodermal
epithelium. Alternatively, a small number of serosal mesothelial cells may not
express the ß-Gal protein, or may express at levels below detection.
Overall, our current data indicate a faithful and specific expression of this
protein in the serosal mesothelium and its descendants. However, the current
methods, which have been previously used in related studies
(Cai et al., 2003
;
de Lange et al., 2004
;
Jiang et al., 2002
;
Kawaguchi et al., 2002
),
follow populations of Wt1-expressing cells and are not intended to address
matters of clonal differentiation. Those analyses must await methods to mark
individual progenitors.
Origin of endothelial cells during gut vessel development
Earlier studies had demonstrated that endothelial cells are produced from
the PE in heart development (Mikawa and
Gourdie, 1996; Perez-Pomares
et al., 2002
). Although we detect endothelial cells in small, but
consistent numbers in our lineage studies in the gut and heart, the majority
of endothelial cells in these vessels are not marked by our genetic labeling
system. The original lineage studies of Mikawa and Gourdie
(Mikawa and Gourdie, 1996
)
suggest that angioblasts are a minor population within the PE of the heart. A
similar situation could be true for the serosal mesothelium. One explanation
for this result is that angioblasts are associated with coelomic mesothelia
but are not truly part of the epithelium. In this case, angioblasts may never
express mesothelial markers such as Wt1 and, thus, may be largely undetected
in our experimental model. Several authors have suggested that migratory
angioblasts originating from the liver primordium are intermingled with the
PE, and that the PE is composed of both epithelial and mesenchymal cells
(Nahirney et al., 2003
;
Perez-Pomares et al., 1997
;
Perez-Pomares et al., 1998
).
These cells would be marked by direct retroviral or vital dye labeling, as
employed in previous PE experiments
(Mikawa and Gourdie, 1996
;
Perez-Pomares et al., 2002
).
Alternatively, if angioblasts are truly part of the advancing mesothelium, it
is possible that they never express Wt1 or do not express Wt1-Cre at
sufficient levels to be detected in our system.
Coupling vasculogenesis with the formation of coelomic mesothelium
Previous studies and our current data suggest a recurring relationship
between the formation of coelomic mesothelia, either epicardial or serosal,
and the delivery of vasculogenic cells to developing organs
(Dettman et al., 1998;
Kaufman and Bard, 1999
;
Manasek, 1969
;
Manner, 1993
;
Mikawa and Gourdie, 1996
;
Perez-Pomares et al., 2002
;
Perez-Pomares et al., 1998
;
Viragh and Challice, 1981
;
Vrancken Peeters et al.,
1999
). Both the heart and the alimentary canal are initially
devoid of mesothelia (Manasek,
1969
; Manner,
1993
; Meier,
1980
), but are housed within a common coelom. This coelom is later
subdivided into pericardial and peritoneal cavities by the downward growth of
the septum transversum (Kaufman and Bard,
1999
). Mesothelial precursors of the epicardium and pericardial
coelom arise in association with the septum transversum as it bisects the
common coelom (Viragh and Challice,
1981
). We can only speculate that a similar population of
mesothelial precursors is delivered to the developing gut and peritoneal
cavity, as its origins are unclear. The mesothelial precursors of the
pericardial cavity have been shown to arise from the dorsal aspect of the
coelom (Nahirney et al.,
2003
), and appear to migrate over the developing organs.
Interestingly, our data suggest that the serosal mesothelium also forms
dorsally in the peritoneal cavity, at the mesentery close to the urogenital
ridges. Nevertheless, we would like to stress that, at present, we have no
data providing evidence towards this mechanism. In the heart and the gut, EMT
from the mesothelium produces cells that migrate to developing vessels, where
described molecular mechanisms of vasculogenesis may regulate cell
differentiation (Carmeliet et al.,
1996
; Cleaver and Melton,
2003
; Hellstrom et al.,
1999
; Jain, 2003
;
Lammert et al., 2003
). In
addition, it is important to note that both epicardial and serosal mesothelia
produce non-vasculogenic progeny that reside within the heart and the gut
(Figs 5,
6)
(Mikawa and Fischman, 1992
;
Munoz-Chapuli et al., 2002
;
Perez-Pomares et al., 1997
;
Perez-Pomares et al., 1998
).
Thus, although potential variation may exist, it appears that the vertebrate
embryo employs elements of a common or conserved program for the generation of
vessels to coelomic organs rather than any wholly variant mechanism in their
development.
Coupling vascular development to the formation of mesothelia varies from
blood vessel formation in other regions of the embryo. In the limbs, body wall
and extraembryonic tissues, vasculogenic mesenchyme is thought to arise from
locally derived mesenchyme (Cleaver and
Krieg, 1999; Gerhardt and
Betsholtz, 2003
; Hirschi and
Majesky, 2004
; Majesky,
2003
; Saint-Jeannet et al.,
1992
). The present data, along with previous studies on coronary
development, suggest that vasculogenic mesenchyme is `delivered' to organs
within the coelom via its encapsulating epithelium later in development.
Although known molecular signaling mechanisms are likely to regulate the
angioblast/mesenchyme interaction, we propose that coupling the production of
vasculogenic cells to the formation of coelomic mesothelium constitutes a
distinct yet conserved cellular mechanism in blood vessel development.
Clinical relevance of the serosal mesothelium and its contribution to blood vessel formation
Surgeons have long used the omentum, the serosal mesothelium and its
connective tissue companion, to repair injured blood vessels and intestines
with good success (Bertram et al.,
1999; Matoba et al.,
1996
; Roa et al.,
1999
; Sterpetti et al.,
1992
). Although the cellular basis of this reparative function is
unknown, it is interesting to speculate that serosal mesothelium may serve as
a source of diverse cell types in injury repair. A previous study from our
group has shown that mesothelial cell lines can produce vasculogenic cells
after stimulation with specific growth factors, suggesting a retention of
embryonic potential (Wada et al.,
2003a
). Thus, it is possible that the serosa may provide a natural
source of divergent cells to be used in the repair of damaged adult
structures. Finally, a relatively uncommon developmental syndrome, called
`Apple Peel Bowel', producing intestinal atresia has been reported
(Federici et al., 2003
;
Pumberger et al., 2002
;
Waldhausen and Sawin, 1997
).
Interestingly, this atresia or intestinal wasting is associated with regional
loss of the serosa and its associated blood vessels, and is suggestive of a
mechanistic relationship between the generation of coelomic mesothelia and
vascular development.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/23/5317/DC1
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