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First published online 16 April 2008
doi: 10.1242/dev.020958
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1 Wallenberg Laboratory, Sahlgrenska University Hospital, University of
Gothenburg, Gothenburg, Sweden.
2 Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology,
University of Gothenburg, Gothenburg, Sweden.
3 Institute of Biomedicine, Electron Microscopy Unit, University of Gothenburg,
Gothenburg, Sweden.
4 Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
Author for correspondence (e-mail:
per.lindahl{at}wlab.gu.se)
Accepted 17 March 2008
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-lacZ. There was, however, no
migration of SMCs from the ventral to the dorsal side of the vessel. Moreover,
the lateral plate mesoderm-derived cells in the ventral wall of the aorta were
replaced by somite-derived cells at E10.5, as indicated by reporter gene
expression in Meox1-cre/Rosa 26 double transgenic mice. Examination
of reporter gene expression in adult aortas from Hoxb6-cre/Rosa 26
and Meox1-cre/Rosa 26 double transgenic mice suggested that all SMCs
in the adult descending aorta derive from the somites, whereas no contribution
was recorded from lateral plate mesoderm.
Key words: Vascular smooth muscle cell, Aorta, Lateral plate mesoderm, Paraxial mesoderm, Cell origin
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Majesky,
2007). Neural crest cells (NCs) give rise to vascular SMCs in the
cephalic region and in the cardiac outflow region, and are required for proper
patterning of the great vessels (Hutson and
Kirby, 2003; Jiang et al.,
2000). The contribution of NCs extends to the fifth somite in
chicken and to the ligamentum arteriosus in mice
(Jiang et al., 2000;
Le Lievre and Le Douarin,
1975). The secondary heart field gives rise to SMCs in the root of
the aorta and pulmonary trunk (Waldo et
al., 2005). The pro-epicardial organ contributes to SMCs in the
coronary arteries (Ali et al.,
2003; Gittenberger-de Groot et
al., 1998; Mikawa and Gourdie,
1996), and ventrally emigrating neural tube cells contribute to
SMCs in the great vessels and the coronary vessels in chicken
(Ali et al., 2003). The
mesothelium finally gives rise to SMC in mesenteric vessels in mice
(Wilm et al., 2005).
In posterior parts of the body, vascular SMCs are assumed to derive
primarily from splanchnic lateral plate mesoderm
(Gittenberger-de Groot et al.,
1999), but the precise source of these cells has not been
rigorously determined. Observations of the early pattern of SMC contractile
protein expression in mouse and quail aorta suggest that vascular SMC
differentiation is induced in mesenchymal cells that line the endothelium
(Hungerford et al., 1996;
Takahashi et al., 1996). The
contractile proteins are first expressed on the ventral side of the vessel,
and it has been hypothesized that SMCs are induced only in the ventral area of
the aorta and later migrate to populate the lateral and dorsal areas
(Hungerford et al., 1996). This
model has gained support from studies of Edg1 knockout mice, which
selectively lack SMCs on the dorsal side of the aorta at embryonic day (E)
12.5 but develop SMCs on the ventral side as normal
(Liu et al., 2000). Two recent
reports challenge this view. Paraxial mesoderm was shown to contribute to SMCs
in the descending aorta in mice and chicken
(Esner et al., 2006;
Pouget et al., 2006). However,
the extent of this contribution is not clear.
In this investigation, Hoxb6-cre transgenic mice
(Lowe et al., 2000) were
crossed with Rosa26 (R26) reporter mice
(Soriano, 1999) to track cells
of lateral plate mesoderm origin. Meox1-cre/R26 double transgenic
mice were used to track cells of paraxial mesoderm origin
(Jukkola et al., 2005), and
SM22-lacZ mice were used to determine SMC
differentiation by expression of the SMC marker SM22
(Zhang et al., 2001). We
specifically focused on the contribution of lateral plate mesoderm to SMCs in
the descending aorta.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) were provided by Michael R. Keuhn (Division of Basic
Sciences, National Cancer Institute, National Institutes of Health, Bethesda,
MD). R26 reporter mice (Soriano,
1999) were provided by Henrik Semb (Section of Endocrinology, Stem
Cell Center, Lund University, Sweden). Animals were cross-bred to generate
Hoxb6-cre/R26 reporter mice. SM22-lacZ mice
(Zhang et al., 2001) were used
to track the early presence of SMC. Meox1-cre/R26 reporter mice
(Jukkola et al., 2005) were
used to track somite-derived cells. All transgenic lineages have a mixed
genetic background. Animals were housed under barrier conditions in the
transgenic core facility of University of Gothenburg, in accordance with the
local ethical committee guidelines (Dnr. 213-2000, Dnr. 289-2003, Dnr.
230-2006). The morning of vaginal plug detection was counted as E0.5.
X-gal staining
lacZ expression in E8.5-E10.5 embryos
E8.5-E10.5 embryos were dissected and immediately fixed at 4°C for 2
hours in 0.1 M phosphate buffer (pH 7.3) with 0.2% glutaraldehyde, 1.5%
formaldehyde, 5 mM EGTA (pH 7.3) and 2 mM MgCl2. The embryos were
washed for 3x10 minutes in phosphate-buffered saline (PBS), whole-mount
stained at 37°C overnight in 0.1 M phosphate buffer (pH 7.3) with 2 mM
MgCl2, 0.01% sodium deoxycholate, 0.02% IGEPAL CA-630 (Sigma
#I-8896), 5 mM potassium hexacyanoferrate(III), 5 mM potassium
hexacyanoferrate(II) and X-gal (Fluka #16665, 1 mg/ml staining solution),
embedded in paraffin and sectioned at 5 µm.
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lacZ expression in postnatal animals
Postnatal day 2 (P2) mice were stained using different techniques: organs
were dissected, fixed at 4°C for 4 hours in 0.1 M phosphate buffer (pH
7.3) with 0.2% glutaraldehyde, 1.5% formaldehyde, 5 mM EGTA (pH 7.3) and 2 mM
MgCl2, and whole mount stained as described for E8.5-E10.5 embryos.
Slices of the kidney (1 mm) were fixed and stained en bloc as described for
the embryos. Whole pups (unfixed) were mounted in Tissue-Tek OCT compound
(Sakura Finetek, Torrence, CA) at -80°C and cryosectioned at 10 µm. The
sections were fixed in 0.1 M phosphate buffer (pH 7.3) with 0.2%
glutaraldehyde, 1.5% formaldehyde, 5 mM EGTA (pH 7.3) and 2 mM
MgCl2 at room temperature for 15 minutes and stained as described
for E11.5 embryos.
lacZ expression in dissected adult aortas
Adult mice (n=2 Hoxb6-cre/R26 double transgenic males,
3-4 months old; n=2 Meox1-cre/R26 double transgenic females,
2 months old) were anesthetized and the aorta was washed with physiological
NaCl solution. Aortas were dissected from fat and surrounding tissues. They
were immediately fixed at 4°C for 4 hours, washed, whole-mount stained,
paraffin embedded and sectioned, as described for the embryos.
Immunohistochemical staining
E11.5 embryos were triple labeled for Acta2, CD31 and
lacZ-expressing cells. Embryos were dissected, fixed, mounted,
sectioned and stained for lacZ activity as described above
(lacZ expression in E11.5 embryos). Antigen retrieval was
accomplished by trypsin incubation [0.025% in 0.05 M Tris-Cl (pH 7.8) at
37°C for 20 minutes (Trypsin EDTA, Gibco #25200)]. The following primary
antibodies were used: mouse anti-human Acta2 clone 1A4 (DakoCytomation #M0851)
and rat anti-mouse CD31 (PECAM-1) (BD Biosciences Pharmingen #553370).
Secondary antibodies used were: rabbit anti-mouse (biotinylated)
(DakoCytomation #E0354) and goat anti-rat-alexa568 (Molecular
Probes #A11077). All antibodies were diluted 1:200 in blocking buffer. Acta2
staining was completed by incubation in FITC-conjugated ExtrAvidin (Sigma
#E2761) (1:200 in blocking buffer). Slides were mounted in ProLong Gold
antifade reagent (Invitrogen #P36930).
Realtime RT-PCR
Embryos were harvested at indicated time-points and totRNA was extracted
according to standard protocols. Meox1 mRNA was amplified with primers from
Applied Biosystems (Assay-on-demand Mm00440285_m1) at cycling conditions
recommended by the manufacturer. Ct values were normalized for amplification
efficacy, and against GAPDH (Assay-on-demand Mm99999915_g1). Data were
presented as relative expression compared with the E9.5 level. Error bars
represent 95% confidence interval (n=3 per time-point).
Transmission electron microscopy
C57BL/6 mouse embryos at E9.5 were fixed overnight by immersion in 2.5%
glutaraldehyde, 2% paraformaldehyde, 0.02% sodium azide in 0.05 M sodium
cacodylate buffer (pH 7.2-7.4). After washing in 0.15 M sodium cacodylate
buffer, specimens were fixed for 2 hours in 1% osmium tetroxide with 1%
potassium hexacyanoferrate(II) in 0.1 M cacodylate. The embryos were rinsed in
water and contrasted en bloc in 0.5% uranyl acetate in water for 1 hour. They
were dehydrated and infiltrated with epoxy resin (Agar100) according to
standard protocols.
Hoxb6-cre/R26 mouse E9.5 embryos were prepared for electron
microscopy following staining with X-gal, as described above. Transverse body
sections were obtained with a Leica VT1000 tissue slicer (Leica Microsystems
GmbH, Wetzlar, Germany) at 50 µm. Selected tissue slices were fixed for 2
hours in 1% osmium tetroxide and contrasted en bloc in 0.5% uranyl acetate in
water for 1 hour. They were dehydrated in ethanol and infiltrated in plastic
resin as described earlier (Masahira et
al., 2005), followed by flat embedding in epoxy resin.
Light and electron microscopy sections were obtained with a Reichert Ultracut E ultramicrotome (Reichert AG, Vienna, Austria). Ultrathin sections were contrasted with uranyl acetate and lead citrate before examination in a Zeiss 912AB electron microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Zambrowicz et al., 1997). We
investigated the expression pattern of the Hoxb6-cre-activated
R26 reporter gene in detail in mouse embryos at E8.5-E9.0. The
reporter had an anterior demarcation at the 12th somite (7th cervical somite),
and was expressed throughout the length of the embryo. In cross-sections,
reporter expression was found in both somatic and splanchnic lateral plate
mesoderm at all investigated levels (Fig.
1A-C). Ectopic expression was detected in gut epithelium and
extra-embryonic mesoderm. At the posterior end of the embryo, the expression
was more widespread with staining in paraxial mesoderm cells and in the neural
plate (Fig. 1D,E).
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is one of the earliest SMC markers in mice
(Zhang et al., 2001),
SM22-lacZ mice were stained for
SM22-driven lacZ expression to detect the earliest
stages of SMC differentiation. SM22-lacZ expression
was detected at E8.5 adjacent to the dorsal aorta, but the staining was
confined to the anterior part of the embryo. No staining was observed in the
regions that stained positive for the Hoxb6-cre-activated R26
reporter at this stage. The first SM22-lacZ-expressing cells in the
descending aorta were detected at E9.0, in the splanchnic mesoderm facing the
coelom and in cells situated between the initially paired dorsal aortas
(Fig. 1F-H).
SMCs of lateral plate mesoderm origin are confined to the ventral wall of the descending aorta
Side-by-side comparison of staining patterns in stage-matched E9.5
SM22-lacZ and Hoxb6-cre/R26 reporter mouse
embryos was performed to map the contribution of lateral plate mesoderm to the
SMC population in the descending aorta. SM22-lacZ was
expressed in a layer of cells around the circumference of the dorsal aorta in
the forelimb bud region and adjacent segments
(Fig. 2A). In more posterior
positions, the expression was confined to the ventral and lateral walls, and
eventually only ventral walls, of the paired dorsal aortas
(Fig. 2B and 2C). The
Hoxb6-cre/R26 reporter was expressed in splanchnic and somatic
mesoderm but not in intermediate mesoderm
(Fig. 2D). All cells on the
ventral side of the aorta (or paired aortas in more posterior positions)
expressed the reporter, including the
SM22-lacZ-expressing cells
(Fig. 2A-C). The reporter was
also expressed in cells in the lateral wall of the aorta and to a lesser
extent in the dorsal wall (arrows in Fig.
2A-C).
Ultra-thin sections were prepared from lacZ-stained Hoxb6-cre/R26 reporter mice at E9.5 to resolve the identity of the lateral and dorsal cells with transmission electron microscopy (TEM). The lacZ staining was confined to endothelial cells facing the luminal side of the aorta (Fig. 2E-G). No staining was seen in peri-endothelial cells in the dorsal or lateral wall of the aorta. In the ventral wall, the staining was found in both endothelial cells and in peri-endothelial cells.
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Lateral plate mesoderm-derived SMCs are replaced at E11.5
The expression pattern of the R26 reporter gene was examined in
Hoxb6-cre/R26 reporter mouse embryos at E11.5 to follow the fate of
lateral plate mesoderm-derived cells in the descending aorta. A single layer
of lacZ-expressing cells was found in the vessel wall, whereas the
surrounding tissues were unstained. The lateral plate mesoderm cells that
occupied the ventral side of the aorta at E9.5 had been replaced by other
cells. The aorta was surrounded by large numbers of lacZ-negative
cells in the lung bud and liver regions
(Fig. 4A), and by four or five
layers of lacZ-negative cells in more posterior regions
(Fig. 4B).
Triple staining for Acta2, the endothelial marker CD31, and the R26 reporter gene showed consistent lacZ expression in endothelial cells (Fig. 4C). SMCs were in general not stained (Fig. 4C,D). A few cases of lacZ-expressing SMCs were, however, detected in a discrete region where lateral plate mesoderm-derived mesenchymal cells were still found adjacent to the aorta (Fig. 4E).
Lateral plate mesoderm cells do not contribute to the adult descending aorta
We examined the aortas from adult Hoxb6-cre/R26 reporter mice for
contribution of lateral plate mesoderm cells. Endothelial cells throughout the
length of the descending aorta expressed the R26 reporter gene,
whereas SMCs in the anterior and middle part of the vessel were unstained
(Fig. 5A-D). By contrast, SMCs
in the posterior part of the descending aorta expressed the R26
reporter (Fig. 5E,F). The
transition from lacZ-negative to lacZ-expressing SMCs was
gradual and occurred in a region anterior to the renal arteries. The results
could indicate that lateral plate mesoderm contributes to the SMCs in the
posterior part of the descending aorta, but the significance of this finding
is not clear. Hoxb6-cre activates the R26 reporter gene in
some ectopic tissues in the posterior part of the embryo at E9.0-E9.5,
including paraxial mesoderm (Fig.
1G,H), and the lacZ-expressing cells may derive from
these tissues.
Somite-derived cells replace lateral plate mesoderm-derived SMCs in the ventral wall of the aorta at E10.5
Paraxial mesoderm contributes to SMCs in the aorta in mice and chicken
(Esner et al., 2006;
Pouget et al., 2006).
Meox1-cre/R26 double transgenic mice were investigated for somite
contribution to the dorsal aorta in order to identify the cells that replace
the lateral plate mesoderm-derived SMCs at E11.5. Meox1 is expressed
in pre-somitic paraxial mesoderm and in the somites
(Candia et al., 1992), and
Meox1-cre has been shown to activate the R26 reporter gene
in somites and somite-derived tissues from E8.5-E9.5 in mice
(Jukkola et al., 2005).
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At E10.5, the expression domain of the reporter had expanded towards the ventral side of the embryo and reporter-expressing cells had migrated along the ventrolateral wall of the aorta to circumvent the vessel (Fig. 6E-J). The lacZ-negative cells that were situated on the ventral side of the aorta at E9.5 had been replaced by three or four layers of lacZ-expressing cells in the Meox1-cre/R26 reporter mice (Fig. 6E-H). We conclude that the invasion of Meox1-cre/R26-expressing cells in the area ventral to the aorta at E10.5 correlates with the loss of lateral plate mesoderm-derived cells at E11.5. In the posterior part of the embryo, Meox1-cre/R26-expressing cells were still confined to the dorsal and dorsolateral wall of the aorta (Fig. 6I,J), which suggests that the dorsal-to-ventral migration of somite-derived cells is initiated in the anterior part of the embryo and progresses towards the posterior end.
Ectopic expression of the Meox1-cre/R26 reporter in cardiac outflow tract and kidneys in postnatal mice
The expression pattern of the endogenous Meox1 gene has not been
systematically documented after E10.5. In order to investigate somite
contribution to the postnatal and adult aorta, the temporal pattern of
Meox1 expression after somitogenesis and the spatial pattern of
Meox1-cre/R26 reporter gene activation in postnatal mice were
examined to monitor activation of the reporter in non-somite-derived cell
lineages.
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Ectopic expression was recorded in the cardiac outflow tract, in which SMCs
in the ascending aorta and aortic arch expressed the reporter
(Fig. 7F-I). These structures
are known to derive from cardiac neural crest cells. Staining was also seen in
cells that form a branched network of fine caliber on the heart surface
(Fig. 7H). Double staining
against lacZ and Acta2 showed that this structure is separate from
the coronary arteries (Fig.
7I), and we propose that it represents cardiac nerves that also
have neural crest origin (Jiang et al.,
2000). However, the staining pattern indicated expression in
additional cells in the valve plane, including a small number of atrial
myocardial cells, SMCs in the aortic root and SMCs in the pulmonary trunk
(Fig. 7F,G). These cells
probably originate from the secondary heart field
(Waldo et al., 2005).
Expression was also detected in the cortex of the kidney
(Fig. 7J,K). The staining
encompassed mature and developing nephrons, undifferentiated metanephric
mesenchyme, and intra-renal blood vessels, whereas collection ducts lacked
expression. No expression was observed in the pelvic region of the kidney, or
in the ureter (data not shown). The pattern of reporter expression indicates
that the reporter was activated in a subset of intermediate mesoderm, i.e. the
metanephrogenic blastema (Sariola and
Sainio, 1998). Other intermediate mesoderm-derived structures such
as adrenal cortex and gonads were not stained
(Fig. 7L,M).
SMCs in the adult descending aorta originate from the somites
We finally examined the aortas from postnatal and adult
Meox1-cre/R26 double transgenic mice for contribution of
somite-derived cells. The reporter gene was consistently expressed in adult
aortic SMCs from the cardiac outflow tract to the iliac arteries
(Fig. 8A-G). The homogenous
staining of SMCs in the posterior part of the vessel suggests that the
lacZ-expressing SMCs that were present in the posterior part of the
aorta in Hoxb6-cre/R26 reporter mice reflect ectopic expression of
Hoxb6-cre in paraxial mesoderm rather than a contribution from
lateral plate mesoderm.
Postnatal Meox1-cre/R26 mice were investigated for somite contribution to SMCs in the major branches of the descending aorta. The reporter gene was not expressed in coeliac or superior mesenteric arteries, but it was expressed in renal arteries and intercostal arteries (Fig. 8H-J, and data not shown). Somite-derived SMCs occasionally extended a short distance into the coeliac and superior mesenteric vessels, but the seam between the lineages was in principle located to the branch points and SMCs of different origin were not mixed (Fig. 8K,L). SMCs in the renal arteries expressed the reporter from the branch point to the kidney (Fig. 8M). The distal boundary of somite contribution to the intercostal arteries was not determined.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-lacZ. These cells were,
however, replaced by somite-derived cells at E10.5, as indicated by reporter
gene expression in Meox1-cre/R26 double-transgenic mice. Examination
of reporter gene expression in aortas from adult Hoxb6-cre/R26 and
Meox1-cre/R26 transgenic mice revealed that all SMCs derive from the
somites and not from lateral plate mesoderm.
|
). It has been suggested that SMCs in the descending aorta
derive from lateral plate mesoderm on the basis of the early expression
pattern of SMC markers (Hungerford et al.,
1996; Takahashi et al.,
1996). SMC induction has further been proposed to be confined to
the ventral side of the vessel, after which the cells migrate to the dorsal
side (Hungerford et al., 1996).
Our results confirmed that SMC differentiation was induced in lateral plate
mesoderm-derived cells on the ventral side of the aorta at E9.0-E9.5. There
was, however, no migration of SMCs from the ventral to the dorsal side of the
vessel at this time point. Moreover, the lateral plate mesoderm-derived
SM22-lacZ-expressing cells in the ventral wall of the
aorta were replaced by somite-derived cells at E11.5. We could not confirm any
contribution of lateral plate mesoderm to SMCs in the descending aorta in
adult mice, and we conclude that lateral plate mesoderm is not a source of
mature SMCs in the descending aorta. The fate of the early lateral plate mesoderm-derived SMC was not investigated. They could die or migrate away. Endothelial cells retained expression of the Hoxb6-cre/R26 reporter gene in adult animals, which argues against a concomitant shift from lateral-plate mesoderm-derived to somite-derived cells in the endothelial population. However, the adult endothelial cells were not investigated in sufficient detail to exclude minor changes.
Two recent papers show that paraxial mesoderm contributes to aortic SMCs.
Segmental plate mesoderm (presomitic mesoderm) was grafted from quail to
chicken, and the host aorta was investigated for quail-derived cells
(Pouget et al., 2006). Quail
cells were preferentially found in the endothelium, but they also
differentiated to SMCs in the trunk region. Genetic recombination experiments
in mice showed that skeletal muscle and aortic SMCs share a common progenitor
cell (Esner et al., 2006).
Moreover, cells from the somite were shown to migrate into the space between
the aorta and the gut at E10.5, and contribute to the aortic SMC population on
both the ventral and dorsal sides of the vessel. This is in agreement with our
finding that somite-derived cells replaced the lateral plate mesoderm-derived
SM22- expressing cells in the ventral wall of the
aorta at E10.5. The homogenous expression of the Meox1-cre/R26
reporter gene in SMCs in the adult aorta suggests that somites are the sole
contributors to SMCs in the adult aorta in mice.
|
). The expression is restricted to a subregion of the
endogenous Hoxb6 expression domain. In contrast to the Hoxb6
gene, Hoxb6-cre is not expressed in intermediate mesoderm, thoracic
paraxial mesoderm (sclerotome), lung and stomach mesenchyme, or CNS, apart
from a segment in the midbrain hindbrain junction
(Eid et al., 1993). As
Hoxb6-cre is not a lineage marker for lateral plate mesoderm, it was
crucial to establish the exact pattern of reporter gene activation. Our
investigation showed that the R26 reporter was activated in both
somatic and splanchnic mesoderm with an anterior expression border at the 12th
somite (7th cervical somite) at E8.5-E9.0. The expression was homogenous and
seemed to encompass all cells in these compartments. The
Hoxb6-cre/R26 reporter mouse model is therefore suitable for
investigating the contribution of lateral plate mesoderm to the aorta
posterior of the 12th somite. Ectopic expression was observed in gut
epithelium and in extra-embryonic tissues, but these structures are not likely
to contribute to aortic SMCs. In the most posterior part of E9.0 embryos, we
observed ectopic expression in paraxial mesoderm, which makes it difficult to
determine the origin of Hoxb6-cre/R26 reporter-expressing cells in
the posterior part of the animal. The overlapping expression of
Hoxb6-cre/R26 and Meox1-cre/R26 reporter genes in SMCs in
the posterior part of the adult aorta is, however, consistent with a somite
origin of these cells.
The Meox1-cre transgene was inserted in the first intron of the
endogenous Meox1 gene, and its expression is controlled by the
endogenous Meox1 promoter
(Jukkola et al., 2005). Meox1
is a lineage marker for paraxial mesoderm, and expression of the
Meox1-cre/R26 reporter gene was confined to somites and
somite-derived tissues at E11.5 (Jukkola
et al., 2005). In postnatal mice, the reporter was also expressed
in the ascending aorta and aortic arch that derive from cardiac neural crest
(Jiang et al., 2000), and in
the aortic root, pulmonary trunk and structures in the heart valve plane that
probably derive from the secondary heart field
(Waldo et al., 2005). Reporter
gene activity in these sites can be traced back to expression of
Meox1 in branchial arch mesenchyme, cardiac cushions and truncus
arteriosus at E11.5 (Candia et al.,
1992). Ectopic expression in cardiac neural crest cells precludes
attempts to determine the anterior border of somite-derived SMCs in the aorta
in Meox1-cre/R26 reporter mice. This border has, however, been mapped
in detail in Wnt1-cre/R26 transgenic mice that label neural crest
cells, and occurs just distal to the ligamentum arteriosus
(Jiang et al., 2000).
|
).
Other derivates of intermediate mesoderm did not express the reporter. The
metanephric kidney develops in a posterior position in the embryo at level
with the bifurcation of the common iliac arteries at E12, and subsequently
ascends to a more anterior position in relation to other structures. It is
therefore less likely that lacZ-positive SMCs in the descending aorta
derive from the metanephrogenic blastema, but contribution from this lineage
can not be formally ruled out.
SMCs in the mesenteric blood vessels were recently shown to derive from the
mesothelium, but the proximal limit of this contribution was not defined
(Wilm et al., 2005). The lack
of Meox1-cre/R26 reporter staining in the superior mesenteric and
coeliac arteries suggests that the seam between somite- and
mesothelium-derived SMCs is located at the root of the vessels. However, the
transition from one lineage to the other is not strictly bound to the branch
points, as fields of somite-derived cells sometime extended a short distance
into the superior mesenteric and coeliac arteries. By contrast, SMCs in the
renal arteries and intercostal arteries consistently expressed the
Meox1-cre/R26 reporter. The recruitment of somite-derived SMCs to the
renal and intercostal arteries, but not to the mesenteric arteries, may relate
to the developmental origin of the vessels. The renal and intercostal arteries
develop by angiogenic sprouting from the aorta, whereas the mesenteric and
coeliac vessels are formed by remodeling of the vitelline artery
(Gest and Carron, 2003).
Our lineage-tracing experiments and earlier reports on somite-derived SMCs
call for a new model concerning the origin of SMCs in the descending aorta
(Fig. 9). Lateral-plate
mesoderm cells on the ventral side of the aorta express SMC marker genes at
E9.0-E9.5. Shortly thereafter, SMC induction occurs in somite-derived cells on
the dorsal side of the aorta. At E10.5, somite-derived cells migrate along the
lateral and ventral walls of the aorta into the zone between the aorta and the
gut, and replace the lateral plate mesoderm-derived SMCs in the ventral wall
of the aorta. The adult descending aorta is entirely populated by
somite-derived SMC. The new view on the ontogeny of aortic SMCs has
implications for vascular biology. The Edg1 knockout phenotype and
proposed function of Edg1 requires re-interpretation. Mice deficient for
Edg1 lack SMCs in the most dorsal aspect of the aorta, and the
phenotype has been assigned to defective migration from the ventral wall
(Liu et al., 2000). This
explanation is not consistent with our findings as we failed to detect any
movements of SMCs from the ventral to the dorsal part of the aorta during
aortic wall development. An alternative explanation is that dorsal SMCs, or
dorsal SMC progenitor cells, are more sensitive to the loss of Edg1
compared with ventral SMCs. The Edg1 example illustrates that detailed
morphological information is necessary to work out the molecular mechanisms
that mediate SMC differentiation.
In summary, we show that SMC differentiation is induced in lateral plate mesoderm-derived cells in the ventral wall of the descending aorta at E9.5 in mice. However, this population is replaced by somite-derived cells at E10.5, and there is no contribution of lateral plate mesoderm cells to SMCs in the adult descending aorta.
Note added in proof
After this work was submitted, a report was published that describes a
similar population of transient primitive SMC in the avian aorta.
Transplantation experiments using the quail-chick system showed that
sclerotomal cells replaced primitive SMCs in the ventral wall of the vessel
(Wiegreffe et al., 2007). This
indicates that the formation of a transient SMC population is evolutionary
conserved.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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