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First published online January 25, 2006
doi: 10.1242/10.1242/dev.02226
1 CNRS URA 2578, Department of Developmental Biology, Pasteur Institute, 28 rue
du Dr Roux, 75 724 Paris Cedex 15, France.
2 Stem Cell Research Institute, Dibit, H.S. Raffaele, Via Olgettina 58, 20132
Milano, Italy.
3 Department of Biology, University of Milan, Via Celoria 26, 20133 Milano,
Italy.
4 Institute of Cell Biology and Tissue Engineering, San Raffaele Biomedical
Science Park of Rome, Via Castel Romano 100/2, 00128 Rome, Italy.
Author for correspondence (e-mail:
margab{at}pasteur.fr)
Accepted 14 December 2005
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-cardiac actin gene. A rare intragenic recombination event results
in a functional nlacZ sequence, giving rise to clones of
ß-galactosidase-positive cells. Periendothelial and vascular smooth
muscle cells of the dorsal aorta are the main cell types labelled,
demonstrating that these are clonally related to the paraxial mesoderm-derived
cells of skeletal muscle. Rare endothelial cells are also seen in some clones.
In younger clones, arising from a recent recombination event, myotomal
labelling is predominantly in the hypaxial somite, adjacent to labelled smooth
muscle cells in the aorta. Analysis of Pax3GFP/+ embryos
shows that these cells are Pax3 negative but GFP positive, with fluorescent
cells in the intervening region between the aorta and the somite. This is
consistent with the direct migration of smooth muscle precursor cells that had
expressed Pax3. These results are discussed in terms of the paraxial mesoderm
contribution to the aorta and of the mesoangioblast stem cells that derive
from it.
Key words: Dorsal aorta, Skeletal muscle, Smooth muscle, Clonal analysis, LaacZ, Pax3
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and
mesoangioblasts (De Angelis et al.,
1999). The latter are stem cells that express some endothelial
markers and give rise to a number of mesodermal derivatives, including
skeletal muscle and smooth muscle (Minasi
et al., 2002). This raises the possibility of a lineage
relationship between these muscles and prompted us to investigate the origin
of smooth muscle cells in the dorsal aorta.
At embryonic day (E) 8 in the mouse, the initial structure of the dorsal
aorta is present as two tubes extending under the neural tube and notochord,
along the anteroposterior axis of the embryo. Fusion of the tubes, in the
central region of the trunk, takes place progressively towards the
extremities, to give the single midline dorsal aorta. The nascent tube is
formed from endothelial cells (Drake and
Fleming, 2000). Mural cells, expressing smooth muscle markers,
differentiate under the endothelial cell layer, which faces into the lumen. In
the avian dorsal aorta, the first mural smooth muscle cells appear ventrally
(Hungerford et al., 1996).
Pericytes are a class of mural cell that characteristically lie within the
basal lamina of the endothelium and constitute the smooth muscle component of
capillaries, whereas thicker blood vessels, such as the dorsal aorta,
accumulate additional outer layers of vascular smooth muscle cells
(Gerhardt and Betsholtz,
2003).
Smooth muscle is derived from mesoderm, except at certain sites, such as in
the head and aortic arches, where it originates from the neural crest
(Le Lievre and Le Douarin,
1975). It is thought that, during vascular development, smooth
muscle cells are recruited from surrounding mesenchyme and induced to
differentiate by factors produced by the endothelial cells of the vessel
(Hirschi et al., 1998).
However, endothelial and smooth muscle cells may derive from a common
Flk1-expressing progenitor (Yamashita et
al., 2000; Ema et al.,
2003). Furthermore, labelling experiments have suggested that
endothelial cells in the dorsal aorta can transdifferentiate into smooth
muscle (DeRuiter et al., 1997).
The mesoangioblast stem cells may correspond to an endothelial/pericyte
intermediate cell type (Cossu and Bianco,
2003).
Endothelial cells in the dorsal aorta have been shown to derive from two
distinct mesodermal sources. In both birds and mammals, it has been proposed
that there may be a common progenitor cell, the hemangioblast, derived from
splanchnic mesoderm, that gives rise to the endothelial cells of the vessel
wall and to hematopoetic cells (Jaffredo
et al., 1998; Nishikawa et
al., 1998). Grafting experiments in avian embryos have shown that
somites give rise to endothelial cells in the dorsal aorta; this paraxial
mesoderm contribution was restricted to a dorsolateral location, whereas the
ventral floor was colonized by cells derived from splanchnic mesoderm, at the
stages examined (Pardanaud et al.,
1996).
Somites form from paraxial mesoderm as segmented structures, following an
anteroposterior developmental gradient, on either side of the neural tube,
from about E8 in the mouse. Under the influence of signals from surrounding
tissues, cells in the somite acquire different mesodermal identities
(Tajbakhsh and Buckingham,
2000). Ventrally, mesenchymal cells of the sclerotome will give
rise to the bone of the vertebral column and ribs. Dorsally the initial
epithelial structure of the somite is retained as the dermomyotome, which
gives rise to dorsal derm and to all the skeletal muscles of the body. The
first skeletal muscle to form is the myotome. Muscle progenitor cells
delaminate from the dermomyotome and migrate under this epithelium where they
differentiate. Initially, this process takes place from the epaxial
dermomyotome, which is adjacent to the axial structures of the neural tube and
notochord. Later, cells from the other, hypaxial, extremity of the
dermomyotome also contribute to the myotome. Other muscle progenitor cells
migrate out from the hypaxial dermomyotome to form, for example, the muscles
of the limb. Dermomyotomal cells are characterized by the expression of Pax3,
an important regulator of myogenesis. Endothelial cells are also associated
with somites, from which they migrate into the body and limbs. Somite
transplantation experiments in avian embryos have shown that angioblasts are
derived from all somitic compartments
(Noden, 1989;
Wilting et al., 1995). More
recently lineage tracking, using retrovirus vectors in the chick embryo, has
shown that endothelial and skeletal muscle cells in the limb are derived from
a common progenitor cell, labelled in the hypaxial dermomyotome
(Kardon et al., 2002). It has
been suggested that the mesenchymal cells of the avian sclerotome can be
recruited to give rise to the smooth muscle of blood vessels formed in this
region of the embryo (Christ et al.,
2004).
In order to examine a possible lineage relationship between cells in the
dorsal aorta and the skeletal muscle cells of the myotome in the mouse embryo,
we adopted a genetic approach that permits retrospective clonal analysis
(Bonnerot and Nicolas, 1993;
Nicolas et al., 1996). This
employs a laacZ reporter that contains a duplication of the
lacZ coding sequence under the control of regulatory sequences
directing expression to the tissues of interest. In the embryo, a rare
intragenic recombination event will remove the duplication to give
lacZ, which encodes a functional ß-galactosidase (ß-gal)
protein when the gene is expressed. A common progenitor cell that has
undergone such a recombination event will give rise to ß-gal+
cells that are clonally related. We have used mice in which we had targeted
the -cardiac actin gene with a nlaacZ reporter
(Meilhac et al., 2003), in
order to carry out such a clonal analysis in the myocardium. In addition to
the heart, -cardiac actin is also expressed in embryonic skeletal
muscle and in the dorsal aorta (Sassoon et
al., 1988).
The retrospective clonal analysis presented in this paper shows that cells in the dorsal aorta and in the myotome have a common clonal origin. The properties of the common progenitor cells are discussed. Based on the analysis of Pax3GFP/+ mice, we propose that GFP-labelled progenitor cells migrate from the somite to the dorsal aorta. We also document the spatiotemporal characteristics of clones in the dorsal aorta, in terms of cell type and position. Most of them are smooth muscle cells, but occasional labelled endothelial cells are present in the clones, in keeping with the existence of a common vascular progenitor.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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c-actinnlaacZ1.1/nlaacZ1.1 line,
in which a nlaacZ sequence is targeted to the -cardiac actin
gene, used for clonal analysis, was as described by Meilhac et al.
(Meilhac et al., 2003). The T4
transgenic line expresses nlacZ under the control of 9 kb of genomic
sequence, upstream of -cardiac actin
(Biben et al., 1996). The
Pax3GFP/+ mouse line was obtained by targeting a DNA
sequence encoding GFP to an allele of Pax3
(Relaix et al., 2005).
Pax3GFP/+ mice carrying the T4 transgene were obtained by
crossing males of the T4 line with Pax3GFP/+ mice. The
ROSA26R-lacZ line was as described by Soriano
(Soriano, 1999) and
Ht-PA-Cre transgenic mice as by Pietri et al.
(Pietri et al., 2003). Embryos
in which the conditional ROSA26 allele had been activated were
obtained by crossing the two mouse lines.
Production and description of clones
The nlaacZ sequence produces a truncated ß-galactosidase
(ß-gal) protein, which is deprived of enzymatic activity. It will only
give rise to a ß-gal-positive cell if it undergoes an internal
recombination event, which removes the duplication and the stop codons
generated by it. This is a spontaneous event, which occurs during mitosis at a
low frequency (Bonnerot and Nicolas,
1993). Descendants of a cell in which this has occurred will be
detected provided that they express the -cardiac actin gene. Clones
that had been obtained by crossing heterozygous
c-actinnlaacZ1.1/+ males
(Meilhac et al., 2003) were
used to analyze labelling in the dorsal aorta at E10.5. Additional clones at
E10.5 and E9.5 were produced by crossing superovulated wild-type females
(C57/B6SJL) with homozygous
c-actinnlaacZ1.1/nlaacZ1.1 males, as
described by Meilhac et al. (Meilhac et
al., 2004b). At E10.5, a total of 729, and at E9.5, 449,
c-actinnlaacZ1.1/+ embryos were
analyzed. Dissected embryos were fixed for 30 minutes in 4% paraformaldehyde
(PFA) followed by staining in X-Gal solution at 37°C overnight [0.4 mg/ml
X-Gal in 2 mM MgCl2, 0.02% NP-40, 0.1 M PBS, 20 mM
K4Fe(CN)6, 20 mM K3Fe(CN)6]. After
X-Gal staining, the liver, gut and other tissues were removed to expose the
dorsal aorta. Embryos were stored at 4°C in 4% PFA.
Statistical establishment of clonality
The intragenic recombination that converts nlaacZ into
nlacZ is a spontaneous, heritable and random event. The frequency of
its occurrence can therefore be analyzed by the fluctuation test of Luria and
Delbrück (Luria and Delbrück,
1943). This parameter has been estimated to be about
3.6x10-6 per cell and per division in the myocardial lineage
of the c-actinnlaacZ1.1/+ line
(Meilhac et al., 2004a). The
fluctuation test of Luria and Delbrück also predicts that the number of
independent recombinations follows a Poisson distribution. Based on the number
of observations in the dorsal aorta (see
Table 1), we can calculate
that, at E10.5, a single case of two independent recombination events within
the dorsal aorta is the maximum expected. No multiple independent
recombination events are expected at E9.5. We can therefore conclude that
ß-gal+ cells are clonally related.
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Conformity of a frequency to a theoretical law was assessed by the
classical 
2 test, calculating the parameter
2=(O-C2)/C, where O is the observed frequency and C
the calculated frequency (1 degree of freedom).
In the case of the heart, which has a much larger number of -cardiac
actin-expressing cells at E10.5, the predicted frequency of double
recombination events for this target tissue is much higher
(Meilhac et al., 2003). We
cannot therefore study clonality between the heart and dorsal aorta (23
embryos show staining in the heart and dorsal aorta, but with an expected
number of 26 labelled embryos that correspond to a double recomination event)
at this stage, as we can for the myotome and dorsal aorta, based on the
statistical analysis.
Histology and immunohistochemistry
Embryos were embedded in gelatin/sucrose and 10 µm thick sections were
obtained using a cryostat. Before immunostaining, slides were washed three
times in PBS and permeabilized with blocking solution (5% lamb serum, 1% BSA,
0.02% Tween). Incubation with the primary antibody was for 2 hours at room
temperature, followed by washing with PBS, and a 1-hour incubation with the
secondary antibody coupled to the fluorochrome.
To visualize nuclei, sections were incubated with Bisbenzimide H33342. Sections were observed and photographed using a Zeiss Axioplan microscope equipped with an Axiocam camera. Optical sections were performed using a Zeiss ApoTome system.
Antibodies used were as follows: anti-ß-galactosidase, rabbit
polyclonal, used at 1:200 dilution; anti--smooth muscle actin, mouse
monoclonal, 1:400 (Sigma); anti-CD-31/PECAM, rat monoclonal, 1:200 (Pharmingen
BD Bioscience); anti-laminin, rabbit polyclonal, 1:400 (Sigma); anti-GFP,
rabbit polyclonal, 1:700 (Biovalley), anti-Pax3, mouse monoclonal, 1:50
(Developmental Studies Hybridoma Bank). Fluorochrome-conjugated goat secondary
antibodies were used at 1:300 dilution (Alexa-Fluor 488 or 594 anti-mouse or
anti-rabbit IgG, Molecular Probes).
In situ hybridization
For in situ hybridization, embryos were fixed overnight in 4% PFA in PBS at
4°C. After fixation, embryos were washed in PBS, incubated in 15% sucrose
and embedded into 7.5% gelatin. Sections (12 µm) were obtained using a
cryostat. The in situ hybridization protocol, used with a digoxigenin-labelled
riboprobe, was as described by Henrique et al.
(Henrique et al., 1995), with
modifications
(http://www.hhmi.ucla.edu/derobertis/).
In situ hybridization and immunodetection of GFP were performed on adjacent
serial sections. Sections were observed and photographed using a Zeiss ApoTome
system. The Pax1 riboprobe was kindly provided by Dr Schughart
(Institute of Mammalian Genetics, Neuherberg, Germany).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-cardiac actin in the dorsal aorta-cardiac actin-expressing cells in the dorsal
aorta, we used a transgenic mouse line, T4, that expresses nlacZ
under the control of regulatory sequences of the -cardiac actin gene
(Biben et al., 1996). At
embryonic day (E) 10.5, ß-galactosidase-positive (ß-gal+)
cells are observed in the heart, somites and dorsal aorta
(Fig. 1A,B), where the
endogenous gene is also expressed (Sassoon
et al., 1988). In the somites, labelled cells are present in the
skeletal muscle of the myotome (Fig.
1C,D). At this stage (40 somites), the caudal limit of
ß-gal+ cells in the dorsal aorta is at the level of somite 30
(where somite 1 is the most anterior and therefore most mature), whereas
labelled cells in the myotome are detectable up to about somite 34.
To characterize the -cardiac actin-expressing cells of the dorsal
aorta, we used double immunostaining with antibodies to ß-gal, and to
-smooth muscle actin or CD31/PECAM, which mark smooth muscle or
endothelial cells, respectively. In Fig.
1C,D, -smooth muscle actin labelling of cells in the dorsal
aorta and myotomes is shown. Not all -smooth muscle actin-positive
cells in the myotome are ß-gal+
(Fig. 1D), which probably
reflects the later onset of -cardiac actin expression, as skeletal
muscle cells differentiate. In the dorsal aorta, at E10.5, the
ß-gal+ cells represent a subpopulation (
10%) of the cells
that express this smooth muscle marker. About 60% of cells are -smooth
muscle actin positive, of which about 45% are periendothelial cells in the
pericyte position within the basal lamina of the endothelium
(Fig. 1E-H) and 15% are
vascular smooth muscle cells. A similar distribution of ß-gal+
cells is seen between these two mural cell types. The remaining 40% of cells
express the endothelial marker CD31. Very few (0.2%) of these endothelial
cells are ß-gal+ (Fig.
1I,J). The cell composition and extent of ß-gal labelling is
similar in the anterior region where the dorsal aorta bifurcates, or in the
central part of the trunk where it is a single tube.
At E9.5 (25 somite stage; Fig.
1K,L), ß-gal+ cells are present in the anterior
branches of the dorsal aorta and within the wall of the fused tube, extending
caudally to the level of about somite 20. The dorsal aorta is smaller at this
stage with fewer labelled cells, but the proportion of smooth muscle
actin-positive cells that are ß-gal+ remains at about 10%. Of
the remaining 40% of endothelial cells, about 0.6% are ß-gal+
at E9.5. Again the different cell types are similarly distributed on the
anteroposterior axis. By E11.5, ß-gal+ cells are no longer
detectable in the dorsal aorta, indicating that the expression of
-cardiac actin is a transitory phenomenon in this structure.
Clones of -cardiac actin-expressing cells in the dorsal aorta also colonize the myotome
The introduction of a nlaacZ reporter into the -cardiac
actin gene made it possible to carry out a retrospective clonal analysis on
cells that express this gene (Meilhac et
al., 2003; Meilhac et al.,
2004a). ß-gal+ cells, in which a functional
nlacZ sequence is present as a result of a rare intragenic
recombination event, are observed in the myotomes and dorsal aorta of
-cardiac actinnlaacZ/+ embryos. In order to examine
a possible clonal relationship between labelled cells in these two structures,
729 embryos from the -cardiac actinnlaacZ/+ line
were examined at E10.5, and 449 at E9.5. The numbers of embryos with
ß-gal+ cells in the myotome and dorsal aorta are shown in
Table 1. The low frequency of
embryos with labelled cells in the dorsal aorta, 5% at E10.5 and 2% at E9.5,
indicates that these cells result from a rare recombination event and are
clonally related (see Materials and methods). It is striking that many of
these embryos also have ß-gal+ cells in the myotome. The
probability that labelled cells in the dorsal aorta and myotome arise from two
independent recombination events in the same embryo is very low
(Table 1, see statistical
analysis). We therefore conclude that there are -cardiac
actin-expressing cells in the dorsal aorta that are clonally related to cells
in the myotome.
Classification of clones with ß-gal+ cells in the dorsal aorta
Retrospective clonal analyses of cells in the myotome have already been
described at E11.5 (Nicolas et al.,
1996; Eloy-Trinquet and
Nicolas, 2002a; Eloy-Trinquet
and Nicolas, 2002b). We have used a similar classification to
describe the characteristics of myotomal clones that also have labelled cells
in the dorsal aorta. The main parameter taken into account, which reflects the
age of the clone, was the extent to which ß-gal+ cells are
distributed along the anteroposterior axis, and whether they colonize somites
on either side of the axis. An axial extension of seven somites was taken as
the cut off point between long and short clones, based on previously observed
clonal distributions at E11.5
(Eloy-Trinquet and Nicolas,
2002a). This extension probably reflects the number of somites
prefigured in the presomitic mesoderm. The number of labelled cells in a clone
is also an important indicator of age, as a more ancient recombination event
will generate more cells that potentially express the nlacZ reporter
when the -cardiac actin gene is transcribed.
Four different categories of clones with labelled cells in the dorsal aorta were distinguished. (1) Long clones in which labelled cells are present in myotomes that extend over seven or more somites on the anteroposterior axis, with a mono- or bilateral distribution of ß-gal+ cells. (2) Short clones, with a somite extension of less than seven, with a mono- or bilateral distribution of labelled cells. (3) Single somite clones in which ß-gal+ cells are present in the myotome of a somite, on one side of the axis. (4) Clones of ß-gal+ cells in the dorsal aorta, but not the myotomes. Examples of the different categories of clones are shown in Fig. 2 and the results are summarized in Fig. 3. At E10.5, all four categories of clones are observed.
Most long clones have a bilateral distribution. In this category, the
progenitor cell in which the recombination event took place preceded the onset
of segmentation and the introduction of bilateralization in the presomitic
mesoderm. Recombination probably occurred in the self-renewing mesodermal
population of the primitive streak. A clone such as E10.5-419, which has a
very high number of labelled cells (as shown in
Fig. 3A for the dorsal aorta),
including extensive labelling in all compartments of the heart, probably
reflects an early recombination event, probably predating the onset of
gastrulation. Short clones are also necessarily derived from a nlacZ
progenitor cell that arose before segmentation. The number of labelled cells
in the dorsal aorta of these clones, like those of the long clones, tends to
be higher when the labelled cells also have a longer extension on the axis of
the aorta. Single somite clones, however, tend to have fewer labelled cells in
the dorsal aorta, reflecting a more recent origin, probably after the onset of
somitogenesis. In this case, and in clones that have no labelled cells in the
somite, ß-gal+ cells in the dorsal aorta are restricted to the
level of one or two somites. The average number of ß-gal+
cells per `segment', at the level of each pair of somites, in the dorsal aorta
(Fig. 3A, grey box) is
remarkably constant in all categories of clones with labelled cells in the
myotome. Both Student's t-test and the Snedecor test indicate that
the distribution of values per segment in different classes of clones with
expression in the somites is not significantly different
(Fig. 3B). When the total
number of myotomal clones was analysed, the same categories of clones with
ß-gal+ cells in the myotome were found as those previously
described (Eloy-Trinquet and Nicolas,
2002a). This suggests that myotomal clones that also colonise the
dorsal aorta do not have distinct characteristics, but derive from a similar
progenitor cell pool. At E9.5, fewer clones were analyzed but most of these
are in the single somite or aorta only classes
(Fig. 3C). The absence of long
clones partly reflects the smaller number of somites and mature myotomes
expressing -cardiac actin at this stage; a clone such as E9.5-68 may
correspond to another clonal category at later stages. Examples of the three
categories found are shown in Fig.
2B.
The nature of cells in the dorsal aorta that are derived from progenitor cells common to myotomal muscle
We have analyzed the location of clones of ß-gal+ cells in
the dorsal aorta. Examples are shown in
Fig. 4. Labelled smooth muscle
cells are observed in dorsal (Fig.
4A,B), ventral (Fig.
4C,D,G,H), and lateral positions (data not shown), both in a
periendothelial position (Fig.
4A-D), and in the outer layers of the wall of the aorta or in the
blood vessels that derive from it (Fig.
4G,H). Rare ß-gal+ endothelial cells, protruding
into the lumen, are detected (Fig.
4E,F). At E10.5 (Fig.
5A), the distribution of cell type varies with the age of the
progenitor cell. Occasional ß-gal+ endothelial cells are
observed in long and short clones. In these older clones, periendothelial
smooth muscle cells predominate, whereas, in the other two categories of
clone, vascular smooth muscle cells are more frequent, in the outer layers of
the vessel. These observations are presented schematically in
Fig. 5B. They provide evidence
for the existence of a common progenitor for periendothelial and vascular
smooth muscle cells, and suggest that endothelial cells may also derive from a
common vascular progenitor cell. At E9.5
(Fig. 5C), fewer clones were
available for analysis. Most ß-gal+ cells are in the
periendothelial position, reflecting the immaturity of the aorta, which has
relatively few outer vascular smooth muscle cells at this stage. However a few
labelled endothelial cells are also detected at E9.5. In
Table 2, the relative positions
of different labelled cell types around the circumference of the dorsal aorta
are summarized as a percentage for each category of clone. There is no notable
difference in cell type distribution or the localization of aortic cells along
the axis.
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). We have generated a Pax3 allele with a
GFP reporter that marks Pax3-expressing cells as being GFP
positive and also, because of GFP stability, marks cells that had previously
expressed Pax3 (Relaix et al.,
2005). As shown in Fig.
6, GFP+ cells are present in the somite, but are also
detected in the region between the somite and the dorsal aorta
(Fig. 6D,E), with which they
are also associated (Fig. 6H). The Pax3 antibody, however, only marks cells in the somite, notably in the
epithelial structure of the dermomyotome, and also migrating myogenic
progenitor cells derived from the dermomyotome
(Fig. 6G,J). The majority of
periendothelial and vascular smooth muscle cells in the dorsal aorta are
labelled with GFP (Fig. 6H),
which co-localizes with -smooth muscle actin
(Fig. 6K). The
Pax3GFP/+ line was crossed to the T4 transgenic line so
that ß-gal+ cells expressing the -cardiac
actin-nlacZ transgene could be monitored. As seen in
Fig. 6H,K,
ß-gal+ cells in the dorsal aorta are GFP+. Cells
labelled in the clonal analysis, which is dependent on the expression of
-cardiac actin, are therefore mainly derived from
Pax3-expressing cells.
Pax3 is also expressed in the dorsal neural tube
(Fig. 6E) and marks neural
crest cells derived from this structure that will form, for example, the
dorsal root ganglia (Fig. 6E).
It is therefore possible that the GFP+ cells observed outside of
the somite are of neural crest origin. In the absence of Pax3, neural crest
and its derivatives (Fig. 6F)
are severely compromised. However, in Pax3 mutant embryos,
GFP+ cells are still detected in the region between the somite and
the dorsal aorta (Fig. 6F,I), and labelled cells, which co-localize with -smooth muscle actin, are
present in the dorsal aorta (Fig.
6L). This therefore strongly suggests that the GFP+
cells in the dorsal aorta are not of neural crest origin and that, as
expected, neural crest cells do not give rise to vascular smooth muscle in the
trunk (Le Douarin and Kalcheim,
1999). Further confirmation was provided by an experiment in which
the conditional ROSA26R-lacZ reporter
(Soriano, 1999) was crossed
with a mouse line carrying a Cre-recombinase transgene under the control of
regulatory sequences for the human tissue plasminogen activator
(Ht-PA) gene, which is a marker of neural crest cells
(Pietri et al., 2003). Neural
crest cells and their derivatives, such as the sympathetic ganglia in the
vicinity of the dorsal aorta, expressed the ROSA26-lacZ reporter, but
the dorsal aorta in the trunk was negative (see Fig. S1A,B in the
supplementary material). Therefore, neural crest does not contribute to this
structure. However, in the Pax3 mutant, where the hypaxial
dermomyotome is affected, the wall of the aorta is thinner
(Fig. 6L) than in
Pax3GFP/+ embryos (Fig.
6K), and the number of -cardiac
actin-nlacZ-expressing cells is reduced by a factor of three.
Pax3 is expressed in presomitic mesoderm, just prior to
somitogenesis, and throughout the early epithelial somite
(Fig. 6O). Part of the GFP
labelling seen ventrally in the more mature somites probably corresponds to
sclerotomal cells derived from earlier progenitors in which Pax3 was
previously transcribed. Using Pax1 expression as a marker of early
sclerotome (Christ et al.,
2004), we see co-localization with GFP (see Fig. S1C-E in the
supplementary material). However, strongly labelled GFP+ cells are
located more ventrally, contiguous laterally with the hypaxial dermomyotome
and extending towards the dorsal aorta.
Endothelial cells, at E10.5, identified by their position adjacent to the lumen, are not GFP+ (Fig. 6H,K). Because they are the first cells to form the dorsal aorta, we also examined Pax3GFP/+ embryos at E8.5. At this stage endothelial cells, marked by CD31/PECAM staining, are not labelled by GFP (Fig. 6M,N), even when they are adjacent to GFP+ cells in the early somites (Fig. 6O). Immature epithelial somites have numerous GFP- cells (Fig. 6N), and, more posteriorly, where somites have not yet formed, Pax3 is not expressed in the unsegmented paraxial mesoderm (Fig. 6M), and Pax3(GFP) does not therefore mark their derivatives, which may include endothelial cells of the dorsal aorta.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-cardiac actin gene, which was targeted with the nlaacZ
reporter for this clonal analysis, is expressed in a subpopulation of cells in
the dorsal aorta, most of which express smooth muscle markers. We observe
ß-gal+ cells in the dorsal aorta over a short time period.
Association between smooth and cardiac actin isoforms is seen in other
developing muscles. Overlapping expression with smooth muscle actin is
observed in early skeletal muscle
(Woodcock-Mitchell et al.,
1988), and it is notable that in the hearts of -cardiac
actin mutant mice, smooth muscle actin is upregulated
(Kumar et al., 1997). We have
distinguished two types of smooth muscle-expressing mural cells in the dorsal
aorta, based on their position. The lineage relationship between pericytes and
vascular smooth muscle cells has not been clear
(Gerhardt and Betsholtz,
2003); however, our clonal analysis demonstrates that the two
types of mural cells in the dorsal aorta can derive from a common progenitor.
The T4 line shows that -cardiac actin is also expressed in some
endothelial cells and we see rare labelled endothelial cells in some clones.
The presence of endothelial cells in the same clone as periendothelial and
vascular smooth muscle cells indicates that they share a common progenitor. At
E10.5, only a few long and short clones show this phenomenon, indicating that
the common progenitor is present before somitogenesis. At E9.5, labelled
endothelial and smooth muscle cells are seen in a couple of `single somite'
clones. However, at this stage fewer somites have formed and such clones may
have subsequently evolved into an older clonal category. Consistent with an
early common progenitor, endothelial cells of the dorsal aorta appear to be
laid down before Pax3(GFP) expression and the onset of somitogenesis. The
observation that endothelial cells are Pax3(GFP) negative, whereas most mural
smooth muscle cells are positive, does not support the proposal of a
significant transdifferentiation of endothelial to smooth muscle cells in the
dorsal aorta, previously made for avian embryos
(DeRuiter et al., 1997).
Previous evidence for a common vascular progenitor came from experiments with
Flk1-positive mouse embryonic stem cells which were shown to differentiate
into vascular smooth and endothelial cell types on injection into the chick
embryo, or when analyzed under clonal conditions in vitro
(Yamashita et al., 2000;
Ema et al., 2003). We now show
that such a common vascular cell progenitor can exist in vivo.
|
).
|
-cardiac actin is only expressed in a minority of
endothelial cells, it is difficult to predict the importance of the paraxial
mesoderm contribution to this cell type. However grafting experiments, in
which quail somites are introduced into the chick embryo, have shown that
endothelial cells in the dorsal aorta derive from paraxial mesoderm and that
endothelial progenitor cells are still present in somites. In these
experiments, quail endothelial cells were mainly located dorsally
(Pardanaud et al., 1996),
whereas the rare labelled endothelial cells identified in our clonal analysis
were situated ventrolaterally, indicating that the paraxial mesoderm
contribution is not limited to the dorsal wall of the aorta. Our Pax3(GFP)
observations indicate that, in the mouse embryo, endothelial cells in the
dorsal aorta are not derived from somitic cells that express Pax3. If
they originate from paraxial mesoderm, as suggested by the clonal analysis,
this must be prior to the expression of Pax3.
Somitic origin of cells in the dorsal aorta
Clonally related cells in the dorsal aorta do not show a random
distribution, but tend to be located adjacent to labelled somites. This
segmental tendency is notable in more recent clones. Labelled cells in the
dorsal aorta in single somite clones, or in clones where only the aorta is
labelled, maintain a discrete distribution not extending beyond the limits of
one, or at most two, somites. Furthermore, statistical analysis shows that
there is no significant difference in the distribution of labelled cells per
`segment' in the dorsal aorta in different categories of clone. This supports
the possibility of `segmental' restriction in the clonal contribution, with no
dispersion on the anteroposterior axis, as the number of labelled cells per
`segment' would be expected to decrease with the diminishing age and size of
the clones if there was dispersion along the axis. This is consistent with a
colonization of the smooth muscle compartment of the dorsal aorta by cells
derived from adjacent somites.
The results of the clonal analysis, together with examination of
(Pax3)GFP-positive cells at E10.5, suggests the presence of multipotent
progenitor cells located in the hypaxial dermomyotome that can give rise to
the skeletal muscle of the hypaxial myotome and to the mural smooth muscle
cells of the dorsal aorta. More recent single somite clones support this
model. In these clones, but also in short clones, there is a marked
association between preferential labelling of the hypaxial myotome and the
presence of labelled cells in the dorsal aorta, whereas in clones which do not
colonize the dorsal aorta the epaxial and hypaxial myotome show similar
labelling. Even in older long clones with cells in the dorsal aorta, a
hypaxial bias is observed. This is consistent with the early segregation of
progenitor cells for the medial (epaxial) and lateral (hypaxial) myotome,
indicated by a clonal analysis in the mouse embryo at E11.5
(Eloy-Trinquet and Nicolas,
2002b). A minority of clones with labelled cells in the dorsal
aorta show epaxial myotomal labelling. This is the case for some long clones,
where the progenitor was present well before somitogenesis. The extensive
Pax3(GFP) labelling of smooth muscle cells in the dorsal aorta indicates that
the precursors of these cells, as well as those of the myotome, are in the
somite. Epaxial labelling is also seen in some short and single somite clones,
suggesting that the common progenitor, present in the epaxial somite, can also
give rise to cells that migrate from this location to the dorsal aorta.
GFP-positive cells are not labelled with the -smooth muscle actin
antibody (nor with that for PECAM) in the region between the somite and the
wall of the aorta, indicating that differentiated cells do not migrate, and it
is the precursors of mural smooth muscle cells that leave the somite. These
cells no longer express Pax3, based on antibody staining, but did so
previously, as they are GFP positive. Unlike skeletal muscle progenitor cells,
which require Pax3 to delaminate and migrate to the limb, (Pax3)GFP-positive
cells continue to contribute to the dorsal aorta in Pax3 mutant
embryos, although to a reduced extent. In Pax3 mutants, the hypaxial
dermomyotome undergoes apoptosis and this reduction of smooth muscle cells is
therefore consistent with their derivation from this part of the somite
(Tajbakhsh and Buckingham,
2000).
|
|
). However,
the clonal analysis presented here clearly shows that at least part of the
smooth muscle component of the dorsal aorta, marked by -cardiac actin,
is clonally related to skeletal muscle cells of the myotome. Whereas in the
case of older clones, the progenitor is present in presomitic mesoderm, in
single somite clones the common progenitor is probably located in the somite,
necessarily in the dermomyotome rather than in the sclerotomal compartment,
which does not give rise to skeletal muscle
(Tajbakhsh and Buckingham,
2000). Pax3(GFP) labelling of cells in the dermomyotome is
intense, whereas only residual GFP is seen in cells of the sclerotome,
reflecting previous expression throughout the epithelial somite. The relative
intensity of the GFP staining and its location compared with Pax1
transcripts, which mark sclerotomal cells, suggest a dermomyotomal origin for
GFP-positive smooth muscle cells in the dorsal aorta. Neural crest cells
represent another potential source of Pax3-GFP cells. However, GFP-positive
cells are still present in the dorsal aorta in the Pax3 mutant, where
neural crest is compromised, and a lineage tracing experiment with a neural
crest-specific Cre mouse line confirms that neural crest does not contribute
to the smooth muscle of the dorsal aorta in the trunk.
The dermomyotome as a source of multipotent progenitor cells
Experiments in the chick embryo have indicated that the dermomyotome is a
source of multipotent cells, with a common progenitor for derm and skeletal
muscle (Ben-Yair and Kalcheim,
2005), and for the migrating population that contributes to the
skeletal muscle and endothelial cells of the limb
(Kardon et al., 2002). Our
results now suggest that it is also a source of progenitor cells that give
rise to skeletal and smooth muscle. This is particularly evident in single
somite clones with labelled cells in the dorsal aorta and hypaxial myotome.
Clones with labelled cells only in the dorsal aorta, or only in the myotome,
may derive from a precursor cell in the dermomyotome that has already
segregated from the skeletal/smooth muscle lineage.
The cells that we have followed in the dorsal aorta present interesting
parallels with the mesoangioblast stem cell isolated from this structure
(De Angelis et al., 1999;
Minasi et al., 2002). These
cells express -cardiac actin and Pax3 (G.C., unpublished). In
vivo transplantation experiments of mouse dorsal aorta into the chick embryo
showed that mouse cells contributed both to blood vessels and to adjacent
muscle fibres (Minasi et al.,
2002). This has led to the suggestion that the vasculature may be
a source of muscle progenitor cells during development
(De Angelis et al., 1999;
Cossu and Mavilio, 2000). One
can speculate that the mesoangioblast may correspond to a progenitor cell from
the hypaxial dermomyotome that has retained multipotent properties.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/4/737/DC1
|
ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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