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First published online 28 January 2009
doi: 10.1242/dev.029942
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INSERM ERI 25, Muscle and Pathologies, 34295 Montpellier Cedex 05, France and University Montpellier I, EA4202, 34295 Montpellier Cedex 05, France.
* Author for correspondence (e-mail: Pascal.de-Santa-Barbara{at}inserm.fr)
Accepted 5 January 2009
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: FGF pathway, Scleraxis, Gut development, Tendon, Visceral smooth muscle, Chick
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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).
The motility of the GI tract is ensured by the correct coordination of the
visceral smooth muscle cells (SMC) and the autonomous ENS. The ENS originates
from neural crest cells that migrate from the dorsal region of the neural tube
and colonize the whole gut to establish its innervation
(Wallace and Burns, 2005).
SMCs derive from the splanchnopleural mesoderm that will form the
undifferentiated visceral mesenchyme
(Roberts, 2000). Few have
investigated the molecular mechanisms involved in the differentiation of the
visceral mesenchyme into SMCs. SMCs are present in both vascular and digestive
systems; however, the digestive tract is the most abundant contributor of SMCs
in humans (Gabella, 2002). In
the chick, the gizzard (muscular stomach or antrum) has a thick layer of
smooth muscle that facilitates mechanical digestion, whereas the
proventriculus (glandular stomach or fundus), which develops anteriorly to the
gizzard, has only a very thin smooth muscle layer
(Roberts, 2000). The
intestines show modest development of smooth muscle layers. In the avian
stomach, SMC differentiation is observed from embryonic day (E) 9. Thus, the
chick stomach offers an ideal model in which to elucidate the molecular
mechanisms that control visceral SMC differentiation. In the GI tract,
Bmp4, a ligand that belongs to the transforming growth factor β
(TGFβ) superfamily, is expressed in the mesenchyme of the whole chick gut
with the exception of the gizzard (Roberts
et al., 1998). When overexpressed, Bmp4 causes a
reduction in the thickness of the smooth muscle layer of the stomach,
demonstrating a regulatory role in gut muscle growth
(Roberts et al., 1998).
Conversely, the homeotic gene Bapx1 is expressed only in the chick
gizzard mesenchyme and acts as a repressor of Bmp4, therefore
modulating gizzard smooth muscle development
(Nielsen et al., 2001). In
addition, we have investigated the function of the BMP pathway during visceral
SMC differentiation and found that aberrant modulation of BMP activity altered
this process (de Santa Barbara et al.,
2005).
In order to identify factors that trigger and control the differentiation of visceral SMC, we carried out a microarray screen to isolate candidate genes. We identified Scleraxis, a member of the basic-helix-loop-helix (bHLH) family of transcription factors, which is expressed in tendon cells of the stomach adjacent to the visceral SMC. We then used the avian retroviral system to specifically misexpress or inactivate Scleraxis in the stomach mesenchyme, and showed that Scleraxis expression defines the intermuscular tendon domains that are established in close association with the visceral SMC.
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MATERIALS AND METHODS |
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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).
Retroviral misexpression studies
The Fgf8 (Brent and Tabin,
2004), sFgfR2b (Mandler and
Neubuser, 2004) and GFP
(Moniot et al., 2004) viral
constructs have been previously described. ShScleraxis associated
with the mouse U6 promoter and full-length avian Scleraxis cDNA were
cloned into the shuttle vector Slax and then subcloned into the RCAS(A)
vector. Full-length avian Scleraxis cDNA was cloned in frame in the
Slax-Engrailed vector and then subcloned into the RCAS(A) vector. All vectors
were transfected into avian DF-1 cell lines, and viruses harvested and titered
using standard techniques. To target the presumptive stomach mesenchyme,
misexpression experiments were performed on stage 9-10 embryos, as previously
described (Moniot et al.,
2004).
Primary cell cultures derived from stomach mesenchyme
Gizzards from stage 25 (referred to as E5 gizzards) were harvested in PBS
solution. After collagenase treatment (Sigma) at room temperature for 12
minutes, we isolated the mesenchymal layer using fine forceps (Simon-Assman
and Kédinger, 2000). Individual mesenchymal cells were plated on dishes
and kept in culture for 24 (E5+1D) and 72 hours (E5+3D) in DMEM, 10% fetal
bovine serum in the absence or presence of the different avian
retroviruses.
Expression analyses
In situ hybridization experiments on whole tissues/embryos and paraffin
sections were carried out as previously described
(Moniot et al., 2004).
Different chick templates for antisense riboprobes were obtained by PCR
amplification using specific primer sets (details of primers are available on
request). The following plasmids were used: 
SMA, Type I
Collagen, Fgf7, Fgf10, Fgfr1
(Edom-Vovard et al., 2001),
Fgfr2 (Brent and Tabin,
2004), Fjx (Yamaguchi
et al., 2006), Scleraxis (gift from D. Duprez),
Sox10 (Moniot et al.,
2004) and Tenomodulin. Immunohistochemistry was performed
on paraffin sections using polyclonal antibodies against SMA
(Sigma) and Phospho-Histone H3 Ser10 (Upstate), and monoclonal antibodies
against Type I Collagen, GAG and Decorin [Developmental Studies Hybridoma Bank
(DSHB)].
In cellulo in situ hybridization was performed as previously described
(Gregoire et al., 2006).
Methyl violet staining was used as described
(Bi et al., 2007).
Immunofluorescence was performed using monoclonal antibodies directed against
Tenascin (DSHB) and Type I Collagen (DSHB), and polyclonal antibodies against
Caldesmon (Sigma), Desmin (Sigma) and Sox9
(Moniot et al., 2004). The
Alexa 488 anti-mouse and Alexa 555 anti-rabbit secondary antibodies
(Invitrogen) were used, and nuclei were stained with DAPI (Molecular Probes).
Cells were mounted in FluorSave reagent (Calbiochem).
For microarray experiments and quantitative RT-PCR amplification, RNAs were extracted with the RNeasy Kit (Qiagen). Biotinylated complementary RNAs were hybridized to the Affymetrix GeneChip Chicken Genome Arrays using standard manufacturer's protocols (Affymetrix, IRB, CHU Montpellier, France). Fluorescence intensities were quantified and analyzed using the GCOS software (Affymetrix; see Table S1 in the supplementary material). Scleraxis expression was quantified by quantitative RT-PCR amplification using LightCycler technology (Roche Diagnostics; 95°C for 10 seconds, 60°C for 5 seconds, 72°C for 10 seconds). PCR primers are available on request. mRNA values were determined by LightCycler analysis software (version 3.1), according to the standard curves. Data were represented as the relative mean level of Scleraxis expression relative to 18S standard expression.
For electron microscopy, tissues were immersed in a solution of 3.5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.4) overnight at 4°C. They were then washed and post-fixed in 1% osmic acid plus 0.8% potassium ferrocyanide in the dark at room temperature for 2 hours. After washing, tissues were dehydrated in graded ethanol solutions and embedded in EmBed 812 DER 736. Thin sections (85 nm; Leica-Reichert Ultracut E) were collected. Sections were counterstained with uranyl acetate and lead citrate, and observed using a Hitachi 7100 transmission electron microscope at the CRIC facility (C. Cazevieille, Montpellier, France).
Photography
Images of whole-mount tissues and paraffin sections were collected with a
Nikon DXM1200 camera connected to a Nikon Multizoom AZ100 microscope.
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RESULTS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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;
Niessen et al., 2005;
Mericskay et al., 2007). We
also identified a new cluster composed of genes (i.e. Scleraxis, Decorin,
Tenomodulin and Type XII Collagen) that are associated with the
development and differentiation of tendon tissues
(Tozer and Duprez, 2005).
In this study, we focused on Scleraxis, a member of the bHLH family of
transcription factors previously reported as an early marker of tendons and
tenocytes (Edom-Vovard et al.,
2001; Schweitzer et al.,
2001). We next examined the expression profile of
Scleraxis during GI tract development and found it expressed in two
specific subdomains of the stomach mesenchyme from E6 to E9, and at the
boundaries of these two domains after SMC differentiation (E12;
Fig. 1B). At E9,
Scleraxis was expressed also in the caecum, a structure that
separates the colon from the small intestine (see Fig. S1 in the supplementary
material).
The mesenchyme of the avian embryonic stomach is composed of visceral SMC
(that expresses SMA, a SMC-specific factor) and intercalated
ENS cells (positive for Sox10) to allow autonomous contraction.
However, the expression of Scleraxis and SMA or
Sox10 was mutually exclusive (Fig.
1C), suggesting that Scleraxis is expressed neither in
visceral SMC nor in ENS cells. Analyses by light microscopy of the embryonic
gizzard showed a homogenous visceral mesenchymal structure with migrating ENS
cells in the outer layer at E6 (Fig.
1D). Conversely, at E12, the gizzard consisted of two
well-differentiated smooth muscle areas both associated with two connective
structures (Fig. 1D), which, at
E9, appeared to be close to the differentiating SMC
(Fig. 1E). These connective
tissues were positive for Scleraxis, Tenomodulin (another gene
identified by our microarray screen) and other tendon markers, such as
Type I Collagen (Ros et al.,
1995), Four-jointed
(Yamaguchi et al., 2006) and
Decorin, all of which showed an expression pattern overlapping with that of
Scleraxis (Fig. 1C,E)
in these two connective tissues. Conversely, Scleraxis and SMA
were detected in two specific and mutually exclusive domains
(Fig. 1E). Furthermore, we
found that, in the mouse, Scleraxis expression was limited to two
small domains in the antrum that correspond to the avian gizzard
(Fig. 1F).
An early study highlighted the intimate relation between muscle bundles and
these two connective structures in the adult avian gizzard
(Watzka, 1932); two centra
tendinea were observed in the adult gizzard and were characterized as being
rich in collagen fibrils (McLelland,
1979; Gabella,
1985). Attachment of two skeletal muscles through a central
structure was also described in the diaphragm. This was considered to be
another category of tendon and was named an intermuscular tendon
(Ackerman and Greer, 2007;
Murchison et al., 2007). Taken
together, these data indicate that, in the stomach, Scleraxis
exhibits a restricted expression pattern that defines two tendons closely
associated to the two visceral smooth muscles
(Fig. 1F). We define them as
intermuscular tendons.
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;
Edom-Vovard et al., 2002). In
order to identify the FGF ligand(s) and receptor(s) that could be responsible
for the initiation of Scleraxis expression in E6 stomach, we
quantified by RT-PCR their levels in E6 gizzards, and detected robust
expression of eight FGF ligands and three FGF receptors
(Fig. 2A). Whole-mount in situ
hybridization revealed that Fgf7, Fgf10, Fgfr1 and Fgfr2
were expressed in the gizzard mesenchyme from E6
(Fig. 2B; see also Fig. S2 in
the supplementary material). The other detected FGF ligands were expressed
mainly in the epithelial layer (our unpublished data). We then investigated
whether the FGF signaling pathway could regulate Scleraxis
expression. With this aim, we perturbed the FGF pathway by misexpressing an
inhibitory form of the mouse FGFR2 IIb receptor (referred to as sFgfR2b) that
is secreted and preferentially sequesters the FGF ligands 1, 3, 7 and 10
(Mandler and Neubuser, 2004),
and then monitored Scleraxis expression by in situ hybridization and
quantitative RT-PCR (Fig.
2C,D). In the gizzard, infection with RCAS-sFgfR2b retroviruses
led to a downregulation of Scleraxis expression and a size reduction
of the two specific domains (Fig.
2C), whereas expression of RCAS-GFP, a negative control, had no
effect on Scleraxis (compare Fig.
2C with Fig. 1B).
Efficient retroviral infection of the mesenchyme was confirmed by
immunohistochemistry using antibodies against the avian retroviral GAG protein
(3C2), or by in situ hybridization using a riboprobe against the avian
retroviral envelop gene (env; data not shown). We monitored
the Scleraxis mRNA level by quantitative RT-PCR and observed a strong
decrease in RCAS-sFgfR2b in the stomach; this finding was supported also by
the observed reduction of Fgf10 mRNA in this condition
(Fig. 2D). Conversely, ectopic
activation of the FGF signaling pathway along the GI tract following
RCAS-Fgf8 misexpression caused multiple morphological defects, mainly
in the proventriculus, the gizzard and the caecum
(Fig. 2E; see also Fig. S3 in
the supplementary material). RCAS-Fgf8-infected stomachs showed
ectopic expression of Scleraxis associated with other tendon markers,
such as Type I Collagen and Tenomodulin
(Fig. 2E; Figs S4 and S5 in the
supplementary material). Moreover, in RCAS-Fgf8-infected stomachs, we
observed reduced expression of SMA in the areas of ectopic expression
of the tendon markers (Fig. 2E;
Fig. S3 in the supplementary material). To analyze the effect of FGF
activation on cell proliferation, we used antibodies directed against
phosphorylated Histone 3B (PH3), a standard cell cycle marker of the G2/M
transition (Fig. 2F). We did
not observe significant differences in the number of proliferative cells in
the gizzard SMCs following infection of RCAS-Fgf8 or RCAS-GFP,
indicating that FGF activation does not induce a global upregulation of the
mitotic potential. These data suggest that the FGF signaling pathway controls
positively the establishment of tendons in the developing stomach, and
recruits mesenchymal cells towards a tendon fate.
Tendon structures are essential for the development of the stomach
Scleraxis belongs to the bHLH family of transcription factors and is one of
the rare transcription factors expressed at the onset of tendon development
(Edom-Vovard et al., 2001;
Schweitzer et al., 2001). In
order to investigate more directly the role of Scleraxis in stomach
development, we used a loss-of-function technique that involved delivering
ShScleraxis to the chick stomach in vivo
(Fig. 3A,B)
(Harpavat and Cepko, 2006).
First, we tested the efficiency of RCAS-ShScleraxis in primary cells
derived from stomach mesenchyme and observed a robust reduction of
Scleraxis mRNA (Fig.
3C) without any side effect on the identity of the infected cells
(data not shown). In embryonic stomachs, RCAS-ShScleraxis led to a
strong downregulation of Scleraxis expression
(Fig. 3D) and to some minor
defects, such as a smaller gizzard and a straight proventriculus
(Fig. 3D). Strong retroviral
infection was correlated with decreased expression of Scleraxis, Type I
Collagen and Tenomodulin
(Fig. 3E), suggesting a
reduction in size of the tendon domains in the developing stomach. Conversely,
we observed an increase of the territory labeled by SMA, a SMC marker
(Fig. 3E).
We next used a gain-of-function approach and ectopically misexpressed
full-length Scleraxis along the GI tract
(Fig. 4). Although we
misexpressed Scleraxis in different regions of the digestive system,
we observed effects only in the stomach
(Fig. 4). Scleraxis
misexpression induced a gross phenotype characterized by an aberrantly dilated
gizzard associated with a curved proventriculus
(Fig. 4A).
RCAS-Scleraxis expressing cells were highly concentrated and
organized around territories of endogenous expression
(Fig. 4A), whereas the muscle
domains were strongly reduced, as revealed by an SMA riboprobe
(Fig. 4A). Although
RCAS-Scleraxis expression was correlated with SMA inhibition,
we did not observe an ectopic induction of Type I Collagen, as we had
in the case of RCAS-Fgf8 misexpression (compare
Fig. 4B with
Fig. 2E).
Scleraxis is considered to be a gene activator and recently Type I
Collagen has been identified as a target in tendon fibroblasts
(Lejard et al., 2007).
Therefore, we thought that by converting Scleraxis into a transcriptional
repressor, we could block the expression of target genes and counteract its
activator function. With this aim, we fused the Engrailed repressor domain in
frame to Scleraxis and misexpressed RCAS-Scleraxis-Engrailed
in the stomach to repress endogenous Scleraxis function. In embryonic
stomachs, RCAS-Scleraxis-Engrailed led to some minor defects,
comparable to those observed in RCAS-ShScleraxis stomachs (data not
shown). Type I Collagen expression was strongly inhibited in the
infected tendon domain and this inhibition was associated with defects in the
tendon structure (Fig. 4C).
Recently, others genes known to be expressed specifically in the tendons, such
as Tenomodulin and Type XIV Collagen, were demonstrated to
be targeted by Scleraxis in the limb
(Shukunami et al., 2006;
Murchison et al., 2007). In
contrast to the limb tendon, we found that Type XIV Collagen was not
expressed in the intermuscular tendon but only in gastric ENS cells (see Fig.
S6 in the supplementary material). Using quantitative RT-PCR to analyze the
expression levels of Tenomodulin, we observed that Scleraxis
misexpression in the gizzard upregulated Tenomodulin, whereas ectopic
expression of Scleraxis in the proventriculus did not
(Fig. 4D). Moreover,
Scleraxis-Engrailed expression in the gizzard strongly repressed
Tenomodulin expression (Fig.
4D). These findings demonstrate that, during the development of
the intermuscular tendon, Scleraxis is upstream of Type I Collagen
and Tenomodulin, and strongly suggest that Type I Collagen
and Tenomodulin are in vivo targets of Scleraxis in the stomach.
However, Scleraxis alone cannot induce ectopic expression of Type I
Collagen, suggesting the necessity of an additional interacting
partner(s).
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In summary, our data indicate that tendon structures are important for the regulated development of the stomach, and that their differentiation is closely coordinated with that of visceral smooth muscles.
Undifferentiated stomach mesenchymal cells give both SMCs and tendon cells
We showed that tendon and smooth muscle domains are closely associated in
the stomach and that activation of the FGF pathway in the anterior and
posterior parts of the stomach mesenchyme induces ectopic tendon domains
(Fig. 2), suggesting that
modulation of this pathway in the visceral mesenchyme is sufficient to pattern
the tendon. To evaluate the capacity of undifferentiated visceral mesenchyme
to give rise to tendon cells, we set up primary cell cultures derived from
stage 25 gizzards before the onset of Scleraxis expression in the
stomach. Throughout the study, we refer to stage 25 gizzard mesenchyme as E5
gizzard mesenchyme. With this aim, we adapted a reliable technique that
allowed us to enzymatically separate the gizzard mesenchyme from its
endodermal counterpart (Fig.
6A) (Simon-Assman and Kédinger, 2000). Next, E5 mesenchymal
cells were cultured for one (E5+1D) and three days (E5+3D). At E5+1D, we
observed that some isolated, scattered cells were positive for
Scleraxis expression; at E5+3D, we observed the presence of several
colonies that were all positive for Scleraxis expression
(Fig. 6B). All
Scleraxis-positive colonies were also visualized by staining with
Methyl Violet, a specific histological tendon dye
(Fig. 6C)
(Bi et al., 2007). We then
analyzed these colonies and found that they strongly expressed Type I collagen
and Tenascin (tendon markers) (Tozer and
Duprez, 2005), faintly expressed Desmin (mesenchymal derived cell
marker) but did not express Caldesmon (SMC marker) or Sox9 (chondrocyte
marker) (Lefebvre et al.,
1997) (Fig. 6D).
Conversely, we observed robust expression of Caldesmon and Desmin in the cells
adjacent to the colonies, suggesting that visceral SMC organized around these
structures (Fig. 6D).
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These data suggest that E5 gizzard undifferentiated mesenchyme can differentiate into at least two distinct cell types: visceral SMC and tendon cells. These data also indicate that the FGF pathway and Scleraxis are essential for the formation and differentiation of intermuscular tendons.
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DISCUSSION |
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SUMMARY
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DISCUSSION
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). The
rectococcygeus muscle, for instance, is attached to the anterior surface of
the second and third coccygeal vertebrae, and is inserted onto the posterior
surface of the rectum through anchoring tendons. In this study, we describe
two tendons that connect two smooth muscle bundles in the stomach. The
connection of two muscle bundles through a connective structure, which is
defined as an intermuscular tendon, has been previously described; for
example, in the diaphragm (Ackerman and
Greer, 2007). The localization of two intermuscular tendons in the
stomach suggests that their physiological role is to diminish the high tension
generated by the periodic contraction of the powerful stomach smooth muscle
structures. In this study, we found that some specific tendon markers (i.e. Scleraxis, Tenomodulin, Four jointed, Type I Collagen and Decorin) are associated with the development and differentiation of these structures. However, other limb tendon-specific markers are not expressed. Indeed, a putative target gene of Scleraxis, Type XIV Collagen is not expressed in the intermuscular tendons but is expressed in the gastric ENS cells. These molecular differences between intermuscular tendons and force-transmitting or anchoring tendons suggest that the generation of tendon structure or the organization of the tissue attachment in these tendon subgroups requires molecular modulation.
Determination of the intermuscular tendon domain
At E6, Scleraxis expression defines two domains that give rise to
the tendon structures of the stomach (Fig.
7). These two domains are induced by the mesenchymal FGF signaling
pathway. We also demonstrate that inactivation of Scleraxis
expression extinguishes the tendon domains, whereas ectopic expression blocks
the differentiation of the visceral mesenchyme into SMC. Tendons are defined
as the connective tissues that connect/attach muscle to bone (musculoskeletal
system) and muscle to muscle (diaphragm system)
(Tozer and Duprez, 2005;
Ackerman and Greer, 2007).
Tendon cells can have different embryological origins; for instance, body wall
tendons derive from one specific somitic compartment, the syndetome
(Brent et al., 2003), and
craniofacial tendons from cranial neural crest cells
(Crane and Trainor, 2006). No
embryonic origin was demonstrated for the intermuscular tendons in the
diaphragm (Ackerman and Greer,
2007). The digestive tract mesenchyme derives from the
splanchnopleural mesoderm and is also colonized by neural crest-derived
(Le Douarin and Teillet, 1973;
Barlow et al., 2008) and
mesothelium-derived vascular SMC (Wilm et
al., 2005) cells. Chick/quail chimera experiments demonstrated
that colonization by neural crest-derived cells of the stomach gives rise to
ENS cells (Le Douarin and Teillet,
1973) and not to the tendon domains we identified in this work. An
early study of the muscle-tendon structures in the avian gizzard noted the
intimate relationship between muscle bundles and tendons in this organ, and
suggested that both derive from the same cell type
(Watzka, 1932). Our finding
that mesenchymal FGF signaling triggers Scleraxis expression and,
consequently, the determination of the two tendon domains in vivo and in
primary cell culture suggests that, upon FGF activation, selected stomach
mesenchymal cells are primed to differentiate into tendon cells and to express
Scleraxis.
Differentiation of the intermuscular tendon structure
As previously reported (Brent and Tabin,
2004; Edom-Vovard et al.,
2002), we found that the mesenchymal FGF signaling pathway
positively regulates Scleraxis expression and induces differentiation
of the two tendon structures and the expression of differentiation markers,
such as Type I Collagen and Tenomodulin, in different parts
of the digestive tract. Conversely, Scleraxis is essential for the development
of the tendons and Tenomodulin expression. However,
Scleraxis misexpression alone cannot induce ectopic expression of
Type I Collagen, whereas the transcriptional repressor
Scleraxis-Engrailed inhibits Type I Collagen expression in vivo.
Scleraxis is a bHLH transcription factor that can interact with the bHLH
factor E47 to regulate the expression of Type I Collagen in tendon
fibroblasts (Lejard et al.,
2007). All these data suggest that Scleraxis requires additive
transcription factors or interacting partners to control the differentiation
process initiated by activation of the FGF pathway. We can also imagine that
Scleraxis proteins present in the musculoskeletal or in the intermuscular
tendons might interact with different tissue-specific partner(s) to reinforce
these tissue specificities. In addition, other signaling pathways activated by
FGF can cooperate with Scleraxis to induce tendon differentiation. Different
studies showed that the FGF-WNT regulatory network controls mesenchyme
development. Recently, mesenchymal FGF signaling has been reported to regulate
β-catenin-mediated WNT signaling in lung mesenchyme
(Yin et al., 2008). The
canonical Frizzled receptor Fz8 is expressed in the stomach
(Theodosiou and Tabin, 2003)
and its expression pattern overlaps with that of Scleraxis, which
suggests that Scleraxis and the WNT canonical pathways are induced after
activation of the FGF pathway. In future studies we will try to identify the
WNT ligand that interacts with Fz8 and that might enable the cooperation with
Scleraxis.
In summary, we show that the vertebrate stomach harbors two tendon domains located in the antrum/muscular stomach, which are closely associated with the visceral smooth muscle structures. The possible physiological function of these domains might be to ensure elasticity of the stomach during contraction of the two massive visceral muscles.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/5/791/DC1
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Footnotes |
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SUMMARY
INTRODUCTION
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RESULTS
DISCUSSION
REFERENCES |
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