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First published online 22 March 2006
doi: 10.1242/dev.02336
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6ß1-laminin interactions regulate early myotome formation in the mouse embryo
1 Department of Animal Biology and Centre for Environmental Biology, Faculty of
Sciences, University of Lisbon, 1749-016 Lisbon, Portugal.
2 Gulbenkian Institute of Science, 2781-901 Oeiras, Portugal.
3 Department of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam,
The Netherlands.
4 Stem Cells and Development, Department of Developmental Biology, CNRS URA
2578, Pasteur Institute, 25 rue du Dr Roux, 75724 Paris Cedex 15,
France.
5 Molecular Genetics of Development, Department of Developmental Biology, CNRS
URA 2578, Pasteur Institute, 25 rue du Dr Roux, 75724 Paris Cedex 15,
France.
Author for correspondence (e-mail:
solveig{at}fc.ul.pt)
Accepted 22 February 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6ß1 integrin, a
laminin receptor, suggesting that integrin 6ß1-laminin
interactions are required for myotomal laminin matrix assembly. Blocking
6ß1-laminin binding in cultured wild-type mouse embryo explants
resulted in dispersion of Myf5-positive cells, a phenotype also seen in
Myf5nlacZ/nlacZ embryos. Furthermore, inhibition of
6ß1 resulted in an increase in Myf5 protein and ectopic myogenin
expression in dermomyotomal cells, suggesting that 6ß1-laminin
interactions normally repress myogenesis in the dermomyotome. We conclude that
Myf5 is required for maintaining 6ß1 expression on myogenic
precursor cells, and that 6ß1 is necessary for myotomal laminin
matrix assembly and cell guidance into the myotome. Engagement of laminin by
6ß1 also plays a role in maintaining the undifferentiated state of
cells in the dermomyotome prior to their entry into the myotome.
Key words: Integrin, Laminin, Mouse embryo, Myotome, Dermomyotome, Extracellular matrix, Myf5
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Kahane et al.,
1998; Venters et al.,
1999; Gros et al.,
2004). Epaxial myotome formation is dependent on the myogenic
regulatory factor (MRF) Myf5. In Myf5nlacZ/nlacZ
(Myf5-null) mouse embryos, the epaxial MPCs delaminate from the
dermomyotome but fail to form the myotome
(Tajbakhsh et al., 1996).
Rather, MPCs accumulate around the epaxial edge of the dermomyotome or migrate
aberrantly. The myotome is normally delimited by a laminin-rich basement
membrane that separates it from the underlying sclerotome
(Tosney et al., 1994;
Bajanca et al., 2004). The role
of this laminin-rich matrix during myotome formation is unknown, but,
strikingly, it does not form in Myf5nlacZ/nlacZ embryos
(Tajbakhsh et al., 1996).
Laminins are a family of extracellular matrix (ECM) glycoproteins, composed
of , ß and 
chains, which associate to form a trimeric
cross-like structure. To date, five , three ß and three
chains have been described that associate to form 12 different laminin
isoforms (reviewed by Tunggal et al.,
2000; Ekblom et al.,
2003). Laminins, together with collagen type IV, perlecan and
nidogen, are the major components of basement membranes. Studies using C2C12
myoblasts and embryonic stem cells suggest that laminin matrix formation is
dependent on specific interactions with cellular receptors
(Sasaki et al., 1998;
Fleischmajer et al., 1998;
Colognato et al., 1999;
Henry et al., 2001). These are
thought to be dystroglycan, for an initial recruitment of laminin molecules,
and ß1 integrins, supporting their assembly
(Colognato et al., 1999;
Henry et al., 2001).
Furthermore, assembly of the laminin matrix is essential for the organisation
of the remaining basement membrane components into a continuous basement
membrane (Yurchenco et al.,
2004; Yurchenco and Wadsworth,
2004).
Cells sense their ECM environment through cell-surface receptors, the most
common of which belong to the integrin family
(Miranti and Brugge, 2002).
Integrins are heterodimers composed of an and a ß subunit, which
together determine the ligand specificity of each integrin
(Hynes, 1992) (reviewed by
van der Flier and Sonnenberg,
2001). The extracellular region of an integrin binds to specific
ECM ligands, while the intracellular region interacts with the cytoskeleton
and associated proteins, and can modulate numerous intracellular signalling
pathways, regulating processes such as survival, proliferation, migration and
differentiation (reviewed by Miranti and
Brugge, 2002; Danen and
Sonnenberg, 2003). Integrin expression patterns change during the
course of skeletal muscle development
(Gullberg et al., 1998;
Bajanca and Thorsteinsdóttir,
2002; Bajanca et al.,
2004; Cachaço et al.,
2005), suggesting constant modulation of cell-ECM interactions
during the different stages of myogenesis. Although the exact roles of these
cell-ECM interactions in vivo are not well understood, it is clear that
ß1 integrins play a role in skeletal muscle development. When the
ubiquitously expressed ß1A subunit is substituted by the striated muscle
specific splice variant ß1D (van der
Flier et al., 1997), primary myogenesis is impaired, leading to a
reduction in muscle mass (Cachaço et
al., 2003). Furthermore, when the integrin ß1 gene
(Itgb1) is inactivated specifically in myogenic cells, mutant animals
die at birth with highly underdeveloped muscles
(Schwander et al., 2003).
Recently, we described the expression patterns of several ß1 integrins
during myotome formation in the mouse. The laminin receptor, 6ß1,
is expressed in epithelial somites and in the dermomyotome, and remains
strongly expressed on MPCs that colonise the epaxial myotome
(Bajanca et al., 2004). In the
present study, we analysed the normal pattern of laminin matrix assembly in
the mouse myotome. We then addressed the issue of why myotomal laminin
assembly fails in Myf5nlacZ/nlacZ embryos. We show that
the major basement membrane components are present around Myf5-null
cells, but 6ß1 integrin is absent from MPCs when they delaminate
from the dermomyotome. Blocking 6ß1 binding to laminin in cultured
explants of wild-type mouse embryos resulted in dispersion of Myf5-expressing
MPCs, a phenotype resembling the one observed in
Myf5nlacZ/nlacZ embryos. Surprisingly, when binding of
6ß1 to laminin was blocked, we observed an increase in
Myf5-expressing cells and ectopic myogenin expression in the dermomyotome.
Together, our results suggest that 6ß1-laminin interactions are
involved not only in myotome formation, but that they also prevent precocious
myogenesis in the dermomyotome.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Owing to its proximity to Myf5, within the same locus, the
Mrf4 gene is also affected in these mutants. These
Myf5nlacZ/nlacZ embryos are thus functional double
knockouts for Myf5 and Mrf4 in the early embryo
(Kassar-Duchossoy et al.,
2004). In normal embryos, Mrf4 mRNA transcripts only
start being detected at E9.5, so Myf5 protein is the only MRF driving epaxial
myotome formation before that time. Furthermore, in Myf5 mutant mice
where the Mrf4 locus is not affected, Mrf4 expression is not
sufficient to rescue normal epaxial myotome development
(Kassar-Duchossoy et al.,
2004). Thus, it is unlikely that defects in epaxial myotome
formation observed in Myf5nlacZ/nlacZ embryos are due to
the absence of Mrf4. Rather, they should be due to the absence of
Myf5 protein.
Embryos were collected from crossings between
Myf5nlacZ/+ animals. The day of the vaginal plug was
designated as embryonic day (E) 0.5. Embryos were staged as described by
Houzelstein et al. (Houzelstein et al.,
1999). DNA was isolated from yolk sacs and genotyping was carried
out as described (Tajbakhsh et al.,
1997). Wild-type embryos were obtained from crossings of outbred
Hsd:ICR(CD-1) mice (Harlan Interfauna Iberica).
Immunohistochemistry and ß-galactosidase staining
Immunohistochemistry on cryostat (Bright Clinicut) sections (10 or 30
µm) and whole mounts was performed as described by Bajanca et al.
(Bajanca et al., 2004). Primary
antibodies were: anti-ß-galactosidase (Promega), anti-EHS-laminin
(Sigma), anti-collagen type IV (Chemicon), anti-perlecan (domain IV;
Chemicon), anti-ß1 integrin (L-16; Santa Cruz Biotechnology),
anti-myogenin (F5D; Santa Cruz Biotechnology) and anti-laminin 5 chain
(Miner et al., 1997) (a gift
from J. Miner) all diluted 1:100; anti-laminin 1 chain (AL-4; Chemicon)
diluted 1:20; anti-Myf5 (C-20; Santa Cruz Biotechnology) diluted 1:2000;
anti-myosin (F-59; a gift from F. Stockdale) diluted 1:20; and anti-desmin
(D3; Developmental Studies Hybridoma Bank) and anti-laminin ß1 chain
(78B3) (Sonnenberg et al.,
1986) as undiluted supernatants. The polyclonal anti-EHS laminin
antibody raised against laminin 1 (chain composition 1ß11)
recognises the 1, ß1 and 1 chains, but may also crossreact
with other laminins (Paulsson,
1994).
Secondary antibodies were Alexa Fluor 568-conjugated anti-mouse IgG, Alexa
Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 546-conjugated
anti-rabbit IgG, all F(ab')2 fragments (Molecular Probes), diluted
1:1000; FITC-conjugated anti-rat IgG and anti-goat IgG (Sigma), diluted 1:100.
Some slides were stained with 4',6-Diamidino-2-phenylindole (DAPI,
Sigma). ß-Galactosidase staining with X-gal substrate was performed as
described by Hogan et al. (Hogan et al.,
1986).
Experimental interfering with 6ß1 integrin-laminin interactions
Wild-type E8.0-E9.5 embryos were collected in cold culture medium with 10
mM HEPES and immediately processed. The head region above the otic vesicles
and the viscera were removed, but all structures surrounding the somites were
left intact. Explants were prepared from two littermates with the same number
of somites, one being the control explant and the other the experimental one.
All explants were placed on a Millipore filter (pore size 0.8 µm) in
DMEM:Ham's F12 medium (Gibco) supplemented with 4 mM glutamine, 1 mM sodium
pyruvate, 50 U/ml penicillin and 8 mg/ml streptomycin, and cultured at
37°C in a 5% CO2 atmosphere
(Kil et al., 1998;
Webb et al., 2002). The GoH3
rat monoclonal antibody against the 6 integrin subunit
(Sonnenberg et al., 1987;
Sonnenberg et al., 1990)
specifically blocks the binding of 6ß1 to laminin, without causing
activation of the integrin (e.g.
Sonnenberg et al., 1988;
Sonnenberg et al., 1990;
Burrows et al., 1995;
Falk et al., 1996;
Jiang et al., 2001). Pilot
experiments were performed with several different concentrations of GoH3. A
1:10 dilution of the supernatant and 20 µg/ml of purified antibody provided
the most reproducible results and were used in all experiments. Control
explants were cultured in: (1) medium only, (2) medium with a 1:10 dilution of
the supernatant containing the rat monoclonal antibody CA5 against integrin
7 (absent at this stage) (Bajanca et
al., 2004) or (3) medium with 20 µg/ml of JsE3 (rat monoclonal
antibody against an irrelevant 50 kDa cell surface antigen)
(Sonnenberg et al., 1986).
Incubation periods ranged from 14 to 24 hours. Penetration of the GoH3
antibody was verified by exposing some GoH3-treated explants to a
FITC-conjugated anti-rat IgG antibody. Immunoreactivity was observed in the
interior of the explants, on cells of the dermomyotome and myotome among
others (data not shown).
Control experiments were performed to determine the quality of the culture
conditions: immediately fixed (i.e. not cultured) explants, control explants
and experimental explants were compared in terms of apoptosis levels (TUNEL
assay, Roche), proliferation (phospho-H3 immunohistochemistry; Upstate) and
cell and tissue morphology (phalloidin and DAPI staining). Although apoptosis
levels were slightly increased in cultured explants compared with immediately
fixed ones, no difference was observed between control explants and explants
cultured with GoH3. There was no difference between the proliferation levels
and morphology of 6-negative embryonic structures between explant
types, indicating that GoH3 did not have non-specific effects.
Imaging
Sections processed for immunohistochemistry were photographed using an
Olympus DP50 digital camera coupled to an Olympus BX60 microscope equipped
with Normaski optics and epifluorescence. Optical z-series of whole
mount embryos and thick cryostat sections were obtained in a Leica SP2
confocal microscope. Embryos stained with X-gal were photographed using an
Olympus Camedia C-4040 digital camera coupled to a Wild M8 stereomicroscope.
Images were edited in Adobe Photoshop 7.0.
Quantification of Myf5 and myogenin immunoreactivity in dermomyotomes was performed using the ImageJ software on unprocessed images of embryo sections. The mean grey-level intensity of dermomyotomes (including or excluding lips) and neural tube was calculated for each section. The neural tube was selected to measure nonspecific fluorescence as it has a similar cell density, but has no specific immunoreactivity for Myf5 and myogenin. To compensate for differences in fluorescence intensity among embryos and sections, we computed a ratio of `mean fluorescence intensity for dermomyotome/neural tube' for each section. These ratios represent the n-fold increase of Myf5 or myogenin in the dermomyotome compared with the neural tube. Fluorescence intensity was measured in 7-26 sections from each control (n=3 for Myf5; n=4 for myogenin) and GoH3-treated (n=5 for Myf5; n=4 for myogenin) explant and a nested analysis of variance was used to test for differences between control and GoH3-treated explants.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (data not shown). As the first MPCs delaminate from the
epaxial dermomyotome to form the myotome, a laminin matrix starts being laid
down (Fig. 1A,B). As more cells
enter the myotome, this matrix becomes sheet like, separating the epaxial
myotome from the sclerotome (Fig.
1C,D). The myotomal laminin matrix, detected with antibodies
against laminin 5-, 1- and ß1-chains, is then found along
the full extent of the epaxial myotome
(Fig. 1E-G). Immunoreactivity
for the laminin 2-chain was not detected in the myotome at E9.5 (data
not shown). Therefore the basement membranes delimiting the dermomyotome
dorsally and separating the myotome from the sclerotome at E9.5 contain
laminin 1 (1ß11) and laminin 10 (5ß11),
but not 2-chain laminins (laminin 2, laminin 4 and laminin 12).
Co-immunohistochemistry for laminins and Myf5 shows that Myf5-positive
cells are often in close contact with the laminin-containing myotomal basement
membrane (arrows in Fig. 1F,G).
Confocal sectioning through a sagittal plane confirms that Myf5-positive cells
are rare in the area of the myotome closest to the dermomyotome
(Fig. 1H) and that most
Myf5-positive cells within the myotome are located near the myotome-sclerotome
interface (Fig. 1I-K)
(Venters et al., 1999), the
area where the myotomal laminin matrix is localised
(Fig. 1E-G,L-M). Most
myogenin-positive cells are located away from the myotome-sclerotome interface
(compare progression from Fig.
1H-J) (Venters et al.,
1999) and, thus, far from the laminin matrix. However, the central
band of myogenin-positive nuclei reaches this interface in the middle of the
myotome (arrowheads in Fig.
1I-K). It is notable that most of the Myf5-positive cells
localised at this interface are positioned rostral and caudal to the central
area containing myogenin-positive nuclei
(Fig. 1H-K). Cells
co-expressing Myf5 and myogenin are preferentially found centrally, but close
to the sclerotome (arrowheads in Fig.
1I,J), suggesting that cells are beginning myogenic
differentiation at this location. Interestingly, in this area, the laminin
matrix is discontinuous (Fig.
1L,M), suggesting that it is an area where new laminin matrix is
assembled to accommodate for the medial growth of the myotome.
Myogenin-positive cells located in this area are not full length (arrowheads
in Fig. 1L), but are
elongating, as shown by desmin labelling at the cell extremities (arrows in
Fig. 1M), which are in contact
with the assembled laminin matrix at the myotome-sclerotome interface (arrows
in Fig. 1M).
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).
Thus, the absence of laminin could contribute to the phenotype of
Myf5nlacZ/nlacZ embryos. We first determined whether laminin was present near Myf5-null cells at early stages of myotome development and confirmed that assembled laminin is absent both near the ß-galactosidase (ß-gal)-positive cells and at the sclerotome interface in E9.5 Myf5nlacZ/nlacZ embryos (compare Fig. 2B,E with 2A,D).
The absence of a laminin-rich basement membrane adjacent to
Myf5-null cells could be due to one of several causes (reviewed by
Ghosh and Stack, 2000;
Tunggal et al., 2000). First,
production of laminin chains might not occur, suggesting that Myf5, directly
or indirectly, regulates transcription of laminin (Lam) genes. Second,
intracellular assembly or secretion of the laminin trimer might be affected.
Finally, extracellular assembly of laminin molecules into a basement membrane
might be impaired. A high magnification of cryosections immunolabelled for
ß-gal and laminin shows the presence of laminin near ß-gal-positive
MPCs in Myf5nlacZ/nlacZ embryos
(Fig. 2C). This indicates that
laminin molecules are produced and secreted but are not assembled.
In normal embryos, the myotomal basement membrane is not only rich in laminin (Fig. 2A,D), but also in collagen type IV (Fig. 2F) and perlecan (Fig. 2H). Although a continuous basement membrane is not formed in Myf5nlacZ/nlacZ embryos, there is evident immunoreactivity for laminin, perlecan and collagen type IV (Fig. 2E,E',G,G',I,I'). In fact, collagen type IV (Fig. 2G,G') and perlecan (Fig. 2I,I') form patches among Myf5-null MPCs (Fig. 2G,I). However, ß-gal-positive Myf5-null cells disperse through this matrix (Fig. 2G,I), which, unlike the normal basement membrane, does not appear to restrict cells successfully within the myotomal space. Our results thus suggest that the major basement membrane components are present in the myotomal area of Myf5nlacZ/nlacZ embryos but a functional basement membrane fails to assemble.
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6ß1 integrin expression when they detach from the dermomyotome6ß1 is strongly expressed by
early epaxial MPCs (Fig. 3A,C)
(Bajanca et al., 2004). This
integrin is normally present on dermomyotomal and myotomal cells
(Fig. 3A,C) but, although
present on dermomyotomal cells in Myf5nlacZ/nlacZ embryos,
it is undetectable on most MPCs (ß-gal-positive cells) that have
delaminated from the epaxial lip of the dermomyotome (arrows in
Fig. 3B,D). These results
suggest that, while (or soon after) delaminating from the dermomyotome,
Myf5-null MPCs are not able to maintain 6ß1 on their cell
surface, and thus lose the ability to interact with extracellular laminin
through this integrin. This may result in: (1) the impairment of laminin
assembly; and, in the absence of a basement membrane barrier, (2) cell
dispersal.
Blocking 6ß1-laminin binding perturbs myotome formation
We hypothesised that the defect in myotome colonisation and laminin matrix
formation in Myf5nlacZ/nlacZ embryos could be due to a
failure of MPCs to bind laminin. To test this hypothesis, we used a well
characterised integrin 6-blocking antibody (GoH3) to inhibit
specifically 6ß1 binding to laminin in wild type (i.e.
Myf5-expressing) embryo explants cultured in vitro.
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When E8.0 embryo explants are cultured with GoH3 for 14 hours, the Myf5-positive MPCs that delaminated from the dermomyotome are found near the neural tube instead of being positioned in the myotomal area (arrows in Fig. 4B). When slightly older (E9.5) embryo explants were cultured with GoH3 for 24 hours, a dramatic scattering of Myf5-positive cells was observed (compare Fig. 4G with 4J) and very few Myf5-expressing cells are found in the central area of the myotome (bracket in Fig. 4J). Interestingly, this distribution pattern of Myf5-positive cells is very similar to the distribution of MPCs in Myf5nlacZ/nlacZ embryos (compare Fig. 4J with 4F).
The pattern of desmin immunoreactivity is also disorganised in GoH3-treated explants compared with controls. Some dispersed cells upregulate desmin but elongated cells were not detected among them (Fig. 4K,L). The same pattern is detected in the central myotomes, suggesting that the differentiation pattern of cells that were already there at the time of the addition of the antibody is perturbed. Myosin immunoreactivity is unperturbed in GoH3-treated explants (data not shown), suggesting that fully elongated myocytes are unaffected by GoH3.
We hypothesised that the myotomal laminin matrix might constitute a barrier restraining myogenic cells inside the myotomal space. The immunoreactivity for laminin is discontinuous and diffuse in GoH3-treated embryos both in the myotomal basement membrane (compare Fig. 4D with 4C) and at the epaxial lip (arrowheads in Fig. 4C,D).
Our results thus suggest that 6ß1-laminin interactions are
necessary for the normal translocation of MPCs from the dermomyotome into the
myotome, for the normal differentiation of cells in the myotome and for
laminin assembly at the myotome-sclerotome interface.
Blocking 6ß1-laminin binding leads to a precocious activation of the myogenic programme in dermomyotomal cells
We noticed that in embryo explants cultured with GoH3, the expression of
Myf5 was often unusually widespread in the dermomyotome (compare
Fig. 4A with 4B), raising the
possibility that dermomyotomal cells might be differentiating precociously. To
address this, we cultured explants of E9.0 embryos with GoH3 for 14 hours and
tested for the expression of Myf5 and myogenin. Control explants showed a
normal expression pattern for these proteins
(Fig. 5A,C). Myf5 is expressed
in the myotome and in dermomyotomal lips just prior to MPC delamination and
entry into the myotomal space (Ott et al.,
1991; Venters et al.,
1999) and myogenin is upregulated in myotomal cells
(Fig. 5A,C). Embryos incubated
with GoH3, showed a striking increase in Myf5 expression and, surprisingly, an
ectopic expression of myogenin in the dermomyotome
(Fig. 5B,D). This effect is not
restricted to the epaxial lip in this experiment, but extends towards the
hypaxial dermomyotome. Quantification of the fluorescence intensity in the
whole dermomyotome showed an increase in both Myf5 and myogenin fluorescence
in the presence of GoH3 (Fig.
5E). The increase in myogenin fluorescence was significant
(P=0.009), but the increase in Myf5 fluorescence, although
considerable, did not score statistically significant (P=0.09). As
Myf5 is normally expressed in the dermomyotome lips, we reasoned that
excluding lips in our measurements would better identify whether a difference
exists in the remaining areas of the dermomyotome. Measurements were made for
both Myf5 and myogenin (Fig.
5F), and there was a statistically significant difference between
control and GoH3-treated explants (P=0.03 for Myf5; P=0.01
for myogenin). We therefore conclude that 6ß1-laminin interactions
normally repress myogenesis while cells are in the dermomyotome.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6ß1 by MPCs colonising the myotome, which strongly
suggests that the cause of the failure of laminin matrix assembly and the
consequent cell dispersal in Myf5nlacZ/nlacZ embryos is
the absence of this integrin on myotomal cells. Blocking
6ß1-laminin binding in mouse embryo explants not only confirmed
these conclusions, but in addition revealed an unexpected role for
6ß1-laminin interactions in the repression of myogenesis in the
dermomyotome.
The relationship between myogenic cells and laminin changes with their progressive maturation
Myogenic cells interact with laminin at several phases of their
differentiation programme (schematised in
Fig. 6). MPCs in the epaxial
dermomyotomal lip detach from their basement membrane when undergoing an
epithelium to mesenchyme transition, but soon after entering the myotomal
space, they accumulate electron dense basement membrane material on the side
facing the sclerotome (Tosney et al.,
1994). This agrees with our detection of patchy laminin
immunoreactivity between early epaxial MPCs and the sclerotome
(Fig. 6A). Thus, we suggest
that these first MPCs facilitate the assembly of laminin very soon after they
enter the myotomal space and the observation that laminin fails to assemble in
Myf5nlacZ/nlacZ embryos reinforces this conclusion. After
the initial deposition of the laminin matrix, the myotome grows in thickness
and, consequently, new extracellular material has to be added to enable an
increase in the surface area of the basement membrane. The identification of
areas of unassembled laminin at the myotome-sclerotome interface, midway
between rostral and caudal edges of the myotome
(Fig. 6B), suggests that the
basement membrane is growing at this point.
As the myotome grows and myotomal cells advance in their differentiation
programme, their interaction with the laminin matrix changes. The great
majority of young (Myf5-positive, myogenin-negative) cells that enter the
myotome are found near the myotome-sclerotome interface, i.e. closely apposed
to the pre-existing, assembled laminin matrix (see
Fig. 6B), suggesting that they
use this matrix as a migration substrate. More differentiated (Myf5- and
myogenin-positive) cells are generally found in a central position near the
myotome-sclerotome interface (i.e. midway between rostral and caudal
dermomyotomal lips) (Venters et al.,
1999), an area where the laminin matrix is discontinuous (see
Fig. 6B). We speculate that the
loss of contact with assembled laminin might play a role in myogenin
activation and initiation of myocyte elongation observed in this area (step 4;
Fig. 6B). Alternatively, this
may be an indirect effect resulting from a unique growth factor environment at
this site.
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;
Venters et al., 1999). Myosin
is present in fully elongated cells, which are localised far from the laminin
matrix, while the expression pattern of desmin shows that the tips of
elongating cells contact this matrix. We thus propose that, as new myogenic
cells are being added, intercalating between the myotome and its laminin
matrix, the elongating cells are displaced, most probably only retaining
contact with the laminin matrix through their tips
(Fig. 6B).
Assembly of the myotomal basement membrane depends on Myf5 and 6ß1-laminin interactions
The formation of the myotomal laminin matrix is severely affected in
Myf5nlacZ/nlacZ embryos
(Tajbakhsh et al., 1996) (this
study). However, dotted immunoreactivity for laminin and patchy
immunoreactivity for collagen IV and perlecan was present around
Myf5-null cells. A scaffold of assembled laminin is a prerequisite
for the subsequent integration of the other basement membrane components into
a mature basement membrane (Henry et al.,
2001; Yurchenco et al.,
2004; Yurchenco and Wadsworth,
2004). Thus, our results suggest that although the production of
basement membrane components is unaffected in
Myf5nlacZ/nlacZ embryos, the assembly of a laminin matrix
is greatly impaired, resulting in the absence of the myotomal basement
membrane.
Laminin polymeric networks can be induced to self-assemble in vitro
(Yurchenco et al., 1985;
Cheng et al., 1997;
Yurchenco and Cheng, 1993),
but there is growing evidence that ß1 integrins are involved in the
assembly of laminin-based basement membranes in vivo (reviewed by
Tunggal et al., 2000;
Yurchenco et al., 2004).
Embryos null for the integrin ß1 subunit gene (Itgb1) die early
(at E5.5) and basement membranes are not detected
(Stephens et al., 1995;
Li et al., 2002). Furthermore,
conditional inactivation of Itgb1 in skeletal muscle cells
(cre/skeletal -actin promoter) leads to discontinuities in muscle
basement membranes (Schwander et al.,
2003). Inactivation of integrin subunits tends to produce
more subtle phenotypes, probably owing to compensation by other related
-subunits (Hynes,
1996). However, in embryos null for the 6 subunit gene
(Itga6), laminin deposition in neural tissues is abnormal
(Georges-Labouesse et al.,
1998) and newborn Itga3-null mice display severe defects
in several epithelial basement membranes
(Kreidberg et al., 1996;
DiPersio et al., 1997). In
addition, embryos null for both Itga6 and Itga3 present
basement membrane discontinuities and cell detachment
(De Arcangelis et al.,
1999).
The most likely ß1 integrin to mediate ES cell basement membrane
assembly is 6ß1 (Henry et al.,
2001). Here, we show that Myf5nlacZ/nlacZ
embryos display a dotted pattern of laminin immunoreactivity around MPCs and
that MPCs fail to maintain the 6ß1 integrin after they delaminate
from the dermomyotome. This strongly suggests that the absence of
6ß1 on Myf5-null MPCs is the cause of their failure to
assemble laminin.
Colonisation of the myotome is dependent on 6ß1-laminin binding
We demonstrate that 6ß1-laminin binding is also important for
the colonisation of the myotome by MPCs. In explants placed in culture with
GoH3 at E9.5, myotome formation had already been initiated and thus some
laminin matrix was present in these myotomes before the beginning of the
culture period. In spite of the presence of this laminin matrix, MPCs
scattered and failed to colonise the myotomal space. This shows that an
interaction between 6ß1 and laminin is not only important for
laminin matrix assembly during early myotome formation, but also serves to
correctly position MPCs in the myotomal space.
The abnormal staining pattern for desmin suggests that the inhibition by
GoH3 leads to a disturbance of elongating myocytes. Our experiments do not
allow us to determine whether this effect is due to a detachment of the tips
of elongating myocytes or whether it is due to the inability of
differentiating cells to initiate their elongation (see
Fig. 6B; longitudinal view). By
contrast, fully elongated myocytes do not appear to be affected by incubation
with GoH3. In fact, these myocytes also express 4ß1, which
co-localises with its ligand fibronectin, at their tips
(Bajanca et al., 2004), so they
may not exclusively depend on 6ß1 for the maintenance of their
elongated phenotype. Interestingly, 6ß1-laminin interactions have
been implicated in embryonic retinal neurite outgrowth
(de Curtis and Reichardt,
1993) but whether a similar mechanism occurs in elongating
myocytes remains an unanswered question.
Inhibition of 6ß1-laminin binding leads to activation of myogenesis in the dermomyotome
Our results show that inhibiting the binding of dermomyotomal
6ß1 to laminin leads to an activation of the myogenic programme in
that both Myf5 and myogenin are upregulated. Furthermore, the most significant
upregulation of Myf5 occurs in the central myotome. We propose that the
antibody perturbation experiments mimic the disengagement between
6ß1 and laminin that normally only occurs epaxially (at the
epaxial lip). It is commonly believed that the environment at the epaxial lip
is such that inducing signals (e.g. Wnts, Shh) override repressive signals
(e.g. BMPs) leading to the induction of myogenesis (reviewed by
Currie and Ingham, 1998;
Cossu and Borello, 1999;
Tajbakhsh and Buckingham,
2000; Pownall et al.,
2002). By contrast, in other regions of the dermomyotome where
myogenic progenitor cells are also present (see
Relaix et al., 2005;
Kassar-Duchossoy et al., 2005;
Gros et al., 2005; Ben-Yair et
al., 2005) repressive signals override potential inducing signals at the
stages under study. Our results suggest that the experimental disengagement
between 6ß1 and laminin in those regions of the dermomyotome
pushes the equilibrium such that repressive signals no longer prevail. This
strongly implicates 6ß1 disengagement from laminin as one of the
factors that drive myogenesis epaxially.
The observation that chick epiblast cells activate MyoD after dissociation
in culture, has led to the suggestion that myogenesis is normally repressed in
the embryo (George-Weinstein et al.,
1996) (see Cossu et al.,
1996; Currie and Ingham,
1998). Interestingly, perturbation of ß1 integrin-laminin
interaction has been shown to lead to precocious differentiation in other
systems. Precursors of cerebellar granule cells are attached to laminin in the
meningeal basement membrane by 6ß1 and 7ß1, and under
those conditions they proliferate and remain undifferentiated
(Pons et al., 2001;
Blaess et al., 2004).
Differentiation normally occurs when these cells detach from the basement
membrane and migrate into the deeper layers of the cerebellum. Conditional
inactivation of Itgb1 in the central nervous system resulted in
impaired laminin matrix formation by cerebellar granule cell precursors,
reduced cell proliferation and precocious expression of differentiation
markers (Blaess et al., 2004).
These data suggest that 6ß1 and/or 7ß1 binding to
laminin normally prevents the differentiation of these cells and that their
detachment from laminin causes differentiation. During pancreas development,
the proliferation of pancreatic insulin-producing ß cell precursors is
dependent on their binding to laminin 1 via 6ß1, and
differentiation occurs when this binding is blocked by the addition of GoH3 to
organ cultures (Jiang et al.,
2001). Interestingly, no alterations in ß-cell
differentiation were detected in Itga6-null embryos
(Jiang et al., 2001). Thus, at
least in this system, blocking 6ß1 function has a more severe
effect than inactivation of the Itga6 gene. It is possible that other
laminin receptors (e.g. the closely related 3ß1 or 7ß1
integrins) are upregulated in the absence of Itga6 expression (see
Hynes, 1996).
6ß1-laminin interactions have been shown to promote
differentiation in some cell types, including skeletal muscle
(Sastry et al., 1996) and lens
(Walker and Menko, 1999;
Walker et al., 2002). However,
in these two situations, the differentiation-promoting effect was attributed
to the presence of the 6A variant. The 6 subunit exists in two
splice variant forms, 6A and 6B
(Hogervorst et al., 1991;
Tamura et al., 1991), but only
the 6B variant is present during early (E8.5-E9.5) myotome formation
(Thorsteinsdóttir et al.,
1995). There is evidence that the two 6 variants modulate
6ß1 signalling in different ways. For example 6Aß1,
but not 6Bß1, activates ERK
(Wei et al., 1998;
Ferletta et al., 2003). We
suggest that the binding of laminin by the 6Bß1 integrin
contributes to a signalling pathway that represses myogenesis in the
dermomyotome and that the detachment of MPCs from the dermomyotomal laminin
matrix permits the activation of the myogenic programme during the early
stages of myotome development. Whether this signalling pathway is dependent
only on engagement of 6Bß1 or whether synergism with a pathway
activated by growth factors occurs
(Colognato et al., 2002;
Blaess et al., 2004) (reviewed
by Danen and Sonnenberg,
2003), remains to be determined.
|
ACKNOWLEDGMENTS |
|---|
5
and myosin antibodies respectively. Anti-desmin (D3; D. A. Fischman) was from
DSHB developed under the auspices of NICHD and maintained by the University of
Iowa. We thank Luís Marques and Ingrid Kuikman for help with some
experiments, Jorge Palmeirim for statistical advice, Leonor Saúde for
reading the manuscript and Prof. E. G. Crespo for constant support. This study
was financed by Fundação para a Ciência e a Tecnologia
(FCT)/FEDER via POCTI/BCI/47681/2002 (to S. Thorsteinsdóttir and F.B.)
and by FP6/EU Network of Excellence, Cells into Organs, of which M.B., F.B.
and S. Thorsteinsdóttir are members. M.B. and S. Tajbakhsh laboratories
were financed by the Pasteur Institute, CNRS and A.F.M. F.B. was supported by
a PhD grant SFRH/BD/1359/2000 (FCT/FEDER) and by the FP6/EU Network of
Excellence, Cells into Organs. |
Footnotes |
|---|
|
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