* Howard Hughes Medical Institute and Center for Cancer Research, Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139; Department of Veterans Affairs and Department of Neurology and
Neurological Sciences, Stanford University School of Medicine, Stanford, California 94305; § Department of Pathology, Tufts
University Schools of Medicine and Veterinary Medicine, Boston, Massachusetts 02111; and
Department of Cell Biology and
Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
5-deficient mice die early in embryogenesis
(Yang et al., 1993
). To study the functions of
5 integrin later in mouse embryogenesis and during adult life
we generated
5
/
;+/+ chimeric mice. These animals contain
5-negative and positive cells randomly
distributed. Analysis of the chimerism by glucose-
6-phosphate isomerase (GPI) assay revealed that
5
/
cells contributed to all the tissues analyzed.
High contributions were observed in the skeletal muscle. The perinatal survival of the mutant chimeras was
lower than for the controls, however the subsequent life
span of the survivors was only slightly reduced compared with controls (Taverna et al., 1998
). Histological
analysis of
5
/
;+/+ mice from late embryogenesis to adult life revealed an alteration in the skeletal muscle structure resembling a typical muscle dystrophy. Giant fibers, increased numbers of nuclei per fiber with altered position and size, vacuoli and signs of muscle
degeneration-regeneration were observed in head, thorax and limb muscles. Electron microscopy showed an
increase in the number of mitochondria in some muscle
fibers of the mutant mice. Increased apoptosis and immunoreactivity for tenascin-C were observed in mutant
muscle fibers. All the alterations were already visible at
late stages of embryogenesis. The number of altered
muscle fibers varied in different animals and muscles and was often increased in high percentage chimeric
animals. Differentiation of
5
/
ES cells or myoblasts showed that in vitro differentiation into myotubes was achieved normally. However proper adhesion and survival of myoblasts on fibronectin was
impaired. Our data suggest that a novel form of muscle
dystrophy in mice is
5-integrin-dependent.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE interactions of extracellular matrix (ECM)1 components with each other or with cell surface receptors have an important role in many biological processes such as embryonic development, wound healing,
malignant transformation and many others (Hynes, 1990,
1992
; Ruoslahti, 1991
; Hynes and Lander, 1992
; Giancotti and Mainiero, 1994
). Cell-ECM interactions are mediated
by cell surface receptors called integrins. Integrins are
transmembrane glycoproteins which consist of noncovalently linked heterodimers each composed of an
and a
chain (Hynes, 1992
).
5
1 integrin is a specific receptor, which binds to the
arginine/glycine/aspartic acid region of one of the most
common ECM molecules, fibronectin (FN) (Pytela et al.,
1985).
5
1 is involved in many cellular processes including cell proliferation and oncogenic transformation (Plantefaber and Hynes, 1989
; Giancotti and Ruoslahti, 1990
;
Schreiner et al., 1991
), cell survival (Varner et al., 1995
; Zhang et al., 1995
), cell migration (Akiyama et al., 1989;
Giancotti and Ruoslahti, 1990
), assembly of FN-rich matrices (Fogerty et al., 1990
), wound healing (Guo et al.,
1991
), T cell activation (Shimizu and Shaw, 1991
), and gene
expression (Werb et al., 1989
). It also plays an important
role during embryogenesis (Yang et al., 1993
). Indeed, the
5 subunit is expressed at high levels during embryogenesis in Xenopus (Whittaker and De Simone, 1993), chicken
(Muschler and Horwitz, 1991
) and mouse (Goh et al.,
1997
); its expression is more restricted in adult tissues
(Muschler and Horwitz, 1991
).
Development of skeletal muscle is a multistep process
that starts when the somitic mesoderm differentiates into
the dermamyotome. Soon after that, primary myoblasts
proliferate and migrate to their peripheral locations where
they differentiate into postmitotic multinucleated myotubes, the primary myotubes (primary fusion). The migration of secondary myoblasts that align with the primary myotubes leads to the formation of secondary myotubes
(secondary fusion). Finally the myotubes specialize as fast
or slow contracting fibers and become striated and innervated (Kelly and Rubinstein, 1994). Several adhesion molecules, integrins, cadherins, and immunoglobulin superfamily members are thought to be involved in myogenesis
(Knudsen et al., 1990). The extracellular matrix of skeletal
muscle consists of a basal lamina around every myotube
and interstitial connective tissue (endomysium) between
the fibers. Collagens and fibronectins are abundant in the
endomysium whereas the basal lamina contains type IV
collagen, laminin, heparan sulfate proteoglycan, entactin (nidogen), and fibronectins (Sanes, 1994
).
and
integrin
subunits are expressed on skeletal muscle cells at different
times and subcellular locations.
1,
3,
5,
6, and
v are
highly expressed during muscle development and downregulated after full differentiation (Bronner-Fraser et al.,
1992
; Duband et al., 1992
; Enomoto et al., 1993
; McDonald et al., 1995
).
7 integrin is abundant through all
stages of muscle development (Bao et al., 1993
), whereas
4 integrin expression rises during secondary myogenesis
then is not expressed anymore (Rosen et al., 1992
).
v subunit is concentrated at the costameres and at the myotendinous junction (MTJ);
3 is localized to the MTJ whereas
5 is present in adhesion plaque-like structures along the
myotube.
7 is concentrated at the MTJ but can also be
detected at the neuromuscular junction and along the sarcolemmal membrane. The
1 subunit is present on myoblasts and on muscle fibers along the entire membrane
with maximum concentrations at the MTJ and costameres
or Z discs (Bozyczko et al., 1989
).
A key question is what are the functions of these various
integrins in muscle biology? One way to address this question is via genetic elimination of specific integrins. 5-deficient mice die at approximately day 10 or 11 of gestation
(Yang et al., 1993
). The null embryos have pronounced defects in posterior trunk and yolk sac mesodermal structures. No somites, a kinky neural tube, and vascular defects are observed in the posterior end. To study the
functions of the
5 molecule after day 10 or 11 and in adult animals, we generated
5
/
embryonic stem (ES) cells
and injected them into wild-type (WT) blastocysts to obtain
5
/
;+/+ chimeric animals. Here we present data
on the characterization of these chimeras.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Growth, Selection, and Differentiation of ES Cells
ES cells, D3 line (Doetschman et al., 1985), were grown as described previously (George et al., 1993
).
5 heterozygous ES cells were obtained as
described in Yang et al. (1993)
. One heterozygous
5 ES cell line, clone
47, was expanded and selected with 4-5 mg/ml G418 (GIBCO BRL,
Gaithersburg, MD). After 7-9 d of selection, drug-resistant clones were
picked and expanded on feeder cells. Half of the cells from each clone
were frozen in 10% DMSO in fetal bovine serum and half were lysed for
extraction and analysis of DNA. From three independent selection experiments 5 clones that were null for
5 from Southern blot analysis were obtained (154, 162, 194, 201, and 305). Further Southern blot analysis of one of these clones (154) also showed that the vicinity of the mutated genomic
region was not altered during the selection (data not shown). Some heterozygous clones that did not become null after G418 selection were used
as control clones (152, 155, 98) as well as D3 wild-type cells and ES cells
heterozygous for P and E selectins (Robinson et al., 1997
). All these cells
had characteristics of wild-type cells.
To generate chimeric mice, ES cells were prepared and injected into
C57BL6 blastocysts as described by George et al. (1993) and Bradley et al.
(1987). Chimeric progeny were identified by coat color 1 wk after birth.
Before and around birth the animals were screened by PCR for the presence of the neo gene and by glucose-6-phosphate isomerase assay (see below). Differentiation of ES cells followed the protocol of Yang et al.
(1996)
. The embryoid bodies were analyzed for muscle differentiation after 15-30 d of culture in a leukemia inhibitory factor-free medium. Differentiated cultures were stained with an antibody against skeletal muscle
myosin heavy chain (MY32; Sigma Chemical Co., St. Louis, MO) as described (Yang et al., 1996
).
DNA Extraction, Southern Blot, and PCR Analysis
DNA was extracted from ES cells or myoblasts as described in Yang et al.
(1996). Southern blot analyses were performed as described in Yang et al.
(1993)
. PCR analysis for the neo gene was performed on tail DNA as described in Taverna et al. (1998)
.
Glucose-6-Phosphate Isomerase Analysis of Tissue
Glucose-6-phosphate isomerase (GPI) analysis was performed on extracts
of different tissues, e.g., limb or pectoral muscle, as described in Yang et al.
(1996). Densitometric analysis of the GPI assays was performed using either 1 D-multilane scan or Spot Denso scan of the IS-1000 Digital Imaging
System (Alpha Innotech, San Leandro, CA). The percentage of ES cell-derived (129Sv) isoform of GPI was calculated as described in Yang et al.
(1996)
.
Histological Analysis
Pregnant mothers were killed towards the end of pregnancy and embryonic day E16-E18 embryos were analyzed. Embryos, newborns, 1-4 wk-old mice, or several month-old animals were killed in a CO2 chamber, opened ventrally and dorsally, and then the opened carcasses were immersed in 10% formalin (3.7% formaldehyde in phosphate-buffered saline) and kept in fixative until processed. In some cases embryos or newborns were cut transversely into four portions, each portion embedded in mounting medium (OCT compound; Miles Laboratories, Elkhart, Milwaukee, WI) and immediately frozen in liquid nitrogen-cooled isopentane. 6-µm sections were cut. The same freezing procedure was used to generate sections from dissected limb or pectoral muscle of adult mice. Transverse or longitudinal paraffin-embedded or frozen sections were processed for hematoxylin and eosin (H&E) or phosphotungstic acid hematoxylin (PTAH) staining according to the supplier's protocol (Sigma Chemical Co.).
Apoptosis
Apoptotic cells were analyzed in the animals using terminal transferase biotinylated-dUTP nick-end labeling (TUNEL) on paraffin sections from E17 or E18 formalin-fixed embryos as described by Morganbesser et al. (1995). To analyze apoptosis in vitro, myoblasts (see below) were plated on mouse laminin-1 (GIBCO BRL) or FN for 2 h before being trypsinized and resuspended in buffer containing 1 µg/ml annexin-FITC as described by the manufacturer's protocol (Zymed, South San Francisco, CA). After a 10-min incubation, the cells were washed, stained with 1 µg/ml Hoechst 33342, and then mounted on a slide. In five separate fields, both the total number of cells and the number of annexin-positive cells were counted.
Immunohistochemistry
To analyze ECM molecules, 6-µm paraffin-embedded or frozen sections
from E18 embryos were used. Frozen sections were also used to analyze
the distribution of 5 integrin. Paraffin-embedded sections from overnight
formalin-fixed embryos were treated with trypsin (0.1%) for 30 min at
room temperature and then stained as described in George et al. (1993)
.
Frozen sections from unfixed embryos were stained with the same protocol except that they were not pretreated with trypsin and the incubations
were shortened to 15 min for primary and secondary antibodies. Primary
antibodies were used at 1:100 dilution: rabbit polyclonal anti-LM (Sigma Chemical Co.); rabbit polyclonal anti-collagen IV (Becton Dickinson Labware, Franklin Lakes, NJ); rabbit polyclonal anti-entactin (nidogen), a gift
of A. Chung (University of Pittsburgh, Pittsburgh, PA); rabbit 24 polyclonal anti-FN (Mautner and Hynes, 1977
); rat monoclonal anti-tenascin-C (Sigma Mtn-12; Sigma Chemical Co.); rat monoclonal anti
5 integrin (PharMingen, San Diego, CA). Secondary antibodies were used at
1:200 dilution of FITC-conjugated goat anti-rabbit or anti-rat (Biosource
International, Camarillo, CA).
Isolation of Myoblasts
Limb muscles from neonatal chimeric mice (/
;+/+ or +/
;+/+) were
dissociated to isolate pure populations of myoblasts as described in Rando
and Blau (1994)
. Primary cultures were plated on laminin-1-coated dishes
and grown in growth medium consisting of Ham's F-10 nutrient mixture
(BioWhittaker, Walkersville, MD), 20% fetal bovine serum (FBS) (BioWhittaker), 2.5 ng/ml basic fibroblast growth factor (bFGF) (Promega
Corp., Madison, WI), and penicillin (200 U/ml)/streptomycin (200 µg/ml)
(GIBCO BRL). ES cell-derived myoblasts were purified by maintaining
the cells in G418 (50 µg/ml) for at least 2 wk. To induce differentiation,
myoblast cultures were maintained in medium consisting of Dulbecco's
modified Eagle's medium supplemented with 2% horse serum and penicillin/streptomycin. For analysis of differentiation of each cell population,
the differentiated cultures were fixed with MeOH (
20°C) and stained
with Hoechst (1 µg/ml). The fusion index was determined microscopically
as the ratio of the number of nuclei in cells with three or more nuclei to
the total number of nuclei. 10 random fields in each of three separate cultures were counted at 40× magnification, with each field having between
50 and 100 nuclei.
Retroviral Infection
5 cDNA was introduced into
5-deficient myoblasts by retroviral-mediated gene transfer (Guan et al., 1990
) using a human
5 cDNA insert (Argraves et al., 1987
). Retroviral infection of myoblast populations was performed as described previously (Rando and Blau, 1994
). To ensure a high
level of infection, cells were infected on three successive days, which gives
>90% infection (Rando and Blau, 1997
). To control for the infection procedure,
5-deficient myoblasts were infected with a retrovirus lacking a
cDNA insert. Expression of
5 in retrovirally transduced cells was examined by Western blot analysis (see below).
Cell Adhesion
Cells were plated on 5 µg/ml LM-1, 5 µg/ml FN (both from GIBCO BRL), or collagen type I (100 µg/ml; Sigma Chemical Co.) and allowed to attach for 1 h at 37°C. Unattached cells were removed by washing. Adherence was then assessed by hemacytometer counts of attached cells after trypsinization.
Western Blot Analysis
Myoblasts were lysed in RIPA buffer. Proteins (50 µg) were electrophoresed on 7.5% SDS-PAGE gels under nonreducing conditions then transferred to nitrocellulose membranes (Schleicher & Schuell, Inc., Keene,
NH). The membranes were probed with an antibody to 5 integrin (1:5,000;
Chemicon, Temecula, CA) followed by a peroxidase-linked donkey anti-
rabbit secondary antibody (1:10,000; Amersham Corp., Arlington Heights,
IL). The enhanced chemiluminescence (ECL) system was used to visualize the bound secondary antibody.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of Chimeric Mice
ES cells heterozygous for 5 integrin were selected in the
presence of high concentrations of G418 (4-5 mg/ml) to
obtain homozygous clones. Southern blot analysis indicated that five different
5
/
ES cell clones were generated (Fig. 1). The five clones were injected into WT
C57BL6 blastocysts. Control chimeras were produced by
injecting clones heterozygous for
5 integrin or for P and E selectins (Robinson et al., 1997
) or WT ES cells (D3)
into WT C57BL6 blastocysts. Chimeric animals had both
black (from C57BL6) and agouti (from 129Sv-derived ES
cells) coat color.
|
For embryos and newborns the chimerism was determined by PCR analysis for the neo gene (Taverna et al.,
1998) in combination with a GPI assay for quantitation
(Yang et al., 1996
). The chimerism in adults was evaluated
by the coat color. A representative population of chimeric
animals obtained using
5
/
or control ES cells was the
following (Table I): at day 17 or 18 of pregnancy, 56% (76 out of 135) of the C57BL6 blastocysts injected with five independent
5
/
clones developed as chimeric embryos,
whereas only 22% (31 out of 139) of the C57BL blastocysts injected with control ES cells (D3 or
5 +/
clones)
developed as chimeric embryos. The number of embryos
in uterus (chimeric and nonchimeric) was similar in both
kind of injections; therefore the higher number of
5
/
;
+/+ chimeric embryos indicates a better contribution of
5
/
ES clones in the blastocysts; i.e., the null ES clones are highly competent, more so than the parent and control
ES cells, presumably as a result of the subcloning involved
in their selection. In contrast, at weaning (4-wk-old animals) 18% (37 out of 205) of
5
/
;+/+ and 15% (39 out
258) of control chimeras were observed. The marked decrease of
5
/
;+/+ chimeras from 56% at E17 or E18
to 18% at weaning suggests that not all the chimeras
present in uterus or born can survive. Indeed we found
that many
5
/
;+/+ newborn chimeras died within the
24 h after delivery. The lower survival rate for
5-null chimeras suggested requirements for
5 integrin in the perinatal development of certain tissues or organs.
|
Developmental Capacities of 5-null ES Cells
The chimerism of various tissues was analyzed by GPI assay in adult animals (see Table II). The contribution of
129Sv cells in some organs was lower than expected from
coat color in both mutant and control chimeras. This was
probably due to a disadvantage of 129Sv cells in specific
organs. However the 5-null cells contributed equally as
well as did the control ES cells (Table II). We analyzed the
populations of mature 129Sv-derived T and B lymphocytes by FACS® analysis and observed equal contributions
in mutant and in control chimeras (data not shown). Clone
154 was tested for germ-line transmission of the
5-null allele: germline transmission was obtained from both a male
and a female chimera indicating the presence of both
5
/
sperm and oocytes. We conclude that
5
/
cells
contribute to all tissues analyzed, albeit in somewhat different percentages.
|
Muscle Defects in Animals Chimeric for 5
Histological analysis of E16-E18 embryos, newborns,
young, and adult animals revealed no major defects. The
chimeras were normal in size, weight, and appearance and
showed no obvious defects in behavior (walking, climbing,
swimming, or mating). The only defects we detected consistently were structural alterations in skeletal muscles.
Approximately 40% of the 150 5
/
;+/+ chimeric embryos and newborn animals analyzed showed abnormal
skeletal muscles in the head, thorax, or limb (i.e., muscles
derived from several different embryonic origins
neural
crest, lateral mesoderm, somites). Chimerism varying from
10 to 90% was observed for both
5
/
;+/+ chimeras
and control animals. Embryos with the most noticeable alterations had high chimerism as evaluated by GPI analysis
of either the upper portion of the right posterior leg (including cartilage, muscle, and skin) or the dissected limb muscle of that leg. Examples of these alterations are illustrated in Fig. 2. Fig. 2, B and C show transverse sections
through the thorax (B) or limb (C) of E18
5
/
;+/+
chimeric embryos and they illustrate irregularity in the
sizes of the fibers with presence of giant fibers, central nuclei, and vacuoles. Increased collagen deposition between
fibers and fiber degeneration were also observed in muscle
from E18
5
/
;+/+ chimeric embryos (Fig. 2, E and F).
Sections of the thoracic muscles of control E18 embryos
(Fig. 2, A and D) showed good regularity in the muscle
structure and no fiber degeneration or collagen accumulation could be observed. Analysis of
5
/
;+/+ chimeras
in the first month after birth confirmed the presence of
muscle degeneration and regeneration in the skeletal muscles. Fig. 2, H and I show examples of degeneration in the
head muscles of a 9-d-old chimera. Variability in fiber size,
central nuclei, empty spaces in the fibers, and increase of
connective tissue (especially evident in Fig. 2 I with PTAH staining) were observed. A control muscle is shown in Fig.
2 G. Signs of muscle degeneration-regeneration were still
visible in some adult animals (several months old). Many
fibers with central nuclei (Fig. 2 K, limb) ring fibers indicative of fiber degeneration and regeneration (Fig. 2 L,
limb) and infiltration of adipose tissue (data not shown)
were found in head, thorax, and limb muscles. Control
limb muscle is shown in Fig. 2 J. The diaphragms and the heart of
5
/
;+/+ chimeric mice appeared normal at all
stages (data not shown), perhaps because the heart and diaphragm generally showed a lower 129Sv (ES cell) contribution than other muscles for both
/
and control chimeras (Table II). Alternatively, it is possible that mice
with a high percentage of
5
/
cells in the diaphragm
are among those that die early. Ultrastructural analysis of
limb muscle of E17 embryos revealed an increase in the
number of mitochondria in some fibers of mutant chimeric
mice (Fig. 3, B and C) compared with control chimeras
(Fig. 3 A). It is possible that the fibers with more mitochondria correspond to the giant fibers we observed by
histology.
|
|
The distribution of 5 integrin in mutant and control
embryos was investigated by immunohistochemistry.
5
integrin immunoreactivity was found around each muscle
fiber and at some MTJ in control animals (Fig. 4, A and
B). In mutant embryos a conspicuous decrease of
5 integrin staining was found around many muscle fibers; however, fibers completely negative for
5 were rare (Fig. 4 C)
due to the fact that null and wild-type myoblasts fuse together.
|
The composition of the ECM in the muscles of chimeric
mice was analyzed by immunohistochemistry. Fibronectin,
the ligand for 5
1 integrin, was present around and between all the fibers of both mutant and control chimeras.
No major, consistent differences in intensity were observed between giant- and regularly sized fibers of mutant
chimeras (data not shown). The basement membrane components, nidogen (entactin), collagen IV, and laminin-1
were also analyzed and no differences in staining were observed between mutant and control chimeras (data not
shown). Expression of tenascin-C (TN-C) has been correlated with muscle regeneration in several muscular dystrophies (Settles et al., 1995). In normal muscle it is present
only at the MTJ and on the nerves crossing the muscle (Chiquet and Fambrough, 1984
). The analysis of TN-C in
E17 or E18 embryos showed an enhanced deposition of
TN-C around and between some muscle fibers of
5
/
;
+/+ chimeric embryos compared with controls (Fig. 5 A),
suggesting a similar process of degeneration-regeneration as in other myopathies.
|
In mdx/mdx dystrophic mice, degeneration of fibers is
sometimes preceded by apoptosis (Matsuda et al., 1995;
Sandri et al., 1995
; Tidball et al., 1995
). We investigated
whether the same is true in
5
/
;+/+ chimeric mice.
Fig. 5 B shows that, although almost no apoptosis was observed in E18 control chimeras, some E18 mutant animals
(seven out of 10 chosen at random) showed an increase of
apoptotic fibers in various muscles (Fig. 5 B, dark fibers).
Myogenesis In Vitro in the Absence of 5 Integrin (ES
Cells and Myoblasts)
Our in vivo data suggest that muscle differentiation can
occur even in almost complete absence of 5-positive myoblasts (GPI of the limb ~95%). However, long-term integrity of the muscle is compromised. We examined the ability of
5-null ES cells to differentiate into myoblasts and
myotubes. Three null and two heterozygous (control) ES
cell clones were grown in suspension to produce embryoid
bodies. The embryoid bodies were then plated on gelatin-coated coverslips to induce differentiation. The differentiated cultures were stained with an antibody against skeletal muscle myosin heavy chain. 42% of the wells (19 out of
45) with
5-null embryoid bodies (all three clones) and
28% of the wells (14 out of 50) with control embryoid bodies stained positively. The degree of differentiation (fusion, myosin expression) appeared similar in control and
mutant cultures (Fig. 6 A).
|
To test further the role of 5 integrin in muscle cell adhesion, growth, differentiation, and survival we isolated
primary myoblast cultures from control and mutant chimeric mice. Myoblasts derived from
5-null or heterozygous ES cells were selected by culture in G418. The purity
of the cell populations was analyzed by Southern blot
analysis (data not shown) (Yang et al., 1993
, 1995) and the
expression of
5 integrin was analyzed by Western blot
analysis (Fig. 7 A). Control (+/
) myoblasts expressed
5 integrin whereas
5-null myoblasts were negative for
5
expression (Fig. 7 A). When maintained in low-serum medium, both control (+/
) and mutant (
/
) myoblasts
differentiated into multinucleated myotubes (Fig. 6 B),
and the rate and extent of myotube formation was similar
in the two populations (Fig. 6 C). Control and
5-deficient myoblasts grown on laminin-1 had similar doubling times
(data not shown) and displayed similar morphologies (Fig.
7 B). However, the phenotypes of the two populations
were very different when plated on fibronectin, the ligand
for the
5
1 integrin receptor. Whereas
5-expressing
cells attached and spread readily on fibronectin,
5-deficient cells adhered poorly, showed very little spreading, and displayed a rounded morphology, typical of poor cell-
substrate adherence (Fig. 7, B and C). As further evidence
that the different substrate requirements were indeed due
to
5 deficiency (and not to some other property of the
5-deficient cell population), we restored
5 expression to
the
5-deficient cells by retrovirus-mediated gene transfer.
A control population of
5-deficient cells was infected
with a retrovirus expressing only the neo gene. Expression of
5 in rescued cells (but not in control infected cells) was confirmed by Western blot analysis (Fig. 7 A). The rescued cells appeared to adhere as well to fibronectin as did
wild-type cells (Fig. 8, A and B).
|
|
Because of the apoptosis we observed in vivo (Fig. 5 B)
and since interference with integrin-mediated adhesion
has been reported to cause apoptosis (Frisch and Francis,
1994; Zhang et al., 1995
; Goh et al., 1997
), we tested for
apoptosis in the myoblasts cultured on various substrates.
Whereas
5-expressing cells showed similar percentages
of annexin-positive cells on fibronectin or laminin-1,
5-deficient cells showed a fourfold increase in annexin-positive cells on fibronectin compared with laminin-1 (Fig. 9).
Clearly, the absence of
5 expression increases the propensity of the cells to undergo apoptotic cell death when
FN is the major component of the ECM.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results presented here show that 5-null cells can participate in a wide variety of differentiative processes; animals with a high degree of chimerism in many organs survive and reproduce. The only defect which we observed
consistently in chimeric animals containing a significant
proportion of
5-null cells was a novel form of muscular
dystrophy. This allowed us to focus on the differentiation and survival of muscle cells and tissues and the dependence of these processes on
5
1 integrin.
Earlier results have shown blockade of migration of
myoblasts and of their differentiation into myotubes by inhibitory antibodies against various integrin subunits, including 1 (Jaffredo et al., 1988
; Menko et al., 1987),
4
(Rosen et al., 1992
) or
7 (Echtermeyer et al., 1996
; Yao
et al., 1996
). However, in vitro differentiation of ES cells
and myoblasts lacking
1 or
4 proceeds normally (Yang et al., 1996
; Brakebush et al., 1997) and
1-null and
4-null cells can participate in myogenesis in chimeric mice
(Faessler and Meyer, 1995
; Yang et al., 1996
). In the experiments reported here, we obtained similar results for
5-null cells. In vitro, neither
5-null ES cells nor
5-null
myoblasts showed any deficit in myogenesis and, in chimeric mice, muscles with a high proportion of
5-null cells
could form. These results demonstrate that
5
1 integrin
is not essential for the proliferation, migration, or differentiation of myoblasts, myotubes, and skeletal muscles. However, in contrast with the results for
4-null chimeras
(Yang et al., 1996
), we observed a significant level of abnormalities in the skeletal muscles of the
5-null chimeras
(Figs. 2-4). These muscles showed many characteristics of
muscular dystrophy, including giant fibers, central nuclei,
vacuoles, fibrosis, and fiber degeneration. Later in life we
observed signs of regeneration such as ring fibers. We also
observed increased apoptosis and ectopic expression of TN-C (Fig. 5) as has been reported for other forms of muscular dystrophy (Settles et al., 1996
).
These results are reminiscent of the observations of
Mayer et al. (1997) on
7 integrin-deficient mice, which
also exhibit muscular dystrophy, and of recent reports of
deficiencies in
7
1 in human and murine muscular dystrophies (Hodges et al., 1997
; Mayer et al., 1997
; Vachon
et al., 1997). It appears that these two integrins,
5
1 and
7
1, one a receptor for fibronectin and the other a receptor for laminin, are both necessary for long-term integrity
of myotubes, although not for their initial development.
5 is found localized at adhesion plaques (McDonald
et al., 1995
) and at the MTJ (Fig. 4);
7 is concentrated at
the MTJ (McDonald et al., 1995
) where tendons attach.
Because these two integrins are present at the points in the
fibers where mechanical stress occurs, it suggests an anchoring function of these molecules. Two Drosophila mutants show a similar situation; in myospheroid and inflated
(mutations affecting integrin subunits) muscle differentiation occurs in the absence, respectively, of
PS or
PS2 integrins; however, on contraction, the muscles detach from
their attachments (Volk et al., 1990
; Brabant et al., 1993).
We can imagine that, in the absence of
7
1 or
5
1, important points of adhesion are lost or weakened and therefore contraction leads to damage to the myotubes. The increase in the number of mitochondria in some fibers of the
5
/
;+/+ chimeric mice could suggest that, when the fibers cannot function normally, they tend to hypercontract and they need more ATP that requires the formation of a
higher number of mitochondria. Other possibilities for the
increase in mitochondria include altered differentiation
and compensation for the reduced level of
5 integrin.
It is noteworthy that many forms of muscular dystrophy
arise from defects in connections to the extracellular matrix. That includes the classical muscular dystrophies arising from defects in dystrophin and its transmembrane linkage via dystroglycans to laminins and in the laminins
themselves (Campbell, 1995). The novel form of muscular
dystrophy which we describe here differs from the others,
including those caused by
7 integrin deficiencies, in having no obvious connection with laminins.
5
1 has no
affinity for laminins and is believed to be specific for
fibronectin. The
5-null myoblasts show defects in adherence and survival on fibronectin substrates but behave
normally on laminin (Fig. 7).
The fact that the muscle defect of the 5-chimeric mice
is visible at a very early age (embryonic and postnatal life)
and is more attenuated later in life might be due to the
high expression, and probable importance, of
5 in embryonic and postnatal muscle followed by later downregulation (McDonald et al., 1995
). In vitro data show that overexpression of
5
1 in myoblasts promotes proliferation and inhibits differentiation, suggesting a proliferative function of this integrin (Sastry et al., 1996
). It is possible that,
in the chimeras,
5 is particularly important when a high
rate of proliferation is occurring. However, since our mice
are chimeras and the muscle fiber is a fusion of
5-null and
wild-type cells we cannot exclude the possibility that the
defect is partially rescued by the presence of wild-type
cells. Another possible reason for amelioration of the phenotype in later life could be gradual replacement of
5-null cells by wild type during regeneration. Consistent with
this possibility is the appearance of ring fibers in the older
muscles, indicating some fiber regeneration.
We favor the hypothesis that the dystrophy arises from
defects in the myofibers themselves, as discussed above. In
particular, the time of onset during fetal life corresponds
with the period when 5
1 is known to be strongly expressed in muscle cells and the parallels with the muscle
defects seen in Drosophila integrin mutants are suggestive.
However, we cannot rule out the possibility that defects or
deficits in other
5-null cells, such as interstitial fibroblasts, neurons or Schwann cells or vascular endothelial cells, could contribute to the phenotype observed. We do
know that
5-null fibroblasts can assemble FN matrix and
migrate and adhere normally (Yang and Hynes, 1996
) and
proliferate normally in vitro (Goh, K.L., and R.O Hynes,
unpublished data) which argues against a causal defect in
fibroblasts without eliminating that possibility.
The reasons for the degenerative changes observed in
the muscles deficient in 5
1 remain unclear, as indeed is
the case for other muscular dystrophies. Several possible
explanations can be imagined. As mentioned above, if
5
1 (and
7
1) are important for maintaining mechanical connections between the myotubes and adjacent structures (e.g., tendons), disruption of the weakened linkage
under contraction is a likely initiating cause. Perhaps less
likely in this case is a general weakening of the cell surface structure comprising submembranous cytoskeleton connected to the basal lamina. Another possibility is that the
apoptosis (Fig. 5) could be a causative event rather than a
secondary consequence. Precedent exists for cells' being
dependent on specific integrin-matrix adherence for cell
survival (Zhang et al., 1995
) and such dependences include
dependence on
5
1-fibronectin interactions (Zhang et al.,
1995
). However, our in vitro data somewhat argue against this idea without ruling it out. The
5-null myoblasts do indeed show increased apoptosis when plated on fibronectin
but do not when plated on laminin (Fig. 9). Myotubes are
surrounded by a basal lamina rich in laminin, although
also containing fibronectin. Thus, although it is possible
that adherence to fibronectin via
5
1 is specifically necessary for myotube survival, it seems more likely that any
such requirement for attachment to basal lamina is satisfied by connection to laminin via
7
1 or via dystroglycans. Whatever the detailed cause-effect relationships
leading to fiber degeneration and muscular defects in the
mice, the results reported here reveal a novel form of muscular dystrophy.
![]() |
Footnotes |
---|
Address correspondence to R. Hynes, Massachusetts Institute of Technology, 77 Massachusetts Ave., E17-227, Cambridge, MA 02139. Tel.: (617) 253-6422. Fax: (617) 253-8357. E-mail: rohynes{at}mit.edu
Received for publication 26 May 1998 and in revised form 12 August 1998.
The authors wish to acknowledge the excellent technical assistance of K. Mercer and D. Crowley for histology; M. Cummiskey and V. Evans for
blastocyst injections; J. Trevithick for immunohistochemistry and P. Reilly
for electron microscopy (all from Massachusetts Institute of Technology,
Cambridge, MA). We thank A. Chung for the anti-entactin antibody. We
are grateful to M. DiPersio and R. Chiquet-Ehrismann for critical reading
of the manuscript and B. Bader for helpful discussion.
D. Taverna was supported by Swiss National Foundation, Ciba Geigy
Foundation and European Molecular Biology Organization. R.O. Hynes
is a Howard Hughes Medical Institute Investigator. This work was supported by the Howard Hughes Medical Institute, by a Program of Excellence (POE) grant (PO1 HL41484) from the National Heart, Lung and
Blood Institute and by grants from the Muscular Dystrophy Association
and the Department of Veterans Affairs (RAG 9502-010) to T.A. Rando.
![]() |
Abbreviations used in this paper |
---|
ECM, extracellular matrix; ES, embryonic stem; FN, fibronectin; GPI, glucose-6-phosphate isomerase; H&E, hematoxylin and eosin; LM, laminin; MTJ, myotendinous junction; PTAH, phosphotungstic acid haematoxylin; TN-C, tenascin-C; WT, wild-type.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Akiyaa, S.K., S.S. Yamad, W.T. Chen, and K. Yamada. 1989. Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration matrix assembly, and cytoskeletal organization. J. Cell Biol. 109: 863-875 [Abstract]. |
2. | Argraves, W.S., S. Suzuki, H. Arai, K. Thompson, M.D. Pierschbacher, and E. Ruoslahti. 1987. Amino acid sequence of the human fibronectin receptor. J. Cell Biol. 105: 1183-1190 [Abstract]. |
3. |
Bao, Z.Z.,
M. Lakonishok,
S. Kaufman, and
A.F. Horwitz.
1993.
![]() ![]() |
4. | Bozyczko, D., C. Decker, J. Muschler, and A.F. Horwitz. 1989. Integrin on developing and adult skeletal muscle. Exp. Cell. Res. 183: 72-91 |
5. | Brabant, M.C., and D.L. Brower. 1993. PS2 integrin requirements in Drosophila embryo and wing morphogenesis. Dev. Biol. 157: 49-59 |
6. | Bradley, A. 1987. Production and analysis of chimeric mice. In Teratocarcinomas and Embyonic Stem Cells: A Practical Approach. E.J. Robertson, editor. IRL Press, Oxford, UK. 113-151. |
7. |
Brakebusch, C.,
E. Hirsch,
A. Potocnik, and
R. Faessler.
1997.
Genetic analysis
of ![]() |
8. |
Bronner-Fraser, M.,
M. Artinger,
J. Muschler, and
A.F. Horwitz.
1992.
Developmentally regulated expression of ![]() |
9. | Campbell, K.P.. 1995. Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage. Cell. 80: 675-679 |
10. | Chiquet, M., and D.M. Fambrough. 1984. Chick myotendinous antigen. I. A monoclonal antibody as a marker for tendon and muscle morphogenesis. J. Cell Biol. 98: 1926-1936 [Abstract]. |
11. | Doetschman, T.C., H. Eistetter, M. Katz, W. Schmidt, and R. Kemler. 1985. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87: 27-45 |
12. |
Duband, J.L.,
A.M. Belkin,
J. Syfrig,
J.P. Thiery, and
V.E. Koteliansky.
1992.
Expression of ![]() |
13. |
Echtermeyer, F.,
S. Schoeber,
E. Poeschl,
H. von der Mark, and
K. von der
Mark.
1996.
Specific induction of cell motility on laminin by ![]() |
14. |
Enomoto, M.I.,
D. Boettinger, and
A.S. Menko.
1993.
![]() |
15. |
Faessler, R., and
M. Meyer.
1995.
Consequences of the lack of ![]() |
16. |
Fogerty, F.J.,
S.K. Akiyama,
K.M. Yamada, and
D.F. Mosher.
1990.
Inhibition
of binding of fibronectin to matrix assembly sites by anti-integrin (![]() ![]() |
17. | Frisch, S.M., and H. Francis. 1994. Disruption of epithelial cell matrix interactions induces apoptosis. J. Cell Biol. 124: 619-626 [Abstract]. |
18. |
George, E.L.,
E.N. Georges,
R.S. Patel-King,
H. Rayburn, and
R.O. Hynes.
1993.
Defects in mesodermal migration and vascular development in fibronectin-deficient mice.
Development (Camb.)
119:
1079-1091
|
19. | Giancotti, F.G., and F. Mainiero. 1994. Integrin-mediated adhesion and signaling in tumorigenesis. Biochim. Biophys. Acta 1198: 47-64 |
20. |
Giancotti, F.G., and
E. Ruoslahti.
1990.
Elevated levels of the ![]() ![]() |
21. |
Goh, K.L.,
J.T. Yang, and
R.O. Hynes.
1997.
Mesodermal defects and cranial
neural crest apoptosis in ![]() |
22. | Guan, J.L., J.E. Trevithick, and R.O. Hynes. 1990. Retroviral expression of alternatively spliced forms of rat fibronectin. J. Cell Biol 110: 833-847 [Abstract]. |
23. | Guo, M., L.T. Kim, S.K. Akiyama, H.R. Gralnick, K.M. Yamada, and F. Grinnel. 1991. Altered processing of integrin receptors during keratinocyte activation. Exp. Cell Res. 195: 315-322 |
24. |
Hodges, B.L.,
Y.K. Hayashi,
I. Nonaka,
W. Wang,
K. Arahata, and
S.J. Kaufman.
1997.
Altered expression of the ![]() ![]() |
25. | Hynes, R.O. 1990. Fibronectin. Springer-Verlag, New York. 546 pp. |
26. | Hynes, R.O.. 1992. Integrins: versatility, modulation and signaling in cell adhesion. Cell 69: 11-25 |
27. | Hynes, R.O., and A.D. Lander. 1992. Contact and adhesive specificities in the associations, migrations and targeting of cells and axons. Cell 68: 303-322 |
28. | Jaffredo, T., A.F. Horwitz, C.A. Buck, P.M. Rong, and F. Dieterlen-Lievre. 1988. Myoblast migration specifically inhibited in the chick embryo by grafted CSAT hybridoma cells secreting an anti-integrin antibody. Development (Camb.). 103: 431-446 [Abstract]. |
29. | Kelly, A.M., and N.A. Rubinstein. 1994. The diversity of muscle fiber types and its origin during development. In Myology. A.G. Engel and C. Franzini-Armstrong, editors. McGraw-Hill, New York. 119-133. |
30. | Knudsen, K.A.. 1990. Cell adhesion molecules in myogenesis. Curr. Opin. Cell Biol 2: 902-906 |
31. | Matsuda, R., A. Nishikawa, and H. Tanaka. 1995. Visualization of dystrophic muscle fibers in Mdx mouse by vital staining with evans blue: evidence of apoptosis in dystrophin-deficient muscle. J. Biochem 118: 959-964 [Abstract]. |
32. | Mautner, V., and R.O. Hynes. 1977. Surface distribution of lets protein in relation to the cytoskeleton of normal and transformed cells. J. Cell Biol 75: 743-768 [Abstract]. |
33. |
Mayer, U.,
G. Saher,
R. Faessler,
A. Bornemann,
F. Echtermeyer,
H. von der
Mark,
N. Miosge,
E. Poeschl, and
K. von der Mark.
1997.
Absence of integrin ![]() |
34. |
McDonald, K.A.,
M. Lakonishok, and
A.F. Horwitz.
1995.
![]() ![]() |
35. | Menko, A.S., and D. Boettiger. 1987. Occupation of the extracellular matrix receptor, integrin, is a control point for myogenic differentiation. Cell 51: 51-57 |
36. | Morgenbesser, S.D., N. Schreiber-Agus, M. Bidder, K.A. Mahon, P.A. Overbeek, J. Horner, and R.A. DePinho. 1995. Contrasting roles for c-myc and L-myc in the regulation of cellular growth and differentiation in vivo. EMBO (Eur. Mol. Biol. Organ.) J. 14: 743-796 [Abstract]. |
37. |
Muschler, J.L., and
A.F. Horwitz.
1991.
Down regulation of the chicken ![]() ![]() |
38. | Plantefaber, L.C., and R.O. Hynes. 1989. Changes in integrin receptors on oncogenically transformed cells. Cell 56: 281-290 |
39. | Pytela, R., M.D. Piershbacher, and E. Ruoslahti. 1994. Identification and isolation of a 140 Kd cell surface glycoprotein with properties expected of a fibronectin receptor. Cell 40: 191-198 . |
40. | Rando, T.A., and H.M. Blau. 1994. Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J. Cell Biol. 125: 1275-1284 [Abstract]. |
41. | Rando, T.A., and H.M. Blau. 1997. Methods for myoblast transplantation. Methods Cell Biol 52: 261-272 |
42. | Robinson, S.D., P.S. Frenette, H. Rayburn, M. Cummiskey, M. Ullman-Culleré, D.D. Wagner, and R.O. Hynes. 1997. Generation of double- and triple-deficient selectin mice. Microcirculation 4: 131 . |
43. | Rosen, G.D., J.R. Sanes, R. LaChance, J.M. Cunningham, J. Roman, and D.C. Dean. 1992. Roles for the integrin VLA-4 and its counter receptor VCAM-1 in myogenesis. Cell 69: 1107-1119 |
44. | Ruoslahti, E.. 1991. Integrins. J. Clin. Invest 87: 1-5 |
45. | Sandri, M., U. Carraro, M. Podhorska-Okolov, C. Rizzi, P. Arslan, D. Monti, and C. Franceschi. 1995. Apoptosis, DNA damage and ubiquitin expression in normal and mdx muscle fibers after exercise. FEBS Lett. 373: 291-295 |
46. | Sanes, J.R. 1994. The extracellular matrix. In Myology. A.G. Engel and C. Franzini-Armstrong, editors. McGraw-Hill, New York. 242-260. |
47. |
Sastry, S.K.,
M. Lakonishok,
D.A. Thomas,
J. Muschler, and
A.F. Horwitz.
1996.
Integrin ![]() |
48. | Schreiner, C., M. Fisher, S. Hussein, and R.L. Juliano. 1991. Increased tumorigenicity of fibronectin receptor deficient Chinese hamster ovary cell variants. Cancer Res 51: 1738-1740 [Abstract]. |
49. | Settles, D.L., R.A. Cihak, and H.P. Erickson. 1996. Tenascin-C expression in dystrophin-related muscular dystrophy. Muscle Nerve. 19: 147-154 |
50. |
Shimizu, Y., and
S. Shaw.
1991.
Lymphocyte interactions with extracellular matrix.
FASEB (Fed. Am. Soc. Exp. Biol.) J.
5:
2292-2299
|
51. |
Taverna, D.,
M. Ullman-Cullerè,
H. Rayburn,
R.T. Bronson, and
R.O. Hynes.
1998.
A test of the role of ![]() |
52. |
Tidball, J.G.,
D.E. Albrecht,
B.E. Lokensgard, and
M.L. Spenser.
1995.
Apoptosis precedes necrosis of dystrophin-deficient muscle.
J. Cell Sci.
108:
2197-2204
|
53. |
Vachon, P.H.,
H. Xu,
L. Liu,
F. Loechel,
Y. Hayashi,
K. Arahata,
J.C. Reed,
U.M. Wewer, and
E. Engvall.
1995.
Integrins (![]() ![]() |
54. |
Varner, J.A.,
D.A. Emerson, and
R.L. Juliano.
1995.
Integrin ![]() ![]() |
55. | Volk, T., L.I. Fessler, and J.H. Fessler. 1990. A role for integrin in the formation of sarcomeric cytoarchitecture. Cell 63: 525-536 |
56. | Werb, Z., P.M. Tremble, O. Behrendtsen, E. Crowley, and C.H. Damsky. 1989. Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression. J. Cell Biol. 109: 877-889 [Abstract]. |
57. |
Whittaker, C.A., and
D.W. DeSimone.
1993.
Integrin ![]() |
58. |
Yang, J.T., and
R.O. Hynes.
1996.
Fibronectin receptor functions in embryonic
cells deficient in ![]() ![]() ![]() |
59. |
Yang, J.T.,
H. Rayburn, and
R.O. Hynes.
1993.
Embryonic mesodermal defects
in ![]() |
60. |
Yang, J.T.,
T.A. Rando,
W.A. Mohler,
H. Rayburn,
H.M. Blau, and
R.O. Hynes.
1996.
Genetic analysis of ![]() |
61. |
Yao, C.C.,
B.L. Ziober,
R.M. Squillace, and
R.H. Kramer.
1996.
![]() |
62. |
Zhang, Z.,
K. Vuori,
J.C. Reed, and
E. Ruoslahti.
1995.
The ![]() ![]() |