Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario, Canada L8S 4K1
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Abstract |
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To gain insight into the regeneration deficit
of MyoD/
muscle, we investigated the growth and
differentiation of cultured MyoD
/
myogenic cells.
Primary MyoD
/
myogenic cells exhibited a stellate
morphology distinct from the compact morphology of wild-type myoblasts, and expressed c-met, a receptor
tyrosine kinase expressed in satellite cells. However,
MyoD
/
myogenic cells did not express desmin, an
intermediate filament protein typically expressed in
cultured myoblasts in vitro and myogenic precursor
cells in vivo. Northern analysis indicated that proliferating MyoD
/
myogenic cells expressed fourfold
higher levels of Myf-5 and sixfold higher levels of
PEA3, an ETS-domain transcription factor expressed
in newly activated satellite cells. Under conditions that
normally induce differentiation, MyoD
/
cells continued to proliferate and with delayed kinetics yielded
reduced numbers of predominantly mononuclear myocytes. Northern analysis revealed delayed induction of
myogenin, MRF4, and other differentiation-specific markers although p21 was upregulated normally. Expression of M-cadherin mRNA was severely decreased
whereas expression of IGF-1 was markedly increased in
MyoD
/
myogenic cells. Mixing of lacZ-labeled
MyoD
/
cells and wild-type myoblasts revealed a
strict autonomy in differentiation potential. Transfection of a MyoD-expression cassette restored cytomorphology and rescued the differentiation deficit. We interpret these data to suggest that MyoD
/
myogenic
cells represent an intermediate stage between a quiescent satellite cell and a myogenic precursor cell.
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Introduction |
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THE myogenic regulatory factors (MRFs)1 form a
group of four basic helix-loop-helix transcription
factors consisting of MyoD, Myf-5, myogenin, and
MRF4. The MRFs have been demonstrated to play pivotal
roles in the determination and differentiation of myogenic
precursors into mature skeletal muscle (Weintraub et al.,
1991; Rudnicki and Jaenisch, 1995
). Gene targeting experiments have provided much insight into understanding
MRF function in vivo. The introduction of null mutations
in all four factors into the germline of mice has revealed
the existence of a hierarchical relationship among the
MRFs and has defined two functional groups of MRFs
(Braun et al., 1992
; Rudnicki et al., 1992
, 1993
; Nabeshima et al., 1993
; Patapoutian et al., 1995
; Zhang et al., 1995
).
The primary MRFs, MyoD and Myf-5, appear to be required for myogenic determination, whereas the secondary MRFs, myogenin and MRF4, are required later in the
developmental program as differentiation factors (Megeney and Rudnicki, 1995
; Rudnicki and Jaenisch, 1995
).
Satellite cells, the stem cells of adult skeletal muscle, reside beneath the basal lamina of adult skeletal muscle
closely juxtaposed against muscle fibers. Satellite cells
arise around day 17 of development and are believed to
represent a unique myoblast lineage distinct from embryonic and fetal lineages (Cossu et al., 1985; Bischoff, 1994
).
Satellite cells make up 2-7% of the nuclei associated with
a particular fiber and the proportion varies with age and
particular muscle group. Satellite cells are normally mitotically quiescent, but are activated (i.e., initiate multiple rounds of proliferation) in response to stress induced by
weight bearing or other trauma such as injury. The descendants of the activated satellite cells, myogenic precursor
cells, undergo multiple rounds of division prior to fusing to
existing or new fibers. Satellite cells appear to form a population of stem cells that are distinct from their daughter
myogenic precursor cells as defined by biological and
biochemical criteria (Bischoff, 1994
). The total number of
satellite cells in muscle remains relatively constant, suggesting that a capacity for self-renewal in the satellite cell compartment maintains the population of quiescent cells
(Bischoff, 1994
).
Quiescent satellite cells express no detectable MRFs.
Upon activation and entrance into the cell cycle, MyoD is
rapidly upregulated concomitantly with proliferating cell
nuclear antigen, a marker for cell proliferation, whereas
myogenin is expressed last during the time associated with
fusion and differentiation (Smith et al., 1994; Yablonka-Reuveni and Rivera, 1994
). Analysis of gene expression by
RT-PCR of individual satellite cells in cultured intact muscle fibers at times after their activation substantiates that
quiescent satellite cells express no detectable MRFs but do express c-met, a receptor tyrosine kinase (Cornelison
and Wold, 1997
). Moreover, these experiments reveal that
activated satellite cells first express either Myf-5 or MyoD
followed soon after by coexpression of Myf-5 and MyoD.
After proliferation, myogenin and MRF4 are expressed in
cells beginning their differentiation program (Cornelison
and Wold, 1997
).
The regeneration deficit observed in MyoD/
muscle
strongly supports the assertion that MyoD plays an essential role in regulating the satellite cell myogenic program
(Megeney et al., 1996
). Muscle regeneration is severely deficient in MyoD
/
mice and compound mutant mice
lacking both MyoD and dystrophin (designated mdx: MyoD
/
) exhibit severe myopathy leading to premature
death (Megeney et al., 1996
). Muscle regeneration in
MyoD
/
muscle is characterized by an almost complete
absence of proliferative myogenic precursor cells. However, electron microscopic examination of MyoD-deficient muscle reveals increased numbers of satellite cells and the
number of primary myoblasts recovered is increased 2.5-fold in MyoD
/
muscle and 13-fold in mdx:MyoD
/
muscle. Taken together, our data suggest a model in which
upregulation of MyoD is required for satellite cells to enter the myogenic precursor cell proliferative phase that
precedes terminal differentiation. In the absence of MyoD,
myogenic stem cells undergo an apparent increase in numbers as a consequence of an increased propensity for self-renewal rather than progression through the myogenic differentiation program (Megeney et al., 1996
).
To investigate the function of MyoD during the course
of satellite cell activation, we established primary myogenic cultures from wild-type and MyoD-deficient adult
muscle. Importantly, these experiments were performed
with newly established low-passage primary cultures. Our
analysis strongly supports the hypothesis that MyoD is required for satellite cells to progress efficiently through the
myogenic precursor cell developmental program. Moreover, our data suggest that MyoD/
myogenic cells represent an intermediate developmental stage between quiescent satellite cells and myogenic precursor cells.
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Materials and Methods |
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Isolation of Primary Myoblasts, Cell Culture, and Immunohistochemistry
Satellite cell-derived primary myoblasts were isolated from adult lower
hindlimb muscle from 2-3-mo-old mice as described previously (Megeney
et al., 1996), with the exception that hepatocyte growth factor (10 ng/ml;
R&D Systems Inc.) and heparin (5 ng/ml; Sigma Chemical Co.) were included in the growth medium for the first 48 h after the plating of the final
cell preparation, and supplemented with FGF2 (2.5 ng/ml FGF2) thereafter. The primary cultures were maintained on collagen-coated dishes in
Ham's F10 (GIBCO BRL) supplemented with 20% FCS, 200 U/ml penicillin, 200 µg/ml streptomycin, and 0.002% Fungizone (GIBCO BRL). The
medium was changed daily and cultures were routinely passaged 1:3 as they reached 60-70% confluence. To maintain the primary characteristics of the cells, all experiments were performed using cultures that had undergone between four and seven passages. Differentiation medium consisted
of DME supplemented with 5% horse serum and antibiotics as described
above (GIBCO BRL).
The extent of culture purity and differentiation was determined by subjecting the purified myoblasts to c-met, desmin, and myosin heavy chain
(MHC) immunostaining. Briefly, myoblasts in growth medium were fixed
in 4% paraformaldehyde in PBS (PFA), stained with anti-c-met antibody
SP260 (Santa Cruz Biotechnology), and antidesmin antibody DE-U-10
(DAKO). MHC expression was detected by fixing differentiated cultures
with 90% methanol and staining with MF20 mAb (Bader et al., 1982). Immunostaining was similarly performed with anti-
-catenin antibody sc-1496 (Santa Cruz Biotechnology) and anti-M-cadherin antibody sc-6470
(Santa Cruz Biotechnology). Immunostaining with anti-c-met and anti-
-catenin antibodies were detected with fluorescein-conjugated anti-goat
antibodies (Sigma Chemical Co.), and photographed on a Zeiss Axiophot
microscope equipped with a UV source and FITC detection filters.
Desmin, M-cadherin, and MHC staining was visualized using a HRP-coupled secondary antibody (Bio-Rad Laboratories) in PBS containing 0.6 mg/ml diaminobezidine (Sigma Chemical Co.).
Differentiation Time Course and Growth Rate Measurements
Growth rate analysis was determined by [3H]thymidine incorporation of
three independent isolates of wild-type and MyoD/
cultures seeded in
24-well plates at 104 cells per well in growth medium (each in triplicate).
For day 0, the cells were cultured 24 h in growth medium before addition of 2 µCi of [3H]thymidine for 2 h. Incorporation of [3H]thymidine was
normalized to protein concentration as determined by Bradford assay.
The remaining wells were exposed to differentiation medium and labeled
with [3H]thymidine for 2 h on sequential days.
To assay the differentiation potential of wild-type and MyoD/
cultures, 105 low passage cells were seeded into 35-mm dishes in growth medium and cultured for an additional 24 h (day 0) before addition of differentiation medium. On subsequent days (1-5), the cultures were fixed and
immunostained for MHC with antibody MF20. To establish the differentiation potential of the cultures, at least 1,000 nuclei from MF20-positive
cells were counted from several random fields. The percentage of differentiated cells was calculated as: (nuclei within MF20-stained myocytes/total
number of nuclei) × 100; or the fusion index calculated as: (MF20-stained
myocytes containing
2 nuclei/total number of nuclei) × 100. 5-bromo-2'-deoxyuridine (BrdU) incorporation assays on cultured cells were performed (cell proliferation kit; Amersham Pharmacia Biotech). All experiments were performed in triplicate on three independent wild-type and MyoD
/
isolates.
Northern and Western Analysis
To analyze the expression of the MRFs and of differentiation-specific
markers, total RNA from low passage cells in growth (day 0) or differentiation medium (days 1-5) was isolated (Birnboim, 1988) and subjected to
Northern analysis (Maniatis et al., 1982
). Replicate filters were sequentially hybridized to MRF-specific cDNAs as well as
-cardiac and
-skeletal actin, and acetylcholine receptor
subunit probes (Rudnicki et al.,
1993
). Dr. Paul Hastings (McGill University) kindly provided the M-cadherin probe. The Musk cDNA probe was obtained by RT-PCR of C2C12
RNA. The
-catenin and PEA3 probes were kindly provided by Drs. Rolf
Kemler (Max Planck Institute) and John A. Hassell (McMaster University), respectively. Western analysis with rabbit anti-Myf-5 antibody C-20
(Santa Cruz Biotechnology), mouse anti-MyoD antibody 5A8 (PharMingen), and mouse antimyogenin antibody F5D (Developmental Studies
Hybridoma Bank) was performed on extracts prepared from cultures in
growth medium as described previously (LeCouter et al., 1996
).
Mixing of LacZ-expressing MyoD/
and
Wild-Type Cultures
Early passage MyoD/
cultures were lipofected with a 1:10 ratio of
PGK-LacZ-MAR and PGK-Puro plasmids, and stable transformants were
pooled after 10 d of selection in 2 µg/ml puromycin. Plasmid PGK-LacZ-MAR contains the phosphoglycerate kinase 1 promoter expressing nls-lacZ and a chicken lysozyme matrix attachment region to confer high level
site-independent expression (Phi-Van and Stratling, 1996
). Primary cells
were plated with an initial density of 105 cells per 60-mm well, in ratios of
1:0, 1:4, 4:1, and 0:1 of MyoD
/
to wild-type cells. Duplicate cultures
were grown overnight in growth medium before exposing to differentiation medium for 5 d. Wells were washed with PBS, fixed in 2% formaldehyde/0.4% glutaraldehyde followed by X-Gal staining. Cells were postfixed with 90% methanol for 7 min, rinsed with PBS and 0.3% Triton X, before immunostaining with antibody MF-20 as described above.
Transfection with MyoD Plasmid and Generation of Stable MyoD+ Pools
Low passage subconfluent cultures of MyoD/
cells were transfected
with pEMSV-MyoD/PGK-Puro or PGK-Puro alone by lipofectamine (GIBCO BRL), according to the manufacturer's instructions. The MyoD
expression plasmid carries the murine MyoD cDNA driven by the EMSV
promoter and enhancer, as well as a puromycin resistance cassette. The
cultures were refed 24 h after transfection and daily with growth medium
containing 2 µg/ml puromycin (Sigma Chemical Co.) for 10 d. The resulting colonies (>200) were pooled and expanded for further analysis. MyoD
expression was evaluated by Western blot analysis (LeCouter et al., 1996
)
using anti-MyoD mAb 5A8 (PharMingen). The differentiation and fusion
potentials of MyoD-expressing pools were assayed as described above.
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Results |
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Altered Cellular Phenotype of MyoD/
Myogenic Cells
To gain insight into the role of MyoD in satellite cell activation, low passage primary cultures were isolated from
2-3-mo-old wild-type and MyoD/
mice to facilitate the
generation of highly purified satellite cell-derived cultures
and preclude the inclusion of neonatal myoblasts. Cultures
were grown for 48 h in the presence of hepatocyte growth
factor and thereafter in medium supplemented with FGF2
to allow the rapid recovery of high numbers of low passage primary myoblasts as described previously (Allen et al.,
1995
).
As suggested by our previous observations (Megeney
et al., 1996), MyoD
/
cultures displayed a stellate flattened morphology with an enlarged cytoplasm and extended cytosolic processes. By contrast, wild-type cells
were highly refractile under phase-contrast microscopy
and displayed the rounded morphology and small compact cytoplasm characteristic of primary myoblasts (see Figs. 1
and 8).
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Quiescent and activated satellite cells in vivo express the
receptor tyrosine kinase c-met as do cultured myoblasts
(Allen et al., 1995; Cornelison and Wold, 1997
). Proliferating myogenic precursor cells in vivo and myoblasts in vitro
express the intermediate filament desmin. However, satellite cells do not express desmin (George-Weinstein et al.,
1993
). Therefore, primary cultures were immunostained
with antibody reactive with c-met and desmin to assess
their developmental status. Virtually 100% of the primary cells derived from both wild-type and MyoD
/
animals
expressed high levels of c-met as detected by indirect immunofluorescence. Fibroblast cell lines did not express
c-met (not shown). Therefore, the isolation procedure
generated highly purified cultures of myogenic cells (Fig. 1
a). In contrast to the uniform expression of c-met, only
6.2 ± 5.4% of MyoD
/
myogenic cells expressed the
myoblast marker desmin, whereas 89 ± 4.2% of wild-type
cells expressed desmin (Fig. 1 b). These data are consistent
with the notion that MyoD
/
myogenic cells represent
an intermediate developmental stage between a satellite
cell and myogenic precursor cells.
Reduced Differentiation Potential of MyoD/
Myogenic Cultures
To evaluate the differentiation potential of the MyoD/
myogenic cultures the extent of myogenic differentiation
was assessed at the cellular level by immunostaining cultures fixed at daily intervals after mitogen withdrawal with
antibody MF20 reactive with MHC (Fig. 2 a). Importantly,
this analysis was performed on three independently isolated wild-type and MyoD
/
primary myogenic cultures.
After MF20 immunostaining, the proportion of MHC-positive cells and the fusion index of both wild-type and MyoD
/
cultures were assessed (Fig. 2, b and c).
|
Under growth conditions, MyoD/
myogenic cells exhibited about a 100-fold reduction in the rate of spontaneous differentiation (0.10 ± 0.14%) compared to wild-type
cells (10 ± 1.4%) (see day 0 in Fig. 2, a and b). Consistent
with this observation, MyoD
/
cultures displayed a severe defect in their ability to differentiate and form multinucleated myotubes after mitogen withdrawal (Fig. 2 a).
Differentiated wild-type myocytes displayed the typical elongated and multinucleated morphology, whereas differentiated MyoD
/
myocytes were primarily mononuclear and retained a fibroblastic stellate cytomorphology
(Fig. 2 a).
About 50% of the cells in wild-type cultures after 24 h in
differentiation medium had begun to undergo terminal
differentiation as assessed by MHC immunostaining (Fig.
2, a and b). The number of differentiated wild-type myocytes continued to accumulate in a linear manner reaching
~94% 5 d after serum withdrawal (Fig. 2, a and b). By
contrast, 24 h after mitogen withdrawal, the number of differentiated myocytes in MyoD/
cultures remained below the limit of detection (Fig. 2, a and b). The number of
differentiated MyoD
/
myocytes began to accumulate
after 3 d and reached ~70% 5 d after serum withdrawal
(Fig. 2, a and b).
Enumeration of differentiated myocytes containing two
or more nuclei in these cultures (i.e., fusion index) revealed a marked reduction in fusion capacity in MyoD/
cultures (Fig. 2 c). The fusion index of differentiated wild-type cultures was ~90% 5 d of differentiation with an
average of 4.6 ± 0.3 nuclei per myocyte. By contrast, by
day 5 only about 15% of MyoD
/
myocytes contained 2 nuclei, with an average of 1.2 ± 0.2 nuclei per myocyte
(Fig. 2, a and c).
The rate of cell-cycle withdrawal after induction of differentiation was assessed by measuring [3H]thymidine incorporation at daily intervals after transfer into differentiation medium (Fig. 3 a). In growth medium, [3H]thymidine
labeling experiments revealed that MyoD/
cells exhibited an apparent twofold higher growth rate than
wild-type cells (Fig. 3 a). Direct cell count experiments
in growth medium revealed that numbers of primary
MyoD
/
cells accumulated ~1.5-fold faster than wild-type cells (not shown). Moreover, MyoD
/
cells displayed a 1.6-fold increase in mitotic index as determined
by BrdU incorporation (Fig. 3 b). However, this increase
in apparent growth can be partly accounted for by the reduced rate of spontaneous differentiation of mutant (0.10 ± 0.14%) versus wild-type cells (10.0 ± 1.4%) (see day 0, Fig. 2, a and b). In addition, interpretation of these results
is also confounded by other potential variables: differences in the proportion of cells temporarily withdrawn
from the cell-cycle, differences in rates of apoptosis, and
differences in cell-cycle kinetics. Therefore, additional
analyses are required to determine whether primary
MyoD
/
myogenic cells exhibit altered cell-cycle kinetics relative to primary wild-type myoblasts.
|
Wild-type myoblasts and MyoD/
myogenic cells
both exhibited about a twofold increase in the rate of
[3H]thymidine incorporation 24 h after mitogen withdrawal (Fig. 3 a). This apparent increase in DNA synthesis
may reflect a characteristic of primary cells analogous to
the transient increase in cell proliferation observed after
the IGF-I treatment of myoblasts under culture conditions
that induce differentiation (Engert et al., 1996
). After the
first day of mitogen withdrawal, wild-type myoblasts exhibit a rapid withdrawal from the cell cycle as evidenced
by the low levels of [3H]thymidine incorporation (Fig. 3 a).
By contrast, the rate that MyoD
/
myogenic cells incorporated [3H]thymidine continued to increase during the 5 d
after transfer into differentiation medium. Similar results
were obtained using three independent preparations of
low passage primary myogenic cultures.
Taken together, these experiments suggested that
MyoD/
myogenic cells displayed continued proliferation under conditions of low mitogens that normally induce cell-cycle withdrawal and terminal differentiation of
wild-type myoblasts. To investigate cell proliferation under conditions of growth and differentiation, cultured cells
were exposed to BrdU for 4 h followed by immunodetection of nuclear localized BrdU incorporated during
DNA synthesis (Fig. 3 b). Wild-type primary myoblasts in
growth medium exhibited a 20% rate of BrdU incorporation, whereas MyoD
/
myogenic cells exhibited a 32%
rate of labeling. After 5 d of differentiation, 93% of nuclei
in wild-type cultures expressed MHC and 5.5% were labeled by BrdU. In contrast, after 5 d of differentiation,
49% of cells in MyoD
/
cultures expressed MHC and
17% were labeled by BrdU (see Fig. 2 b). Interestingly,
subconfluent cultures of MyoD
/
cells in differentiation
medium exhibited up to 20% rates of BrdU incorporation,
whereas confluent cultures exhibited as low as 5% BrdU
labeling (not shown).
Considered together, these data indicate that MyoD/
myogenic cells continue to proliferate under low-mitogen
conditions that normally induce terminal differentiation of
wild-type myoblasts and suggest that MyoD
/
cells exhibit contact inhibition of growth during differentiation.
The observation that MyoD
/
cells exhibit an enhanced
proliferative potential under conditions that normally induce differentiation strongly supports the notion that
MyoD
/
myogenic cells exhibit an increased propensity
for self-renewal rather than progression through the differentiation program.
Analysis of Muscle-specific Gene Expression
To gain further insight into the differentiation defect in
MyoD/
primary cultures, total RNA was prepared
from both wild-type and MyoD
/
cultures in growth
medium and at daily intervals after induction of differentiation. Northern blot analysis was performed using a panel
of muscle-specific probes.
The expression of the MRFs was investigated to elucidate the regulatory relationships and the potential for
functional compensation in the absence of MyoD. Northern analysis of the expression pattern of the four MRFs
confirmed that MyoD was expressed at high levels in wild-type myoblasts and was absent in the MyoD-deficient
myogenic cells. Furthermore, densitometric analysis and
normalization to 18S rRNA revealed that MyoD was
somewhat downregulated during the differentiation of
wild-type cells (Fig. 4 a). Previously, we observed a 3.5-fold increase in Myf-5 mRNA in newborn and adult muscles in vivo (Rudnicki et al., 1993). We similarly observed a fourfold increase in Myf-5 mRNA in growing MyoD
/
myogenic cells relative to wild-type myoblasts, supporting
the hypothesis that MyoD negatively regulates Myf-5 expression (Fig. 4 b). In wild-type cells, Myf-5 mRNA levels
decreased about twofold after differentiation, whereas in
MyoD
/
cultures, Myf-5 mRNA levels were upregulated about twofold after mitogen withdrawal (Fig. 4 b). In
addition, consistent with the observed 100-fold reduction in the rate of spontaneous differentiation, MyoD
/
myogenic cells expressed fivefold lower levels of myogenin
mRNA in growth medium relative to wild-type myoblasts
(Fig. 4 c). After the induction of differentiation, the relative levels of myogenin mRNA in MyoD
/
cells remained significantly reduced relative to wild-type cultures (Fig. 4 c). In wild-type cells, the level of MRF4 mRNA
remained unchanged until day 2 of differentiation and
was thereafter upregulated about sevenfold. By contrast,
MRF4 mRNA was upregulated in differentiating MyoD
/
cultures to levels approximately two- to threefold lower than that of wild-type cells (Fig. 4 d).
Western analysis of MRF expression in lysates of primary cultures in growth medium confirmed the absence of
MyoD protein in MyoD/
cells (Fig. 4 e). Interestingly,
Myf-5 protein was upregulated >10-fold in MyoD
/
cells, suggesting that posttranscriptional mechanisms may
contribute to this increase (see Fig. 4, b and e for comparison). As suggested by the Northern analysis (Fig. 4 c),
myogenin protein was absent in MyoD
/
cells in growth
medium, whereas low levels were detected in wild-type
myoblasts (Fig. 4 e).
To assess the differentiation kinetics at the level of gene
expression, Northern analysis was performed with a panel
of muscle-specific markers. Analysis of mRNA levels for
differentiation-specific markers revealed a pattern consistent with overall delayed kinetics of differentiation in
MyoD/
myogenic cells. For example, low levels of
-skeletal and
-cardiac actin mRNAs were detected in wild-type cells in growth medium and these increased rapidly after mitogen withdrawal. By contrast, MyoD
/
cells in growth medium expressed no detectable sarcomeric actin mRNA (Fig. 5, a and b). After induction of
differentiation
-skeletal actin mRNA increased about
threefold in wild-type cells, whereas in MyoD
/
cells the levels increased ~65% of wild-type levels by day 5 (Fig. 5
a). Although lower than that of
-skeletal actin, the levels
of
-cardiac actin were found to increase in a similar pattern (Fig. 5 b). Cultured MyoD
/
myogenic cells displayed a twofold reduction in levels of acetylcholine receptor
subunit (AchR
) mRNA in growth medium.
After the induction of differentiation, a 5-fold increase
was observed in wild-type cultures compared to an ~2-fold increase in MyoD
/
cells (Fig. 5 c) for a net 10-fold
relative difference.
|
The protein coding for M-cadherin, a muscle-specific
adhesion molecule, has been suggested to be expressed in
quiescent satellite cells as well as to play an important role
during myoblast differentiation and fusion (Irintchev et al.,
1994; Pouliot et al., 1994
; Zeschnigk et al., 1995
). However, RT-PCR analysis only detects M-cadherin expression in a small number of quiescent satellite cells suggesting that M-cadherin may not be useful as a marker for
satellite cells (Cornelison and Wold, 1997
). In wild-type
cells, M-cadherin mRNA was detected at low levels in
growth medium, M-cadherin increased fivefold by day 2 of
differentiation, and was subsequently downregulated (Fig.
5 d). By contrast, MyoD
/
myogenic cells in growth medium expressed no detectable M-cadherin mRNA. However, low levels were detectable after mitogen withdrawal.
Immunohistochemical detection of M-cadherin on cells in
growth medium confirmed the reduced level of protein detectable on MyoD
/
cells relative to wild-type myoblasts (Fig. 1 d). The reduced levels of M-cadherin expression observed in MyoD
/
myogenic cells may account in
part for the differentiation deficiency as M-cadherin appears to be required for efficient myogenic differentiation
and fusion (Zeschnigk et al., 1995
).
The receptor tyrosine kinase Musk has been suggested
to be expressed in activated satellite cells (DeChiara et al.,
1996), and therefore may provide an additonal marker for
early myogenic cells. Northern blot analysis with a Musk-specific probe revealed the expression of three distinct
isoforms in differentiating myogenic cells (Fig. 6 a). In
wild-type cultures in growth medium, the large mRNA
(isoform 1) was not expressed, the midsize mRNA (isoform 2) was expressed at intermediate levels, and the small
mRNA (isoform 3) was expressed at higher levels. Induction of differentiation of wild-type cultures resulted in upregulation of isoforms 1 and 2, but little change in isoform
3. By contrast, MyoD
/
myogenic cells in growth medium expressed no detectable expression of Musk mRNA
isoforms 1 and 2, and low levels of isoform 3. After induction of differentiation of MyoD
/
cultures, delayed upregulation of isoforms 1 and 2 was observed. Therefore,
these data suggest that Musk is upregulated in a differentiation-dependent manner during muscle regeneration.
|
Adhalin, a dystrophin-associated protein also known as
-sarcoglycan, is upregulated during myoblast differentiation and is required for fully functional myofibers (Roberds et al., 1994
; Liu et al., 1997
). Human loss-of-function
mutations in adhalin results in limb girdle childhood autosomal recessive muscular dystrophy (SCARMD) (Roberds et al., 1994
). The delayed differentiation in the limb
girdles evident in MyoD
/
embryos (Kablar et al., 1997
)
raised the possibility that adhalin may represent a MyoD target gene. Northern analysis revealed that adhalin is
completely absent in growing MyoD
/
cells (Fig. 5 f).
At the onset of differentiation, adhalin was slightly downregulated in wild-type cells, whereas it steadily increased
in MyoD
/
cultures reaching ~50% of wild-type levels
(Fig. 6 b). Therefore, these data substantiate the delayed differentiation of MyoD
/
myocytes, but do not elucidate whether adhalin is specifically upregulated in the
MyoD-induced embryonic lineage that gives rise to hypaxial musculature.
Analysis of Growth-associated Gene Products
To investigate the continued proliferation of MyoD/
cells in differentiation medium, we examined the mRNA
expression levels of several growth-associated proteins
that have been demonstrated to play important roles in
the control of myoblast differentiation. The plakoglobin-related protein,
-catenin (Butz et al., 1992
), is believed to
play important roles in cellular growth and morphogenesis in response to cellular adhesion and Wnt signaling (Miller
and Moon, 1996
; Barth et al., 1997
). Primary wild-type
myoblasts expressed abundant
-catenin mRNA under
growth conditions. These levels increased threefold after
2 d of differentiation but subsequently decreased (Fig. 6
a). In MyoD
/
cultures,
-catenin mRNA levels were
found to continuously increase and to stabilize at levels that were comparable to that of wild-type cells by day 5 of
differentiation (Fig. 6 a). Detection of
-catenin protein
by immunofluorescence revealed a similar nuclear cytoplasmic distribution in wild-type and mutant myogenic
cells (Fig. 1 c). Therefore, these data do not support a role
for
-catenin in the differentiation deficiency evident in
MyoD
/
myogenic cells.
The PEA3 gene product is upregulated in activated satellite cells in vivo, and has been suggested to be important
for myoblast fusion in vitro (Taylor et al., 1997). Wild-type
primary myoblasts in growth medium expressed very low
levels of PEA3 mRNA. However PEA3 levels increased
about twofold by day 5 of differentiation (Fig. 6 b). By
contrast, MyoD
/
cells in growth medium displayed sixfold higher levels of PEA3 mRNA, which declined
steadily to wild-type levels by day 4 of differentiation (Fig.
6 b). However, this increased level of PEA3 was not associated with enhanced differentiation (see above). Nevertheless, because PEA3 is expressed in activated satellite
cells (Taylor et al., 1997
), these data are consistent with
the notion that primary MyoD
/
myogenic cells represent an intermediate stage between a satellite cell and a myogenic precursor cell.
The p53-inducible cyclin-dependent kinase inhibitor p21/
WAF1 arrests proliferating cells when ectopically expressed (el-Deiry et al., 1993; Harper et al., 1993
) and is
upregulated by MyoD during the differentiation of C2C12
myocytes (Guo et al., 1995
; Halevy et al., 1995
). Moreover,
forced overexpression of p21 in myoblasts drives induction
of differentiation-specific genes (Skapek et al., 1995
). Unexpectedly, we observed no significant differences in p21
mRNA levels between wild-type and MyoD
/
cells, in
growth medium or during differentiation (Fig. 6 c). Therefore, the normal induction of p21 in MyoD
/
myogenic
cells after mitogen withdrawal suggests that p21 induction
is not MyoD-dependent and that p21 requires MyoD to
positively stimulate differentiation.
The insulin-like growth factor, IGF-I, stimulates the
proliferation and inhibits differentiation of cultured myoblasts (Quinn and Roh, 1993; Ewton et al., 1994
; Engert et al.,
1996
). To assess whether IGF-I expression was altered in
the absence of MyoD, we analyzed expression levels in
primary myogenic cultures. Wild-type myoblasts in growth
conditions expressed low levels of IGF-I mRNA and these
levels were rapidly extinguished after mitogen withdrawal (Fig. 6 d). By contrast, MyoD
/
myogenic cells expressed over threefold higher levels of the small IGF-I
mRNA isoforms 1 and 2 (Yamori et al., 1991
) and these
remained constant after transfer of the cells to differentiation medium (Fig. 6 d). Interestingly, the 7-kb pre-IGF-I
mRNA (isoform 3) was not expressed under growth conditions but was rapidly upregulated after 3 d of differentiation. These observations strongly suggest that MyoD negatively regulates IGF-I expression and raises the possibility
that MyoD is required for the repression of IGF-I expression during normal myogenic differentiation.
The MyoD Mutant Cellular Phenotype Is a Cell Autonomous Deficit
The reduced fusion and continued proliferation of MyoD/
myogenic cells under conditions that normally promote
cell-cycle withdrawal and terminal differentiation can be
hypothesized to be a consequence of cell autonomous attributes. For example, the marked reduction in M-cadherin expression (Figs. 1 d and 6 b) and the overexpression
of IGF-I (Fig. 6 d) in MyoD
/
myogenic cells could both
act to inhibit differentiation. Alternatively, MyoD
/
cells
may have a unique developmental identity that precludes participation in the myogenic precursor cell differentiation
program. To explore these possibilities, we mixed different proportions of wild-type myoblasts with lacZ-expressing MyoD
/
myogenic cells, and induced differentiation
by culturing the cells in 5% horse serum for 5 d (Fig. 7).
Importantly, PGK-lacZ expression is unaffected by terminal differentiation in transfected wild-type myoblasts (not
shown). Strikingly, lacZ-labeled nuclei were never detected within any myotubes containing more than two
nuclei (Fig. 7, b and c). Conversely, the differentiation
of wild-type myocytes was completely unaffected by the
presence of high numbers of MyoD
/
myogenic cells
(compare Fig. 7, b and c, with Fig. 7 d). Taken together,
these data support the notion that Myf-5 expression may
define a distinct cell identity in the satellite cell developmental program.
|
Our analysis suggested that in satellite cell-derived
myogenic cell lineage, important aspects of cytomorphology, differentiation, and ultimately fusion of mononuclear
cells into myotubes are highly dependent on MyoD activity and cannot be substituted for by other MRFs. To determine whether the observed phenotypic differences between MyoD/
and wild-type primary myogenic cells
were attributable to MyoD, a MyoD-expression plasmid
was introduced into low passage cells and stable pools of
transfectants were analyzed. Western blot analysis indicated that transfected MyoD
/
cells (termed MyoD+)
expressed the exogenous MyoD protein (Fig. 8 a). By densitometry, the levels were found to be approximately
threefold lower than in wild-type cells but similar to
MyoD levels in C2C12 myoblasts. As expected, untransfected or PGK-Puro transfected MyoD
/
cells did not
express MyoD protein (Fig. 8 a). In growth medium, pools of stable MyoD+ cells displayed an almost complete reversion of the fibroblastlike phenotype and exhibited a
rounded compact cytomorphology similar to wild-type
cells (Fig. 8 b). Transfer of MyoD+ cultures into differentiation medium resulted in increased numbers of MF20-positive differentiated myocytes that displayed the elongated bipolar multinucleated myotube morphology typical
of wild-type myocytes (Fig. 8 b). After 3 d in low serum
medium, the MyoD+ pools displayed fusion indices that
were comparable to wild-type cultures and approximately
fivefold higher than MyoD
/
cells (Fig. 8 c). Differentiated MyoD+ myocytes contained 2.5 ± 0.4 nuclei on average, differentiated wild-type myocytes contained 2.7 ± 0.1 nuclei on average, and differentiated MyoD
/
myocytes
contained only single nuclei (Fig. 8 c). Lastly, transfection
of MyoD
/
cells with a Myf-5 expression plasmid did
not rescue cytomorphology or differentiation potential
(not shown). Taken together, these observations suggest
that expression of MyoD is necessary and sufficient to reestablish the progression of MyoD
/
myogenic cells
through the differentiation program.
![]() |
Discussion |
---|
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---|
Analysis of muscle regeneration in MyoD/
mice led to
the suggestion that MyoD is required for satellite cells to
efficiently give rise to proliferative myogenic precursor
cells (Megeney et al., 1996
). To characterize the phenotype of MyoD-deficient myoblasts and to gain insight into
the role of MyoD in the activation and differentiation of
satellite cells, an in-depth characterization of MyoD
/
primary myoblast cultures was undertaken. The establishment and propagation of stable cell lines can potentially
result in aneuploidy as well as the introduction of additional mutations, which are necessary for immortalization
and continuous proliferation in culture. To avoid such
anomalies, all of our analyses were carried out using newly
established low-passage primary myoblast cultures. Our
characterization of early passage primary cultures reported here differs from the analysis of later passage
MyoD
/
cultures (>15 doublings) that exhibited decreased growth, readily formed multinucleated myotubes,
and grew in an FGF2-independent manner (Megeney et al.,
1996
; data not shown). Therefore, these results underscore
the importance of characterizing early passage (<10 doublings) primary cultures before any adaptation to growth in tissue culture conditions.
Primary MyoD/
myogenic cells exhibited a fibroblastlike cytomorphology, and expressed c-met and high
levels of PEA3 and IGF-I, but did not express desmin,
M-cadherin, or Musk. Transfer of primary MyoD
/
cells
into differentiation medium resulted in formation of reduced numbers of mononuclear myocytes and delayed induction of differentiation-specific markers. Mixing experiments revealed that MyoD
/
cells did not influence the
differentiation of wild-type myocytes and did not fuse with
differentiating myotubes. Forced expression of MyoD rescued both the cytomorphology and the differentiation
deficit. Taken together, these data suggest that Myf-5
expressing cells represent an intermediate between a quiescent satellite cell and a proliferating myogenic precursor cell.
Northern blot analysis revealed that MyoD/
myogenic cells expressed fourfold higher levels of Myf-5 mRNA
and Western analysis revealed an even greater increase in
Myf-5 protein. Similar findings are observed for the in vivo
expression of Myf-5 in the muscle of MyoD-deficient mice
(Rudnicki et al., 1992
). Interestingly, delayed muscle differentiation has also been reported during limb development of MyoD
/
embryos in which Myf-5 expressing
myogenic processors arrive in the limb but differentiate
with markedly delayed kinetics (Kablar et al., 1997
). Embryos lacking MyoD display normal development of trunk
musculature in the body proper, whereas muscle development in limb buds and branchial arches is delayed by
~2.5 d. In contrast, embryos lacking Myf-5 display normal
muscle development in limb buds and branchial arches,
but exhibit a marked delay in development of trunk muscles. Although MyoD-mutant embryos exhibit delayed
development of limb musculature, the migration of Pax-3-expressing cells into the limb buds and subsequent induction of Myf-5 in myogenic precursors occurs normally.
These data, together with the observed regeneration deficit in MyoD
/
muscle, indicate that MyoD and Myf-5
cannot fully substitute for each other during embryogenesis and in satellite cells, and suggest that Myf-5 and MyoD
activate discrete subsets of target genes that differentially define myogenic cell identity.
Forced expression of MyoD in a variety of cell lines induces growth arrest even in the absence of differentiation
(Olson, 1992). Consistent with this, myogenic cells lacking
MyoD displayed inefficient withdrawal from the cell cycle
in response to low mitogens. However, the cell-cycle inhibitor p21 was upregulated to similar levels in wild-type
and MyoD
/
cells in growth medium and following induction of differentiation. In C2C12 myoblasts, expression
of p21 appears to be directly induced by MyoD upon cell-cycle arrest and terminal differentiation (Guo et al., 1995
; Halevy et al., 1995
; Skapek et al., 1995
). Moreover, forced
expression of p21 arrests proliferating cells (el-Deiry et al.,
1993
; Harper et al., 1993
), and induces the terminal differentiation of C2C12 myoblasts (Skapek et al., 1995
). Therefore, the relationship between cell-cycle control and differentiation appears to be uncoupled in primary MyoD
/
myogenic cells. Interestingly, depending on context, p21
expression can either arrest cells via inhibiting cdk activity,
or promote cell division by acting as a cdk4/cyclin D1 assembly factor (LaBaer et al., 1997
). Future characterization of cell cycle control in mutant cells should elucidate
this aspect of the MyoD
/
myogenic cell phenotype.
The PEA3 transcription factor was observed to be expressed at about sixfold higher levels in MyoD-deficient
myoblasts under growth conditions. The ETS-domain
transcription factor PEA3 (Xin et al., 1992) is rapidly induced after muscle damage and forced expression of PEA3
stimulates myogenesis in vitro when overexpressed in
satellite cell-derived cultured myoblasts (Peterson and
Houle, 1997
; Taylor et al., 1997
). These data led to the suggestion that PEA3 is an important regulator of activated
satellite cell function (Taylor et al., 1997
). The lack of correlation between increased expression of PEA3 and differentiation potential in MyoD
/
myogenic cells supports
the assertion that MyoD
/
myogenic cells have an identity distinct from wild-type myoblasts. Interestingly, overexpression of PEA3 is correlated with increased metastatic potential of mammary adenocarcinomas (Trimble
et al., 1993
). Moreover, overexpression of PEA3 in cultured cells directly induces the upregulation of a subset of
matrix metalloproteases (Hassell, J.A., personal communication). After muscle damage, activated satellite cells
readily cross the basal lamina and are capable of migrating
from surviving to damaged areas (Hughes and Blau, 1990
;
Phillips et al., 1990
). By contrast, cultured primary myoblasts injected into muscle exhibit a very poor ability to
migrate away from the injection site (Fan et al., 1996
; Gussoni et al., 1997
). Therefore, it will be of interest to determine whether MyoD
/
cells are more invasive than
their wild-type counterpart.
M-cadherin had been suggested to be expressed in satellite cells (Irintchev et al., 1994). However, recent RT-PCR
analysis indicates that a small minority of satellite cells express M-cadherin mRNA (Cornelison and Wold, 1997
).
Consistent with this we observed markedly reduced expression of M-cadherin in MyoD
/
myogenic cells relative to wild-type cells (see Figs. 1 d and 5 b). This observation raises the possibility that MyoD directly or indirectly regulates the expression of surface adhesion molecules
involved in fusion and differentiation. Interestingly, incubation of antagonistic M-cadherin peptides or antisense
RNA inhibits both myoblast fusion and cell-cycle withdrawal in conditions that normally promote differentiation
(Zeschnigk et al., 1995
). Therefore, these data suggest that
cell-cycle withdrawal during terminal differentiation also
involves cell-cell interactions.
Cellular adhesion is clearly linked to regulation of proliferation, migration, and differentiation. For example,
expression of dominant negative cadherin inhibits proliferation and stimulates terminal differentiation of human
epidermal keratinocytes (Zhu and Watt, 1996). Moreover,
integrin and cadherin synergistically inhibit migration and
promote the aggregation of myoblasts (Huttenlocher et al.,
1998
). Forced overexpression of an effector of adhesion mediated signaling integrin-linked kinase (ILK) in intestinal epithelial cells and mammary epithelial cells induces
the activity of G1/S cyclin/Cdks, downregulates E-cadherin expression, induces nuclear translocation of
-catenin, and results in increased invasiveness (Radeva et al.,
1997
; Novak et al., 1998
). Therefore, examination of integrin and cadherin function in MyoD
/
myogenic cells
should elucidate the role of adhesion in the control of proliferation and migration in early myogenic precursors.
High level expression of IGF-I in L6 myoblasts stimulates proliferation and inhibits differentiation, whereas
lower levels of IGF-I stimulate both proliferation and differentiation (Quinn and Roh, 1993; Ewton et al., 1994
;
Lefaucheur and Sebille, 1995
; Engert et al., 1996
). A striking feature of MyoD
/
cells was the relatively high expression of IGF-I mRNA and its continued upregulation
during differentiation. In contrast, wild-type myoblasts expressed lower levels of IGF-I in growth conditions and
these levels decreased during differentiation. It is interesting to speculate that increased IGF-I expression in
MyoD
/
cultures contributes to the observed differentiation delay. As IGF-I levels rapidly decrease in wild-type
cultures induced to differentiate, an interesting hypothesis
is that MyoD is required to downregulate IGF-I at the onset of differentiation. In MyoD
/
myogenic cells, continued IGF-I expression could result in an autocrine loop
that acts to promote proliferation. However, the presence
of MyoD
/
myogenic cells did not inhibit the differentiation of wild-type primary myoblasts raising the possibility
that expression of other components of the IGF-I signaling pathway are downregulated in wild-type myoblasts.
These data further underscore the assertion that MyoD
/
myogenic cells are distinct from wild-type myoblasts.
Continuous myoblast cell lines lacking MyoD exhibit
somewhat similar traits in comparison to primary MyoD/
myogenic cells. For example, C2C12 cells expressing antisense MyoD RNA, display increased Myf-5 expression,
decreased IGF-II expression, and are defective in differentiation (Montarras et al., 1996
). The brain tumor-derived
BC3H1 myoblast cell line (Taubman et al., 1989
) expresses
Myf-5 but does not express MyoD, and exhibits a differentiation deficit with reduced ability to form multinucleated
myotubes (Brennan et al., 1990
). However, unlike primary
MyoD-deficient myogenic cells, BC3H1 myocytes in differentiation-inducing medium exhibit normal upregulation of myogenin together with normal induction of subsets of MHC isoforms and other differentiation-specific markers (Taubman et al., 1989
; Miller, 1990
; Brennan et al.,
1990
). Forced expression of myogenin in BC3H1 cells is
unable to rescue the differentiation deficit, whereas forced
expression of MyoD confers competency for myogenic differentiation (Brennan et al., 1990
). Forced expression of a
functional MyoD protein in MyoD-deficient cells was sufficient to revert the MyoD
/
cytomorphology and rescue the differentiation defect as evidenced by a dramatic
increase in fusion index of MyoD+ cells. Therefore, these
data support the assertion that lack of MyoD results in a
cell-autonomous deficit in the satellite cell differentiation
program. Taken together, the parallels observed between
primary MyoD
/
myocytes and continuous myoblast
cell lines deficient in MyoD support a unique set of functions for MyoD that cannot be substituted for by Myf-5.
The muscle regeneration deficit in MyoD/
muscle
suggests that expression of MyoD is required for satellite
cells to efficiently form differentiation-competent myogenic precursor cells (Megeney et al., 1996
). RT-PCR
analysis reveals that activated satellite cells first express either Myf-5 alone or MyoD alone, before coexpressing Myf-5 and MyoD, and subsequently progressing through
the myogenic program (Cornelison and Wold, 1997
). Our
analysis of the phenotype of primary MyoD
/
myogenic
cells can be interpreted to suggest that MyoD
/
myogenic cells represent an intermediate stage between a quiescent satellite cell and a myogenic precursor cell. Together, these data suggest the hypothesis that expression
of Myf-5 alone may define an intermediate developmental
stage that provides a mechanism for satellite cell self-renewal. In this model, activated satellite cells expressing
only Myf-5 could undergo cell division and either return to
quiescence by downregulating Myf-5, or alternatively upregulating MyoD and progressing through the myogenic
program (see Fig. 9). Clearly, further analysis of the developmental potential and phenotype of primary MyoD
/
myogenic cells may present a unique opportunity to investigate the early myogenic program of satellite cells.
|
![]() |
Footnotes |
---|
Received for publication 20 July 1998 and in revised form 8 January 1999.
Address correspondence to Dr. Michael Rudnicki, Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario, Canada L8S 4K1. Tel.: (905) 525-9140. Fax: (905) 521-2995. E-mail:
rudnicki{at}mcmaster.ca
M.A. Rudnicki is a research scientist of the National Cancer Institute of
Canada, and a member of the Canadian Genetic Disease Network of Excellence. L.A. Sabourin is a postdoctoral fellow of the Medical Research
Council of Canada, and P. Seale is supported by an National Science and
Engineering Research Scholarship.
This work was supported by grants from the National Institutes of Health and the Muscular Dystrophy Association to M.A. Rudnicki.
![]() |
Abbreviations used in this paper |
---|
BrdU, 5-bromo-2'-deoxyuridine; MHC, myosin heavy chain; MRF, myogenic regulatory factors.
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