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DEVELOPMENTAL DYNAMICS 211:141–152 (1998)
Alteration in Myosatellite Cell Commitment
With Muscle Maturation
JIWEI YANG,1 ROBERT KELLY,2 MOLLY DAOOD,3 MARTIN ONTELL,1 JON WATCHKO,3
and MARCIA ONTELL1*
1Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
2Department of Molecular Biology, Pasteur Institute, Paris, France
3Department of Pediatrics and Magee Womens Research Institute, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania
ABSTRACT
Myosatellite cells are myoblasts
found between the basal lamina and sarcolemma of
myofibers of postnatal mice. The extent to which
these cells are programmed, upon differentiation, to
express isoforms of contractile protein genes specific to the type of fiber with which they are associated has been evaluated in vitro using myosatellite
cells derived from the soleus and the extensor digitorum longus muscles (EDL) of 4-day-old and adult
transgenic mice, which express nuclear localizing
b-galactosidase (nlsb-gal) under the control of the
promoter and 38 enhancer of the gene encoding fast
myosin light chain 3F (MLC3F) (Kelly et al. [1995] J.
Cell Biol. 129:383–396). Cultures were allowed to
differentiate either as myocytes (mononucleated
cells), to prevent possible modification of the myosatellite phenotype by other myonuclei in mosaic myotubes, or as myotubes. Transgene expression was
age related, with 90% and 70% of the myocytes
derived from the neonatal EDL and soleus muscles
(muscles that had not yet achieved their mature
phenotype), respectively, having nuclei encoding
b-gal; 61% and 32% of the myocyte nuclei derived
from myosatellite cells of the adult EDL (a fast
muscle) and the adult soleus muscle (a mixed muscle
containing many slow myofibers), respectively, expressed this transgene. Because myosatellite cells
found in adult muscles are the progeny of those
found in the neonate, an alteration of myosatellite
cell commitment to express this transgene occurs
with muscle maturation. Because expression of the
transgene in neonatal and adult muscle in vivo
reflects the expression of the endogenous MLC3F
gene (Kelly et al. [1995] J. Cell Biol. 129:383–396), it is
likely that expression of the transgene by differentiated myosatellite cells reflects the extent of commitment of these cells to produce MLC3F. A hypothesis
is presented that MLC3F is widely expressed in
developing muscles but eliminated in myofibers that
undergo maturation toward a slower phenotype.
Dev. Dyn. 1998;211:141–152. r 1998 Wiley-Liss, Inc.
Key words: myosatellite cell; commitment;
MLC3F transgene; myogenesis; in
vitro
r 1998 WILEY-LISS, INC.
INTRODUCTION
The formation of skeletal muscle fibers during in
utero development, during muscle regeneration, or in
culture involves the fusion of mononucleated myoblasts
to form multinucleated myofibers. In developing avian
muscle, in which diversity among myotubes is present
from the time myotubes first form (Miller et al., 1985),
it has been demonstrated that there are discrete populations of embryonic and fetal myoblasts (Bonner and
Hauschka, 1974; Miller, 1992; Stockdale, 1992; DeCusella Angelis et al., 1994). Moreover, three types of
embryonic myoblasts can be identified based on the
myosin heavy chain (MHC) isoforms produced by myotubes formed by clonal cultures of these myoblasts
(Miller et al., 1985). In developing mammals, in which
the primary myotubes initially display homogeneity of
their MHCs (Narusawa et al., 1987), differences in
cultured embryonic and fetal myoblasts in terms of
their MHC accumulations (Vivarelli et al., 1988; Smith
and Miller, 1992), reactions to tumor promoters (Cossu
et al., 1988), and reactions to TGF-b (De-Cusella et al.,
1994) have also been reported.
The myoblasts found in postnatal muscle are termed
myosatellite cells (Mauro, 1961). It has been suggested
that these cells are of different lineage from myoblasts
found in developing muscles (c.f., Cossu and Molinaro,
1987). These relatively quiescent, mononucleated cells
found between the sarcolemma and basal lamina of
postnatal muscle fibers are the source of additional
myonuclei for growing muscle fibers. When muscle is
injured, myosatellite cells are stimulated to proliferate;
depending on the extent of the trauma, they may
participate in myofiber repair or may fuse with each
other to form new muscle fibers.
In the chick, it has been suggested that populations of
myosatellite cells differ depending on the type of fiber
(i.e., fast versus slow muscle fibers) with which they are
associated (Feldman and Stockdale, 1991; Hartley et
Grant sponsor: National Institutes of Health; Grant number:
AR36294; Grant sponsor: Wellcome Traveling Fellowship; Grant
sponsor: EC Biotechnology; Grant number: PL 950228.
*Correspondence to: Marcia Ontell, Department of Cell Biology and
Physiology, University of Pittsburgh School of Medicine, South BST—
Room 313, Pittsburgh, PA 15261. E-mail: montell@pop.pitt.edu
Received 22 July 1997; Accepted 24 October 1997
142
YANG ET AL.
al., 1991; Bourke et al., 1995; McFarland et al., 1997).
Recently, there has been interest in determining
whether the myosatellite cells found in mature mammalian muscles also comprise a heterogeneous population.
One way to study this in vivo is to evaluate regenerated
myofibers that form de novo, by the fusion of myosatellite cells. If, upon differentiation, myosatellite cells are
predetermined to accumulate fiber type specific isoforms of contractile proteins, it can be expected that
regenerated myofibers will express the same contractile proteins as the original fibers with which the
myosatellite cells were associated, even in the absence
of innervation. When fast or slow muscles regenerate in
situ, in the presence of their respective nerves, they
accumulate transcripts for contractile protein mRNAs
similar to those found in the original myofibers. However, in the absence of innervation, regenerated slow
muscles accumulate similar transcripts (Esser et al.,
1993) and proteins (Whalen et al., 1990) to those found
in fast muscles. These regeneration studies suggest
that myosatellite cells are not predetermined, at least
in terms of contractile protein gene expression. In
contrast, when head muscles of the cat, which express a
superfast MHC, are minced and allowed to regenerate
in the bed of a limb muscle that normally contains
fibers with type IIb MHC, the regenerated muscle
expresses only superfast MHC (Hoh and Hughes, 1991),
suggesting that head muscle myosatellite cells are
committed. Using retroviral lineage marked myosatellite cells injected into muscles of growing rats, Hughes
and Blau (1992) observed that single clones of myoblasts can contribute to fibers of different types in vivo.
They concluded that de novo myotube formation results
in the expression of the MHC isoform of the muscle
fibers from which the myosatellite cells were derived;
when myosatellite cells fuse with existing myofibers,
any intrinsic commitment of myosatellite cell nuclei to
particular programs of gene expression is overridden by
extrinsic signals (e.g., signals emanating from the
endocrine and nervous systems or from other nuclei
within the mosaic myofiber).
By establishing cultures of myosatellite cells and
determining the phenotype of the myotubes that they
form upon differentiation, one can study the commitment of myosatellite cells in the absence of innervation
and without the influence of endocrine factors. In
differentiated cultures derived from cat jaw muscle,
myotubes fail to expresses superfast MHC (Hill et al.,
1989). Similarly, differentiated cultures of human
muscle do not show diversity of myosatellite cells based
on MHC isoform accumulation (Cantini et al., 1980;
Cho et al., 1993; Mouly et al., 1993; Edom et al., 1994).
However, Dusterhoft and Pette (1993) have demonstrated by immunocytochemistry that myotubes formed
in vitro by rat myosatellite cells derived from the fast
tibialis anterior muscle almost exclusively contain fast
MHCs; 10% of the myotubes formed by myosatellite
cells of the slow soleus muscle coexpress fast and slow
MHC (with the remainder expressing only the fast
isoform). In those myotubes coexpressing fast and slow
isoforms, the slow myosin often accumulates around
specific myonuclei, whereas the fast isoform is more
widely distributed. This suggests that these fibers are
formed from a heterogeneous population of myosatellite cells. Rosenblatt et al. (1996) evaluated myotubes
formed in cultures derived from explants of single,
adult mouse muscle fibers of known fiber type. They
reported that MHCb/slow positive myotubes are more
frequently found in cultures established from myosatellite cells associated with type I myofibers; however, a
substantial number of myotubes established from myosatellite cells associated with type IIa and a lesser
number of myotubes established from type IIb myofibers also contain this isoform. Thus, although there
appears to be a tendency toward commitment of the
myosatellite cells of rodent muscle related to the type of
myofiber with which it is associated, all myosatellite
cells associated with a given fiber type do not have the
same commitment to the expression of particular MHCs.
A more effective way of evaluating the diversity of the
myosatellite cells in vitro is to allow these cells to
differentiate as mononucleated myocytes. This will
prevent possible alterations in the programmed phenotype of individual myosatellite cells by other myonuclei
in multinucleated, mosaic myotubes. In the present
study, myosatellite cell diversity has been evaluated
using cultured primary myoblasts derived from the
extensor digitorum longus muscle (EDL), a 99% fast
twitch muscle in the adult mouse (Thomas et al., 1984),
and the soleus muscle, a 40–60% slow twitch muscle of
the adult mouse (Wirtz et al., 1983; Wigston and
English, 1992). The cultures have been derived from
both newborn and adult muscles of transgenic mice
which express nuclear localizing b-galactosidase (nlsbgal) under the control of the myosin light chain 3F
(MLC3F) promoter and 38 enhancer (Kelly et al., 1995).
MLC3F is one of four alkali MLCs commonly found in
rodent skeletal muscle and is preferentially associated
with fast fibers in the adult mouse. The relative concentration of MLC3F is highest in type IIb, intermediate in
type IIx (IId), and lowest in type IIa myofibers (Wada
and Pette, 1993; Zardini and Parry, 1994). During in
utero development, the transgene is activated in myotubes of hindlimb muscles from 11.5 days of embryonic
development, 3.5 days before high level expression of
the endogenous gene. However, during the fetal period
and in the adult, transgene expression in striated
muscle reflects the expression of the endogenous MLC3F
gene (e.g., it is downregulated in adult muscles, such as
the soleus muscle and the diaphragm, that have few
type IIb fibers) (Kelly et al., 1995). We have determined
the frequency of finding mononucleated myocytes with
b-gal–positive myonuclei and the frequency of finding
myotubes with b-gal–positive myonuclei in differentiated cultures of myosatellite cells derived from newborn and adult transgenic mice. These frequencies have
been compared with the frequency with which myofibers with b-gal–positive nuclei are found in intact EDL
MYOSATELLITE CELL COMMITMENT
143
2C,D). Quantitative analyses revealed that in the newborn EDL and soleus muscles and in the adult EDL,
,90% of the myofiber profiles that contained sublaminal nuclei had nuclei that contained b-gal (i.e., reacted
with X-gal staining solution). The frequency of finding
b-gal–positive myonuclei in the adult soleus muscle
was less than one half (t-test; P , 0.001) of that
observed in the adult EDL (Fig. 3).
MLC3F lacZ Transgene Expression in Myocytes
Derived From Newborn and Adult Mice
Fig. 1. Whole mount, b-gal–stained EDL and soleus muscles of
neonatal and adult MLC3F-nlslacZ-E transgenic mice. All muscles were
exposed to the X-gal staining solution for 1.5 hr, the minimal time for
reaction product to appear in the adult soleus muscle. The staining in the
newborn soleus (Neo Sol) appeared to be slightly less intense than that of
the newborn EDL (Neo EDL). The Neo EDL stained with the same intensity as
the Adult EDL. Little reaction product is seen in the adult soleus (Adult Sol)
muscle, even after prolonged staining. Scale bar 5 1 mm.
and soleus muscles of the transgenic mouse. This has
enabled us to determine the extent to which myosatellite cells are committed to a particular muscle phenotype as a result of the type of fiber with which they are
associated. It has also permitted us to attempt to
evaluate whether the commitment is present at birth
(i.e., before the emergence of the muscle fiber’s mature
phenotype) or whether it develops during the postnatal
period.
RESULTS
MLC3F lacZ Transgene Expression in Newborn
and Adult Whole Muscles
Whole newborn and adult muscles of transgenic mice
(MLC3F-nlacZ-E), which express nlsb-gal under the
control of the MLC3F promoter and 38 enhancer (Kelly
et al., 1995), were incubated with X-gal staining solution. The newborn and adult EDL and the newborn
soleus muscle demonstrated reaction product ,20 min
after exposure to X-gal staining solution; in contrast,
the adult soleus muscle required 1.5 hr of exposure
before any reaction product was detectable. The intensity of staining after 1.5 hr was similar for the neonatal
and adult EDL. The neonatal soleus muscle was slightly
less intensely stained than the EDL, and only minimal
staining of the adult soleus muscle was detected (Fig.
1). Observations of transverse sections stained with
X-gal suggested that there was a similar frequency of
myofiber profiles (i.e., cross-sections of muscle fibers)
containing b-gal–positive nuclei relative to the total
population of fiber profiles containing sublaminal nuclei (myonuclei or myosatellite cell nuclei) in the neonatal EDL and soleus muscles (Fig. 2A,B); this was less in
the adult soleus (Fig. 2E,F) than in the adult EDL (Fig.
In primary cultures established from the adult (8 to
12 weeks old) and neonatal EDL and soleus muscles of
the transgenic mice, ,1% of the myoblasts on plates
exposed to proliferation media expressed the transgene. This percentage was similar to the percentage of
cells on these plates that reacted with antibody to
striated muscle MHC (not shown). For studies in which
the myoblasts were allowed to differentiate as myocytes
(mononucleated cells), replicate plates were seeded at
low density. After 6 days in differentiation media, one
plate was reacted with an antibody specific for skeletal
muscle myosin (MF20; Bader et al., 1982) and with
Hoechst 33258 (Fig. 4C,D) while the others were reacted with X-gal staining solution and Hoechst 33258
(Figs. 4A,B,E,F, 5). Myocytes with b-gal–positive myonuclei and myocytes whose nuclei failed to stain with
X-gal were found in all differentiated cultures (Figs.
4A,E, 5A,C). The nuclei of b-gal–negative myocytes
were readily visible when the cells were viewed with
fluorescence optics that included a Hoechst cube (Figs.
4B,F, 5B,D). To correct for the possibility that myoblasts had not differentiated into myocytes after 6 days
in differentiation media and to account for the few
fibroblasts that were present in the culture, the myosin
antibody stained plates were used to determine the
percentage of mononucleated cells that had differentiated into myocytes (Fig. 4C,D).
By determining the frequency of myocytes with b-gal–
positive myonuclei versus the total number of mononucleated cells (b-gal–positive and b-gal–negative cells)
and by correcting for the percentage of mononucleated
cells that had not differentiated into myocytes, it was
possible to calculate the ratio of myocytes that expressed, or failed to express, the transgene. Ninety
percent of the myocytes cultured from the neonatal
EDL were b-gal–positive, whereas only 70% of those
from the newborn soleus were positive (t-test, P ,
0.001) (Fig. 3). In contrast, in cultures of adult muscle,
61% of the myocytes found in EDL-derived cultures and
32% of the myocytes in soleus-derived cultures exhibited reaction product after X-gal staining (t-test, P ,
0.001; Fig. 3).
MLC3F lacZ Transgene Expression in Cultured
Myotubes Derived From Newborn
and Adult Muscles
For studies in which the myosatellite cells were
allowed to differentiate as myotubes (defined as MHC-
144
YANG ET AL.
Fig. 2. Sections of neonatal (A,B) and adult (C–F) EDL (A,C,D) and
soleus (B,E,F) muscles seen in Figure 1. There is no difference in the
frequency of finding fibers with b-gal–positive nuclei (black nuclei) in the
neonatal EDL (A) and soleus (B) muscles. Sections of the adult EDL (C)
and soleus (E) muscle stained with X-gal are shown. Only the nuclei
containing b-gal are visible with brightfield optics. There are significantly
more fibers with b-gal–positive nuclei in the adult EDL (C) than in the adult
soleus (E) muscles. Figures D and F are of the same sections seen in C
and E, respectively, viewed with fluorescence optics. The white dots seen
in D and F are nuclei stained with propidium iodide. The white arrows in C
and E indicate myofibers that have nuclei that are not b-gal–positive.
White arrows in D and F indicate the same fibers seen in C and E (arrows),
respectively. Nuclei in these fibers are stained with propidium iodide
(arrows, D and F), but these nuclei do not stain with X-gal (C and E). By
comparing brightfield and fluorescence images and by focusing with
phase optics (not shown), it is possible to determine the number of myofiber
profiles containing b-gal–positive nuclei relative to the total populations of
myofiber profiles that contain sublaminal nuclei. Scale bars 5 50 µm.
MYOSATELLITE CELL COMMITMENT
Fig. 3. Percentage of myofibers, myocytes, and myotubes with b-gal–
positive nuclei. The percentage of fiber profiles with nuclei that contained
b-gal in intact EDL (open bar) and soleus (filled bar) muscles of neonatal
and adult MLC3F-nlslacZ mice, percentage of myocytes in differentiated
cultures derived from myosatellite cells of the EDL and soleus muscles of
neonatal and adult transgenic mice, and percentage of myotubes with
b-gal-positive myonuclei in differentiated cultures derived from the myosatellite cells of the EDL and soleus muscles of newborn and adult transgenic
mice are shown. Asterisks indicate significant differences at P , 0.001
between the EDL and soleus muscles of a given group. N, neonatal; A,
adult.
positive cells with two or more myonuclei), replicate
plates were seeded at high density. After 6 days in
differentiation media, in cultures derived from the
neonatal EDL and soleus muscles (Fig. 6A,6B), 90% of
the myotubes were b-gal–positive (Fig. 3). A similar
percentage of the myotubes in adult EDL cultures (Fig.
6C) contained b-gal–positive myonuclei; only 55% of the
myotubes derived from the adult soleus muscle (Fig.
6D) reacted with X-gal staining solution (Fig. 3).
Endogenous MLC3F Expression in Cultured
Myotubes Derived From Transgenic Mice
Because there is some disagreement as to whether
MLC3F is routinely produced in differentiated cultures
of mammalian muscle (Rubinstein and Holtzer, 1979;
Whalen et al., 1979; Caravatti et al., 1982; Cox et al.,
1990; Edom et al., 1994) and some concern that the
accumulation of muscle-specific proteins in differentiated cultures depends on culture conditions (Dusterhoft and Pette, 1993), the ability of differentiated
myotubes in our system to accumulate MLC3F was
evaluated with polyacrylamide gel electrophoresis
(PAGE). MLC3F was found in differentiated cultures of
the adult and neonatal EDL and soleus muscles grown
under the same conditions as those used for the b-gal
assay (Fig. 7).
DISCUSSION
This study establishes for the first time that there is
diversity among adult mouse myosatellite cells in terms
of determination of MLC gene expression, based on the
145
type of myofiber with which the myosatellite cells are
associated. The present study evaluates the expression
of MLC3F using primary cultures derived from muscles
of transgenic mice which express nlsb-gal under the
control of the MLC3F promoter and 38 enhancer (Kelly
et al., 1995). (In intact muscles, the expression of this
transgene mimics the expression of the endogenous
MLC3F gene in adult mice [Kelly et al., 1995].) Heterogeneity of myosatellite cells in vitro is displayed in
cultures derived from muscles of postnatal day 4 mice.
This is before the time that the fibers of intact muscles
express their mature phenotype. However, the neonatal
myosatellite cells retain plasticity, so that with muscle
maturation, some myosatellite cells (or their progeny)
lose the ability to spontaneously express the MLC3F
transgene when they undergo differentiation in vitro.
The extent to which there is diversity among myosatellite cells is difficult to assess in cultures which
differentiate as myotubes, because the phenotype of a
mosaic myotube is likely to reflect that of the majority
of myoblasts that participated in that myotube’s formation (Dusterhoft and Pette, 1993). By evaluating the
expression of the MLC3F transgene in cultures in
which myosatellite cells have differentiated as mononucleated myocytes, more accurate quantification of
the percentage of myosatellite cells programmed to
express the MLC3F transgene has been possible. The
frequency with which myotubes that contain nuclei
expressing this transgene are found in vitro has also
been evaluated. In comparing the frequency with which
cultured myocytes and myotubes express the transgene, it must be remembered that in multinucleated
myotubes in vitro (as in multinucleated myofibers in
the intact muscles), nlsb-gal is capable of migrating for
distances .350 µm from the nucleus that encodes it to
enter noncoding myonuclei (Yang et al., 1997). This
would predict a higher frequency of myotube or myofiber profiles with b-gal–positive myonuclei than myocytes with b-gal–positive myonuclei. This is precisely
the observation made in the present study.
At 4 days postnatal, there are similar frequencies of
myofiber profiles with b-gal–positive myonuclei in the
intact EDL and soleus muscles of MLC3F-nlslacZ-2E
mice. There are no published studies comparing the
frequency of myofibers expressing MLC3F in these two
muscles during the neonatal period. However, in a
recent study (Washabaugh et al., in press), it has been
determined by using competitive polymerase chain
reaction (PCR) that the number of molecules of MLC3F
per nanogram of total RNA is the same in the soleus
and EDL muscles of the mouse at birth. The frequency
of myofibers with b-gal–positive myonuclei decreases
by ,50% with age in the soleus muscle, but there is no
age-related effect on this frequency in the EDL muscle.
This is in keeping with the alterations in muscle
phenotype that occur as these two muscles mature. At
birth, these muscles do not contain any adult fast
MHCs, but at 4 days postnatal transcripts for MHCIIb—
but not other adult fast MHC mRNAs—can be demon-
146
YANG ET AL.
Fig. 4. Differentiated cultures of myosatellite cells derived from neonatal EDL (A–D) and soleus (E,F) muscles that contained myocytes. Plates
have been exposed to either X-gal solution and Hoechst dye (A,B,E,F) or
MF20 antibody that has been visualized with CY3-labeled second
antibody (C) and Hoechst dye (D). A: There are many b-gal–positive
myocytes (mononucleated cell) in the EDL derived cultures, and only a
few b-gal–negative myocytes (arrow). The double arrowheads indicate
myotubes, which have been excluded from the quantification. B: Same
culture as seen in A. When a myonucleus contains significant amounts of
product after X-gal staining, Hoechst staining of that myonucleus is
quenched. The arrow indicates the nucleus of the myocyte seen in A
(arrow) that lacked reaction product. By comparing figures such as A and
B, it is possible to determine the relative number of myosatellite cells that
expressed the transgene upon differentiation. C: All the differentiated
myocytes in the culture react with MF20 antibody. D: Same culture as in
C. Arrows indicate nuclei of mononucleated cells that failed to differentiate
into myocytes (MF20-negative cells). By comparing paired micrographs
such as C and D, it is possible to calculate the percentage of mononuclear
cells that have differentiated into myocytes. These data have been used to
correct the calculation of the frequency of myocytes having b-gal–positive
nuclei. E: In cultures derived from the neonatal soleus muscle, the
frequency of b-gal–negative myocytes (arrows) is greater than for similar
EDL-derived cultures (A). F: Same culture as in E. Arrows indicate the
same myocytes, with b-gal–negative myonuclei, as seen in E (arrows).
Scale bar 5 50 µm.
strated by in situ hybridization (Ontell, unpublished
data). During the perinatal period, the EDL and soleus
muscles contain a considerable amount of the develop-
mental isoform MHCperinatal (MHCpn). It has been
suggested that heterodimers consisting of MLC1F and
MLC3F as well as homodimers of MLC3F are associ-
MYOSATELLITE CELL COMMITMENT
147
Fig. 5. Differentiated cultures of myosatellite cells from the adult EDL
(A,B) and soleus (C,D) that contain myocytes, stained with X-gal (A,C)
and with Hoechst dye (B,D). Arrows in A and C indicate mononucleated
cells whose nuclei fail to express b-gal. B and D are of the same cultures
seen in A and C, respectively. The arrows (B and D) indicate nuclei of
myocytes that were a-gal–negative in A and C (arrows). The double
arrowheads in C and D indicate myotubes that were excluded from the
quantification. The frequency of b-gal–positive myocytes in cultures
established from adult muscles is less than for cultures of neonatal
muscles (compare 5A and 4A, and 5C and 4E). Cultures of the adult
soleus muscle (C) had a lower frequency of b-gal–positive myocytes than
cultures of the adult EDL (A). Scale bar 5 50 µm.
ated with this isoform (c.f., d’Albis and Butler-Browne,
1993). This would account for relatively high levels of
expression of the transgene at 4 days postnatal. Although the MHCpn is lost in both muscles with age, the
EDL undergoes a selective decrease in the percentage
of fibers that contain MHCb/slow, and fast MHCs
become the predominant isoforms (Whalen et al., 1984).
In contrast, the decrease in MHCpn in the soleus
muscle is accompanied by an increase in MHCb/slow
and the appearance of MHCIIa. In adult muscle, MLC3F
is preferentially associated with fibers containing MHCIIb and to a lesser extent with fibers containing
MHCIIx (MHCIId) and MHCIIa (Wada and Pette,
1993; Zardini and Parry, 1994). Thus, the decreased
frequency of myofiber profiles with b-gal–positive nuclei in the soleus muscle with age reflects the decreased
accumulation of MLC3F in that muscle. In contrast, the
higher percentage of fiber profiles containing b-gal–
positive myonuclei in the adult EDL muscle is consistent with that muscle’s fast phenotype and its accumulation of MLC3F.
The present study provides the first evaluation of the
commitment of myosatellite cells associated with myofi-
bers that have not achieved their adult phenotype.
Evaluation of myocyte cultures derived from myosatellite cells of neonatal muscle clearly demonstrates that a
significant proportion of the myosatellite cells in both
the EDL and soleus muscles express the MLC3F transgene upon differentiation. However, the frequency is
significantly higher in the neonatal EDL. How does this
finding fit with the observation that the frequency of
myofiber profiles with myonuclei which contain b-gal in
the intact neonatal muscles is similar? One hypothesis
is that the neonatal cultures are derived from a single
source of myoblasts (myosatellite cells), whereas the
intact muscle fibers contain nuclei derived from two or
possibly three different populations of myoblasts (embryonic or fetal myoblasts 1 myosatellite cells, or a combination of all three types of myoblasts) with different
commitments. An alternate hypothesis would be that a
larger percentage of the myonuclei that contain b-gal in
the intact neonatal EDL are nuclei that express this
fusion protein, while many of the nuclei in the neonatal
soleus muscle that are b-gal–positive are nonexpressing nuclei that have received nlsb-gal by translocation
of the fusion protein or its message from the region of
148
YANG ET AL.
Fig. 6. Differentiated cultures of myosatellite cells from neonatal EDL
(A) and soleus (B) muscles and adult EDL (C) and soleus (D) muscles that
contained myotubes. The majority of the myotubes in both of the cultures
derived from neonatal muscles have b-gal–positive myonuclei (A,B).
Fig. 7. PAGE of myosins extracted from differentiated cultures of the
adult and neonatal EDL and soleus muscles (lanes 2–5). Myosin extracted from the intact EDL (lane 1) served as a standard for identification
of the various fast MLC isoforms. Samples were of differentiated cultures
(myotubes) derived from the myosatellite cells of the adult soleus muscle
(lane 2), adult EDL (lane 3), neonatal soleus muscle (lane 4), and
neonatal EDL (lane 5). All cultures accumulated MLC3F protein (arrows).
the expressing nuclei (Yang et al., 1997). In the present
study, data derived from cultures of myosatellite cells
that differentiated as myotubes fail to support the first
Arrows (A,B) indicate the few myotubes that do not have b-gal–positive
myonuclei. The frequency of finding myotubes with b-gal–negative
myonuclei (arrows in C and D) is higher in cultures derived from the adult
soleus muscle (D) than from the adult EDL (C). Scale bar 5 50 µm.
hypothesis, in that the frequency of finding myotubes
with b-gal–positive myonuclei is similar in differentiated cultures of neonatal myosatellite cells and in
muscle fibers of intact neonatal muscles, despite the
differences in the populations of myoblasts that formed
them. Supporting the second hypothesis is the observation that although both neonatal cultures that have
differentiated as myocytes and those that have differentiated as myotubes have been established by the same
population of myosatellite cells, the frequency of myotubes with b-gal–positive myonuclei is similar to the
frequency of myocytes with b-gal–positive nuclei for
cultures derived from the EDL but greater than the
frequency of myocytes with b-gal–positive nuclei for
cultures derived from the soleus muscle. Again, a major
difference between the myocytes and the myotubes is
the ability of b-gal or its mRNA to migrate from
expressing to nonexpressing myonuclei in mosaic myotubes (Yang et al., 1997).
Evaluation of myocyte cultures of adult muscles
demonstrates a more striking diversity in the commitment of myosatellite cells derived from the soleus and
EDL muscles to express the MLC3F transgene than is
present in corresponding neonatal myocyte cultures. In
cultures derived from neonatal muscles, only 10% of the
myosatellite cells of the EDL and 30% of the myosatel-
MYOSATELLITE CELL COMMITMENT
lite cells of the soleus muscle fail to express the
transgene upon differentiation. In contrast, 39% and
68% of the myosatellite cells of the adult EDL and
soleus muscles, respectively, fail to express the MLC3F
transgene upon differentiation. For the soleus muscle,
the change in the frequency of myoblasts committed to
express the MLC3F transgene which occurs with age
roughly parallels the changes in frequencies of myofiber profiles with b-gal–positive myonuclei in the intact
muscles (Fig. 3). For the EDL, the age-related decrease
in the frequency of myosatellite cells committed to
express the transgene occurs in the absence of any
significant age-related change in the frequency of finding myofiber profiles with b-gal–positive myonuclei.
Here, too, it could be postulated that fewer of the
b-gal–positive myonuclei present in the adult EDL
(compared with the neonatal EDL) are nuclei expressing the fusion protein, and that more of them receive
b-gal by translocation of the fusion protein or its mRNA
from expressing myonuclei in the same myofiber. Because myosatellite cells found in adult muscle are
derived from the population of myosatellite cells found
in the neonate, it is clear that any commitment that the
myosatellite cells of the newborn may have to express
MLC3F is plastic, and with muscle maturation that
commitment is altered.
The results of the cell culture experiments with adult
muscles clearly indicate a marked heterogeneity of the
myosatellite cells of adult muscles in terms of transgene expression. Given that the EDL muscle of the
mouse accumulates significantly more MLC3F (a fast
isoform) than does the soleus muscle and that the
frequency of finding b-gal–positive myocytes in differentiated cultures of the EDL is twice that of the soleus
muscle, the heterogeneity in the expression of the
MLC3F transgene (which is expressed in adult muscle
in a pattern similar to the endogenous gene [Kelly et
al., 1995]) appears to be related to fiber type. To further
evaluate the extent to which the adult myofiber of this
transgenic mouse may imprint or change the commitment of its population of myosatellite cells, it will be
necessary to establish cultures from single transgenic
myofibers of known fiber type and to evaluate the
myocytes that form when its myosatellite cells undergo
differentiation (c.f., Rosenblatt et al., 1996).
To understand the significance of heterogeneity among
the myosatellite cells of the neonate in terms of the
expression of the MLC3F transgene, it is important to
understand the pattern of expression of the endogenous
MLC3F during development. High levels of accumulation of MLC3F mRNA are detected with in situ hybridization in the developing crural muscles of the mouse
before 14 days gestation (E14) (Ontell et al., 1993), at a
time when primary myofibers (but not secondary myofibers) are present in crural muscles (Ontell and Kozeka,
1984; Ontell et al., 1988). At this stage, there is no
preferential accumulation of this transcript among or
within any of the crural muscles (Ontell et al., 1993). At
E17, there is enhanced accumulation of the endogenous
transcript in the more mature anterior compartment
149
muscles (including the EDL) than the less mature
posterior compartment muscles (including the soleus
muscle). However, at birth (with the maturation of the
posterior compartment muscles), the intensities of signal for MLC3F mRNA in the two compartments are
similar with in situ hybridization, and the numbers of
molecules of MLC3F mRNA per nanogram of total RNA
isolated from the EDL and soleus muscles are also
similar (Washabaugh et al., in press). This indicates
that expression of the endogenous gene before birth is
not related to fiber type and that it is only during the
postnatal period that the expression of this gene becomes fiber-type–specific. If this is true, then MLC3F
can be regarded as a developmental isoform whose gene
is actively transcribed in most (if not all) muscle fibers
during in vivo development and whose transcription is
downregulated postnatally in fibers that develop a
‘‘slower’’ phenotype.
The diversity of myofibers in adult muscle, based on
the accumulation of different isoforms of musclespecific proteins, adenosinetriphosphatase activity, and
muscle contractile characteristics, is well established.
In adult muscle it has been clearly demonstrated that
the phenotype of a muscle fiber is plastic, in that it may
be altered by changing the pattern of muscle activity
(c.f., Pette and Vrbova, 1985). It would be of interest to
determine the effect of changing a mature muscle’s fiber
type (by altering muscle activity) on the population of
myosatellite cells associated with that muscle.
The results of the present study are consistent with
the hypothesis that muscle phenotype correlates with
myosatellite cell’s commitment to express specific isoforms of contractile proteins upon differentiation. As
neonatal muscles alter their phenotypes toward the
adult fiber type, the commitment of the myosatellite
cells to express the MLC3F is altered, resulting in a
selective loss of commitment of the myosatellite cells of
slow muscle fibers to express the transgene. The mechanism by which muscle phenotype directly or indirectly
influences commitment of myosatellite cells remains to
be determined.
EXPERIMENTAL PROCEDURES
The EDL and soleus muscles were removed from
newborn (2–4 days postnatal) and adult transgenic
mice (MLC3F-nlacZ-E; killed by an anesthetic overdose) that express nlsb-gal under the control of a 2-kb
MLC3F promoter and 38 enhancer (Kelly et al., 1995).
Some muscles were used to determine the frequency of
finding b-gal–positive nuclei in intact muscles; others
served as the source of myosatellite cells for in vitro
studies.
Intact Muscles
Newborn and adult EDL and soleus muscles (five
muscles for each group) were fixed in 4% paraformaldehyde (1 hr at 4°C), washed twice in phosphate-buffered
saline (PBS), incubated in a staining solution [2 mM
MgCl2, 0.1% Tween-20, 5 mM K3Fe(CN)6, 5 mM
150
YANG ET AL.
K4Fe(CN)6, and 0.1% 5-bromo-4-chloro-3-indolyl-bgalactopyranoside (X-gal) (which was dissolved in dimethyl sulfoxide)] at 37°C for 1.5 hr (Sanes et al., 1986),
cryoprotected in 15% sucrose, and frozen in liquid-N2–
cooled isopentane. Serial, transverse cryosections
(10-µm thick) were restained (with X-gal solution) for 3
hr (to ensure that nuclei in the center of the section had
sufficient exposure to the staining solution) and then
stained with propidium iodide (to permit visualization
of nuclei that failed to exhibit blue reaction product
after exposure to X-gal staining solution). Sections was
viewed with a Nikon FXA microscope (Tokyo, Japan)
equipped with brightfield, phase, and fluorescence optics. One thousand muscle fiber profiles (i.e., crosssections of muscle fibers) observed in five different
transverse sections per muscle were evaluated. Fiber
profiles containing sublaminal nuclei (myonuclei or
myosatellite cells) were scored according to whether the
nuclei they contained were b-gal–positive or b-gal–
negative, and the ratio of b-gal–positive myofibers to
the total fiber population that contained sublaminal
nuclei was determined.
Evaluation of Primary Myosatellite
Cell Cultures
Adult and neonatal muscles were excised and transferred into dishes containing Ham’s F10 serum free
medium. For cultures of adult muscles, the same
muscles from both legs were pooled and minced. For
cultures of neonatal muscles, all of the soleus muscles
and all of the EDL muscles obtained from pups in a
single litter (litter size, 6–12 pups) were separately
pooled. Six cultures were evaluated for each group.
Isolation of myosatellite cells and culture conditions
were as described in Ontell et al. (1992). Briefly,
muscles were incubated at 37°C for 20 min in 0.025%
collagenase in Puck’s saline G and centrifuged (1000
rpm 3 5 min). The pellet was resuspended in 10 ml of
complete medium (Ham’s F10 supplemented with 15%
v/v donor horse serum) and seeded into 100-mm gelatincoated dishes. Bovine basic fibroblast growth factor
(b-FGF) was added (4 ng/ml, twice daily), and the
dishes were incubated at 37°C in a water-saturated
atmosphere containing 5% CO2. After 48 hr, the medium containing the suspended muscle debris was
removed and centrifuged, and the pellet was resuspended in 10 ml of complete medium. After preplating
the suspended fragments for 20 min, they were transferred into a 100-mm gelatin-coated dish. (Preplating
enhances the ratio of myoblasts to fibroblasts in primary culture [Richler and Yaffe, 1970].) Basic-FGF (4
ng/ml) was added to the cultures twice a day. Fortyeight hours after transfer, the cultures were refed with
complete medium plus b-FGF. On day 3, cultures were
briefly (,10–20 sec) treated with 0.025% collagenase at
37°C, a procedure that favors the release of myoblasts
while leaving fibroblasts still attached to the plate.
Cells were centrifuged and resuspended in complete
medium. Three sets of plates were established from the
suspension, with the following purposes.
1. To confirm that proliferating myoblasts derived from
MLC3F-nlacZ-E mice did not express the transgene
in our culture system. To achieve this goal, myosatellite cells were plated at a density of 1 3 105 cells per
60-mm dish and grown for 24–48 hr in complete
medium in the presence of b-FGF. Plates were then
fixed in either 2% paraformaldehyde or AFA fix (63%
ethanol, 3.2% formaldehyde, and 750 mM acetic
acid). Paraformaldehyde fixed plates were stained
with X-gal for 3 hr at 37°C. Cells on AFA fixed plates
were permeabilized with 0.1% Triton, nonspecifically blocked by 1% bovine serum albumin (BSA) in
PBS at 37°C for 30 min, and incubated at room
temperature with MF20 (an antibody to skeletal
muscle myosin; Bader et al., 1982) for 1 hr, followed
by an incubation with goat anti-mouse-IgG CY3
conjugate. By staining with MF20, it was possible to
determine the percentage (if any) of myosatellite
cells that differentiated into myocytes under our
‘‘proliferating’’ conditions. If the expression of the
transgene required differentiation, only those cells
that expressed MHC would be expected to be b-gal–
positive.
2. To evaluate myosatellite cell commitment to a specific phenotype in cultures of differentiated, mononucleated myocytes. To favor the formation of differentiated mononucleated cells (myocytes), the plates
were seeded at low density (1 3 104 cells per 60-mm
dish) and grown overnight in complete medium.
Cultures were then switched to differentiation medium (Ham’s F10/5% horse serum with 1.9 mg/ml of
insulin) and fed once every other day for 6 days.
Plates were fixed, treated as described above, and
reacted with Hoechst 33258 to facilitate counting of
all mononucleated cell on the plate. Plates were
observed with a Nikon FXA microscope equipped for
brightfield and fluorescence microscopy. For data
collection, 20 different fields for each plate were
randomly chosen. From the MF20 reacted plates,
the percentage of mononucleated cells that had not
become differentiated myocytes (under the differentiation conditions described above) relative to the
total number of mononucleated cell on the plates
was determined. From the plates stained with X-gal,
the percentage of myocytes with b-gal–positive nuclei relative to the total number of mononucleated
cells on the plate was evaluated. (The few myotubes
present on these plate were not included in any
evaluations.) Because not all myoblasts had undergone differentiation (as judged from MHC immunostaining) and because a few fibroblasts were found
in each plate, it was necessary to correct for the
percentage of undifferentiated cells to determine
what percentage of myosatellite cells expressed the
transgene upon differentiation.
151
MYOSATELLITE CELL COMMITMENT
3. To evaluate myosatellite cell commitment to a specific phenotype in cultures of differentiated, multinucleated myotubes. Cells were seeded at a density
of 1 3 105 cells per 60-mm dish and grown for 48 hr
in complete medium. They were then switched to
differentiation medium for 6 days, fixed, and stained
as described above. For data collection, 20 different
fields on each plate were randomly chosen. The
percentage of myotubes (defined as cells with two or
more nuclei) containing ‘‘blue’’ myonuclei relative to
the total myotube population was determined.
Representative micrographs of sectioned and cultured
material were obtained with a Nikon FXA microscope
equipped with an Optronics CCD camera (Goleta, CA).
This camera was linked to a computer that had Optimas (Seattle, WA) image analysis software.
Statistical Analysis
Analysis of variance (ANOVA) and t-test were used
for statistical analysis. Differences were considered
significant at P , 0.05.
Accumulation of MLC3F in Cultured Myotubes
Polyacrylamide gel electrophoresis (PAGE) was used
to evaluate the accumulation of the MLC3F protein in
cultures that differentiated as myotubes. Cultured cells
were rinsed with PBS and scraped into a myosin
extraction buffer (300 mM NaCl, 100 mM NaH2PO4, 50
mM Na2HPO4, 1 mM MgCl2, 10 mM Na4P2O7, 10 mM
EDTA, and 0.1% b-mercaptoethanol, pH 6.5) and frozen
at 280°C. Cells were thawed, homogenized with a
polytron homogenizer on ice, and allowed to incubate on
ice in a centrifuge tube for 40 min to extract the myosin.
Tubes were centrifuged for 30 min at 4°C at 14,000 3 g.
The supernatant was diluted 1:10 in a low salt buffer (1
mM EDTA and 0.1% b-mercaptoethanol), and the myosin filaments were allowed to precipitate overnight at
4°C. Filaments were pelleted at 14,000 3 g for 30 min,
resuspend in myosin sample buffer (500 mM NaCl and
10 mM NaH2PO4, pH 7.0), and allowed to incubate
overnight at 4°C. The extract was diluted with an equal
volume of sodium dodecyl sulfate (SDS) sample buffer
(62.5 mM Tris-HCl, 2% SDS, 30% glycerol, 0.001%
bromophenol blue, and 5% b-mercaptoethanol, pH 6.8),
boiled for 2 min, and stored at 280°C. A separating gel
consisted of 12.5% acrylamide, and a stacking gel of 5%
acrylamide was used. Approximately 15 µg of protein
was loaded in each lane of the gel. Electrophoresis was
performed at constant current of 20 mA through the
stacking gel and at 25 mA through separating gel. The
gel was then silver-stained (Oakley et al., 1980),
scanned, and processed with an Adobe Photoshop program.
ACKNOWLEDGMENTS
This study was supported by NIH grant AR36294 (to
M.O.). R.K. was supported initially by a Wellcome
Traveling Fellowship and subsequently by EC Biotech-
nology Grant PL 950228. The authors thank Dr. Simon
Watkins, Structural Biology Imaging Center, for his
help.
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