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DEVELOPMENTAL DYNAMICS 209:233–241 (1997)
MRF4 Can Substitute for Myogenin During Early
Stages of Myogenesis
ZHIMIN ZHU1,2 AND JEFFREY BOONE MILLER1,2*
1Neuromuscular Laboratory, Massachusetts General Hospital, Charlestown, Massachusetts
2Program in Neuroscience, Harvard Medical School, Boston, Massachusetts
ABSTRACT
MRF4, myogenin, MyoD, and
Myf-5 are the four members of the basic helix-loophelix family of muscle-specific regulatory factors
(MRFs). We examined whether MRF4 could substitute for myogenin in vivo by determining if the
myofiber- and MRF4-deficient phenotype of myogenin (2/2) mice could be rescued by a myogenin
promoter-MRF4 transgene. When the transgene
was expressed at a physiological level in myogenin-deficient fetuses, we found that expression
of the endogenous MRF4 gene was restored to
normal levels, whereas MyoD levels were unchanged. Thus, MRF4 can participate in a positive autoregulatory loop and can substitute for
myogenin to activate its own promoter. Myogenindeficient fetuses that expressed the transgene
also had more myosin, more and larger myofibers, and a more normal ribcage morphology
than myogenin-deficient littermates without the
transgene. The transgene failed, however, to restore normal numbers of myofibers or viability to
myogenin-deficient mice, because the D1.6 kb
myogenin promoter fragment was not expressed
in most late-forming myofibers. These results
demonstrate that MRF4 is able to substitute for
myogenin to activate MRF4 expression and promote myofiber formation during the early stages
of myogenesis. Dev. Dyn. 209:233–241, 1997.
r 1997 Wiley-Liss, Inc.
Key words: MRF4; myogenin; myogenesis; regulatory loops; transgenic mouse
INTRODUCTION
MRF4, myogenin, MyoD, and Myf-5 constitute the
basic helix-loop-helix family of muscle regulatory factors (MRFs). During development, each of the MRFs
has a unique pattern of expression (Hinterberger et al.,
1991; Buckingham, 1992; Smith et al., 1994). MRF4 is
the last of the MRFs to be expressed in limb and trunk
muscles, appearing about one day later than myogenin
and about two days later than Myf-5 and MyoD. From
studies of transgenic and knock-out mice, it appears
that Myf-5 and MyoD are required for myoblast commitment; myogenin is required for formation of most
myotubes; and MRF4 plays a role in myofiber maturation and formation of particular muscles (Braun et al.,
1992; Rudnicki et al., 1992, 1993; Hasty et al., 1993;
r 1997 WILEY-LISS, INC.
Nabeshima et al., 1993; Braun and Arnold, 1995;
Patapoutian et al., 1995; Venuti et al., 1995; Zhang et
al., 1995; Block et al., 1996).
Studies of MRF function in cultured cells and knockout mice suggest that individual MRFs may have
unique roles in myogenesis (e.g., Block and Miller,
1992; Rudnicki et al., 1993; Rawls et al., 1995; reviewed
Rudnicki and Jaenisch, 1995). Other studies, however,
suggest that MRFs may be interchangeable; for instance, each MRF is capable of converting many types
of non-myogenic cells into myogenic cells in vitro (Weintraub, 1993) and myogenin can substitute for Myf-5 in
vivo (Wang et al., 1996). However, it is not known if
other pairs of MRFs are similarly interchangeable in
vivo. Previous studies have also shown that individual
MRFs regulate each other’s expression, though the
particular cross-regulatory pathways that operate in
vivo in many cases remain to be determined (Weintraub, 1993). In this study we examine how MRF4 and
myogenin participate in cross-regulatory pathways and
whether MRF4 can substitute for myogenin during
myogenesis in vivo.
Based on molecular phylogeny, myogenin is most
closely related to MRF4, whereas Myf-5 is most closely
related to MyoD, leading to the suggestion that the
more closely related pairs of MRFs would be more likely
to be interchangeable (Atchley et al., 1994). Consistent
with this idea, studies of mice that lack two MRFs
suggest that Myf-5 and MyoD can likely substitute for
each other, but not for myogenin, though the cellular
basis of this complementation is not clear (Rudnicki et
al., 1993; Rawls et al., 1995). On the other hand,
myogenin, when under control of the Myf-5 promoter, is
able to substitute for Myf-5 in vivo (Wang et al., 1996).
The cross-regulation and interchangeability of myogenin and MRF4 had not been tested in vivo, though
studies of cultured cells suggested that MRF4 can
activate the myogenin promoter and that these MRFs
may have distinct functional capabilities (Yutzey et al.,
1990; Chakraborty et al., 1991; Block and Miller, 1992;
Black et al., 1995; Naidu et al., 1995).
To find out if MRF cross-regulation and functional
interchangeability extends to myogenin and MRF4 in
*Correspondence to: Dr. Jeffrey B. Miller, Neuromuscular Laboratory, Massachusetts General Hospital, 149 13th Street, Charlestown,
MA 02129. E-mail: Miller@helix.mgh.harvard.edu
Received 15 November 1996; Accepted 26 February 1997
234
ZHU AND MILLER
vivo, we determined if the abnormal phenotype of
myogenin-deficient mice could be rescued by a myogenin promoter-MRF4 transgene. In earlier work (Block
et al., 1996), we generated and characterized myo1565MRF4 transgenic mice in which an ,1.6 kb fragment of
the myogenin promoter (Cheng et al., 1992) was used to
control expression of MRF4 cDNA. This myogenin
promoter fragment does not require myogenin for expression (Cheng et al., 1995) and, up until about
embryonic day 15 (E15), it appears to be expressed in
myogenic cells in the same pattern as the endogenous
myogenin gene (Cheng et al., 1992; Block et al., 1996).
We have now examined how the MRF4-deficient and
myofiber-defective phenotype of myogenin-deficient mice
(Hasty et al., 1993) is altered by expression of the
myo1565-MRF4 transgene. The results show that MRF4
can substitute for myogenin to activate its own promoter and promote myofiber formation during early
stages of myogenesis in vivo.
RESULTS
To analyze MRF cross-regulation and to determine if
MRF4 can substitute for myogenin, we used crossbreeding to introduce a myogenin promoter-MRF4
transgene (myo1565-MRF4, locus 43; Block et al., 1996)
into myogenin (2/2) mice. After analyzing 114 threeweek-old progeny resulting from these crosses, we
found no surviving myogenin (2/2) mice, either with or
without the transgene. All pups that were both myogenin (2/2) and transgene-positive died within a few
minutes of delivery, as do myogenin-deficient neonates
without the transgene (Hasty et al., 1993; Nabeshima
et al., 1993). If the myo1565-MRF4 transgene had
restored viability to myogenin (2/2) mice, we would
have expected ,16 of the 114 survivors to be myogenin
(2/2) and transgene-positive. Though they died at
birth, myogenin (2/2) pups that carried the transgene
appeared to be bigger and better formed, and to have
larger mechanically-evoked limb movements than myogenin (2/2) littermates that did not carry the transgene (not shown).
The failure of the myo1565-MRF4 transgene to prevent death was not due to a general lack of transgene
expression, because MRF4 mRNA and protein were
expressed in myogenin-deficient mice (Figs. 1 and 2).
The myo1565-MRF4 mRNA, which is larger than the
endogenous MRF4 mRNA, was expressed at least from
embryonic day 17 (E17) to birth in myogenin (2/2) mice
(Fig. 1 and not shown). As noted (Hasty et al., 1993), the
amount of endogenous MRF4 mRNA is much lower in
myogenin (2/2) than myogenin-positive fetuses (Fig.
1). However, when the myo1565-MRF4 transgene (locus 43) was expressed in myogenin (2/2) fetuses, the
amount of endogenous MRF4 mRNA was increased to a
level similar to that seen in wild-type littermates (Fig.
1). The combined amount of endogenous and locus 43
transgene MRF4 mRNAs in E18 myogenin-deficient,
transgene-positive mice was at least as great as the
amount of endogenous MRF4 and myogenin mRNAs in
Fig. 1. The myo1565-MRF4 transgene was expressed and increased
endogenous MRF4 expression in myogenin-deficient fetuses. Total RNA
was prepared from limb muscles of E18–19 littermates with the indicated
combinations of myo1565-MRF4 (locus 43) and myogenin expression.
Samples (10 µg) were separated by electrophoresis and hybridized with
probes for MRF4 or myogenin. Probes were of equal specific activity and
autoradiograms were exposed for equal times (1 day). All samples were
analyzed at the same time; some lanes were juxtaposed for presentation.
The transgene was expressed at about equal levels in the presence or
absence of myogenin and produced a MRF4 mRNA that was larger than
that produced from the endogenous gene. MRF4 mRNA was barely
detectable in myogenin (2/2) fetuses, but was abundant when myo1565MRF4 was expressed.
myogenin-positive littermates that have normal muscle
phenotypes (Fig. 1). In contrast, the transgene did not
affect Myf-5 or MyoD expression, because, with or
without the transgene, Myf-5 mRNA was not detectable
and MyoD mRNA was expressed at the same level in
myogenin-deficient and -positive fetuses (not shown).
Consistent with their low level of MRF4 mRNA,
myogenin (2/2) fetuses without the transgene showed
only weak MRF4 immunostaining in a few muscles and
no staining in most muscles (Fig. 2A, D). In contrast,
MRF4 protein was easily detectable in myogenin (2/2)
mice that carried the transgene (Fig. 2B, E). In these
fetuses, MRF4 was expressed in every myofiber that
formed, and strong staining for MRF4 was found in
every muscle examined including the diaphragm, intercostal, thoracic wall, pelvic, brachial, forelimb, hindlimb, head, and neck muscles. In myogenin-positive
controls without the transgene, MRF4 staining varied
in intensity among muscles, with moderate staining in
jaw muscles (Fig. 2C), weak staining in intercostals,
and no staining in the diaphragm (Fig. 2F). From these
initial protein and mRNA analyses, therefore, it appeared that MRF4 was expressed from the transgene at
a physiological level in myogenin (2/2) fetuses, and
that this MRF4 expression activated expression of the
endogenous MRF4 gene, without affecting expression of
the endogenous Myf-5 and MyoD genes.
We also examined whether the transgene affected
expression of pre- and post-synaptic components of the
neuromuscular junction. By RT-PCR, mRNA for the
alpha subunit of AChR, which is the AChR subunit that
binds alpha-Bungarotoxin, was detectable in myogenindeficient and wild-type mice, with or without the transgene (not shown). Furthermore, by staining with alphaBungarotoxin, we found that myofibers in both
myogenin-deficient and myogenin-positive muscles contained single AChR clusters typical of normal neuromuscular junctions, and this normal clustering was not
affected by the transgene (Fig. 2G–I). Also, we examined myogenin-deficient and normal muscles with anti-
MRF4 IN MYOGENIN (2/2) MICE
235
Fig. 2. MRF4 and acetylcholine receptor expression in fetuses with
different combinations of myogenin and myo1565-MRF4 expression.
Sections were made of E18–19 fetuses that were myogenin-deficient (A,
D, G); myogenin-deficient and myo1565-MRF4-positive (B, E, H), and
myogenin-positive (C, F, I). Sections were stained either for MRF4 using a
horseradish peroxidase system so that MRF4-positive nuclei appear dark
(A–F) or for acetylcholine receptor with a fluorescent system so that
receptor clusters appear white (G–I). MRF4 was barely detectable in
diaphragm and jaw muscles of myogenin-deficient fetuses that did not
express the transgene (A, D); whereas it was expressed at high levels in
these muscles of myogenin-deficient littermates that expressed the
transgene (B, E). In myogenin-positive fetuses, MRF4 was expressed at
high levels in jaw muscles, but not at all in the diaphragm (C, F).
Acetylcholine receptor clusters were found in littermates with all combinations of myogenin and transgene expression (G–I). Scale bar 5 40 µm for
A–F and 60 µm for G–I.
bodies to the synaptic vesicle protein, SV2 (Buckley and
Kelly, 1985). SV2 staining was concentrated near AChR
clusters in both myogenin-deficient and myogeninpositive muscles, with or without the transgene (not
shown).
Though expression of myo1565-MRF4 did not completely restore myogenin-deficient mice to wild-type,
the transgene did produce a partial rescue of the
myogenin-deficient phenotype. First, limbs of E18–19
myogenin-deficient fetuses that expressed the transgene contained several times more myosin heavy chain
(MHC) protein than the limbs of myogenin-deficient
littermates without the transgene (Fig. 3A). However,
even this increased amount of MHC was much less
than found in limbs of myogenin-positive fetuses, which
contained high levels of MHC with or without the
transgene (Fig. 3A).
Transgene expression in myogenin-deficient mice also
improved myofiber morphology. In the absence of the
transgene, the MHC-expressing cells in E18–19 myogenin (2/2) fetuses have a very wide range of morphologies. Some muscles of myogenin (2/2) fetuses, e.g., the
diaphragm (Fig. 4D, G) and body wall muscles, contained
MHC-expressing cells that were mostly small, mononucleated, and not striated, whereas other muscles,
e.g., the intercostal (Fig. 4A) and most limb muscles,
contained a mixture of similarly small myofibers and
more normal-sized myofibers that were multinucleated
and striated. In contrast, when the transgene was
expressed in myogenin (2/2) fetuses, such very small
MHC-expressing cells were no longer present in the
diaphragm (Fig. 4E, H), body wall, or intercostal (Fig.
4B) muscles; rather these muscles contained multinucleated, striated, and apparently normal diameter myofi-
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ZHU AND MILLER
Fig. 3. Effects of myo1565-MRF4 expression on the myosin heavy
chain content of myogenin-deficient muscles. A: Expression of myo1565MRF4 increased the total amount of myosin in myogenin-deficient limb
muscles. Crude extracts were prepared from limbs of E18–19 littermates
that had the indicated combinations of myo1565-MRF4 (locus 43) and
myogenin expression. Equal amounts (10 µg) of total protein were
analyzed in each lane by SDS-PAGE and immunoblotting with mAb F59,
which reacts with all isoforms of mouse MHCs. MHC was more abundant
in the limbs of myogenin-deficient fetuses that expressed the transgene
than in myogenin-deficient littermates that did not express the transgene,
though it was still less abundant than in myogenin-expressing limbs. B:
With or without the transgene, myogenin-deficient muscles had more slow
MHC and less perinatal MHC than myogenin-expressing muscles. MHC
was purified from the crude extracts described in A, and equal amounts
(0.5 µg) of total MHC were analyzed in each lane by SDS-PAGE and
immunoblotting. As indicated, all MHC isoforms were detected and equal
sample loading was confirmed with mAb F59; the slow MHC isoform was
detected with mAb BA-F8; and the perinatal MHC isoform was detected
with mAb BF-34. All samples were analyzed on the same gel and
immunoblot; some lanes were juxtaposed for presentation.
bers, which were similar to myofibers in the same
muscles of myogenin-positive fetuses (Fig. 4C, F, I).
In addition, very large diameter myofibers were
found in myogenin-deficient fetuses that expressed
myo1565-MRF4 but not in myogenin-deficient littermates without the transgene. We identified and measured the diameters of the largest myofibers in sections
of E18 littermates. The mean diameter 6 SD was
12.8 6 2.2 µm for myogenin (2/2) myofibers (n 5 112),
18.2 6 3.5 µm (n 5 123) for myofibers that were
myogenin-deficient but expressed myo1565-MRF4, and
16.1 6 3.4 µm (n 5 129) for myogenin-positive myofibers. The largest fibers in myogenin-deficient muscles
were significantly smaller (P , 0.01) than those in
either myogenin-positive or myogenin-deficient, transgene-positive muscles.
Though expression of the transgene improved myofiber morphology, it did not restore normal numbers of
myofibers. Most muscles of myogenin-positive fetuses,
e.g., the diaphragm and intercostals (Fig. 4C, F), contained many more myofibers than did the same muscles
in myogenin (2/2) littermates that expressed myo1565MRF4 (Fig. 4B, E). Thus, when compared to myogeninpositive muscles, the myofibers in many myogenin
(2/2), transgene-negative muscles were fewer in number and poorly formed; whereas myofibers in the same
myogenin (2/2), transgene-positive muscles, though
still few in number, were well formed.
Expression of the myo1565-MRF4 transgene also
improved, albeit incompletely, the abnormal ribcages of
myogenin-deficient neonates (Fig. 5). As noted by Hasty
et al. (1993), the ribs of myogenin-deficient fetuses are
short, nearly straight, and connect perpendicularly to
the sternum and vertebrae, whereas ribs in myogeninpositive fetuses are longer, markedly curved, and connect to the sternum and vertebrae at acute angles (Fig.
5). We consistently (n 5 17) observed that when the
transgene was expressed in myogenin-deficient fetuses,
the ribs were longer and markedly more curved than in
myogenin-deficient littermates without the transgene,
though not as long or curved as in myogenin-positive
littermates (Fig. 5). The ribs in transgene-expressing
mice also attached to the sternum and vertebrae at an
acute angle, that was clearly different from the nearly
perpendicular attachment in myogenin-deficient mice
without the transgene, though not as acute as in
myogenin-positive littermates (Fig. 5).
Further analyses suggested that the transgene specifically failed to improve development of later forming,
slow MHC-negative fibers in myogenin (2/2) mice.
First, limb muscles from myogenin-positive mice contained a smaller proportion of the slow MHC isoform
and more of the perinatal isoform than did limb muscles
from myogenin (2/2) littermates, with or without the
transgene (Fig. 3B). In muscles of all genotypes, the
Adult Fast Type IIA, IIB, or IIX isoforms were not
detectable, whereas the amount of the embryonic MHC
isoform paralleled the amount of total MHC (not shown).
Furthermore, muscles in myogenin-positive fetuses contained a smaller percentage of slow MHC-expressing
myofibers than those in myogenin (2/2), transgenepositive fetuses (Fig. 4, compare panels F and I to
panels E and H). In the E19 diaphragm, only 10.2% (51
out of 500) of the myofibers expressed slow MHC in
myogenin-positive fetuses, whereas 53% (187 out of
353) expressed slow MHC in myogenin (2/2), transgenepositive littermates (P , 0.0001). Slow MHC expression was also found in diaphragms of myogenindeficient littermates without the transgene (Fig. 4G),
but these fibers were poorly formed making it difficult
to determine the percentage that expressed slow MHC.
Slow MHC-expressing myofibers are among the first to
be formed during myogenesis, whereas later forming
myofibers generally express perinatal but not slow
MHC (Condon et al., 1990). Thus, myogenin (2/2)
fetuses, even with the transgene, appear defective in
later stages of myogenesis.
Lack of expression of the myogenin promoter fragment in late stages of myogenesis prevented full rescue
of the myogenin-deficient phenotype by the myo1565MRF4 transgene (Fig. 6). In E18–19 myogenin-positive
fetuses that carried a myo1565-LacZ transgene (Cheng
et al., 1992), we found that LacZ was expressed in only
a subset of the myofibers and muscles (Fig. 6A–D).
(This expression was not fiber type-specific, because
many mixed fiber type muscles such as the diaphragm
showed no staining.) In contrast, we consistently found
237
MRF4 IN MYOGENIN (2/2) MICE
Fig. 4. The myo1565-MRF4 transgene improved myofiber morphology in myogenin-deficient muscles. Sections were prepared of intercostal
(A–C) and diaphragm (D–I) muscles of E19 littermates with the indicated
combinations of myo1565-MRF4 (locus 43) and myogenin expression.
Sections were analyzed either by immunostaining for all MHC with only
mAb F59 (A–C) or by double immunostaining for both all MHC with mAb
F59 (D–F) and slow MHC with mAb BA-F8 (G–I). Myogenin-deficient
muscles that did not express the transgene (A, D) contained large
numbers of very small MHC-expressing cells; in contrast, such small
myocytes were nearly absent from myogenin-deficient muscles that
expressed the transgene (B, E). Diaphragms of myogenin-deficient,
transgene-expressing fetuses (E, H) contained a larger percentage of
slow MHC-expressing myofibers, though fewer myofibers in total, than the
diaphragms of myogenin-positive littermates (F, I). Scale bar 5 45 µm.
the myogenin protein throughout myo1565-LacZ fetuses, even in muscles that failed to express LacZ (Fig.
6E). Near the time of birth, therefore, the myo1565
promoter fragment was expressed in many fewer myogenic cells than the endogenous myogenin gene, whereas
at earlier stages of myogenesis (,E15) expression of
this promoter fragment more closely matches that of
the endogenous gene in myogenic cells (Cheng et al.,
1992; Block et al., 1996).
Finally, we examined how the quantity, as well as the
location, of transgene expression affected the results.
The analyses described above focused on mice that were
heterozygous for locus 43 of the myo1565-MRF4 transgene. We also produced myogenin-deficient fetuses that
were homozygous for locus 43 and found that they
contained about twice as much of both the transgene
and the endogenous MRF4 mRNAs as heterozygotes
(not shown). Despite the increased MRF4 expression,
homozygotes did not show any improvements in phenotype beyond those seen in heterozygotes. Furthermore,
we found that two additional transgene loci, 42-2 and
42-5, were expressed at ,10% of the level of locus 43
(not shown). The low levels of expression from these loci
were sufficient to produce small increases in the amount
of endogenous MRF4 mRNA in myogenin-deficient fetuses (not shown), but were insufficient to produce the
improvements in diaphragm myofibers or the ribcage
that were produced by the much more highly expressed
locus 43 (not shown).
DISCUSSION
Myogenin-deficient fetuses that expressed the myogenin promoter-MRF4 transgene, myo1565-MRF4, had
more myosin, larger myofibers, increased expression of
the endogenous MRF4 gene, and improved ribcage
morphology compared to their myogenin-deficient littermates that did not express the transgene. On the other
hand, the transgene failed to restore normal numbers
of myofibers or viability to myogenin-deficient mice,
apparently because the ,1.6 kb myogenin promoter
fragment was not expressed in most late-forming myofibers. However, based on the improved phenotype of
238
ZHU AND MILLER
Fig. 5. Expression of myo1565-MRF4 improved ribcage morphology
in myogenin-deficient mice. Alcian Blue was used to stain cartilage in
skeletons from E19 littermates with the indicated combinations of
myo1565-MRF4 (locus 43) and myogenin expression. Ribcages of
myogenin-deficient neonates that expressed the transgene (middle) were
larger in diameter, had longer ribs, and showed more normal angles of rib
attachment than ribcages of myogenin-deficient littermates that did not
express the transgene (left). Missing limbs were used for additional
biochemical or histological assays.
Fig. 6. The myo1565-LacZ transgene is not expressed in the same
pattern as the myogenin protein in myogenin-positive fetuses. Sections of
E18–19 myo1565-LacZ fetuses were stained with X-Gal so that LacZpositive cells appears dark (A–D) or by immunofluorescence with an
anti-myogenin mAb so that myogenin-expressing nuclei appear light (E).
Phase contrast (A) and bright-field (B) views show a back muscle in which
a subset of the myofibers expressed LacZ. The additional phase contrast
(C) and bright-field (D) views show a different shoulder muscle in which
none of the myofibers expressed LacZ. Though the muscle shown in C
and D failed to express the myogenin promoter-LacZ transgene, staining
of an adjacent section showed that many myonuclei did contain the
myogenin protein (E). Scale bar 5 30 µm.
MRF4 IN MYOGENIN (2/2) MICE
myogenin-deficient fetuses that expressed the transgene, we conclude that MRF4 is able, at least in early
stages of myogenesis, to functionally substitute for
myogenin.
MRF4 proved able to substitute for myogenin to
increase expression of the endogenous MRF4 gene.
There is a very low level of MRF4 mRNA in the muscles
of myogenin (2/2) fetuses (Hasty et al., 1993), whereas
myogenin-positive fetuses have high levels of MRF4
mRNA. Expression of the myo1565-MRF4 transgene in
myogenin-deficient fetuses led to a large increase in
MRF4 mRNA produced from the endogenous MRF4
gene, but did not affect expression of either MyoD or
Myf-5. Thus, in the presence of the transgene, MRF4
substituted for myogenin to activate, perhaps indirectly, its own promoter, at least in those fibers that
formed in myogenin-deficient, myo1565-MRF4-positive
fetuses. Such positive autoregulation, if found in all
normal myofibers, could maintain the high level of
MRF4 found in adult myofibers that ordinarily have
little or no myogenin (Musaro et al., 1995).
MRF4 was also able to substitute for myogenin in the
development of early-forming myofibers. In the absence
of myogenin, the myosin-expressing cells in some fetal
muscles, notably the diaphragm, were mononucleate
and not striated (Fig. 5, cf. Hasty et al., 1993; Nabeshima et al., 1993); whereas upon MRF4 expression
from myo1565-MRF4 the same muscles contained a
population of large diameter, striated, and multinucleated myofibers even in the absence of myogenin. These
‘‘rescued’’ myofibers were predominantly slow MHCexpressing and thus likely to have been primary fibers
that formed early in development (Condon et al., 1990).
We cannot be sure that MRF4 restored every aspect of
muscle gene expression to these early myogenindeficient fibers; nonetheless, it is clear that MRF4 is
able to substitute for myogenin to markedly improve
early myogenesis.
Alternative approaches are needed to determine
whether MRF4 is similarly able to substitute for myogenin in fetal myogenesis. Though expression of the
myo1565 fragment appears to closely match that of the
endogenous myogenin gene in myogenic cells until E15
(Cheng et al., 1992, 1995; Block et al., 1996), we found
in older fetuses that the myo1565 promoter fragment
was expressed in many fewer muscles and myofibers
than the myogenin protein. Thus, introduction of the
myo1565-MRF4 transgene into myogenin-deficient fetuses did not test whether MRF4 could substitute for
myogenin in the predominantly fast myofibers that
form during later stages of development. Perhaps the
myo1565 fragment lacks regulatory elements necessary for later expression. Alternatively, this fragment
may be more sensitive than the endogenous promoter to
repression by MRF4, which becomes abundant and
appears to repress myogenin expression in neonates
(Zhang et al., 1995). It might be possible, by using
knock-in technology (Wang et al., 1996) or a longer
myogenin promoter fragment (Buonanno et al., 1993;
Yee and Rigby, 1993), to determine if MRF4 substitutes
239
for myogenin in later stages of myogenesis, though even
these approaches may fail if MRF4 represses the myogenin promoter in late fetal cells.
Neuromuscular junctions appeared to form in myogenin-deficient mice. We found by RT-PCR and staining
with alpha-Bungarotoxin that the alpha subunit of the
AChR was expressed in myofibers and assembled into
normal looking clusters even in the absence of myogenin. In contrast, an initial analysis by a less sensitive
technique, northern blotting, suggested that the mRNA
for alpha-AChR was not expressed in myogenindeficient mice (Hasty et al., 1993), leading to the
suggestion that myogenin-deficient mice die due to lack
of neuromuscular junctions (Venuti et al., 1995). However, we observed that myogenin-deficient and wildtype myofibers, with or without the transgene, had
single large AChR clusters. Such clusters indicate
successful innervation, because never-innervated myofibers have multiple small AChR clusters rather than
single large clusters (Gautam et al., 1996). The presence of a properly localized presynaptic protein, SV2,
and motor neuron axons (Brennan et al., 1996) also
suggests that neuromuscular junctions form in myogenin-deficient mice. Whether these synapses are fully
functional remains to be determined, but, if so, it is
likely that myogenin-deficient neonates die because
they have insufficient muscle mass, specifically in the
diaphragm, to breathe successfully.
To what extent are the MRFs functionally interchangeable? In culture, each of the four MRFs is able to
convert non-myogenic C3H10T1/2 cells into myogenic
cells and each is able to trans-activate particular
muscle-specific promoters (Weintraub, 1993), suggesting that MRFs are functionally interchangeable. On the
other hand, some promoters are trans-activated by only
a subset of the MRFs (e.g., Yutzey et al., 1990) and
analyses of MRF-deficient mice show that each MRF
plays a unique role in normal myogenesis (Rudnicki
and Jaenisch, 1995), suggesting that MRFs may not be
interchangeable. Recently, Wang et al. (1996) showed
by knock-in that myogenin can substitute for Myf-5 in
vivo; and we show here that MRF4 can, as so far tested,
substitute for myogenin in vivo. Thus, in at least two
cases, one MRF can substitute for another. Further
work in vivo is needed to determine whether these MRF
pairs are functionally interchangeable in the reverse
order, i.e., Myf-5 for myogenin or myogenin for MRF4.
It is sometimes argued that natural selection must
eliminate genes that encode interchangeable proteins
(Hochgeschwender and Brennan, 1994). Knock-out experiments, however, suggest that some genes in mice
are dispensable, though such conclusions must be
tempered by the lack of natural selection in a laboratory and the limited number of assays performed
(Colucci-Guyon et al., 1994; Galou et al., 1996). On the
other hand, knock-in experiments with engrailed and
MRF genes (Hanks et al., 1995; Wang et al., 1995), as
well as our work, show clearly that some functions can
be carried out by more than one transcription factor.
Further work will show whether these factors are
240
ZHU AND MILLER
completely interchangeable or have a subset of unique
functions. Perhaps the coding regions of potentially
interchangeable proteins can remain in a genome as
long as their regulatory regions produce distinct expression patterns.
EXPERIMENTAL PROCEDURES
Mice
Transgenic mice carrying a myogenin promoter-MRF4
fusion gene—termed myo1565-MRF4—were generated
previously (Block et al., 1996). The myo1565-MRF4
fusion gene includes the rat MRF4 cDNA (Rhodes and
Konieczny, 1989) under control of nucleotides 21565 to
118, relative to the transcription start site, of the
mouse myogenin promoter region (Cheng et al., 1992).
Adult myogenin (1/2) heterozygotes (mygml/1; Hasty
et al., 1993) as well as adult myo1565-LacZ mice
(Cheng et al., 1992) were a gift of E.N. Olson. We
crossed myo1565-MRF4 heterozygotes (Block et al.,
1996) with myogenin (1/2) heterozygotes to generate
mice that were both myo1565-MRF4 (1/2) and myogenin (1/2). These double heterozygotes were then
crossed with myogenin (1/2) mice. Among the progeny
of these crosses were myogenin (2/2) homozygotes, of
which approximately half also carried the myo1565MRF4 transgene.
DNA and RNA Analyses
A PCR assay identified mice that carried myo1565MRF4 (Block et al., 1996), and a Southern blot assay
identified myogenin (2/2), (1/2), and (1/1) mice (Hasty
et al., 1993). To identify endogenous and transgene
MRF4 mRNAs, 10 µg samples of total RNA isolated
(Chomczynski and Sacchi, 1987) from skinned and
deboned hindlimb muscles of E17 to P1 mice were
separated by electrophoresis, transferred to nylon, and
hybridized with a 32P-labeled MRF4 probe (Block et al.,
1996). Probes for myogenin, MyoD, and Myf-5 mRNAs
were also used (Block et al., 1996; Dominov and Miller,
1996). RT-PCR was used to detect mRNA for the alpha
subunit of the acetylcholine receptor (Wheatley et al.,
1992).
Histology and Antibodies
Skeletons of fetal and newborn mice were prepared
and stained with Alcian Blue to detect cartilage (Zhang
et al., 1995). For immunohistology, all muscle isoforms
of myosin heavy chain (MHC) were detected with mAb
F59 (Miller et al., 1985; Miller and Stockdale, 1986a,b;
Miller et al., 1989; Miller, 1990; Smith and Miller,
1992); the slow MHC isoform was detected with mAb
BA-F8 and the perinatal MHC isoform was detected
with mAb BF-34 (Gorza, 1990); and the Adult Fast Type
IIA, IIB, and IIX, as well as the embryonic, MHC
isoforms were detected with specific mAbs (Gorza,
1990). Skeletal muscle isoforms of myosin light chains
(MLC) were detected with mAbs F310 and T14 (Crow et
al., 1983). Myogenin protein was detected with mAb
F5D (Wright et al., 1991). The MyoD, Myf-5, and MRF4
proteins were detected with specific polyclonal antisera
(Smith et al., 1993, 1994). The synaptic vesicle-specific
SV2 protein was detected with mAb 10H, a gift of K.M.
Buckley (Buckley and Kelly, 1985). 5-bromo-4-chloro-3indolyl-b-D-galactoside (X-gal) was used as substrate to
locate b-Galactosidase activity in sections of myo1565LacZ fetuses (Cheng et al., 1995).
Tissue sections were prepared, incubated with primary antibodies, and stained with appropriate fluorescein-, Texas Red-, Cy3-, or horseradish peroxidase-conjugated secondary antibodies (Kachinsky et al., 1994;
Smith et al., 1993, 1994; Block et al., 1996). Sections
from unfixed or paraformaldehyde-fixed tissues were
used to detect MHC isoforms, whereas only paraformaldehyde-fixed tissues were used to detect other antigens
and for multiple staining protocols (Miller et al., 1985;
Smith et al., 1994). As controls, primary antibodies were
omitted from selected sections on a slide. To identify
acetylcholine receptor (AChR) clusters, sections were
incubated sequentially with biotinylated alpha-Bungarotoxin (Molecular Probes, Eugene OR) and Cy3-conjugated streptavidin (Jackson Immunoresearch, Malvern,
PA), with each reagent used at 1 µg/ml for 1 hr at room
temperature. As a control, staining was abolished when
unlabeled alpha-Bungarotoxin was added at 50 µg/ml.
To measure myofiber diameters, sections made through
corresponding regions of littermates were examined
and the largest myofibers were identified in matched
muscles in each section. The diameters of these large
myofibers were measured, mean diameters 6 S.D. were
calculated, and mean differences were examined for
statistical significance by ANOVA and t-test. Differences in fiber type proportions were examined for
significance using Fisher’s exact test and Chi square
with Yates correction using the InStat computer program (v. 1.12, GraphPad Software, San Diego, CA).
ACKNOWLEDGMENTS
We thank Kathleen Buckley, Rick Boyce, Janice
Dominov, S.F. Konieczny, En Li, Jennifer Moss, Craig
Neville, Eric Olson, Ken Rosen, Nadia Rosenthal, Frank
Stockdale, and W.E. Wright for antibodies, reagents
and/or advice. Z.Z. was supported by a NIH postdoctoral fellowship. This work was supported by grants
from the NIH, USDA, and Muscular Dystrophy Association, and an Established Investigatorship from the
American Heart Association to J.B.M.
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