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First published online 11 January 2006
doi: 10.1242/dev.02249
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1 Department of Biochemistry and Molecular Biology, The University of Texas M.
D. Anderson Cancer Center, Houston, TX 77030, USA.
2 Graduate Training Program in Genes and Development, The University of Texas
Graduate School of Biomedical Sciences at Houston, Houston, TX 77030,
USA.
3 Department of Molecular Biology, The University of Texas Southwestern Medical
Center, Dallas, TX 75390, USA.
* Author for correspondence (e-mail: whklein{at}mdanderson.org)
Accepted 14 December 2005
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Skeletal muscle growth, Myogenic bHLH transcription factors, Myogenin, Conditional knockout mice
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Despite
the obvious importance of skeletal muscle, relatively little is known about
the events that govern its growth following birth.
In perinatal (embryonic day 18.5 until birth) and postnatal life, skeletal
muscles grow through hypertrophy - the accretion of muscle protein within
growing fibers - and through the action of a population of muscle stem cells,
the satellite cells, which arise from embryonic somitic progenitors and reside
underneath the basal lamina of the myofiber. At birth, satellite cells, which
have yet to exit the cell cycle, account for 
30% of the nuclei in rodent
limb muscle (Cardasis and Cooper,
1975). During the first few weeks of life, satellite cells fuse to
growing fibers so that the cells eventually account for at least 50% of the
nuclei inside the fiber. As adulthood is reached, muscle growth declines and
the residual satellite cell population accounts for only 6% of the nuclei in
limb muscles (Cardasis and Cooper,
1975). The residual satellite cells become quiescent until
receptor-mediated signaling triggered by exercise or wounding causes them to
re-enter the cell cycle, proliferate and differentiate into muscle
(Seale et al., 2001;
Charge and Rudnicki, 2004).
Thus, postembryonic skeletal muscle formation takes place in two phases: a
rapid, intense growth phase that occurs in perinatal and early postnatal life,
followed by a phase of muscle maintenance and repair adapted for later periods
of life.
Some progress has been made in identifying transcription factors that are
essential for perinatal and postnatal skeletal muscle growth. Pax7, a
paired-box transcription factor, is expressed in satellite cells and is
required for their specification (Seale et
al., 2000). More recent studies have shown that Pax3, a close
relative of Pax7, is also involved in postnatal muscle growth and
Pax3/Pax7-positive progenitor cells appear to be the source of postnatal
satellite cells (Relaix et al.,
2005; Kassar-Duchossoy et al.,
2005). Serum response factor (SRF), a transcription factor that
controls the transcription of muscle genes by interacting with Mrtfa, a member
of the myocardin family of transcriptional co-activators, is also required for
perinatal and postnatal muscle growth (Li
et al., 2005).
The mechanisms by which satellite cells are activated in adult muscle
maintenance and repair are thought to be similar to those underlying the
specification and differentiation of embryonic myoblasts
(Buckingham, 2001;
Seale et al., 2001). In the
embryo, initial muscle development is controlled by the myogenic basic
helix-loop-helix (bHLH) transcription factors in conjunction with the Mef2
MADS-box family of transcription factors
(Yun and Wold, 1996). Myod1,
Myf5, Mrf4 (Myf6 - Mouse Genome Informatics), myogenin and Mef2 isoforms are
all expressed in activated satellite cells in temporal patterns analogous to
those seen in embryonic development
(Cornelison and Wold, 1997).
Among the myogenic bHLH factors, only Myod1 has been implicated in skeletal
muscle formation in postnatal life. Satellite cells isolated from
Myod1-knockout mice fail to fuse and double knockout mice harboring
mutations in the genes encoding Myod1 and dystrophin develop skeletal
myopathies in postnatal life that are not seen in mice lacking only one of the
genes (Megeney et al., 1996;
Sabourin et al., 1999;
Cornelison et al., 2000).
If postnatal muscle growth recapitulates the regulatory events that occur
during embryonic myogenesis, then we would expect the myogenic bHLH factors to
function after birth as they do during embryonic myoblast specification and
differentiation. Myf5, Mrf4 and Myod1 would therefore be predicted to have
functions in specifying postnatal myoblasts with Myf5 and Mrf4 acting upstream
of Myod1, whereas myogenin, Mrf4, and Myod1 would be crucial for the
subsequent differentiation of those myoblasts into myocytes and myofibers
(Yun and Wold, 1996;
Penn et al., 2004;
Kassar-Duchossoy et al., 2004;
Tapscott, 2005). Single-,
double- and triple-knockout mice have been created that harbor null mutations
in up to three of the four myogenic bHLH regulatory genes (reviewed by
Pownall et al., 2002), and
analysis of embryonic skeletal muscle development in these mice has revealed
complex relationships among the four factors
(Rawls et al., 1998;
Valdez et al., 2000;
Kassar-Duchossoy et al.,
2004). For example, Myf5 or Myod1 define independent myogenic
compartments during embryonic development but these are not revealed in single
Myf5 or Myod1 knockout mice
(Kablar et al., 2003). The
conventional model in which Myf5 and Myod1 act together to specify myogenic
fate requires revision in light of recent studies indicating that Mrf4 is also
necessary for myoblast specification
(Kassar-Duchossoy et al.,
2004).
Notably, of the single-knockout mice, only Myog-null mice exhibit
severe skeletal muscle deficiencies, thereby demonstrating that myogenin is
absolutely required for embryonic muscle differentiation
(Hasty et al., 1993;
Nabeshima et al., 1993;
Venuti et al., 1995). Unlike
the other myogenic bHLH factors, myogenin has no redundant or compensatory
mechanisms to replace its function.
Given its essential role in embryogenesis, myogenin might be expected to have an analogous function in satellite cells during postnatal muscle growth. To test this hypothesis, we generated floxed alleles of Myog and used Cre-recombinase mouse lines to delete Myog before and after embryonic muscle development. Our results suggest that myogenin-independent mechanisms can compensate for the loss of myogenin and that these mechanisms are likely to be crucial in regulating postnatal skeletal muscle growth.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) was inserted into the first intron at a unique
BamHI site at position +620 for positive selection. A loxP
sequence was inserted into a unique BsmBI site at position -303 in
the promoter region of Myog. A second construct was generated with a
modified loxP sequence inserted at position +40 in the 5'
untranslated region (UTR) of Myog. The modified loxP
sequence is recognized by Cre recombinase but does not form a hairpin loop and
therefore should not interfere with translational initiation
(Sauer, 1998). For negative
selection, the MC1TK cassette (McMahon and
Bradley, 1990) was inserted into the SalI site of
pBluescript.
Generation of embryonic stem cells and mouse lines
The targeting constructs were electroporated into AB1 embryonic stem (ES)
cells (provided by Richard Behringer, The University of Texas M. D. Anderson
Cancer Center) and positive colonies were selected using G418 for neomycin
resistance and fialuridine (FIAU) for thymidine kinase sensitivity. To
identify targeted ES cells, Southern blot analysis was performed with DNA from
individual colonies following enzymatic digestion with BamHI and
HindIII, and targeted DNA was detected as described by Vivian et al.
(Vivian et al., 1999).
Correctly targeted ES cells were amplified and injected into C57BL/6
blastocysts. The resulting chimeric mice (a mixture of 129 and C57BL/6
strains) were mated to C57BL/6 mice to establish mice harboring the floxed
Myog allele in their germline. Both the loxP promoter
construct and the loxP 5' UTR construct yielded germline mice;
four lines were established for the promoter construct and one line for the
5' UTR construct. In this study, we describe results obtained with the
promoter construct, although the 5' UTR construct gave similar results
(data not shown). The loxP promoter construct is referred to
hereafter as Myogflox.
Mice harboring the Myogflox allele were mated either to
CMV-Cre transgenic mice, which express active Cre recombinase in the
single-cell zygote (Arango et al.,
1999) or to CAGGCre-ERTM transgenic mice, which
ubiquitously express a conditional Cre recombinase that is activated by
intraperitoneal injection of tamoxifen
(Hayashi and McMahon, 2002).
The deleted allele is referred to as Myogflox
. The
genotypes of the mice harboring the Myogflox allele and
either the CMV-Cre or CAGGCre-ERTM transgene were
determined by polymerase chain reaction (PCR) using primers described by
Ovchinnikov et al. (Ovchinnikov et al.,
2000) or by Southern blot hybridization of yolk sac DNA as
described by Rawls et al. (Rawls et al.,
1995). Two milligrams of tamoxifen was injected intraperitoneally
into pregnant females at either E14.5 or E17.5 of pregnancy
(Hayashi and McMahon, 2002).
All mouse experiments were conducted according to the US Public Health Service
Policy on Humane Care and Use of Laboratory Animals. The procedures for use of
mice in this study were approved by The University of Texas M. D. Anderson
Cancer Center Institutional Animal Care and Use Committee.
Histology and immunostaining
Dissected skinned skeletal muscle tissue from hindlimbs, diaphragms and
tongues was prepared for paraffin embedding by fixing in 0.2% gluteraldehyde
and 2% formaldehyde in phosphate-buffered saline (PBS) for 1 hour, rinsing
three times in PBS and fixing in 3.7% formaldehyde overnight. The tissue was
then washed in increasing concentrations (50%, 75%, and 100%) of isopropanol
for 2 hours before embedding in Paraplast (Structure Probe, West Chester, PA).
The embedded tissue was sectioned into 6 µm slices and stained with
Hematoxylin and Eosin. For frozen sections, dissected, skinned muscle tissue
was incubated overnight in 30% sucrose and 0.1 M sodium phosphate buffer (pH
7.2) at 4°C. The tissue was then embedded in OCT (Sakura, Tokyo, Japan)
and rapidly frozen with an ethanol/dry ice mix. The frozen tissue was
sectioned into 10 µm slices that were then fixed in 4%
paraformaldehyde.
Antibody staining was performed using the HistoMouse-Max kit (Zymed, San Francisco, CA) with a 1:50 dilution of anti-myogenin antibody M-225 (Santa Cruz Biotechnology, Santa Cruz, CA) for embryonic skeletal muscle or a 1:250 dilution of anti-myogenin antibody F5D (NeoMarkers, Fremont, CA) for adult skeletal muscle.
Quantitative PCR and reverse-transcriptase PCR
To quantify the extent of Cre recombinase-mediated deletion, dissected tail
or hindlimb muscle was flash-frozen in liquid nitrogen. Genomic DNA with the
genotype Myogflox/flox;CAGGCre-ERTM/+ or
Myog+/+;CAGGCre-ERTM/+ was isolated and used
as a template for quantitative PCR using the iCycler iQ system and iQ SYBR
Green SuperMix (Bio-Rad, Hercules, CA). Cre recombinase-mediated deletion was
intended to remove the first exon of Myogflox so that
Myog primers corresponding to sequences in the first exon of
Myog would amplify fragments specifically representing undeleted
Myogflox. As a control, Myf5 primers were used to
amplify fragments representing the first exon of Myf5, which was
assumed to be unaltered in the genomic DNA. The primer sequences used for this
study are described below. The ratio of undeleted Myogflox
to Myf5 was normalized to the same ratio obtained from genomic DNA of
wild-type mice; this value was the relative fraction of Myog
remaining in the genomic DNA.
For reverse-transcriptase (RT)-PCR of the myogenic regulatory factors, RNA was isolated from dissected muscle tissue using TRI Reagent (Molecular Research Center, Cincinnati, OH). Ten micrograms of RNA was treated with DNase (Invitrogen, Carlsbad, CA), and 1 µg was used as a template for the RT reaction using the SuperScript first-strand synthesis system for RT-PCR (Invitrogen). Each PCR experiment was performed with one tenth of the RT product using the iCycler iQ system and iQ SYBR Green SuperMix (BioRad).
To determine the transcript levels of Igf1, Igf2 and Igfbp2, RNA was isolated from a 30 µm section of hindlimb with skin and fat removed. RNA was isolated using an RNeasy fibrous tissue kit (Qiagen, Valencia, CA). Two micrograms of RNA were used as a template for the RT reaction using Superscript as above. Each PCR reaction was performed with one-fiftieth of the RT product using the Applied Biosystems 7500 Fast real-time PCR system and SYBR Green Master Mix (Applied Biosystems, Foster City, CA).
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Measurement of fiber diameters and body weights
Muscle fiber diameters were calculated by measuring the narrowest diameter
of the fiber with the ruler function of Adobe Photoshop software 7.0.1 (Adobe,
San Jose, CA). Four sections from hindlimbs containing 10 fibers per section
were analyzed from a single 10-week-old mouse for each genotype. Mice were
weighed 10 days after birth and weekly thereafter. Differences between
genotype groups in weights at 6 weeks were analyzed using a two-tailed
Student's t test with significance set at P<0.05.
Statistical analyses
The results were analyzed using a two-sided Wilcoxon rank-sum test (normal
approximation with continuity correction: significance set at
P<0.05). Statistical analyses were performed by Carla Warneke
(Department of Biostatistics and Applied Mathematics, The University of Texas
M. D. Anderson Cancer Center).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). A loxP site was inserted into the promoter region
of Myog and a neomycin cassette with flanking loxP sites was
inserted into the first intron to generate targeted ES cells containing the
Myogflox allele (Fig.
1A,B). Four Myogflox/+ heterozygous mouse
lines were established for further investigation; mice containing the
Myogflox allele are available upon request.
To determine whether functional myogenin protein was expressed from the
Myogflox allele, Myogflox/+ mice were
mated to mice heterozygous for a null allele of Myog
(Myog+/-) to generate mice with a
Myogflox/- genotype
(Fig. 1C). These mice were
viable and fertile, in contrast to the neonatal lethality seen in
Myog-/- mice (Hasty et
al., 1993). Thus, the Myogflox allele was
fully functional, despite the presence of a loxP site in its promoter
and a floxed neomycin cassette in its first intron.
We next determined whether Myogflox behaves as a null
allele following Cre recombinase-mediated deletion. To do this, we mated
Myogflox/flox mice to transgenic mice expressing
CMV-Cre to generate a floxed allele,
Myogflox, that lacked the first exon of
Myog (Fig. 2A).
CMV-Cre mice first express Cre recombinase in the zygote, several
days before the formation of skeletal muscle
(Arango et al., 1999). As
expected, Myogflox/flox mice died as
neonates and had skeletal muscle deficiencies resembling those of
Myog-/- mice (Fig.
2B,C). Diaphragms from E18.5
Myogflox/flox embryos were abnormally thin
and lacked myofibers, closely resembling diaphragms from E18.5
Myog-/- embryos (Fig.
2B). Hindlimbs from E18.5
Myogflox/flox embryos also showed skeletal
muscle abnormalities and, as expected, myogenin protein was not detectable
(Fig. 2C). We also observed a
two- to threefold increase in Myod1 expression using quantitative
RT-PCR (data not shown); this upregulation of Myod1 was not observed
previously in Myog-/- embryos
(Hasty et al., 1993;
Venuti et al., 1995). In
contrast to Myogflox/flox embryos,
corresponding hindlimbs from Myogflox/+ embryos
showed normal skeletal muscle in the hindlimbs and efficient expression of
myogenin (Fig. 2C). These
results demonstrate that the Myogflox allele can be
effectively deleted using the Cre-loxP system.
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If the Myogflox/flox mice were fully
viable, a 1:1 ratio of Myogflox/flox pups to
Myogflox/flox pups would be expected.
However, after either day of tamoxifen injection, we consistently found a
ratio of 2:1 in the offspring from Myogflox/flox;+/+
x Myogflox/flox;CAGGCre-ERTM/+ matings
(Fig. 3A,B). These results
indicated that 50% of the Myogflox/flox mice - those that
inherited the CAGGCre-ERTM transgene and therefore had a
Myogflox/flox genotype - were dying before
P10. It is not clear why these mice did not survive as the
Myogflox/flox mice that did survive were
fully viable, although by P10 they were noticeably smaller than their
Myogflox/flox littermates (see below).
Myogflox/flox and
Myogflox/flox embryos were found at 1:1
ratios at E18.5 (data not shown), suggesting that 50% of the
Myogflox/flox mice died immediately before
birth or early in postnatal life. The reduced viability associated with
Myogflox/flox mice did not appear to be
attributable to defects in skeletal muscle growth (see below) and we did not
find any gender bias in the surviving mice. A possible explanation for the 2:1
ratio was that a dominant modifier gene in the mixed 129/C57BL/6 genetic
background was inherited by 50% of the offspring and enhanced a lethal
phenotype in Myogflox/flox mice. We have not
yet pursued the possibility of a modifier gene, and the basis for the
lethality observed in some of the
Myogflox/flox mice remains unclear.
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/flox genomes was probably an
overestimate because the high cycle threshold values for quantitative PCR with
DNA from Myogflox/flox mice tended to
inflate the final calculation. However, even if 20% of the cells in
Myogflox/flox mice contained genomes with
the Myog gene intact, this would still result in a Myog-null
phenotype. We showed in a previous study that chimeric embryos containing
mixtures of 60% wild-type and 40% Myog-/- myoblasts do not
survive past birth and have skeletal muscle defects identical to those seen in
Myog-/- embryos (Myer
et al., 1997). Additionally, when Myog expression in
embryos containing a low-expressing Myog allele is less than 50% but
greater than 25% of the expression in Myog+/+ control
embryos, the extent of skeletal muscle deficiencies is the same as that seen
in Myog-/- mice
(Vivian et al., 1999).
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/flox mice at 10 weeks after birth
and the extent of Cre-mediated deletion was determined by Southern blot genome
hybridization. Only low levels of the floxed Myog sequence were
detected in hindlimb DNA (Fig.
4B), as was the case with the tail DNA. Moreover, the expression
of Myog transcripts Myogflox/flox
hindlimbs was one-fifth (Fig.
4C, left panel) and one-fiftieth
(Fig. 4C, right panel) of that
of control littermates at 3 days and 2 weeks after birth.
Myogenin protein expression was also greatly attenuated in
Myogflox/flox mice but reduced expression
was not seen immediately. When tamoxifen was injected into pregnant females at
E15.5 and the levels of myogenin expression determined 3 days later,
expression levels were the same in embryos that inherited the
CAGGCre-ERTM transgene as those in embryos that did not (data
not shown). Tail DNA from embryos that inherited the
CAGGCre-ERTM transgene had only 6% of the floxed Myog
sequence compared with control embryos (data not shown). However, when
tamoxifen was injected at E17.5 and the levels of myogenin expression
determined 5 days later (P3), we were unable to detect myogenin expression in
sections of hindlimb muscle from
Myogflox/flox pups
(Fig. 4D, right panel),
although control Myogflox/flox littermates showed robust
expression in myofiber nuclei (Fig.
4D, left panel). In this case, genomic Myog in the
Myogflox/flox mice was 19% of the level in
control mice. These results indicate that sometime between 3 to 5 days after
Myog was deleted, myogenin protein was largely depleted from the
skeletal muscle of Myogflox/flox mice.
Fig. 5A-D) and tongues
(Fig. 5E,F) of 10-week-old mice
was histologically normal regardless of whether the mice had inherited the
CAGGCre-ERTM transgene. Nuclei were correctly positioned at the
periphery of the myofibers and muscle striations appeared grossly normal.
Skeletal muscle from diaphragms was also normal with one exception: the
diaphragms from one pair of Myogflox/flox
littermates were notably thinner than their Myogflox/flox
counterparts (Fig. 5G,H) and
resembled the muscle-deficient diaphragms observed in
Myog-/- embryos. It was difficult to reconcile this
diaphragm defect with the overt behavior of the mice; they breathed normally
and moved about in a normal manner. This was the only instance in which muscle
abnormalities were seen and all other
Myogflox/flox mice examined (n=20)
had normal musculature in their diaphragms
(Fig. 5I,J).
The growth of myofibers in perinatal and postnatal life is associated with
increases in fiber diameter, number of fibers and density of fiber nuclei
(Allen et al., 1979). It was
therefore possible that the loss of myogenin would result in a reduction of
myofiber diameter without affecting muscle histology per se. However, this
proved not to be the case. The diameters of myofibers from hindlimbs of
Myogflox/flox and
Myogflox/flox mice were indistinguishable
from each other (Fig. 5K).
Moreover, the Myogflox/flox mice were as
active as Myogflox/flox control mice and showed no signs
of fatigue or inability to perform routine tasks, including, running, jumping
and grasping. These results indicate that the absence of myogenin did not
alter postnatal skeletal muscle growth or function.
Expression of muscle-specific genes in Myogflox/flox mice
The lack of noticeable skeletal muscle defects in adult
Myogflox/flox mice was unexpected as
deleting myogenin in the germline or zygote leads to severe muscle
deficiencies in embryogenesis and causes neonatal death
(Hasty et al., 1993;
Nabeshima et al., 1993). It is
possible that the loss of myogenin in early postnatal life led to an
upregulation of Mrf4, Myod1 or Myf5. If so, abnormally high
levels of these related transcription factors might functionally compensate
for the loss of myogenin. Using quantitative RT-PCR, we determined the levels
of expression of Mrf4, Myod1 and Myf5, and the control,
Mck, in the hindlimbs of Myogflox/flox and
Myogflox/flox mice at 3 days and 2 weeks
after birth. Mck expression is greatly attenuated in skeletal muscle
of Myog-/- embryos
(Hasty et al., 1993;
Rawls et al., 1998) and is
thought to be a direct target of myogenin
(Chakraborty et al., 1991).
However, there were no significant differences in Mck expression
between Myogflox/flox and
Myogflox/flox mice
(Fig. 6). This result implies
that other myogenic bHLH factors had replaced myogenin in maintaining
Mck expression in postnatal life.
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), but we observed less than twofold downregulation of
Mrf4 expression in Myogflox/flox
mice compared with Mrf4 expression in
Myogflox/flox control mice
(Fig. 6). The lack of notable
downregulation of Mck and Mrf4 expression indicates that the
loss of myogenin did not have a major impact on skeletal muscle gene
expression in postnatal life.
Previous studies have shown that Myod1 and Myf5
expression levels are not significantly different in
Myog-/- mice compared with wild-type mice
(Hasty et al., 1993;
Venuti et al., 1995). We found
a twofold and 1.5-fold upregulation in Myod1 expression in the
skeletal muscle of Myogflox/flox mice at 3
days and 2 weeks after birth, respectively, compared with
Myogflox/flox control mice
(Fig. 6). Although the
differences in Myod1 expression between
Myogflox/flox mice and
Myogflox/flox mice were significant, the extent of the
upregulation was probably too low to contribute to functional compensation. In
a previous study, we showed that overexpressing Myod1 from a strong
constitutive promoter was not sufficient to restore normal skeletal muscle
differentiation in Myog-null ES cells
(Myer et al., 2001), a
conclusion consistent with in vitro studies showing that Myod1 preferentially
activates early, and myogenin late, muscle genes
(Bergstrom et al., 2002;
Penn et al., 2004).
Fig. 6 also shows that
Myf5 expression was not significantly different in
Myogflox/flox and
Myogflox/flox mice. This expression analysis shows that
the absence of myogenin in postnatal life did not markedly upregulate the
expression of any of the myogenic bHLH factors and suggests that these factors
are unlikely to compensate for the absence of myogenin. Mef2c is also
expressed in postnatal skeletal muscle
(Wang et al., 2001) but
Mef2c expression was not significantly affected by the loss of
myogenin (data not shown).
Small size of Myogflox/flox mice
Despite the normal histological appearance of their skeletal muscle,
Mygflox/flox mice were uniformly smaller
than Myogflox/flox mice.
Fig. 7A shows a representative
image of Myogflox/flox and
Myogflox/flox littermates at 12 weeks after birth. The
Myogflox/flox mouse weighed 30% less than
its Myogflox/flox littermate, although it was not thinner
or wasted, but rather proportionally smaller. To show that the smaller size of
Myogflox/flox mice was the result of a
slower growth rate, littermates produced from a pregnant female injected with
tamoxifen at E17.5, were weighed from 1.5 to 9 weeks after birth. Throughout
this time, Myogflox/flox mice weighed less
than their Myogflox/flox littermates
(Fig. 7B). Moreover, the
smaller size and decreased weight was not specific to this litter, as the
weights of 6-week-old Myogflox/flox and
Myogflox/flox mice from multiple
Myogflox/flox;+/+ x
Myogflox/flox;CAGGCre-ERTM/+ matings were
significantly different (Fig.
7C). The mean weight for Myogflox/flox mice
was 20.0 g, and for Myogflox/flox mice, 17.5
g.
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/flox
mice ate and drank with the same regularity as their
Myogflox/flox littermates. We have not established whether
Myogflox/flox mice consumed the same number
of calories as the wild-type control mice, but they showed no signs of loss of
appetite and they were not forcibly excluded from food or water sources by
their larger littermates. Collectively, these results show that the absence of
myogenin in postnatal life had an unexpected consequence on normal body
growth. The smaller size of Mygflox/flox
mice was clearly a secondary effect of the absence of myogenin because
Myog expression is restricted to skeletal muscle and muscle
precursors throughout life (Cheng et al.,
1993; Cheng et al.,
1995).
Igf1 is expressed in skeletal muscle and has been broadly
implicated in skeletal muscle growth, hypertrophy and regeneration through a
calcineurin-mediated pathway (Musaro et
al., 1999; Grounds,
2002). IGFs, IGF receptors and IGF-binding proteins (IGFBPs)
regulate the growth of many tissues (Baker
et al., 1993; Liu et al.,
1993; Peng et al.,
2003; Fisher et al., 2005). IGFBP2, which is required for IGF1 and
IGF2 activity, is expressed in limb mesoderm and has been recently shown
regulate long bone growth in chicks (Fisher et al., 2005). Upregulation of
Igfbp2 or downregulation of Igf1 and Igf2 might
therefore indirectly regulate body size by affecting bone growth. If myogenin
regulated the expression of the genes encoding these factors, skeletal muscle
from Myog-depleted mice might have altered expression levels compared
with wild-type levels. However, quantitative RT-PCR with RNA isolated from
hindlimb tissue of P14 Myog-depleted mice showed no evidence for a
significant change in the expression of Igf1, Igf2 and
Igfbp2 when compared with expression in the corresponding tissues of
wild-type mice (Table 1).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). This crucial growth period should be exceptionally
sensitive to disruptions in any events that regulate skeletal muscle growth.
Unexpectedly, we found that myogenin, an essential regulator of embryonic
skeletal muscle development, was dispensable for skeletal muscle growth in
postnatal life.
Because myogenin is expressed exclusively in skeletal muscle and
its progenitors, its deletion from the genomes of non-skeletal muscle cell
types should not be relevant. However, Cre recombination did not result in the
complete elimination of myogenin from all genomes, implying that while most
skeletal muscle cells in Myogflox/flox mice
were Myog-null, a few were wild type. However, the
Myogflox/flox mice that we chose to analyze
for skeletal muscle abnormalities had more than 85% of their floxed myogenin
sequences deleted from their genomes, and, in most cases, more than 95%. We
previously showed that chimeric mice composed of 40% or more
Myog-deleted myoblasts cannot support skeletal muscle development
(Myer et al., 1997). It is
therefore unlikely that the few Myog-positive cells that were present
in Myogflox/flox mice would be capable of
suppressing any potential postnatal skeletal muscle defects that might be
associated with Myog-deleted cells.
It is also possible that, in some cells, only one of the floxed
Myog alleles was deleted, thereby resulting in some cells that were
heterozygous for functional Myog, some that were Myog null
and some that were wild type. However, the overall levels of Myog
transcript and protein in Myogflox/flox mice
were still well below those required for embryonic and postnatal skeletal
muscle development, as we showed previously using a hypomorphic,
low-expressing allele of Myog
(Vivian et al., 1999).
Satellite cells are crucial for postnatal muscle growth, and if these cells
were compromised by the loss of myogenin, severe consequences for skeletal
muscle growth would result. Clearly, this was not what we observed. Although
we have not determined directly that floxed Myog sequences were
deleted from the satellite cells of
Myogflox/flox mice, we never observed
myogenin-positive cells in regions where active satellite cells reside. In
addition, we showed that genomic DNA from postnatal skeletal muscle, which
contains both multinucleated myofibers and satellite cells, was deficient in
floxed Myog sequences. Because tamoxifen-induced Cre
recombinase-mediated deletion of Myog was effective in both tail and
hindlimb DNA, it seems unlikely that recombination at the Myog locus
would be selectively inhibited in satellite cells.
The most likely interpretation of our results is that mice lacking
functional levels of myogenin are fully capable of generating skeletal muscle
in postnatal life. Except for their proportionally smaller size,
Myogflox/flox mice behaved identically to
their control littermates. Moreover,
Myogflox/flox mice up to 2 years of age do
not appear to be noticeably different from wild-type mice, except for their
small size.
Embryonic lethality and small body size associated with Myogflox/flox mice
Two unexpected features of Myogflox/flox
mice emerged from our analysis. First, 50% of the
Myogflox/flox mice died between E18.5 and
P10; and second, the Myogflox/flox mice that
did survive were approximately two-thirds the size of wild-type mice of the
same age. Although the basis of the perinatal lethality associated with
Myogflox/flox mice is uncertain, it is
possible that some Myogflox/flox mice
inherited a modifier gene from either the C57BL/6 or 129 genome that uncovered
a cryptic Myogflox/flox phenotype associated
with an essential process in perinatal skeletal muscle growth. Whatever the
explanation for the perinatal lethality, our results show clearly that the
Myogflox/flox mice that do survive beyond
P10 have skeletal muscle that is indistinguishable from that of wild-type
mice.
The proportionally smaller body size and mass of
Myogflox/flox mice when compared with
wild-type mice may reflect a subtle role for myogenin in energy homeostasis
(Carbo et al., 2001). Although
a great deal is known about the role of adipose and nerve tissue in energy
homeostasis, little is known about the role that skeletal muscle might have in
this process (Argiles et al.,
2005). Skeletal muscle accounts for greater than 30% of the energy
expenditure in mice (Smith and Muscat,
2005). Myogenin has been implicated in influencing energy
metabolism in skeletal muscle by inducing a shift of enzyme activity from
glycolytic to oxidative metabolism (Hughes
et al., 1999; Ekmark et al.,
2003). This could indicate that myogenin has broader roles in
regulating genes involved in body-wide homeostasis. However, we found no
substantial alterations in the expression of Igf1, Igf2 and
Igfbp2 in Myog-depleted hindlimbs, suggesting that their
effects on long bone growth are not regulated by myogenin. Nonetheless,
skeletal muscle is a source of many other growth factors and cytokines and
these secreted factors may have functions in communicating globally for
regulating growth (Zoico and Roubenoff,
2002). Myogenin could directly or indirectly control the
expression of genes encoding secreted factors. Myostatin, a member of the
TFGß superfamily, is a potent negative regulator of muscle
differentiation but also appears to modulate adipose metabolism, indicating
that myostatin acts systemically in adult mice to regulate both skeletal
muscle and adipose growth (Zimmers et al.,
2002; Argiles et al.,
2005). In addition to myostatin, interleukin 15, interleukin 16
and TNF
are all expressed in skeletal muscle, and have been implicated
in regulating skeletal muscle and adipose metabolism
(Argiles et al., 2005).
Myogenin-independent mechanisms for postnatal skeletal muscle growth
The role of myogenin as an important regulator of skeletal muscle gene
expression and its relationship with Myod1, Myf5 and Mrf4 are well defined
(Penn et al., 2004;
Blais et al., 2005;
Tapscott, 2005). It was
therefore surprising to find that removing myogenin in early postnatal life
did not interfere with skeletal muscle growth. Our results, however, do not
formally exclude an earlier function for myogenin in establishing the
appropriate genetic program in Pax3/Pax7-positive cells, which are the
precursors of postnatal satellite cells
(Relaix et al., 2005;
Kassar-Duchossoy et al.,
2005). Myogenin might have had a crucial function in satellite
cell development before it was depleted from postnatal
Mygflox/flox mice.
Although an earlier function in satellite cell development cannot be ruled
out, we favor the simpler hypothesis that mechanisms entirely independent of
myogenin are required for postnatal skeletal muscle growth. In fact, the
massive growth in skeletal muscle observed in mice in perinatal and postnatal
life may have only minor dependency on the myogenic bHLH regulators. Pax3 and
Pax7 are likely to be the transcription factors responsible for activating the
downstream differentiation events in postnatal satellite cells
(Relaix et al., 2005;
Kassar-Duchossoy et al.,
2005). Downstream regulators may include SRF and MRTF-A, which
have been shown to be crucial for perinatal muscle growth
(Li et al., 2005). However, it
is unlikely that skeletal muscle growth relies entirely on SRF/MRTF-A-mediated
mechanisms as these factors appear to be involved in controlling protein
accretion within myofibers rather than in regulating satellite cell
proliferation and differentiation.
Mef2 proteins may be major regulators of skeletal muscle growth, based on
comparisons between vertebrate and invertebrate species. Vertebrates depend
much more on myogenic bHLH factors than do invertebrates, which depend mainly
on Mef2 proteins for embryonic muscle development
(Olson and Klein, 1998). The
dependence of vertebrate embryonic skeletal muscle development on the myogenic
bHLH regulators suggests that in the vertebrate lineage, these proteins
evolved specialized functions at multiple steps in the myogenic pathway. Mef2
factors may have retained their importance in perinatal and postnatal skeletal
muscle growth in vertebrates, while the myogenic bHLH factors have evolved
novel regulatory functions in embryonic muscle development that have
supplanted Mef2 factors. It is known that Mef2 proteins act as major
transducers of Ca+2 signaling events and that these events have a
central role in the hypertrophic growth and remodeling of adult skeletal
muscle in response to mechanical load
(Olson and Williams, 2000). It
is therefore possible that postnatal skeletal muscle growth depends more on
Ca2+ signaling and Mef2 proteins than on the myogenic bHLH
factors.
|
ACKNOWLEDGMENTS |
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|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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