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J. Biol. Chem., Vol. 278, Issue 35, 33169-33174, August 29, 2003
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From the UMR 7060 CNRS, Equipe Biologie du développement et de la Différenciation Neuromusculaire, Centre Universitaire des Saints-Pères, Université René Descartes, F-75270 Paris Cedex 06, France
Received for publication, May 7, 2003 , and in revised form, May 30, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-subunit gene in cap animal assays and in endoderm
cells of Xenopus embryos but was unable to activate the expression of
the 
-subunit gene. In embryos, overexpression of a dominant-negative
XMyf5 variant led to the repression of -but not
-subunit gene expression. Conversely, XMyoD and XMRF4 activated
-subunit gene expression but were unable to activate -subunit
gene expression. Finally, all MRFs induced expression of the 
-subunit
gene. These findings strengthen the concept that one MRF can specifically
control a subset of muscle genes that cannot be activated by the other
MRFs. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Regarding the question whether a single gene or an array of genes is under
the transcriptional control of an individual MRF, using the Xenopus
model, we have recently shown that, among the MRFs, myogenin played a unique
role in the transactivation of several structural genes of the contractile
apparatus, e.g. 
-tropomyosin and myosin heavy chain
(6). However, whether such
specificity applies to other MRFs still remains controversial. Xenopus
laevis muscle development provides an attractive model to address this
question since, in this system, primary myogenesis occurs in the absence of
myoblast fusion and is devoid of myogenin expression
(7), thus allowing comparison
of the role of MyoD, Myf5, and MRF4 in the control of early gene
expression.
Myogenesis is characterized by the sequential expression of specific gene
families, including those that mediate communication between motor neurons and
muscle. Synaptic transmission at the neuromuscular junction is mediated
through the nicotinic acetylcholine receptor (nAChR), a pentameric complex of
four homologous subunits with a molar stoichiometry of 2,
, , and or 
(8). Expression of the
nAChR subunits and the distribution of the receptors among muscular
fibers is developmentally regulated. nAChR mRNA levels are highest during
myogenic differentiation. Motor innervation inhibits nAChR expression by
repressing transcription of the nAChR subunit genes, whereas
denervation results in reaccumulation
(9). The nAChR -to
-subunit switch allows the expression of two types of channel, namely an
embryonic channel composed of -, -, -, and
-subunits, distributed throughout the fiber, and an adult channel
containing -, -, -, and -subunits, exclusively
expressed at the motor endplate
(10,
11).
The accumulation to a high density of one type of nAChR at the
neuromuscular junction is the result of transcriptional activation of
AChR subunit genes in subsynaptic muscle nuclei. Members of the MyoD
gene family are likely to play a central role in the regulation of
nAChR subunit expression. All the genes coding for nAChR subunits
contain one or more E-boxes in their regulatory regions, which the MRFs
indifferently recognized and bound
(12). Each MRF is capable of
increasing specifically nAChR subunit promoter activity in transient
cotransfection assays (13,
14). However, MRF4
preferentially activates the expression of the -subunit gene
(15).
To date, no in vivo study has directly addressed the individual
role of each MRF in patterning nAChR subunit expression. In the
present study, we compared the expression patterns of MRFs and
nAChR subunit genes during early Xenopus development,
showing that these expression patterns are temporally correlated. We then
compared the in vivo abilities of MyoD, Myf5, and MRF4 to trigger the
expression of each nAChR subunit gene. Among the MRFs, only XMyf5
specifically induced expression of the -subunit gene in cap animal
assays and in endoderm cells of Xenopus embryos, whereas it was
unable to activate expression of the -subunit gene. In addition,
overexpression of a dominant-negative XMyf5 variant led to repression of
normal -subunit gene expression in embryos. The - and
-subunit genes also discriminated among MRFs for their transactivation
in contrast to the -subunit gene, whose expression was induced by all
MRFs tested.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Plasmid Constructions
For expression in Xenopus embryos, the clones pSP64T-XMyoD,
pSP64T-XMyf5, and pSP64T-XMRF4 have already been described
(16–18).
MRF-FLAG constructs were obtained by a variant of the QuikChange site-directed
mutagenesis method (Stratagene) using forward primers,
5'-CAGCACCATCTATCACGTCTTAGATTATAAAGATGACGATGACAAG-3',
5'-CCGACCAATCTACCATGTGTTAGATTATAAAGATGACGATGACAAG-3', and
5'-TATACAGGAGCTGGTAGAGAATGATTATAAAGATGACGATGACAAG-3' for XMyoD,
Xmyf5, and XMRF4, respectively, and reverse primer
5'-TGAGGCTGGTTTAGTGGTAACCCTTGTCATCGTCTTTATAATC-3'. FLAG sequence
was added in-frame with the last coding codon of each MRF just before the stop
codon. The fidelity of all sequences amplified by the polymerase chain
reaction was verified by DNA sequencing.
The dominant-negative XMyf5-DN was constructed as described previously (19). Briefly, the 891-bp fragment coding for amino acid residues 2–298 of the Drosophila repressor engrailed domain was inserted between the last coding codon and the stop codon by directed cloning (20), a PCR method also developed in our laboratory. The primers used were 5'-TTGCCGACCAATCTACCATGTGTTAGCCCTGGAGGATCGC-3' and 5'-TGAGGCTGGTTTAGTGGTAACCTTAGGATCCCAGAGCAGA-3'. The fidelity of the sequence amplified by PCR was verified by DNA sequencing.
Western Blot Analysis
A FLAG epitope tag introduced at the C terminus of the MRF constructs was
used as a control of the translation efficiency of the synthetic mRNAs. Five
MRF-FLAG-injected embryos were homogenized at stage 8 in 100 µl of buffer
containing 25% glycerol, 50 mM KCl, 50 mM Tris-HCl, pH
8.0, 0.1 mM EDTA, 2 mM dithiothreitol, 2 mM
phenylmethylsulfonyl fluoride, 50 µg/ml–1
aprotinin, and 50 µg/ml–1 leupeptin. Proteins
were extracted after two rounds of centrifugation at 13,500 rpm for 10 min at
4 °C. Proteins were resolved by electrophoresis on an 8% polyacrylamide
gel in the presence of sodium dodecyl sulfate (SDS-PAGE), transferred to a
polyvinylidene difluoride membrane, and reacted with an anti-FLAG monoclonal
antibody M2 (Sigma). Briefly, the membranes were blocked with TBS-T (150
mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% Tween 20)
containing 5% dried milk for 1 h at room temperature; incubated with a 1/2000
dilution of 5 µg/ml M2 in TBS-T with 5% dried milk for 1 h at room
temperature; washed and finally incubated with a 1/4000 dilution of anti-mouse
horseradish peroxidase-conjugated antibody (Transduction Laboratories) in
TBS-T with 5% dried milk. Antibody binding was revealed using ECL according to
the manufacturer's instructions (Amersham Biosciences).
Embryo Injection and Animal Cap Assays
Capped mRNAs of XmyoD, Xmyf5, XMRF4, XEF1-, and XMyf5-DN
(dominant-negative Xmyf5) were synthesized in vitro from linearized
plasmids (XbaI restriction enzyme, New England BioLabs) using the SP6
Message Machine kit (Ambion). Injections were performed using an oil-based
microinjector (nanoject, Drummond). Xenopus embryos were placed in
Modified Marc's Ringer solution with 4% Ficoll. Embryos were initially
bilaterally injected with 2 or 6 ng of capped mRNA (MRFs) or with 7.5 ng of
capped mRNA (Xmyf5-DN) at the two-cell embryo stage. Injected embryos were
transferred to Modified Marc's Ringer solution with antibiotics. The animal
cap assay was performed as described previously
(17). Cap animals were
dissected at the blastula stage (stage 10) and cultured until sibling embryos
reached stage 14–15. Total RNA was extracted from five pooled animal
caps, and expression of the Xenopus AChR subunit genes was detected
by RT-PCR. To determine by in situ hybridization the ability of MRFs
to activate expression of nAChR subunit genes in nonsomitic cells, 32-cell
stage embryos were co-injected with 2 ng of MRF mRNA and 2.5 ng of
n-galactosidase RNA into the D1 blastomere. To determine in
situ the effect of Xmyf5-DN, two-cell stage embryos were unilaterally
injected with 7.5 ng of Xmyf5-DN and 2 ng of capped green fluorescent protein
mRNA as a cell tracer. Following injection, embryos were incubated in 20%
Steinberg's solution until the appropriate stage was reached.
RNA Isolation and RT-PCR Analysis
Total RNA was isolated from Xenopus embryos as described by
Chomczynski and Sacchi (21).
Embryos and larvae were staged according to Nieuwkoop and Faber
(22).
First-strand cDNAs were synthesized from 1 µg of total RNAs with
oligo(dT) priming using Superscript reverse transcriptase (Invitrogen) at 37
°C for 1 h. PCR assays were performed as described elsewhere. 1 µCi of
[32P]dCTP was added to each PCR sample, and the PCR products were
resolved electrophoretically on a 20% polyacrylamide gel. The expression of
ornithine decarboxylase (ODC) was first examined as a control for equal
amplification of cDNAs. The primers used for RT-PCR were
5'-GTCAATGATGGAGTGTATGGATC-3' and
5'-GTGCTCAGGAGAGCGGAATGGA-3' (ODC),
5'-TCCTTCTGGAATACACTGGA-3' and
5'-ACTCGCCATGAAGTTGCTCA-3' (AChR ),
5'-ATGGCAATGTCACGTGGCAC-3' and
5'-TGGAGTTCTTGCGGGACGAGC-3' (AChR ),
5'-ACGTCTTGATAAGCAGTGAT-3' and
5'-TGCTAATGAGTGGAATGGCA-3' (AChR ),
5'-ATGTCCTCGTGTACAATACA-3' and
5'-ATGCTTGACTTTGGTGCTCAG-3' (AChR ),
5'-GATGGTTCCATGTACTGGCTT-3' and
5'-GTAAGAAATAGACGAGAATGCT-3' (AChR ),
5'-AACTGCTCCGATGGCATGATGGATTA-3' and
5'-ATTGCTGGGAGAAGGGATGGTGATTA-3'(XMyoD),
5'-ACTACTACAGTCTCCCAGGACAGA-3' and
5'-AGAGTCTGGAATAGGGAGGGAGCA-3' (XMyf5),
5'-CTTTTACCTGGATGGAG-3' and 5'-TGGTGGAGCTAAGACAT-3'
(XMRF4).
In Situ Hybridization
RNA probes were made by in vitro transcription in the presence of
50 µCi [35S]UTP at 1,200
Ci·mmol–1 (PerkinElmer Life Sciences)
according to the manufacturer's instructions (Promega Biotec, Madison, WI).
Unlabeled UTP was omitted from the reaction medium to achieve synthesis of RNA
probes with a specific activity of 
109
cpm·µg–1.
AChR and templates were obtained by cloning the RT-PCR
fragments into pGEMT (Promega), linearized using SpeI (Roche Applied
Science) and transcribed using T7 RNA polymerase (Promega). cRNA probes were
used as described previously
(7).
Densitometry
In Situ Hybridization—Hybridization signals were quantified
with a computerized image analysis system (Visiolab, 2000, Biocom, Paris,
France). The relative density of silver grains/mm2 was determined
in the somites of five unilaterally injected embryos. Relative density was
measured on an average of 10 sections for each embryo.
RT-PCR—Quantification of band "volume" (summed pixel density) was performed using Image Tool software (University of Texas Health Science Center at San Antonio (UTHSCSA)). A standard curve was created by plotting the pixel density for each dilution of the standard sample as a function of the relative amount loaded. The relative amount of DNA in each test sample was then calculated based on this standard curve.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-subunit, were expressed as early
as stages 12–15, i.e. starting 8 h after the appearance of
XMyoD and XMyf5 transcripts, suggesting a direct gene activation
(5). Comparison of MRF
and nAChR expression at later stages of development proved to be more
complex. mRNA expression decreased from stage 19 for XMyoD and
XMyf5 and from stage 22 for XMRF4, and MRF mRNAs became
undetectable at stage 28. The decrease in the expression of the -,
-, -subunit genes followed the decreasing expression of the MRF
genes, albeit at different rates. Indeed, -subunit gene expression
became undetectable after stage 24, whereas - and -subunit
expression persisted until stage 30. Interestingly, the -subunit gene
displayed a biphasic expression pattern with peaks of expression at stage 17
and stage 22. Finally, - and -subunit gene expression persisted
throughout early development.
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Selective nAChR Subunit Gene Activation by MRFs—To determine
whether the pattern of transcription of each nAChR subunit gene
during development can be attributed to differences in transactivation by
MRFs, we compared the specificity of each MRF for transactivation of
nAChR subunit genes, using the Xenopus animal cap assay. In
this assay, two-cell stage embryos were bilaterally injected at the animal
pole with 2 ng of synthetic XMyoD, XMyf5, or XMRF4 RNA. The same FLAG epitope
tag was inserted at the C terminus of each MRF coding sequence to permit
evaluation of the relative amount of each MRF injected
(Fig. 2C). Animal
poles were dissected at the blastula stage and cultured until the late
gastrula stage for molecular analysis. The animal caps were analyzed by RT-PCR
for the expression of nAChR , , , , and
mRNAs. Since cultured animal pole explants normally form epidermis, any
expression of a muscle gene in these cells solely reflects the activity of the
transcription factor gene injected. As shown in
Fig. 2, mRNAs for the nAChR
subunits were detected only in the MRF-RNA-injected samples, and no signal was
obtained following injection of control XEF1 mRNA. A specific
activation of -subunit mRNA was observed after XMyf5 injection
(Fig. 2A). No signal
was detected for -subunit gene expression with either XMyoD or XMRF4.
-subunit transcription was activated by XMyf5 or XMyoD, but not by
XMRF4, whereas -subunit transcription was activated by XMyoD and XMRF4,
but not by XMyf5. -subunit mRNA was found to be activated in all animal
caps except in the control XEF1. In contrast, expression of the
-subunit gene was not induced by any of the MRFs injected.
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To detect a possible dose effect for the gene activation specificity, we
examined the expression of the nAChR subunit genes in embryos
injected with 6 ng of synthetic MRF RNAs. Although the differences between the
MRF transactivation potentials were less marked, we fully confirmed the data
obtained with the 2 ng-injected embryos. In particular, -subunit gene
expression was activated by each MRF (Fig.
2B). In addition, the preferential role of XMyf5 for
transactivation of the -subunit gene, as well as of XMyf5 and XMyoD for
transactivation of the -subunit gene, were confirmed in these
conditions. Finally, expression of the -subunit gene was not detected at
any dose of MRF injected.
In Vivo -Subunit Gene Activation by XMyf5—To
confirm that MRFs could discriminate between the nAChR subunit
targets in vivo, XMyf5 was overexpressed in a predetermined
blastomere. In the 32-cell stage embryo, the cells derived from the D1
blastomere mainly give rise to endodermal structures
(26). XMyf5 and
n-galactosidase RNAs or XMyoD and n-galactosidase RNAs were
injected into the D1 blastomere of 32-cell stage embryos. At stage 35, the
injected embryos were assayed for the presence of - or -subunits
of the nAChR mRNAs, respectively activated by XMyf5 and by XMyoD, in
endodermal tissue. In situ hybridization analysis revealed a discrete
but significant expression of -subunit transcripts in some of the
endoderm cells derived from the XMyf5-injected blastomere
(Fig. 3). No hybridization
signal was detected in endoderm cells with the nAChR- probe, neither in
the embryos injected with XMyf5 nor in the embryos injected with XMyoD. In
contrast, the myotomal tissue of all the injected embryos displayed high
levels of hybridization with the nAChR- and - probes (data not
shown).
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-Subunit Gene Repression by a Dominant-negative Myf5
Variant—To provide additional evidence for the role of XMyf5 as a
preferential regulator of the -subunit gene, we tested by in
situ hybridization analysis whether a dominant-negative mutant of XMyf5
(XMyf5-DN) could selectively suppress -subunit expression in
vivo without perturbing -subunit expression. Upon unilateral
injection into two-cell stage embryos, XMyf5-DN efficiently reduced
-subunit gene expression (Fig.
4), when this gene is normally activated in the mesoderm. The
reduction of -subunit gene expression was correlated spatially with the
expression of the co-injected lineage tracer green fluorescent protein. In
contrast, the level of -subunit mRNA was not affected by overexpression
of XMyf5-DN. As shown by RT-PCR analysis
(Fig. 5), Xmyf5-dependent
induction of -subunit mRNA was significantly reduced by XMyf5-DN,
whereas expression of the -subunit was unaffected. In these
experiments, two-cell stage embryos were bilaterally injected with XMyf5-DN or
with XEF1 as a control. Taken together, these results provided
additional evidence that -subunit expression was specifically regulated
by XMyf5 protein activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-subunit gene by XMyf5. - and
-subunit gene activation was less specific, excluding only one MRF from
their activator panel. Finally, activation of the -subunit gene implies
a nonspecific effect of the different MRFs.
Overexpression of any nAChR subunit gene in the cap animal assay
is controlled by at least one determination MRF factor, i.e. MyoD or
Myf5. This MRF activation was concomitant with the onset of nAChR
transcription, as revealed by comparing the respective expression patterns.
Furthermore, expression of the nAChR subunit genes was shown to be
dependent on the expression of their corresponding MRF activators since
injection of a dominant-negative XMyf5 altered -subunit gene
expression. These observations reinforce the role of the MRFs in the
initiation of nAChR gene transcription during early development, with
the notable exception of the -subunit gene. However, no valid prediction
can be made on the specific role of MyoD, Myf5, or the combination of both in
promoting the transcription of nAChR genes. Since no difference could
be detected in the expression patterns of the nAChR subunits that
could account for the number and identity of their relevant MRF activators, it
can be speculated that when the two determination factors have an activating
capacity, as for the - and -subunits, gene activation arises from
a competition toward the E-box targets. In this regard, it should be
emphasized that the specificity of the initiation of nAChR gene
expression by the determination MRFs is dose-dependent, as was shown for the
expression of the -subunit and to a lesser extent the -subunit.
Consistent with these data, no difference in the expression of the
nAChR genes was reported in MyoD and Myf5-null mice
(27).
In later developmental steps, each nAChR subunit gene seems to
adopt a unique strategy for the maintenance of its transcriptional activation.
Activation of the -subunit gene during differentiation requires a major
activation by XMyf5, as shown by 1) the results of the cap animal assay, 2)
the results of the in vivo injection in 32-cell embryos, and 3) the
comparison of the expression patterns during development
(Fig. 6A). In
vivo activation of the other subunit genes may require additional
factors, which are likely to be MRF family members in the case of the -
and -subunits (Fig.
6B) or other factors in the case of the - and
-subunits. A functionally significant finding from this study is that
XMRF4 most likely has a cooperative function with XMyoD for the activation of
- and -subunit gene expression at subsequent developmental
steps. The data from the cap animal assays, showing an activation by XMRF4
only in the case of an activation by XMyoD, were highly consistent with the
relative expression profiles of - and -subunit genes and the
expression profiles of XMyoD and XMRF4. All together, these
results suggest that XMRF4 maintains the expression rate of - and
-subunit genes, whose activation is initiated by XMyoD. Thus, XMyoD and
XMRF4 functions may overlap for the transactivation of the nAChR - and
-genes. Furthermore, overexpression of XMyoD in the 32-cell embryos
failed to activate -subunit expression in endoderm cells in
vivo. This might be interpreted as a consequence of an absence of XMRF4.
Thus, a working hypothesis, to be tested, is that expression of the
-subunit gene, effectively triggered by XMyoD in these conditions,
required activation by XMRF4 to display a normal expression level detectable
by in situ hybridization. Our results suggest a functional overlap of
XMyoD and XMRF4 and substantiate the results obtained with the knockout mice.
In contrast to either a MyoD-ora MRF4-null mutant, which
exhibited nearly normal muscle differentiation,
MyoD–/–/MRF4–/–
mouse embryos have severe muscle defects similar to those seen in
myogenin mutants
(28). Rather than a critical
level of myogenic basic helix-loop-helix proteins insufficient to completely
trigger the myogenic program, it can be proposed, in the light of the present
study, that the terminal differentiation process relies in part on the correct
activation of a particular set of genes, specifically activated by
differentiation factors that may act solitarily, such as myogenin, or in
tandem, such as MyoD and MRF4. This hypothesis is strengthened by the results
reported by Cornelison et al.
(29), who examined the genetic
requirements for MyoD for successful satellite cell myogenesis. These authors
have described the dependence of MRF4 expression on MyoD in adult
satellite cells. Severe defects in the muscle regenerating process could be
observed in the absence of both of these MRFs. In contrast,
MyoD–/– embryos were
phenotypically normal and, interestingly, displayed a significant expression
of MRF4 at a level indistinguishable from wild type
(30). As a conclusion,
differentiation defects likely resulted from a simultaneous deficiency in MyoD
and MRF4 activities. Besides, this model accounts for the observation that, in
contrast to myogenin, MyoD and MRF4 possess a functionally interchangeable
helix III domain implicated in the efficient targeting of endogenous
responsive genes (31).
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In contrast to the observation by Liu et al.
(12) that MRF4 bound the
-subunit promoter, no MRF was able to trigger -subunit gene
expression in the cap assay. Thus, these data might reflect a discrepancy
between the DNA binding at nAChR promoters and the efficiency of the
transcriptional activity. In this regard, studies using heterologous systems
have already shown such a discrepancy between MRF binding and transactivation.
For instance, several muscle gene promoters, including creatine kinase, myosin
light chain 1/3, and troponin I, that bound MyoD, myogenin, and MRF4
(4) were not efficiently
activated by MRF4 (32). This
is also the case for the myogenin promoter to which MRF4 binds but does not
activate (33). This feature
applied for all the MRFs since Liu et al.
(12) demonstrated by chromatin
immunoprecipitation assays that the MRFs indifferently bind to the promoters
of AChR subunit genes.
Control of nAChR gene transcription is crucial to the development and maintenance of synapses in muscle. The myogenic factor proteins of the MyoD family are essential molecules in regulating the ongoing rates of nAChR gene transcription during early myogenesis in a specific and reproducible manner. In Xenopus, this precise orchestration of nAChR gene activation occurs with a target specificity. Our results further substantiate the concept that an individual MRF can control a subset of muscle genes that cannot be activated by the other MRFs or with dramatically lower efficiency. This concept opens new ways to assign, at the gene level, a particular role to each MRF in myogenesis.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 33-1-4286-2153; Fax:
33-1-4286-2119; E-mail:
chanoine{at}biomedicale.univparis5.fr.
1 The abbreviations used are: MRFs, myogenic regulatory factors; nAChR,
nicotinic acetylcholine receptor; ODC, ornithine decarboxylase; DN,
dominant-negative; RT, reverse transcription; X, Xenopus.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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