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Expression of Myogenic Regulatory Factors During Muscle
Development of Xenopus: Myogenin mRNA Accumulation
Is Limited Strictly to Secondary Myogenesis
Laboratoire de Biologie du Développement, Université René Descartes, Paris, France
To clarify the acquisition of the
adult muscle pattern in Xenopus laevis, in situ
hybridization and reverse transcriptase-polymerase chain reaction were used to correlate the
time course of gene expression for myogenic
regulatory factors (Myf-5, MyoD, and myogenin)
with the expression of contractile protein (myosin heavy chain; MHC) genes during hindlimb
formation compared with their expression in
dorsal body muscles. After the precocious expression of Myf-5 and MyoD mRNA in limb bud (stage
50), myogenin mRNA strongly accumulated later
at paddle stages (stages 52/53) concomitantly with
the accumulation of both the larval and the adult
MHC mRNAs. In dorsal body muscles, as early as
stage 52, myogenin transcripts accumulated in a
few small, secondary myofibers expressing the
adult MHC mRNA that were located along the
dorsomedial edge, but they were never detected
in the large, primary myofibers of the body expressing the larval MHC mRNA. During metamorphosis, the areas expressing both the adult MHC
and the myogenin transcripts gradually expanded
from the dorsomedial edge to the ventral side of
the dorsal body muscles, accounting for the progression of the secondary ‘‘adult’’ myogenesis described previously (Nishikawa and Hayashi [1994]
Dev. Biol. 165:86–94). This work shows that, in Xenopus, the accumulation of myogenin mRNA is restricted to secondary myogenesis, including the formation of new muscles in developing limbs as well
as in dorsal muscles during body remodeling. This
shows that myogenin is not required for primary
myogenesis, and it suggests a crucial role for myogenin in the terminal differentiation program, including myoblast fusion and the activation of adulttype muscle genes. Dev. Dyn. 1998;213:309–321.
r 1998 Wiley-Liss, Inc.
Key words: myogenesis; myogenic regulatory factors; myogenin; myosin heavy chains;
During the development of most vertebrate skeletal
muscles, multinucleate myotubes are formed by fusion
of many myoblasts (Youn and Malacinski, 1981a). Howr 1998 WILEY-LISS, INC.
ever, this does not occur during the myogenesis of the
primary myotome muscle of Xenopus laevis (Muntz,
1975; Kielbowna, 1980; Youn and Malacinski, 1981b),
in which myoblasts are seen to differentiate into uninucleate myotubes. Later, at the onset of metamorphosis, these develop into multinucleate muscle fibers. For
a long time, the fate of the primary myotomal myofibers
and the origin of the multinucleated secondary myotomal myofibers have been controversial (Kielbowna,
1966, 1980; Muntz, 1975). In 1987, Boudjelida and
Muntz showed that multinucleated primary myotomal
fibers in Xenopus arise from amitotic division of the
primary nuclei and, thus, without cell fusion. More
recently, Nishikawa and Hayashi (1995) showed that
these primary myotomal myofibers die. In this scenario,
secondary, multinucleated myofibers arise from fusion
of recently migrated adult-type myoblasts during metamorphosis (Nishikawa and Hayashi, 1994). Likewise,
limb muscles, which do not appear until the middle of
metamorphosis, are characterized by a classic scheme
of myogenesis, including cell fusion (Muntz, 1975;
Dhanarajan and Atkinson, 1981).
In amphibians, metamorphosis, during which many
new muscles are formed, is also a phase of terminal
muscle differentiation when some muscle structural
genes, such as adult myosin heavy chains (MHCs),
begin to be expressed (Chanoine et al., 1987; Radice and
Malacinski, 1989) and when the full range of adult fiber
types arises (Kay et al., 1988; Schwartz and Kay, 1988;
for review, see Radice et al., 1989).
The discovery of a family of skeletal muscle specific
genes, the MyoD family, which is involved in the early
stages of determination and differentiation of muscle
cells, provided a powerful approach for investigating
important aspects of myogenesis (Sassoon, 1993). Members of the MyoD gene family, including MyoD itself
(Davis et al., 1987), myogenin (Wright et al., 1989),
myogenic regulatory factor 4 (MRF4; Rhodes and Konieczny, 1989; Braun et al., 1990; Miner and Wold,
1990), and Myf-5 (Braun et al., 1989), encode basic
helix-loop-helix (bHLH) proteins (DNA binding tran-
Grant sponsor: Association Française Contre les Myopathies.
*Correspondence to: Pr. C. Chanoine, Laboratoire de Biologie du
Développement, Centre Universitaire des Saints-Pères, Université
René Descartes, 45 rue des Saints-Pères, 75270 Paris Cedex 06,
France. E-mail:
Received 2 June 1998; Accepted 4 August 1998
scription factors) that are able to convert nonmuscle
cells to a muscle phenotype in culture (Choi et al., 1990;
Schäfer et al., 1990) and that can promote the transcription of a number of muscle-specific genes (Weintraub et
al., 1991). In mammals, Myf-5 and MyoD seem to
intervene at the level of the precursor muscle-cell
population, whereas myogenin and probably MRF4
intervene at the level of muscle differentiation (for
review, see Buckingham, 1994).
Xenopus homologs of MyoD (Hopwood et al., 1989;
Scales et al., 1990), Myf-5 (Hopwood et al., 1991),
myogenin, and MRF4 (Jennings, 1992) have been identified, and their temporal and spatial expression has
been analyzed during early embryogenesis (for review,
see Gurdon et al., 1992; Jennings, 1992). Surprisingly,
no myogenin transcript has been detected during early
muscle development in Xenopus, and it has been suggested that myogenin could be expressed later, during
multinucleation of muscle fibers at the time of metamorphosis (Jennings, 1992).
Nothing is known about the expression of the bHLH
regulatory factors during multinucleation of the muscle
fibers in developing Xenopus. For this reason, in this
work, by using in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR), we analyzed the expression of Myf-5, MyoD, and myogenin in
relation to the expression of muscle structural genes in
developing hindlimb and compared it with their expression in dorsal body muscles. Because it has been shown
that myogenin is essential for in vivo myogenesis
(Venuti et al., 1995), it was of particular interest in this
study to analyze the expression of the myogenin gene
during the late events of primary myogenesis and
during the secondary myogenesis, which takes place
during metamorphosis (Nishikawa and Hayashi, 1994).
This work also offers a characterization of the distinct
myogenic cell lineages in Xenopus.
Developing Hindlimb
In Xenopus, hindlimb buds become visible at stage
48. From this stage to stage 52, there was no detectable
hybridization with probes for any of the mRNAs for the
myogenic regulatory factors (MRFs), nor was there any
hybridization with probes for the larval and adult
MHCs in the hindlimbs (not shown). Nevertheless, by
using RT-PCR, transcripts for Myf-5 and MyoD began
to be detectable from stage 50, whereas myogenin
mRNA was first expressed later, at stage 52, a stage
that is characterized by extensive growth of the limb
(Fig. 1). The analyzed transcripts were coreverse transcribed and coamplified in the same reaction with the
ubiquitously expressed elongation factor 1␣ gene (EF1␣; Krieg et al., 1989) serving as an internal control for
the amount of RNA tested and for the RT-PCR reproductibility. Additional controls (see Experimental Procedures) indicated clearly that the generation of PCR
products is strictly dependent on the synthesis of cDNA
during the RT reaction and on the presence of both
Fig. 1. Reverse transcriptase-polymerase chain reaction (RT-PCR)
analysis of the myogenic regulatory factors and the normalizing expressed elongation factor 1␣ (EF-1␣) mRNA during hindlimb development. Stages are those of Nieuwkoop and Faber (1967). Myf-5 and MyoD,
myogenin regulatory factors (MRFs).
forward and reverse primers during subsequent PCR
amplification. From stage 53, there was a dramatic
change in the expression of the genes for the MRFs and
the MHCs in the Xenopus hindlimb (Figs. 1, 2). Transcripts for the three myogenic regulatory factors, Myf-5,
MyoD, and myogenin, were detected in the proximal
parts of the paddle by using in situ hybridization. The
intensity of hybridization was such that the signal for
myogenin ⬎ MyoD ⬎ Myf-5. Simultaneously, the transcripts for larval and adult MHCs were expressed. This
paddle stage corresponds to the time of the first appearance of multinucleated myotubes in the hindlimb
(Muntz, 1975). At stage 54, when the forming digits
began to be observed, all of the transcripts also accumulated in the distal parts of the hindlimb (Fig. 3). In the
following stage 55, there was a dramatic decrease in the
levels of Myf-5 mRNA, which were now only slightly
detectable in the distal parts of the hindlimb (Fig. 4E).
At this stage, a clear proximal-distal gradient in the
expression of larval MHC mRNA was also evident. The
level of signal with the probe for larval MHC was much
less in proximal parts than that in distal parts of the
hindlimb (Fig. 4A). From this stage, the intensity of
hybridization was such that the signal for MyoD ⬎
myogenin ⬎ Myf-5.
Dorsal Body Muscles
From stage 48 to stage 62, the larval MHC mRNA
accumulates in all the large myofibers of the body,
whereas no hybridization signal for the adult MHC and
for myogenin are observed (Figs. 2A, 3B, 5C, 6B). From
stage 52, the adult MHC mRNA begins to accumulate
in a few small myofibers located along the dorsomedial
Fig. 2. A–F: Hindlimb bud at stage 53 reacted with cRNA probes as
indicated. D is a brightfield photomicrograph of the same section that is
shown in darkfield in E. Sections shown A–F are in serial order. All probes
are specific for the transcript indicated. Autoradiographs were exposed to
emulsion for 6 days prior to development for the myosin heavy chains
(MHC) probes [(E3), larval MHC; (A7), adult MHC] and 12 days prior to
development for the MRF probes (Myf-5, MyoD, and myogenin). Arrowhead in A indicates that body muscles strongly react with the larval MHC
(E3) probe. Arrow in C indicates nonspecific background due to the
edge (Fig. 5A,B) and the periphery of the body (Fig. 6A).
These small myofibers do not express the larval MHC
mRNA (Figs. 5C, 6B). From stage 48, by using RT-PCR,
transcripts for MyoD, Myf-5, and myogenin were detected in dorsal body muscles (Fig.7). Because, in a
previous work (Jennings, 1992), no myogenin transcript was detected either in embryos or in adult frogs,
we carried out in situ hybridization analysis to determine the spatial localization of the myogenin mRNA. At
stage 53 (Fig. 5E,F) and at stage 54 (Fig. 3 A,C),
whereas Myf-5 and MyoD transcripts were detected in
all the parts of the dorsal muscles, a more discrete
hybridization signal was observed for myogenin mRNA.
Myogenin transcripts were never detected in the large
‘‘larval’’ myofibers (Figs. 3D, 5D) or in the small ‘‘adult’’
myofibers at the periphery of the body (Fig. 6D). From
stage 48, at high magnification, some isolated mononucleate cells located at the periphery of the large
myofibers (expressing the larval MHC) were seen to
express myogenin mRNA (Fig. 8). From stage 52,
myogenin mRNA was detected in the small muscle cells
expressing the adult MHC that were located along the
dorsomedial edge (Fig. 5D). No particular localization
of Myf-5 or MyoD transcripts was noted in these
myogenin-expressing muscle cells compared with the
‘‘larval’’ fibers (Fig. 5E,F). At stage 61/62, during anatomical metamorphosis, the areas expressing the adult
MHC transcripts gradually expanded from the dorsome-
dial edge to the ventral side of the dorsal body muscle
(Fig. 9B): The new-forming muscle mass strongly expressed the adult MHC mRNA. Myogenin mRNA was
expressed only in these ‘‘adult-type’’ muscle cells (Fig.
9D). Some areas of necrosis were observed in the
‘‘larval’’ parts of the dorsal body muscles, as noted
previously by Nishikawa and Hayashi (1995; see Fig.
9A). All of these results are summarized in Figure 10.
This paper provides a detailed spatial and temporal
analysis of Myf-5, MyoD, myogenin, and MHC gene
expression in developing hindlimb of Xenopus compared with their accumulation in dorsal body muscles
during the same period.
Differential Expression of MHC Genes
in Hindlimb and Dorsal Body Muscles
In Xenopus, it is known that muscle development is
characterized by a transition from larval to adult
MHCs at the time of metamorphosis (Kordylewsky et
al., 1989; Radice and Malacincki 1989). The interesting
work of Nishikawa and Hayashi (1994) greatly contributed to a better understanding of Xenopus myogenesis,
because, by using immunohistochemical analysis, it
showed that, before metamorphosis (stage 53), only a
small number of muscle fibers at the dorsal part of
dorsal muscle expressed adult-type muscle proteins
Fig. 3. A–G: Sections of hindlimb at stage 54 (early digits) and
attached myotomal muscles reacted with probes as indicated. E is a
brightfield photomicrograph of the same section that is shown in darkfield
in D. Sections in A–D are in serial order. G is a brightfield photomicrograph
of the same section that is shown in darkfield in F. F is 100 µm away from
D. Note that the intensity of the signal for Myf-5 is greater in distal parts
than in proximal parts of the hindlimb. Arrowheads in D indicate nonspecific background due to pigment.
(MHC and ␤-tropomyosin). Later, during metamorphosis, the adult-type area gradually expanded from the
dorsal side to the ventral side in correlation with the
degeneration of preexisting larval-type muscle fibers
(Nishikawa and Hayashi, 1995; present study). In
accordance with those authors, we show that, during
pre- and prometamorphosis, the small myofibers located along the dorsomedial edge and the periphery of
the body express adult MHC mRNA. Furthermore, we
show that they do not express the larval MHC transcripts, which are detected only in the large myofibers
of the body. This confirms the hypothesis proposed by
Nishikawa and Hayashi (1994) that the myosin isoform
transition, which occurs in Xenopus myotome at the
onset of metamorphosis, is achieved by new proliferation of adult-type myoblasts and not by a change in
gene expression within the same cell. In contrast, in the
hindlimb, multinucleated myofibers coexpress larval
and adult MHC mRNA, showing a more classic scheme
of myogenesis, like that observed in mammals, which is
characterized by a change in gene expression in the
same myofiber.
Myf-5 and MyoD mRNAs Are Expressed
Precociously During Hindlimb Development
This work shows that MyoD and Myf-5 mRNAs
accumulate precociously during hindlimb development
at stage 50, before the appearance of multinucleated
myotubes. The initial appearance of myogenin mRNA
occurs later, and it strongly accumulates at stage 53
concomitantly with the expression of muscle structural
genes (i.e., MHC genes). The different timing of expression for the individual myogenic factors suggests that
each factor has a distinct role during Xenopus myogenesis. Nevertheless, it appears that, during three types
of myogenesis in Xenopus, including somitogenesis,
regeneration, and limb development (Hopwood et al.,
1989, 1991; Rupp and Weintraub, 1991; Nicolas et al.,
Fig. 4. A–F: Sections of hindlimb at stage 55 reacted with probes as
indicated. D is a brightfield photomicrograph of the same section that is
shown in darkfield in C. Sections in A–F are in serial order. There is a
proximal-distal gradient for the accumulation of Myf-5 and larval MHC
(E3) mRNA. Myf-5 mRNA is still detected only in the distal parts, whereas
the intensity of the signal for the larval MHC is greater in distal parts than
in proximal parts of the hindlimb.
1996, 1998, present study), MyoD and Myf-5 are the
first myogenic factors that are expressed, suggesting
their involvement in muscle determination or/and early
muscle differentiation. Previous functional studies using ectopic expression of these myogenic factors supported this hypothesis (Hopwood and Gurdon, 1990;
Hopwood et al., 1991; Ludolph et al., 1994).
scripts were not detected either in embryos (from stage
1 to stage 47) or in adult frog muscle. From stage 48 to
stage 61/62, corresponding to late larval development of
the myotome, we never detected myogenin mRNA in
the primary myofibers expressing the larval MHC. To
our knowledge, this represents the only case of in vivo
myogenesis without myogenin expression. The work of
Boudjelida and Muntz (1987) stongly supported the
idea that, during this period, the majority of primary
myofibers become multinucleated by amitosis, the nuclei of the uninucleate myotomal myotubes being polyploid up to octaploid in Xenopus (Kielbowna, 1966). In a
recent work, Myer et al. (1997), by using chimeric mice
Myogenin mRNA Accumulates During Secondary
Myogenesis but Not During Primary Pyogenesis
In a previous report (Jennings, 1992), myogenin
mRNA was never detected during early embryogenesis
of Xenopus. Jennings indicated that myogenin tran-
Fig. 5. A–F: Transverse sections of the body at stage 53 reacted with
probes as indicated. The region of the dorsomedial edge at the level of
hindlimb is shown. A is a brightfield photomicrograph of the same section
that is shown in darkfield in B. Sections A–F are in serial order.
Arrowheads indicate the regions reacting with the adult MHC (A7) cRNA
probes. Arrows indicate nonspecific background due to pigment.
containing mixtures of myogenin null and wild type
cells, showed that myogenin is required in vivo to
establish an extracellular environment compatible with
myoblast fusion. The fact that myogenin is not expressed during primary myogenesis of Xenopus could
account for this specific type of myogenesis without cell
fusion, which is characterized by the death of larval
fibers (Nishikawa and Hayashi, 1994, 1995) without
‘‘terminal differentiation’’ (i.e., without expression of
adult structural protein gene). Based on the fact that
Fig. 6. A–D: Transverse sections of the body at stage 53 reacted with probes as indicated. Detail of the
periphery of the body is shown. C is a brightfield photomicrograph of the same section that is shown in darkfield
in A. Arrowheads indicate the regions reacting with the adult MHC (A7) cRNA probes. Arrows indicate
nonspecific background due to pigment.
bHLH myogenic regulatory factors, to at least some
degree, have functionally redundant roles in myogenesis (Weintraub, 1993), it is probable that at least one of
the other bHLH MRFs could play the role of myogenin
during primary myogenesis in Xenopus.
In contrast to the expression observed during primary myogenesis, we showed that myogenin mRNA
strongly accumulates during secondary myogenesis,
including the formation of new muscles in developing
limbs and the new myogenesis process involving the
Fig. 7. Reverse transcriptase-polymerase chain reaction analysis of
the myogenic regulatory factors and standardization-control EF-1␣ mRNA
in dorsal body muscles at stages 48 and 55. Note that the level of
myogenin mRNA is higher at stage 55 than at stage 48.
construction of adult-type fibers during body remodeling. In homozygous null mutant mice with a myogenin
(⫺/⫺) gene, the skeletal muscle displayed a marked
reduction in myofibers (Hasty et al., 1993; Nabeshima
et al., 1993). However, normal numbers of myoblasts
were present, indicating an essential in vivo role for
myogenin in the terminal differentiation of myoblasts
into myotubes. Venuti et al. (1995) reported that, in
mice, the absence of myogenin may affect secondary
myogenesis more severely than primary myogenesis
and suggested that secondary myofibers may require
myogenin-dependent activation of a specific set of genes
that cannot be activated by other MRFs. In Xenopus,
the restricted localization of myogenin mRNA in secondary myofibers, expressing adult contractile protein
genes (i.e., adult MHC), strongly suggests a crucial role
for myogenin in the acquiring of the adult muscle
pattern. The fact that the expression of myogenin and
expression of the adult fast MHC are associated raises
the question of the involvement of this particular
myogenic regulatory factor, myogenin, in the activation
of this MHC gene. In this regard, it should be noted
that, during muscle regeneration of Xenopus, myogenin
and adult fast MHC mRNA begin to be detected at the
same time, during the myoblast-to-myotube conversion
(Nicolas et al., 1996).
In Xenopus regenerating muscles, myogenin mRNA
is expressed transiently (Nicolas et al., 1996), which is
also the case during development, because myogenin
transcripts were not detected in adult limb and axial
muscles (Jennings, 1992; unpublished observations).
The fact that the small adult fibers located at the
periphery of the body were detected precociously during
premetamorphosis, before the anteroposterior progression of the adult type areas (during metamorphosis; see
above) initiated from the dorsomedial edge, supports
the hypothesis that the myogenin gene could be expressed transiently before premetamorphosis in the
periphery of the body.
Fig. 8. Photomicrographs at high magnification showing longitudinal
sections of the body at stage 48 (A) and at stage 54 (B,C) reacted with
myogenin cRNA probes. Arrowheads indicate some isolated cells expressing myogenin mRNA.
What Is the Biological Significance of the
Isolated Mononucleate Cells Expressing
Myogenin mRNA in Dorsal Body Muscles?
The presence of some isolated mononucleate cells
expressing myogenin mRNA in the body gives rise to
several different hypotheses. First, they could repre-
Fig. 9. A–D:Transverse sections of the body at stage 62 reacted with probes as indicated. A is a brightfield
photomicrograph of the same section that is shown in darkfield in B. Sections A–D are in serial order.
Arrowheads indicate the regions reacting with the adult MHC (A7) cRNA probes. Stars in A indicate areas of
sent migrating myoblasts involved in the construction
of adult muscles. However, in all of the species analyzed
to date, proliferating myoblasts do not express myogenin mRNA, which is associated with muscle differentiation during myotube formation (Buckingham, 1994).
On the other hand, they could represent some activated
satellite fusing with primary ‘‘larval’’ myofibers and
contributing to the multinucleation process. Indeed, it
has been established that, in most skeletal muscle, the
satellite cells fuse with their associated muscle fiber to
produce multinucleate cells (Moss and Leblond, 1970).
In Xenopus myotome, Boudjelida and Muntz (1987)
showed that satellite cells are visible first after the
majority of the primary muscle fibers have become
multinucleate. They appear to be in a dormant state,
and their numbers increases up to stage 59. In the same
work, electron microscopic investigation did not support the hypothesis that multinucleation in Xenopus
myotome could result from fusion of the satellite cells
with the fiber. We have shown previously that the
quiescent satellite cells in adult muscle do not express
bHLH myogenic regulatory factor mRNA (Nicolas et al.,
1996), whereas myogenin mRNA was detected transiently in activated, fusing satellite cells following
muscle injury. For this reason, we cannot exclude the
fact that some satellite cells could take part in the
multinucleation process. This hypothesis is not in opposition to the fact that division of the primary myonuclei
by amitosis is responsible mainly for the multinucleation of the majority of the primary ‘‘larval’’ fibers.
Evidence of Distinct Myogenic Programs
in Xenopus
This work showed that myogenesis in Xenopus is
characterized by different myogenic programs in terms
of expression of both myogenic regulatory factors and
contractile protein genes (see Fig. 10). Myogenin is not
expressed during the ‘‘early’’ differentiation, which leads
to primary myofibers expressing the larval MHC. At the
onset of metamorphosis, secondary myogenesis takes
place, giving rise to adult-type fibers expressing myogenin and the ‘‘adult-type’’ muscle genes (Nishikawa
and Hayashi, 1994; this study). The fact that primary
and secondary myogenesis are characterized by both
Fig. 10. Expression of MRFs (MyoD, Myf-5, and myogenin) and MHC
genes in developing hindlimb of Xenopus compared with their expression
in dorsal body muscles. Hatched areas represent transcripts that were
detected during primary myogenesis. Open areas represent transcripts
that were detected during secondary myogenesis. Stages are those of
Nieuwkoop and Faber (1967; N&F).
distinct morphological events (i.e., with or without
myoblast fusion) and distinct expression of contractile
protein genes should be correlated to the absence or the
presence of myogenin expression, respectively. This
probably accounts for different intrinsic properties of
embryonic and adult-type myoblasts. On the other
hand, the differences between primary and secondary
myogenesis in terms of the expression of both myogenic
regulatory factors and contractile protein genes, in
part, may reflect environmental differences. In particular, it is known that thyroid hormone, the levels of
which increase during metamorphosis, up-regulates
the adult fast MHC mRNA (Sachs et al., 1996) and can
modulate the expression of some myogenic regulatory
factors (Hughes et al., 1993). Even if the origin of the
myoblasts involved during secondary myogenesis is not
clear in Xenopus (for review, see Radice et al., 1989), the
fact that the myofibers coexpressed larval and adult
fast MHC during limb formation, whereas the adult
myofibers only expressed the adult fast MHC during
body remodeling, supports the existence of two distinct
myogenic cell lineages.
raised in tap water. Embryos and larvae were staged
according to Nieuwkoop and Faber (1967).
Adult male and female Xenopus laevis were maintained at 22°C in tap water and fed once a week.
Breeding pairs were injected twice at 8-hr intervals
with 100 units or 500 units (for females) and with 50
units or 150 units (for males) of human chorionic
gonadotropin (Paines and Byrne Ltd., London, United
Kingdom). Fertilized eggs, embryos, and tadpoles were
Preparation and Prehybridization
of Tissue Sections
The procedure for fixing, embedding, and sectioning
tissues was the same as that used for mouse embryos
and was essentially the same as described by Wilkinson
et al. (1987) with some modifications (Ontell et al.,
1993). Briefly, tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), dehydrated,
and infiltrated with paraffin. Then, 6-µm-thick serial
sections were mounted on TESPA coated RNase-free
glass slides (Sigma, St. Louis, MO). Sections were
deparaffinized in xylene, rehydrated, digested with
proteinase K, postfixed, treated with dithiothreitol/
iodoacetamine/N-ethylmaleimide (to reduce nonspecific
35S-binding; Zeller and Rogers, 1989), treated with
triethanolamine/acetic anhydride, washed, and dehydrated.
Probe Preparation
The following probes were used to generate antisense
cRNAs: 1) XMyoD template is a 598-nucleotide (nt), 38
fragment (BamHI-EcoRI; position 872–1,469) of
XmyoD2–24 (Hopwood et al., 1989) subcloned in pGEM
4Z (Promega Biotec, Madison, WI), cut with BamHI,
and transcribed with SP6 RNA polymerase. 2) Xmyogenin template is a 522-nt fragment corresponding to a
DNA sequence subcloned into pGEM7Zf(⫹), plamid
pCJMG2 (Jennings, 1992), linearized with SphI, and
transcribed by using SP6 RNA polymerase. 3) pSP73-
XMyf-5–2 template (Hopwood et al., 1991) was linearized by using PstI and was transcribed by using SP6
RNA polymerase. The RNA probe was a 534-nt, 38
fragment (PstI-EcoRI, position 601–1,134). 4) Adult
MHC template is the A7 cDNA clone (Radice and
Malacinski, 1989) cut with ApaI and transcribed with
SP6 polymerase. The RNA probe was a 330-nt, 38
fragment (PstI-EcoRI). A7 transcripts are expressed in
fast-type fibers (Nicolas et al., 1996). 5) Embryonic
MHC template is a 330-nt, 38 fragment (PstI-EcoRI) of
the original E3 cDNA clone (Radice and Malacinski,
1989) subcloned into pGEM4Z (Promega Biotec), cut
with HindIII, and transcribed with SP6 RNA polymerase. E3 transcripts are detected during embryonic as
well as larval development (Radice and Malacinski,
1989). For this reason, in the text, we have named it
larval myosin heavy chain transcript (larval MHC).
cRNA probes were made by in vitro transcription in
the presence of 50 µCi [35S]UTP at 1,200 Ci/mmol (NEN
research product, Boston, MA), according to the manufacturer’s instructions (Promega Biotec). However, unlabeled UTP was omitted from the reaction medium in
order to achieve synthesis of RNA probes with a specific
activity of 109 cpm/µg. Probes were hydrolyzed to an
average of 100 nucleotides by limited alkaline hydrolysis, according to Cox et al. (1984), for efficient hybridization, and used at 50,000 cpm/µl hybridization solution.
Thirty microliters of hybridization solution were loaded
per section.
Hybridization and Washing Procedures
High-stringency conditions for hybridization and
posthybridization were followed. Sections were hybridized overnight at 53°C with posthybridization washing
in 2 ⫻ standard saline citrate, 50% formamide, and 50
mM dithiothrietol at 65°C for 30 min. Autoradiography
was carried out with Kodak NTB-2 track emulsion
(Eastman-Kodak, Rochester, NY), developed in Kodak
D19 developer, and stained lightly with Giemsa.
Evaluation of Hybridization Signal
To compare the intensity of hybridization signal with
a given cRNA probe over time, sections at each stage
were hybridized with the same probe preparation,
washed, dipped into emulsion, exposed, and developed
together. Changes in the level of hybridization signal
with a given cRNA probe over time were evaluated by
taking darkfield photomicrographs with a constant
light intensity and a constant time of exposure of the
film to the light source. When it was desirable to
compare the relative intensity of hybridization signals
of different probes, at a given stage, serial sections of
the hind limb muscles were reacted with the different
probes, processed for autoradiography, and developed
at the same time. A similar protocol using darkfield
photomicrographs (as described above) with a constant
light intensity and a constant time of exposure of the
film to the light source was used to compare the
intensity of signal for these probes at a given develop-
mental stage. Hybridization signals over the muscles
with sense (control) probes were not above the background due to pigment.
For the histological results presented, transverse
frozen sections, 8 µm thick, were used. They were
stained with hematoxylin and eosin.
RNA Extraction
Total RNA was purified by using the method of
Auffray and Rougeon (1980) and was checked by agarose gel electrophoresis and ethidium bromide staining.
RT-PCR was performed in one single tube, according
to Goblet and Whalen (1995) and Lin-Jones and Hauschka (1996).
First-Strand cDNA Synthesis
One microgram of total RNA was used for RT-PCR for
all primer pairs. The RNA was denatured briefly with
25 ng of random primer (Gibco BRL, Grand Island, NY)
at 65°C for 5 min. Then, 1 ⫻ RT buffer (ATGC,
Noisy-le-Grand, France), 1 mM each dNTP, 10 U of
RNasin (Promega), and 200 units of AMV Reverse
Transcriptase (ATGC) were added to a final reaction
volume of 20 µl. RT reactions were incubated for 10 min
at room temperature, for 60 min at 37°C, and for 5 min
at 95°C.
PCR Amplification
The whole RT reaction was diluted to 100 µl final
volume with 1 ⫻ PCR buffer (ATGC). Multiplex PCR
reaction was performed with an MRF-specific set of
primers, with an EF-1␣-specific sets of primers (see
below), and with 2 units of Taq polymerase (ATGC).
Samples were overlaid with paraffin oil (80 µl) and
amplified in a thermocycler (Appligène, Illkirch, France).
The cycling parameters were as follows: The initial
cycle consisted of a 95°C denaturation for 5 min, 1 min
at a 55°C annealing temperature, and 1 min at a 72°C
extension temperature. The remaining cycles were for
30 sec at 95°C, 1 min at 55°C, and 1 min at 72°C, with
the final cycle having a 10 min extension at 72°C.
Due to the different abundance of EF-1␣ and MRF
transcripts, we performed nine PCR cycles with MyoD
or myogenin primer pairs or 12 PCR cycles with Myf-5
primer pairs, before adding the EF-1␣ primers and
continuing amplification for 19 additional cycles. Onefifth of the PCR sample was electrophoresed on 6%
polyacrylamide gels. DNA was transferred onto Hybond-C super membrane (Amersham, Buckinghamshire, United Kingdom). Southern blots were hybridized with MRF- and EF-1␣-radioactive probes. The
amount of EF-1␣ PCR product was quantified by using
a Bio-imaging analyzer and NIH Image software
(Bethesda, MD). This calibration allowed us to adjust
the volumes of the PCR reactions loaded on the polyacrylamide gel.
PCR Primers
XMyf-5. The primer pair 58-ACTACTACAGTCTCCCAGGACAGAG-38 (F) and 58-AGAGTCTGGAATAGGGAGGGAGCAT-38 (R) (positions 524–774; Hopwood et
al., 1991) produced a fragment of 250 base pairs (bp)
from cDNA.
XMyoD. The primer pair 58-AACTGCTCCGATGGCATGATGGATTA-38 (F) and 58-ATTGCTGGGAGAAGGGATGGTGATTA-38 (R) (positions 662–952; Hopwood et
al., 1989) produced a fragment of 289 bp from cDNA.
Xmyogenin. The primer pair 58-CCAGCCCTTATTTCTTTTCAGACCA-38 (F) and 58-AATCCCTGAGCCCTGTAATAAAACC-38 (R) (positions 35–183; Jennings, 1992) produced a fragment of 147 bp from cDNA.
EF-1␣. The primer pair 58-CCTGAATCACCCAGGCCAGATTGGTG-38 (F) and 58-GAGGGTAGTCTGAGAAGCTCTCCACG-38 (R) (positions 1088–1311; Krieg et
al., 1989) produced a fragment of 222 bp from cDNA.
Negative Controls
Negative controls were performed with samples in
which the reverse transcriptase or RNA or one of the
primers was omitted to detect eventual DNA contamination. All of these controls remained constantly negative.
We thank Drs. J.B. Gurdon, C.G.B. Jennings, G.
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