Department of Medicine, Stanford University, School of Medicine, Stanford, CA 94305-5151, USA
* Author for correspondence (e-mail: stockdale{at}stanford.edu)
Accepted 3 April 2003
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SUMMARY |
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Key words: Somite, Myotome, Slow myosin, Innervation, Sonic hedgehog, Chick
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INTRODUCTION |
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The neural tube and notochord are both sources of signals that establish
myogenic cell lineages during avian and mammalian somitic myogenesis
(Buffinger and Stockdale, 1994;
Buffinger and Stockdale, 1995
;
Münsterberg and Lassar,
1995
; Stern et al.,
1995
). Sonic hedgehog (Shh), a product of the notochord and floor
plate (Echelard et al., 1993
;
Krauss et al., 1993
;
Riddle et al., 1993
), has a
key role in somite compartmentalization. Although initially thought to promote
the formation of sclerotome and to antagonize dermomyotome formation
(Fan and Tessier-Lavigne,
1994
), other studies have demonstrated that Shh signaling is also
required for the initiation of myogenesis in somites
(Borycki et al., 1998
;
Borycki et al., 1999
;
Concordet et al., 1996
;
Currie and Ingham, 1996
;
Hammerschmidt et al., 1996
;
Johnson et al., 1994
;
Münsterberg et al., 1995
;
Weinberg et al., 1996
). It has
been proposed that Shh, in combination with members of the Wnt family produced
in the dorsal neural tube and surface ectoderm
(Münsterberg et al.,
1995
; Spence et al.,
1996
; Spörle et al.,
1996
; Stern et al.,
1995
; Tajbakhsh et al.,
1998
), might activate myogenic regulatory factor gene expression
and initiate myotome formation. In addition, Shh affects cell proliferation
and survival (Cann et al.,
1999
; Teillet et al.,
1998
) as well as the expression of specific myotomal muscle
phenotypes (Blagden et al.,
1997
; Cann et al.,
1999
; Du et al.,
1997
).
Skeletal muscle fibers are formed by the fusion of mononucleated myoblasts
and their subsequent differentiation into multinucleated muscle fibers. Two
broad classes of muscle fibers have been defined based on physiological and
structural criteria: rapidly contracting oxidative fibers and slowly
contracting glycolytic fibers. The rate of contraction is particularly
dependent on the specific isoform(s) of the myosin heavy chain (MyHC) family
produced within a myofiber
(Bárány, 1967;
Reiser et al., 1988
). Within
the limb muscles, rapidly contracting muscle fibers express only
MyHCs of the fast class, whereas slowly contracting muscle
fibers frequently express a MyHC of the fast class, in
addition to MyHCs of the slow class, which are designated
slow MyHC 1, 2 and 3 in birds. No detailed study has
examined the expression of the various MyHC isoforms during
myogenesis in the somites.
The best-understood system for generating myofiber diversity is in the
zebrafish, in which slow-MyHC-expressing myofibers appear
before those that express fast MyHC, and the hedgehog family of
signaling molecules is required for slow fibers to form
(Devoto et al., 1996). Shh
initiates slow-fiber formation when overexpressed in paraxial mesoderm of the
zebrafish (Blagden et al.,
1997
; Du et al.,
1997
) and, along with the tiggywinkle and echidna hedgehog
proteins, controls induction of muscle pioneers from the adaxial cell
population (Currie and Ingham,
1996
; Lewis et al.,
1999
). By contrast, little is known about how myotomal fiber
diversity develops in embryos of birds and mammals, or of the relationship
between the first myotomal fibers and subsequent muscle formation in the
vertebrate epaxial musculature. Although Shh has been shown to influence cell
survival and proliferation in the avian myotome
(Cann et al., 1999
;
Teillet et al., 1998
), it is
unclear whether the Shh signaling pathway is instructive for myotomal muscle
fiber type in birds or mammals in vivo.
Here, we have investigated the appearance of the three avian isoforms of slow MyHC during formation and maturation of the myotome in chick embryos. As shown by whole-mount in situ hybridization and RT-PCR analyses using isoform-specific probes, the embryonic fast MyHC (efast MyHC) gene and all three slow MyHC genes are expressed in myotomal fibers. From the onset of expression, mRNA transcripts from the efast MyHC gene are distributed throughout the cytoplasm of myotomal fibers, whereas the mRNA transcripts for all three slow MyHC family members are restricted to the central, nuclear domain. To investigate the mechanism regulating the appearance of the various MyHC isoforms, we used surgical and pharmacological methods to interfere with innervation of the myotome. The expression of efast MyHC and slow MyHCs 1 and 3 in the myotome occurs independently of innervation or signals from the neural tube or notochord. By contrast, the expression of the slow MyHC 2 gene requires functional innervation of the myotome.
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MATERIALS AND METHODS |
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Whole-mount in situ hybridization
Embryos were harvested and processed for whole-mount in situ hybridization
according to the protocol of Nieto and colleagues
(Nieto et al., 1996). After
fixation in 4% paraformaldehyde, embryos were dehydrated overnight in absolute
methanol, rehydrated the following morning in a graded series of methanol-PBT
(PBS + 0.1% Tween-20) washes and treated with 10 µg ml-1
proteinase K (Boehringer Mannheim) at room temperature for 5-30 minutes. After
proteinase-K treatment, the embryos were rinsed in a small volume of PBS and
refixed for 20 minutes in a solution of 4% paraformaldehyde, PBS, 2 mM EGTA,
0.1% Tween-20 and 0.1% glutaraldehyde.
Embryos were hybridized overnight at 70°C with digoxigenin-labeled RNA probes. Unbound probe was removed by multiple washes with TBST (Tris-buffered saline, 0.1% Tween-20) and, following a blocking step, embryos were incubated overnight with alkaline-phosphatase-conjugated anti-digoxigenin Fab fragments (Roche) diluted 1:1000. Unbound antibody was removed by extensive washes with TBST containing 2 mM levamisole prior to visualization with 0.225 mg ml-1 Nitro Blue Tetrazolium (Sigma) and 0.1167 mg ml-1 BCIP (Sigma) dissolved in NTMT (0.1 M Tris, pH 9.5, 50 mM MgCl2, 0.1 M NaCl, 0.1% Tween-20).
Digital images of in situ hybridized myotomes were quantified using Adobe Photoshop 6 software. Multiple points from the central (nuclear domain) and peripheral regions of myotomes cut in sagittal section were measured and the level of blue staining resulting from the alkaline-phosphatase reaction was determined as a proportion of the total (RGB) color.
Immunohistochemistry
To examine the distribution of fast and slow MyHC proteins during myotome
development, cryostat sections were made through HH stage 20-21 embryos that
had been ethanol fixed and embedded in OCT (Tissue-Tek). Frozen sections 10
µm thick were mounted on poly-L-lysine-treated slides and
stained with monoclonal antibodies F59 (specific for fast MyHC isoforms) and
S58 (recognizing slow MyHC 2 and 3 isoforms) using previously described
methods (Crow and Stockdale,
1986; Miller et al.,
1985
). F59 was visualized with a Texas-Red-conjugated anti-mouse
IgG (Vector Laboratories), S58 with a FITC-conjugated anti-mouse IgA (Southern
Biotechnologies) and nuclei with a DAPI counterstain.
The medial and lateral margins of the myotome were determined by
whole-mount immunostaining with a rabbit polyclonal anti-desmin antisera
(Sigma). Embryos that had been previously processed for in situ hybridization
with either the efast MyHC or slow MyHC 3 probes were washed
to remove fixative and stained as previously described
(Kahane et al., 2002).
Outgrowth of neurons from the spinal cord to the developing myotome was
analyzed with monoclonal antibody 16.5H2 (Developmental Studies Hybridoma
Bank) specific for motor neurons or monoclonal anti-neurofilament antibody
NN18 (Sigma). Both primary antibodies were visualized with
Texas-Red-conjugated anti-mouse IgG. To examine the spatial relationship
between neurons and the developing myotome, somite explants were double
stained with both the rabbit anti-desmin antisera and the mouse
anti-neurofilament antibody NN18.
RT-PCR primers and in situ hybridization probes
Isoform-specific primers were made for embryonic and neonatal fast
MyHC, slow MyHCs 1, 2, and 3, and cNkx 2.5 using
sequence from the 3' regions of each gene
(Table 1). Each primer pair was
used in RT-PCR assays to examine the temporal appearance of each isoform in
the myotome. Total RNA was extracted from the three rostral-most somites of
10- to 30-somite embryos and from ED6 wing buds (Qiagen). cDNA was synthesized
from 5 µl of each sample and amplified using the isoform-specific primers
in the presence of [32P]dCTP. Using an annealing temperature of 65°C,
amplification was monitored at 20, 24, 28, 30, 32, 34, 36, 40, 42, 44 and 48
cycles for each developmental stage. At 36 cycles, each primer pair produced
product in the linear phase of amplification, and all subsequent analyses were
conducted using these conditions. Each primer pair produces a fragment of
unique and diagnostic size when analyzed by PAGE and visualized by an
overnight exposure to X-ray film. Amplification products were specific to
input RNA because, in the absence of reverse transcriptase, no signal was
detected.
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Somite explant cultures
Explants were made from HH stage 14 embryos. Segments containing three or
four pairs of somites, the neural tube, notochord and lateral plate were
removed from the embryo at the cervical level and transferred to a
collagen-coated dish in a single drop of medium. In some instances, tungsten
needles were used to separate the neural tube and notochord from somites on
one side of the explant. Both halves, one containing somites alone and the
other neural tube, notochord and somites were incubated overnight at 37°C
in DME containing 5% embryo extract, 10% horse serum, 1% glutamine and 1%
penicillin and streptomycin.
To prevent transmission at the neuromuscular junction in explants containing paired somites, neural tube, and notochord, d-tubocurare (d-tubocurarine chloride) was added to the culture medium at a concentration of 16 µM. Following overnight incubation, explants were fixed in 70% ethanol for immunostaining with antibodies directed against desmin and/or motor neurons, or in 4% paraformaldehyde for in situ hybridization with probes for efast MyHC and all three slow MyHCs.
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RESULTS |
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The temporal appearance of each MyHC gene transcript was also determined by in situ hybridization analysis of HH 13-25 embryos. Using isoform-specific probes, the number of somites expressing each isoform was determined and plotted against the total number of somites that had formed within each embryo (Fig. 2). This confirmed the order of appearance of each MyHC isoform and provided information on the rate at which muscle fibers within the maturing somites began to express each MyHC isoform. The first muscle fibers to express detectable levels of either fast or slow MyHC transcripts were found in myotomes of the rostral-most somites (somites 1-4) around HH stage 14 (22 somites) and, with time, there was a progressive rostral-to-caudal emergence of expression at each developmental stage. As shown by the slope of their lines, the rate of appearance of mRNA transcripts for each gene was approximately the same.
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Intracellular localization of fast and slow MyHC
mRNA initially differs within the myotome
The intracellular location of the MyHC gene transcripts is
markedly different for the fast and slow isoforms. From the
onset of its expression in the myotome, efast MyHC was detected
throughout the width of the myotome, extending from its most rostral to its
most caudal edge (Fig. 3A,C).
By contrast, all three slow MyHC gene transcripts were initially
located exclusively in a stripe, equidistance from the caudal and rostral
edges of each myotome, corresponding to the position of myotomal fiber nuclei
(Fig. 3B). Sagittal sections
through developmentally immature somites demonstrate the restriction of the
slow transcripts to the central nuclear domain within myotomal fibers
(Fig. 3E). The pattern of
nucleus-restricted expression of all slow MyHC transcripts was
maintained for a substantial time during somite/myotome maturation. By HH
stage 18-19, slow MyHC mRNA showed a biregional distribution. The
strongest signal was still restricted to the nuclear domain, with a weaker
signal spanning the width of the fibers
(Fig. 3D). By HH 25, the
distribution of slow MyHC mRNA transcripts became nearly identical to
that of fast MyHC transcripts
(Fig. 3F). A relative
measurement of mRNA distribution along the rostral-caudal axis of the myotome
was made by sampling the intensity of staining at points from the center to
the periphery of sagittally sectioned immature
(Fig. 3E) and mature
(Fig. 3F) somites (see
Materials and Methods). As judged by staining intensity, slow MyHC
mRNA localized predominately to the center of the myotome in immature somites
(78.1±18.8 units), compared to the peripheral regions (33.6±2.2
units). However, as the somite matures, this distinction becomes less
apparent, with the central region of the myotome (37.3±4.9 units)
staining nearly the same as the remainder of the myotome (35.0±2.8
units). Each of the slow MyHC genes showed the same initial pattern
of spatial mRNA location and each underwent the same developmental change in
location of the transcripts as the somites matured (data not shown).
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Effects of innervation on MyHC gene expression in the
myotome
The expression of sMyHC2 is significantly delayed compared with
the expression of sMyHC1 and sMyHC3 as determined by in situ
hybridization assays. This delay prompted an investigation into the
developmental events known to occur immediately prior to the stages of
development at which sMyHC2 expression was first seen (HH 16).
Previously published data suggest that, at HH 14-16, nerve fibers first extend
from the neural tube toward the myotome
(Auda-Boucher et al., 1997
;
Bo et al., 2000
;
Hollyday, 1995
;
Kil and Bronner-Fraser, 1996
;
King and Munger, 1990
;
Meiniel and Bourgeois, 1982
).
It has been demonstrated that myotubes formed in vitro from myoblasts from
slow-MyHC-expressing muscle will express slow MyHC
2 only if innervated (DiMario and
Stockdale, 1997
). Therefore, it was postulated that innervation
played a role in the differentiation or developmentally controlled expression
of myosin within maturating myotomal fibers. To test this hypothesis, we used
both surgical and pharmacological approaches to inhibit innervation.
Explants were made from thick cross-sections through young embryos (HH
15) at the cervical level. Explants of three or four paired somites were
incubated in vitro for 24 hours with or without adjacent neural
tube/notochord. In explants including the neural tube, immunostaining with an
antibody to neurofilament protein after 4 hours of incubation demonstrated
that axons had not grown out from the neural tube, whereas, after an overnight
incubation, axons formed and extended into the myotome
(Fig. 6A,B). Explants double
stained with antibodies to desmin and motor neurons demonstrated that these
axons grew from the neural tube and branched to come into physical contact
with the myotome (Fig.
6C,D).
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To demonstrate that innervation is required to initiate sMyHC2, functional innervation was blocked in explants by exposure to d-tubocurare (Fig. 8). Explants of paired somites, neural tube and notochord grown for 24 hours in the presence of 16 µM d-tubocurare failed to initiate expression of sMyHC2 (Fig. 8D), but the expression of the other slow MyHC genes and of efast MyHC was unaffected (Fig. 8A-C). Control experiments were carried out demonstrating that the outgrowth of nerves from neural tube into the adjacent myotome occurred normally in explants exposed to d-tubocurare (data not shown). With pharmacological blockade by d-tubocurare, diffusible signals from the neural tube/notochord would still be expected to reach myotomal fibers. Thus, the two approaches, surgical and pharmacological prevention of neuromuscular interaction demonstrate that the initiation of sMyHC2 depends on functional innervation of myotome fibers.
Myotomal fiber differentiation
These studies of the temporal and spatial expression of myosin within
myotomal fibers reveal a pattern of differentiation of the first fibers to
form in the myotome. It is apparent from whole-mount in situ hybridization
that slow and fast MyHC mRNAs first accumulate in different
regions of the myotome (Fig.
9A,C). Although efast MyHC transcripts are the first to
appear in fibers and virtually all fibers eventually express slow
MyHC transcripts as well, their respective sites of initiation within the
myotome reveal information about the dynamics of myotome formation. To
determine the sequence of differentiation within the forming myotome, embryos
were assayed by in situ hybridization with probes for efast MyHC or
sMyHC3 and were subsequently immunostained with monoclonal antibodies
to desmin. Desmin is expressed in all myotomal fibers at the outset of
myogenic differentiation (Denetclaw et
al., 1997; Lin et al.,
1994
; Venters et al.,
1999
), and our results show that MyHC genes are expressed
shortly after the myotome forms. Thus, desmin expression in the absence of
MyHC expression provides a marker for the most recently formed
myotomal fibers. The first desmin-positive, efast-MyHC-expressing
fibers appear in the ventrolateral regions of the myotome with
desmin-positive, MyHC-negative fibers extending to the dorsomedial
lip (Fig. 9A). At slightly
later stages, as efast MyHC expression progressively appears in more
medial fibers, slow MyHC co-expression first begins within those
fibers located near the middle of the myotome. The fibers that first express
slow MyHCs are flanked both medially and laterally by fibers
expressing desmin but no slow MyHC
(Fig. 9C). As the myotomes
matured, transcripts of both types of myosin were detected in all the myotomal
fibers. After an initial expansion from the middle of the myotome to the
ventrolateral margin, slow MyHC expression expanded in the dorsal
direction (Fig. 9D), like
efast MyHC expression (Fig.
9B). Finally, by HH 25, both fast and slow MyHC
expression were seen throughout all fibers of the myotome - medially,
centrally and laterally.
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DISCUSSION |
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The progression in the expression of slow myosin heavy chains observed
within the myotome is also found in the expression of myosin heavy chains in
the hypaxial muscles (Crow and Stockdale,
1986; Hoh, 1979
;
Kennedy et al., 1986
).
However, although slow MyHC isoforms appear in myotomal fibers in
rapid succession over a period of a few hours, changes in fibers of hypaxial
muscles occur over a period of days. The significance of the pattern of myosin
heavy chain isoform transitions is not known. These transitions could reflect
different roles for the various isoforms in the assembly of sarcomeres, or
they could be the result of an evolutionary holdover.
Isoform transitions in birds require the up- and downregulation of
individual slow MyHC genes in precise sequence
(Crow and Stockdale, 1986;
Cerny and Bandman, 1987
).
Although work has elucidated some of the factors regulating MyHC gene
expression, including thyroid hormone
(Gustafson et al., 1986
;
Izumo et al., 1986
),
innervation (Pette, 2001
) and
activity (Cerny and Bandman,
1986
; Kennedy et al.,
1986
), exactly how these and other mechanisms interact to regulate
a complex series of isoform changes is not known. Thyroid hormone is perhaps
the best documented agent that regulates individual fast MyHC
isoforms both positively and negatively, depending on the cellular context
(Izumo et al., 1986
;
Morkin et al., 1989
). However,
it is unlikely that thyroid hormone is a factor in the expression of myosin in
the myotome because the changes in slow MyHC occur before the hormone
is produced by the embryo.
Intracellular localization of slow MyHC transcripts
The myotome is initially formed as a single layer of mononucleated muscle
fibers subjacent to the dermomyotome. Within each myotomal fiber, the nucleus
takes up a central location such that, in the primary myotome, the nuclei form
a column extending centrally along the medial-lateral axis of the somite
(Kahane et al., 1998). For
20 hours from the time myosin mRNAs are first detectable in the myotome
(HH 14-16), transcripts for each of the three avian slow MyHC genes
are restricted to the domain where the nuclei are located
(Fig. 3). By contrast,
efast MyHC mRNA does not show this initial intracellular restriction
at any time during fiber formation. By HH 18-19 slow MyHC transcripts
begin to be observed more broadly throughout ventrally located myotomal fibers
in the most-rostral somites. That this phenomenon occurs for all three
slow MyHC genes suggests that it is a true developmentally regulated
process specific for slow MyHC members of the MyHC gene
family. This initial localization of slow MyHC mRNA was not observed
in the mouse myotome for the mammalian homologue,
-cardiac/slow
MyHC (Lyons et al.,
1990
).
The in situ hybridization assay used in this study does not have sufficient
resolution to determine whether slow MyHC transcripts are initially
intranuclear or perinuclear, or are in both locations. We hypothesized that,
if the slow MyHC transcripts are intranuclear, this could be a
mechanism of translational control to regulate the appearance of slow MyHC
protein in the cytoplasm of myotomal fibers. To test this hypothesis, HH 17-21
embryos were split along the midline of the neural tube. One half of the
embryo was then assayed for slow MyHC 3 mRNA by in situ
hybridization, while the other half was assayed immunologically for slow MyHC
protein. We were not able to support the hypothesis, because we found that
slow MyHC protein was detected in myotomal fibers contralateral to those in
which slow MyHC mRNA transcripts were confined to the nuclear domain
(data not shown). It is also possible that slow MyHC transcripts are
actually dispersed in the cytoplasm in fibers of less mature myotomes and are
below the level of detection by whole-mount in situ hybridization.
Alternatively, the slow MyHC transcripts could be located in the
cytoplasm in a perinuclear location and thus be in a position to be
translated. This would require a mechanism whereby all three slow
MyHC mRNA species would contain sequence information restricting the
transcripts to a perinuclear location. Precedence for targeting mRNAs to a
specific cytoplasmic location is found in the work of Singer and co-workers
(Kislauskis et al., 1994), who
have identified a sequence in the 3' UTR of the
-actin
gene, designated the `zipcode' that is responsible for the intracellular
localization of transcripts to the cell periphery. Regardless of the exact
location of the slow MyHC transcripts, all three isoforms show the
same pattern of temporal and spatial distribution within the nuclear domain
and appear to be expressed in the same cells over an extended period of time.
Coincidently, the three slow MyHC genes are linked to a single locus
in the chicken genome (Chen et al.,
1997
), but the timing of their appearance in the myotome suggests
that each is independently regulated.
Innervation-dependent regulation of slow MyHC 2 in the
myotome
The maturation of myotomal muscle requires innervation at an early stage in
its formation. We show that innervation of the myotome is necessary for the
initiation of slow MyHC 2 expression. The addition of d-tubocurare to
explants to block functional innervation prevents sMyHC2 expression
in the myotome. Because pharmacological blockade by d-tubocurare would not
necessarily prevent the release of diffusible signals from the neural
tube/notochord, these observations suggest that it is innervation per se that
is important. Thus, the surgical and pharmacological prevention of
neuromuscular interaction demonstrates that the initiation of sMyHC2
depends on functional innervation of myotome fibers. By contrast, the
expression of sMyHC1, sMyHC3 and efast MyHC is an autonomous
process that is independent of nerve outgrowth. Innervation is also an
important aspect of sMyHC2 expression in limb muscles, in which
muscle cells isolated from chicken limbs and co-cultured with nerves can
initiate sMyHC2 gene expression, whereas muscle cells cultured alone
are not (DiMario and Stockdale,
1997; Lefeuvre et al.,
1996
).
Effects of sonic hedgehog on the myotome
Shh is expressed by cells of the notochord and floor plate of the neural
tube (Echelard et al., 1993;
Krauss et al., 1993
), and has
multiple effects on myogenesis in the somite. Acting in concert with members
of the Wnt family of proteins as an activator of the myogenic determination
genes MyoD and Myf5, sonic hedgehog is important for avian
myotome formation (Borycki et al.,
1998
; Borycki et al.,
1999
; Gustafsson et al.,
2002
; Münsterberg et al.,
1995
; Stern et al.,
1995
; Johnson et al.,
1994
). In the zebrafish, Shh has been assigned an instructive role
in the formation of slow muscle fibers. Adaxial cells, located immediately
adjacent to the notochord, form slow muscle fibers that migrate to a
superficial position in the adult (Devoto
et al., 1996
). Ectopic expression of Shh leads to an expansion of
slow muscle cells at the expense of fast muscle cells
(Blagden et al., 1997
;
Du et al., 1997
;
Norris et al., 2000
). It is
not clear whether this signaling molecule plays an instructive role in the
formation of avian slow muscle fibers
(Cann et al., 1999
;
Stockdale et al., 2002
). In
the chicken, ectopic expression of Shh in the somite leads to an increase in
the expression of slow MyHC 3 as well as an increase in efast
MyHC in the myotome. Surgical removal of axial sources of Shh from chick
embryos prevents myotome formation
(Pownall et al., 1996
),
probably caused by the failure of MyoD or Myf5 to be
expressed in precursors located in the dermomyotome
(Borycki et al., 1998
;
Borycki et al., 1999
;
Münsterberg et al.,
1995
), making moot the question of whether slow muscle fibers can
form in the absence of Shh signaling in birds.
Shh has also been shown to have dramatic effects on cell proliferation and
survival in the somite (Cann et al.,
1999; Marcelle et al.,
1999
; Teillet et al.,
1998
). Somites grown in explant cultures separated from
Shh-producing axial structures show greatly reduced levels of cell
proliferation and greatly increased levels of apoptosis compared with explants
of somites associated with axial structures
(Cann et al., 1999
).
Conversely, the addition of sonic hedgehog to the growth medium prevented
apoptosis and expanded the number of muscle cells in somites cultured without
axial structures, mimicking the effects of the neural tube. In vivo, the
implantation of Shh-expressing cells prevented apoptosis in somites of embryos
lacking axial structures (Teillet et al.,
1998
), and Shh-expressing cells increased proliferation in somites
separated from the notochord and neural tube
(Marcelle et al., 1999
).
Previous work by Amthor and coworkers
(Amthor et al., 1999) showed
that implantation of a Shh-releasing bead into mature somites with well-formed
myotomes increased the expression of MyoD in the epaxial muscle after
24 hours of incubation. In response to Shh-soaked beads, we also observed
locally increased amounts of MyoD mRNA in the somite, particularly in
the epaxial myotome (data not shown) and in MyHC gene expression. The
observed expansion of both fast and slow MyHC expression in
the myotome adjacent to a Shhreleasing bead is consistent with the conclusion
that Shh signals induce precocious differentiation of muscle fibers in the
myotome. The ectopic addition of Shh also leads to hypertrophy of individual
myotomal muscle fibers (Fig.
5), suggesting a previously unknown role for this important
signaling molecule in the regulation of muscle fiber size [for a review of
muscle size, see Patel et al. (Patel et
al., 2002
).
Implications for myotome formation
In the myotome, as in skeletal muscle in general, one of the first
indicators of myoblast differentiation is the expression of the intermediate
filament protein desmin (Denetclaw et al.,
1997; Lin et al.,
1994
; Venters et al.,
1999
), which defines the boundaries of the myotome. Our in situ
hybridization data demonstrate that maturation of myotomal fibers, as defined
by the expression of MyHCs, begins in a subset of desmin-positive
fibers, located ventrolaterally in the myotome
(Fig. 9A). These fibers first
express efast MyHC and, as somites mature, the domain of fast
MyHC expression expands dorsomedially in the myotomes
(Fig. 9B). At the last time
examined (HH 21), even in the most mature somites, desmin-positive
MyHC-negative fibers remained in the dorsomedial region of the
myotome (Fig. 9B). These are
the last fibers to express MyHCs. In a similar way, Duxson and
co-workers (Venters et al.,
1999
) demonstrated in the mouse that there exists a gradient of
increasingly more mature muscle fibers as one proceeds ventrally from the
dorsomedial edge of the myotome.
A few hours after the first appearance of fast MyHC transcripts,
slow MyHC gene expression begins within these same fibers
(Fig. 2). Fibers that first
express slow MyHC transcripts are first seen midway between the
medial and lateral borders of the myotome
(Fig. 9C), in fibers that are
continuing to express efast MyHC. As somites mature, the domain of
slow MyHC expression expands first ventrolaterally and subsequently
dorsomedially (Fig. 9D).
Denetclaw and colleagues (Denetclaw and
Ordahl, 2000) have shown that myoblasts first enter into the
myotome from the dorsomedial lip and only later from the ventrolateral lip. It
is possible that the muscle fibers located ventrolateral to those expressing
slow MyHC are younger than those in the mid-myotome, having entered
from the ventrolateral lip. Such fibers, formed from cells of the
ventrolateral lip, could have initiated expression of efast MyHC but,
because they are younger, have not initiated slow MyHC expression.
Alternatively, it is possible that a signal originating from a restricted
region of the overlying dermomyotome could induce the underlying myotomal
fibers to activate slow MyHC genes. Based on gene expression patterns
and morphology, Spörle (Spörle,
2001
; Spörle et al.,
2001
) has identified a centrally located region in the myotome,
termed the intercalated (dermo)myotome, that is in a similar location to that
of the initial slow MyHC activation and can be characterized by
specific molecular markers (Hadchouel et
al., 2000
; Teboul et al.,
2002
). One marker of the intercalated dermomyotome is the
homeobox-containing gene engrailed
(Spörle, 2001
). In the
zebrafish, engrailed is expressed in muscle pioneers, the first
muscle cells to differentiate within the myotome and a subset of slow-fiber
skeletal muscle precursors (Hatta et al.,
1991
; Devoto et al.,
1996
). However, it should be realized that, unlike the zebrafish,
the myotome of avian embryos does not show the same distinct separation of
fast and slow muscle fibers. Although slow MyHC genes appear to be
expressed first in a subset of myotomal fibers located centrally within the
myotome, during the early stages of development examined here, all (or nearly
all) myotomal fibers eventually show a single phenotype in which both
fast and slow MyHCs are expressed
(Fig. 4).
These observations have implications for the proposed mechanisms of myotome
formation (Fig. 10). The
appearance of myosin gene expression should be a temporal matter with regard
to the maturation of fibers within the myotome and thus should reflect the age
of the fibers. As measured by the expression of MyHC, maturation of
the myotome first occurs ventrolaterally, with an increasing number of fibers
expressing MyHC. This pattern suggests that the youngest fibers are
the most medial ones in the early myotome, leading to the suggestion that the
origin of most myotomal fibers must be the dorsomedial region of the somite.
This conclusion is in agreement with models of early myotome formation, which
have demonstrated that the dorsomedial lip of the dermomyotome is the source
of epaxial myotomal fibers (Christ et al.,
1978; Cinnamon et al.,
2001
; Denetclaw and Ordahl,
2000
; Denetclaw et al.,
1997
; Denetclaw et al.,
2001
; Kahane et al.,
1998
; Kahane et al.,
2002
; Venters and Ordahl,
2002
; Venters et al.,
1999
).
|
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ACKNOWLEDGMENTS |
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REFERENCES |
---|
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---|
Amthor, H., Christ, B. and Patel, K. (1999). A
molecular mechanism enabling continuous embryonic muscle growth - a balance
between proliferation and differentiation. Development
126,1041
-1053.
Auda-Boucher, G., Jarno, V., Fournier-Thibault, C., Butler-Browne, G. and Fontaine-Perus, J. (1997). Acetylcholine receptor formation in mouse-chick chimera. Exp. Cell. Res. 236,29 -42.[CrossRef][Medline]
Bárány, M. (1967). ATPase activity of myosin correlated with speed of muscle shortening. J. Gen. Physiol. Suppl. 50,197 -216.
Blagden, C. S., Currie, P. D., Ingham, P. W. and Hughes, S.
M. (1997). Notochord induction of zebrafish slow muscle
mediated by Sonic hedgehog. Genes Dev.
11,2163
-2175.
Bo, X., Schoepfer, R. and Burnstock, G. (2000).
Molecular cloning and characterization of a novel ATP P2X receptor subtype
from embryonic chick skeletal muscle. J. Biol. Chem.
275,14401
-14407.
Borycki, A.-G., Mendham, L. and Emerson, C. P., Jr
(1998). Control of somite patterning by Sonic hedgehog and its
downstream signal response genes. Development
125,777
-790.
Borycki, A. G., Brunk, B., Tajbakhsh, S., Buckingham, M.,
Chiang, C. and Emerson, C. P., Jr (1999). Sonic
hedgehog controls epaxial muscle determination through Myf5 activation.
Development 126,4053
-4063.
Buffinger, N. and Stockdale, F. E. (1994).
Myogenic specification in somites: induction by axial structures.
Development 120,1443
-1452.
Buffinger, N. and Stockdale, F. E. (1995). Myogenic specification of somites is mediated by diffusible factors. Dev. Biol. 169,96 -108.[CrossRef][Medline]
Cann, G. M., Lee, J. W. and Stockdale, F. E. (1999). Sonic hedgehog enhances somite cell viability and formation of primary slow muscle fibers in avian segmented mesoderm. Anat. Embryol. 200,239 -252.[CrossRef][Medline]
Cerny, L. C. and Bandman, E. R. (1986). Contractile activity is required for the expression of neonatal myosin heavy chain in embryonic chick pectoral muscle cultures. J. Cell Biol. 103,2153 -2161.[Abstract]
Cerny, L. C. and Bandman, E. R. (1987). Expression of myosin heavy chain isoforms in regenerating myotubes of innervated and denervated chicken pectoral muscle. Dev. Biol. 119,350 -362.[Medline]
Chen, Q., Moore, L. A., Wick, M. and Bandman, E. (1997). Identification of a genomic locus containing three slow myosin heavy chain genes in the chicken. Biochim. Biophys. Acta 7,148 -156.
Christ, B., Jacob, H. J. and Jacob, M. (1978). On the formation of the myotomes in avian embryos. An experimental and scanning electron microscope study. Experientia 34,514 -516.
Christ, B., Jacob, M. and Jacob, H. J. (1983). On the origin and development of the ventrolateral abdominal muscles in the avian embryo. An experimental and ultrastructural study. Anat. Embryol. 166,87 -101.[Medline]
Cinnamon, Y., Kahane, N., Bachelet, I. and Kalcheim, C.
(2001). The sublip domain - a distinct pathway for myotome
precursors that demonstrate rostral-caudal migration.
Development 128,341
-351.
Concordet, J.-P., Lewis, K. E., Moore, J. W., Goodrich, L. V.,
Johnson, R. L., Scott, M. P. and Ingham, P. W. (1996).
Spatial regulation of a zebrafish patched homologue reflects the roles of
sonic hedgehog and protein kinase A in neural tube and somite
patterning. Development
122,2835
-2846.
Crow, M. T. and Stockdale, F. E. (1986). Myosin expression and specialization among the earliest muscle fibers of the developing avian limb. Dev. Biol. 113,238 -254.[Medline]
Currie, P. D. and Ingham, P. W. (1996). Induction of a specific muscle cell type by a hedgehog-like protein in zebrafish. Nature 382,452 -455.[CrossRef][Medline]
Denetclaw, W. F. and Ordahl, C. P. (2000). The
growth of the dermomyotome and formation of early myotome lineages in
thoracolumbar somites of chicken embryos. Development
127,893
-905.
Denetclaw, W. F., Jr, Christ, B. and Ordahl, C. P.
(1997). Location and growth of epaxial myotome precursor cells.
Development 124,1601
-1610.
Denetclaw, W. F., Jr, Berdougo, E., Venters, S. J. and Ordahl,
C. P. (2001). Morphogenetic cell movements in the middle
region of the dermomyotome dorsomedial lip associated with patterning and
growth of the primary epaxial myotome. Development
128,1745
-1755.
Devoto, S. H., Melançon, E., Eisen, J. S. and
Westerfield, M. (1996). Identification of separate slow and
fast muscle precursor cells in vivo, prior to somite formation.
Development 122,3371
-3380.
DiMario, J. X. and Stockdale, F. E. (1997). Both myoblast lineage and innervation determine fiber type and are required for expression of the slow myosin heavy chain 2 gene. Dev. Biol. 188,167 -180.[CrossRef][Medline]
Du, S. J., Devoto, S. H., Westerfield, M. and Moon, R. T. (1997). Positive and negative regulation of muscle cell identity by members of the hedgehog and TGF-ß gene families. Cell Biol. 139,145 -156.[CrossRef]
Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J. A. and McMahon, A. P. (1993). Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75,1417 -1430.[Medline]
Eloy-Trinquet, S. and Nicolas, J. F. (2002).
Clonal separation and regionalisation during formation of the medial and
lateral myotomes in the mouse embryo. Development
129,111
-122.
Fan, C.-M. and Tessier-Lavigne, M. (1994). Patterning of mammalian somites by surface ectoderm and notochord: evidence for sclerotome induction by a hedgehog homolog. Cell 79,1175 -1186.[Medline]
Gustafson, T. A., Markham, B. E. and Morkin, E.
(1986). Effects of thyroid hormone -actin and myosin heavy
chain gene expression in cardiac and skeletal muscles of the rat: measurement
of mRNA content using synthetic oligonucleotide probes. Circulation
Res. 59,194
-201.[Abstract]
Gustafsson, M. K., Pan, H., Pinney, D. F., Liu, Y., Lewandowski,
A., Epstein, D. J. and Emerson, C. P., Jr (2002). Myf5
is a direct target of long-range Shh signaling and Gli regulation for muscle
specification. Genes Dev.
16,114
-126.
Hadchouel, J., Tajbakhsh, S., Primig, M., Chang, T. H., Daubas,
P., Rocancourt, D. and Buckingham, M. (2000). Modular
long-range regulation of myf5 reveals unexpected heterogeneity between
skeletal muscles in the mouse embryo. Development
127,4455
-4467.
Hamburger, V. and Hamilton, H. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88,49 -92.
Hammerschmidt, M., Bitgood, M. J. and McMahon, A. P. (1996). Protein kinase A is a common negative regulator of Hedgehog signaling in the vertebrate embryo. Genes Dev. 10,647 -658.[Abstract]
Hatta, K., Bremiller, R., Westerfield, M. and Kimmel, C. B. (1991). Diversity of expression of engrailed-like antigens in zebrafish. Development 112,821 -832.[Abstract]
Hoh, J. F. Y. (1979). Developmental changes in chicken skeletal myosin isoenzymes. FEBS Lett. 98,267 -270.[CrossRef][Medline]
Hollyday, M. (1995). Chick wing innervation. I. Time course of innervation and early differentiation of the peripheral nerve pattern. J. Comp. Neurol. 357,242 -253.[Medline]
Huang, R. and Christ, B. (2000). Origin of the epaxial and hypaxial myotome in avian embryos. Anat. Embryol. 202,369 -374.[CrossRef][Medline]
Izumo, S., Nadal-Ginard, B. and Mahdavi, V. (1986). All members of the MHC multigene family respond to thyroid hormone in a highly tissue-specific manner. Science 231,597 -600.[Medline]
Johnson, R. L., Laufer, E., Riddle, R. D. and Tabin, C. (1994). Ectopic expression of Sonic hedgehog alters dorsal-ventral patterning of somites. Cell 79,1165 -1173.[Medline]
Kahane, N., Cinnamon, Y. and Kalcheim, C. (1998). The origin and fate of pioneer myotomal cells in the avian embryo. Mech. Dev. 74, 59-73.[CrossRef][Medline]
Kahane, N., Cinnamon, Y. and Kalcheim, C.
(2002). The roles of cell migration and myofiber intercalation in
patterning formation of the postmitotic myotome.
Development 129,2675
-2687.
Kavinsky, C. J., Umeda, P. K., Sinha, A. M., Elzinga, M., Tong,
S. W., Zak, R., Jakovcic, S. and Rabinowitz, M.
(1983). Cloned mRNA sequences for two types of embryonic myosin
heavy chains from chick skeletal muscle: I. DNA and derived amino acid
sequence of light meromyosin. J. Biol. Chem.
258,5196
-5205.
Kennedy, J. M., Kamel, S., Tambone, W. W., Vrbova, G. and Zak, R. (1986). The expression of myosin heavy chain isoforms in normal and hypertrophied chicken slow muscle. J. Cell Biol. 103,977 -983.[Abstract]
Kil, S. H. and Bronner-Fraser, M. (1996). Expression of the avian alpha 7-integrin in developing nervous system and myotome. Int. J. Dev. Neurosci. 14,181 -190.[CrossRef][Medline]
King, E. D. and Munger, B. L. (1990). Myotome and early neurogenesis in chick embryos. Anat. Rec. 228,191 -210.[Medline]
Kislauskis, E. H., Zhu, X. and Singer, R. H. (1994). Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype. J. Cell Biol. 127,441 -451.[Abstract]
Krauss, S., Concordet, J.-P. and Ingham, P. W. (1993). A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell 75,1431 -1444.[Medline]
Lefeuvre, B., Crossin, F., Fontaine-Perus, J., Bandman, E. and Gardahaut, M. F. (1996). Innervation regulates myosin heavy chain isoform expression in developing skeletal muscle fibers. Mech. Dev. 58,115 -127.[CrossRef][Medline]
Lewis, K. E., Currie, P. D., Roy, S., Schauerte, H., Haffter, P. and Ingham, P. W. (1999). Control of muscle cell-type specification in the zebrafish embryo by Hedgehog signalling. Dev. Biol. 216,469 -480.[CrossRef][Medline]
Lin, Z., Lu, M.-H., Schultheiss, T., Choi, J., Holtzer, S., DiLullo, C., Fischman, D. A. and Holtzer, H. (1994). Sequential appearance of muscle-specific proteins in myoblasts as a function of time after cell division: evidence for a conserved myoblast differentiation program in skeletal muscle. Cell Motil. Cytoskeleton 29, 1-19.[Medline]
Lyons, G. E., Ontell, M., Cox, R., Sassoon, D. and Buckingham, M. (1990). The expression of myosin genes in developing skeletal muscle in the mouse embryo. J. Cell Biol. 111,1465 -1476.[Abstract]
Machida, S., Matsuoka, R., Noda, S., Hiratsuka, E., Takagaki, Y., Oana, S., Furutani, Y., Nakajima, H., Takao, A. and Momma, K. (2000). Evidence for the expression of neonatal skeletal myosin heavy chain in primary myocardium and cardiac conduction tissue in the developing chick heart. Dev. Dyn. 217, 37-49.[CrossRef][Medline]
Marcelle, C., Ahlgren, S. and Bronner-Fraser, M. (1999). In vivo regulation of somite differentiation and proliferation by sonic hedgehog. Dev. Biol. 214,277 -287.[CrossRef][Medline]
Marti, E., Takada, R., Bumcrot, D. A., Sasaki, H. and McMahon,
A. P. (1995). Distribution of Sonic hedgehog peptides in the
developing chick and mouse embryo. Development
121,2537
-2547.
Meiniel, R. and Bourgeois, J. P. (1982). Appearance and distribution `in situ' of nicotinic acetylcholine receptors in cervical myotomes of young chick embryos. Radioautographic studies by light and electron microscopy. Anat. Embryol. 164,349 -368.[Medline]
Miller, J. B., Crow, M. T. and Stockdale, F. E. (1985). Slow and fast myosin heavy chain content defines three types of myotubes in early muscle cell cultures. J. Cell Biol. 101,1643 -1650.[Abstract]
Morkin, E., Bahl, J. J. and Markham, B. E. (1989). Control of cardiac myosin heavy chain gene expression by thyroid hormone. In Cellular and Molecular Biology of Muscle Development (ed. L. H. Kedes and F. E. Stockdale), pp.381 -389. New York: Alan R. Liss.
Münsterberg, A. E., Kitajewski, J., Bumcrot, D. A., McMahon, A. P. and Lassar, A. B. (1995). Combinatorial signaling by Sonic hedgehog and Wnt family members induces myogenic bHLH gene expression in the somite. Genes Dev. 9,2911 -2922.[Abstract]
Münsterberg, A. E. and Lassar, A. B.
(1995). Combinatorial signals from the neural tube, floor plate
and notochord induce myogenic bHLH gene expression in the somite.
Development 121,651
-660.
Nieto, M. A., Patel, K. and Wilkinson, D. G. (1996). In situ hybridization analysis of chick embryos in whole mount and tissue sections. Methods Cell Biol. 51,219 -235.[Medline]
Norris, W., Neyt, C., Ingham, P. and Currie, P.
(2000). Slow muscle induction by hedgehog signalling in vitro.
J. Cell Sci. 113,2695
-2703.
Ordahl, C. and Le Douarin, N. M. (1992). Two myogenic lineages within the developing somite. Development 114,339 -353.[Abstract]
Patel, K., Christ, B. and Stockdale, F. E. (2002). Control of muscle size during embryonic, fetal, and adult life. Results Probl. Cell Differ. 38,163 -186.[Medline]
Pette, D. (2001). Historical perspectives:
plasticity of mammalian skeletal muscle. J. Appl.
Physiol. 90,1119
-1124.
Pownall, M. E., Strunk, K. E. and Emerson, C. P., Jr
(1996). Notochord signals control the transcriptional cascade of
myogenic bHLH genes in somites of quail embryos.
Development 122,1475
-1488.
Reiser, P. J., Greaser, M. L. and Moss, R. L. (1988). Myosin heavy chain composition of single cells from avian slow skeletal muscle is strongly correlated with velocity of shortening during development. Dev. Biol. 129,400 -407.[Medline]
Riddle, R. D., Johnson, R. L., Laufer, E. and Tabin, C. (1993). Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75,1401 -1416.[Medline]
Schultheiss, T. M., Xydas, S. and Lassar, A. B.
(1995). Induction of avian cardiac myogenesis by anterior
endoderm. Development
121,4203
-4214.
Spence, M. S., Yip, J. and Erickson, C. A.
(1996). The dorsal neural tube organizes the dermamyotome and
induces axial myocytes in the avian embryo.
Development 122,231
-241.
Spörle, R. (2001). Epaxial-adaxial-hypaxial regionalisation of the vertebrate somite: evidence for a somitic organiser and a mirror-image duplication. Dev. Genes Evol. 211,198 -217.[CrossRef][Medline]
Spörle, R., Gunther, T., Struwe, M. and Schughart, K.
(1996). Severe defects in the formation of epaxial musculature in
open brain (opb) mutant mouse embryos.
Development 122,79
-86.
Spörle, R., Hadchouel, J., Tajbakhsh, S., Schughart, K. and Buckingham, M. (2001). Evidence for subdivisions of epaxial somite derivatives. In The Origin and Fate of Somites, Vol. 329 (ed. E. Sanders, J. W. Lash and C. P. Ordahl), pp. 153-165. Amsterdam: IOS Press.
Stern, H. M., Brown, A. M. and Hauschka, S. D.
(1995). Myogenesis in paraxial mesoderm: preferential induction
by dorsal neural tube and by cells expressing Wnt-1.
Development 121,3675
-3686.
Stockdale, F. E., Nikovits, W., Jr and Christ, B. (2000). Molecular and cellular biology of avian somite development. Dev. Dyn. 219,304 -321.[CrossRef][Medline]
Stockdale, F. E., Nikovits, W., Jr and Espinoza, N. R. (2002). Slow myosins in muscle development. Results Probl. Cell Differ. 38,199 -214.[Medline]
Tajbakhsh, S., Borello, U., Vivarelli, E., Kelly, R., Papkoff,
J., Duprez, D., Buckingham, M. and Cossu, G. (1998).
Differential activation of Myf5 and MyoD by different Wnts in explants of
mouse paraxial mesoderm and the later activation of myogenesis in the absence
of Myf5. Development
125,4155
-4162.
Teboul, L., Hadchouel, J., Daubas, P., Summerbell, D.,
Buckingham, M. and Rigby, P. W. J. (2002). The early epaxial
enhancer is essential for the initial expression of the skeletal muscle
determination gene Myf5 but not for subsequent, multiple phases of
somitic myogenesis. Development
129,4571
-4580.
Teillet, M., Watanabe, Y., Jeffs, P., Duprez, D., Lapointe, F.
and Le Douarin, N. M. (1998). Sonic hedgehog is
required for survival of both myogenic and chondrogenic somitic lineages.
Development 125,2019
-2030.
Umeda, P. K., Sinha, A. M., Jakovcic, S., Merten, S., Hsu, H.-J., Subramanian, K. N., Zak, R. and Rabinowitz, M. (1981). Molecular cloning of two fast myosin heavy chain cDNAs from chicken embryo skeletal muscle. Proc. Natl. Acad. Sci. USA 78,2843 -2847.[Abstract]
Umeda, P. K., Kavinsky, C. J., Sinha, A. M., Hsu, H.-J.,
Jakovcic, S. and Rabinowitz, M. (1983). Cloned mRNA
sequences for two types of embryonic myosin heavy chains from chick skeletal
muscle. J. Biol. Chem.
258,5206
-5214.
Venters, S. J. and Ordahl, C. P. (2002). Persistent myogenic capacity of the dermomyotome dorsomedial lip and restriction of myogenic competence. Development 129,3873 -3885.[Medline]
Venters, S. J., Thorsteinsdottir, S. and Duxson, M. J. (1999). Early development of the myotome in the mouse. Dev. Dyn. 216,219 -232.[CrossRef][Medline]
Weinberg, E. S., Allende, M. L., Kelly, C. S., Abdelhamid, A.,
Murakami, T., Andermann, P., Doerre, O. G., Grunwald, D. J. and
Riggleman, B. (1996). Developmental regulation of Zebrafish
MyoD in wild-type, no tail and spadetail embryos.
Development 122,271
-280.
Yutzey, K. E., Rhee, J. T. and Bader, D.
(1994). Expression of the atrial-specific myosin heavy chain
AMHC1 and the establishment of anteroposterior polarity in the developing
chicken heart. Development
120,871
-883.