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First published online June 15, 2006
Journal of Experimental Biology 209, 2432-2441 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02269
Somite formation and expression of MyoD, myogenin and myosin in Atlantic halibut (Hippoglossus hippoglossus L.) embryos incubated at different temperatures: transient asymmetric expression of MyoD
1 Department of Biology, Brattøra Research Centre, Norwegian
University of Science and Technology (NTNU), N-7491 Trondheim,
Norway
2 Institute of Aquaculture Research (AKVAFORSK), PO Box 5010, N-1430
Ås, Norway
* Author for correspondence at present address: BioMar AS, Kjøpmannsgt. 50, N-7484 Trondheim, Norway (e-mail: trine.galloway{at}biomar.no)
Accepted 13 April 2006
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There were no significant effects of temperature on the onset of somitogenesis or number of somites at hatching. The rate of somite formation increased with increasing temperature, and the expression of MyoD, myogenin and MyHC followed the cranial-to-caudal somite formation. Hence, no significant effect of temperature on the spatial and temporal expression of the genes studied was found in relation to somite stage. MyoD, which has subsequently been shown to encode the MyoD2 isoform, displayed a novel bilaterally asymmetric expression pattern only in white muscle precursor cells during early halibut somitogenesis. The expression of myogenin resembled that previously described for other fish species, and preceded the MyHC expression by approximately five somites. Two MyLC2 cDNA sequences were for the first time described for a flatfish, probably representing embryonic (MyLC2a) and larval/juvenile (MyLC2b) isoforms.
Factors regulating muscle determination, differentiation and development have so far mostly been studied in vertebrates with external bilateral symmetry. The findings of the present study suggest that more such investigations of flatfish species could provide valuable information on how muscle-regulating mechanisms work in species with different anatomical, physiological and ecological traits.
Key words: Atlantic halibut, Hippoglossus, flatfish, muscle, temperature, bilateral asymmetry, MyoD, myogenin, myosin heavy chain, myosin light chain
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), which in mammals have been shown to exhibit distinct but
overlapping functions. Whereas MyoD and myf-5 play critical roles in the
determination of multipotent mesodermal stem cells to a myogenic lineage,
myogenin and MRF4 are required for differentiation of myoblasts to myotubes
and subsequently to mature muscle cells
(Ludolph and Konieczny, 1995;
Rudnicki and Jaenisch, 1995).
These transcription factors share a highly conserved basic region mediating
DNA binding, and a helix-loop-helix (HLH) domain essential for dimerization
with the ubiquitously expressed E proteins
(Murre et al., 1989;
Lassar et al., 1991). The
resulting heterodimer has a high affinity for the consensus E-box motif CANNTG
located in the promoter or the enhancer of the majority of muscle-specific
genes and thereby promotes the transcription of these target genes
(Ma et al., 1994;
Puri and Sartorelli, 2000).
During the embryonic development of most fish species, MyoD is first
present in adaxial cells of the presomitic mesoderm
(Weinberg et al., 1996;
Delalande and Rescan, 1999;
Temple et al., 2001;
Xie et al., 2001;
Tan and Du, 2002;
Hall et al., 2003;
Cole et al., 2004;
Zhang et al., 2006), showing
that some mesodermal cells are destined to become muscle cells very early in
organogenesis. The adaxial cells will later become myoblasts, migrate and
mature into slow superficial red muscle fibres, whereas the lateral cells of
the somites form the bulk of fast white muscle fibres
(Devoto et al., 1996).
Myogenin expression succeeds MyoD expression, and activates
fusion of myoblasts and differentiation to multinucleated myotubes
(Rescan, 2001). Subsequently
the maturing muscle fibres start to express genes encoding muscle-specific
contractile proteins, such as myosin heavy chain (MyHC) and myosin light chain
(MyLC) (reviewed in Goldspink et al.,
2001).
Since fish are ectothermic animals and most teleost fish eggs are
fertilised externally, water temperature is one of the most important external
factors regulating developmental rate (Blaxter, 1988), partly by influencing
the transcription rate of hundreds of genes (e.g.
Malek et al., 2004). Egg
incubation temperature affects the size and number of red and white muscle
fibres at hatching, as well as muscle growth throughout larval and juvenile
life in many fish species (Stickland et
al., 1988; Vieira and
Johnston, 1992; Brooks and
Johnston, 1993; Usher et al.,
1994; Johnston et al.,
1995; Nathanailides et al.,
1995; Johnston and McLay,
1997; Alami-Durante et al.,
1997; Johnston et al.,
1998; Galloway et al.,
1998). By contrast, gene expression of the MRFs is affected by
temperature in some fish species (Wilkes
et al., 2001; Xie et al.,
2001) but not in others
(Temple et al., 2001;
Hall et al., 2003;
Cole et al., 2004).
The Atlantic halibut (Hippoglossus hippoglossus L.) is the largest
flatfish in the North Atlantic (Haug,
1990), and is of great commercial value both for fisheries and
aquaculture, owing to its rapid growth and good flavoured flesh. In Norwegian
waters the halibut spawns between December and April, and the mean temperature
at which the embryos develop is presumed to be around 5°C
(Lønning et al., 1982;
Haug et al., 1984;
Kjørsvik et al., 1987),
with 2 and 9°C being close to the lower and upper thermal tolerance
limits, respectively, for these early life stages
(Bolla and Holmefjord, 1988;
Pittman et al., 1989;
Galloway et al., 1999).
Incubation of halibut eggs at experimental temperatures within these thermal
tolerance limits has been shown to have a positive effect on developmental
rate (Pittman et al., 1989;
Galloway et al., 1999) but a
negative effect on muscle mass size at hatching
(Galloway et al., 1999).
However, no information is available for any flatfish species about possible
effects of temperature on the rate of somite development and gene expression
of the underlying regulatory factors. In the present study the muscle-specific
genes encoding the regulatory factors MyoD and myogenin and the structural
proteins MyLC and MyHC were isolated from Atlantic halibut, and the impact of
temperature on their temporal and spatial expression during somitogenesis was
examined.
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Embryos/larvae were sampled from each temperature group at regular
intervals of day-degrees (d° = days x temperature in degrees
Celsius); at 30, 40, 50, 60, 70 and 80 d° after fertilisation and 6 d°
after hatching (=day when >50% of the embryos had hatched). Samples for
reverse transcription (RT)–PCR analyses (N=20 per sampling
point) were frozen on dry ice in Nunc cryotubes and stored at –80°C.
Samples for in situ hybridisation (N=25 per sampling point)
were fixated in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS)
overnight and dechorionated before the embryos were rinsed twice in PBS,
washed in 50% and 75% methanol in PBS and stored at –80°C in 100%
methanol. During dechorionisation the yolk sac was also removed. Samples for
examination of somite stage, standard length and myotome height (N=10
per sampling point) were fixated in a modified Karnovsky fixative, as
described elsewhere (Galloway et al.,
1998).
Cloning of muscle specific genes
Total RNA was extracted from whole embryos at different stages and from
fast (white) and slow (red) skeletal muscle of a 1-year old Atlantic halibut
weighing 45.9 g (14.8 cm in length) using TRIzol (Gibco-BRL, Gaithersburg, MD,
USA). Total RNA (100–500 ng) was reverse transcribed into cDNA using the
Ready-To-Go T-primed First-Strand Kit (Pharmacia Biotech, Uppsala, Sweden).
Initially, partial halibut MyoD and myogenin cDNA sequences
were PCR amplified using degenerate primers against conserved regions. The
MyoD primers myd1 and myd2 were based on the N- and C-terminal
sequences DFYDDPCF and DCLSSIVE, respectively, whereas the myogenin
primers myg1 and myg2 corresponded to the regions MELFET and TSIVDSIT,
respectively. Downstream MyoD and myogenin cDNA sequences
were isolated by 3'RACE (Rapid Amplification of cDNA Ends) combining the
MyoD-specific forward primers myd16 and myd21, or the
myogenin-specific primers myg8 and myg13, with the oligo(dT)-tailed
primer and the adapter primer described by Frohman et al.
(Frohman et al., 1988).
Upstream sequences of halibut MyoD were successfully PCR amplified
from genomic DNA by Genome Walking (Clontech, San Francisco, CA, USA) by
including the specific antisense primers myd38 and myd39 according to the
manufacturer's protocol. Based on the gene structure of zebrafish
MyoD (Du et al.,
2003) and mouse myogenin
(Edmondson et al., 1992), the
primers sets myd16 + myd23 and myg13 + myg15 were used to PCR amplify the two
introns of halibut MyoD and myogenin, respectively, from
genomic DNA (Qiagen DNA isolation kit). Two partial halibut MyLC
cDNAs were isolated by running 3'RACE on pooled embryonic cDNA from
several developmental stages, using the specific forward primer mlc1 encoding
the conserved region MAPKKA. Finally, a partial halibut MyHC cDNA was
amplified from fast muscle RNA by RT–PCR, using the primers mhc1 and
mhc3 encoding the MyHC sequences KNWPWMK and MDLENDK, respectively.
All PCRs were run on a Perkin Elmer 2400 thermal cycler (Cetus, Hayward, CA, USA) by denaturation at 94°C for 2 min followed by 35–40 cycles of amplification at 94°C for 30 s, 58–65°C for 30–50 s depending on the primers, and 72°C for 50 s. The PCR mixture of 12.5 µl contained 0.6 i.u. Taq DNA polymerase (Promega, Southampton, UK) together with 0.2 mmol l–1 of each of the dNTPs and 10 pmol of each primer in standard GeneAmp® PCR buffer. PCR amplicons were visualized by agarose gel electrophoresis and ethidium bromide staining, cloned into pGEM-T Easy vector (Pharmacia Biotech), and sequenced in both directions (Applied Biosystems 377A, Foster City, CA, USA).
Spatiotemporal gene expression
Whole-mount in situ hybridization
cRNA probes for whole-mount in situ hybridization (WM-ISH) were prepared
from plasmids containing halibut MyoD, myogenin or MyHC
cDNAs. The plasmids were linearised using SalI and NcoI
restriction endonucleases, DIG labelled (Roche Diagnostics, GmBH, Mannheim,
Germany), and purified according to the manufacturer's protocol. The labelled
sense and antisense probes were electrophoresed together with standards
included in the labelling kit to evaluate the probe concentrations. Three or
more embryos or larvae per developmental stage from each temperature group
were used for WM-ISH with each antisense RNA probe. Corresponding sense probes
were used as negative controls on one sample per developmental stage from each
temperature group. WM-ISH was performed according to the procedure of Hall et
al. (Hall et al., 2003) with
modifications as follows. Permeabilisation of all embryonic stages was
achieved by digestion in 20 µl ml–1 proteinase K for 15
min, and for newly hatched larvae the conditions were 50 µl
ml–1 for 10 min. Hybridisation with 500 ng
ml–1 digoxigenin (DIG)-labelled cRNA probe in the
hybridisation solution lasted 1–3 days at 70°C. Bound probe was
conjugated to an alkaline-phosphatase-labelled anti-DIG antibody (Roche
1093274) at a dilution 1:8000 in the `Heaven Seven' solution overnight at
4°C. A colour reaction occurred by adding 0.45 mg ml–1
nitroblue tetrazolium (NBT) and 0.175 mg ml–1
5-bromo-4-chloro-3-indolylphosphate (BCIP) to the `Divine Nine' solution and
incubating in darkness overnight at 4°C. Photographs were taken on a Leica
MZ75 binocular microscope using a Nikon Camera Head DS-5M and a Nikon Camera
Control Unit DS-L1. The yolk sac was removed before the embryos/larvae were
photographed.
RT–PCR
The temporal expression of MyoD, myogenin and MyLC were also examined by
RT–PCR using the intron spanning primers described above for amplifying
MyoD and myogenin cDNAs. The MyLC2a and MyLC2b cDNAs, which differed in the
length of the 3' untranslated region (UTR), were coamplified by
3'RACE and separated by gel electrophoresis. From each sampling point,
total RNA of five embryos or larvae was mixed before cDNA synthesis. Proper
first strand cDNA synthesis was verified by PCR amplifying halibut
ß-actin (GenBank accession no. AJ630123) using the primers actb1 and
actb2. These primers, as well as the 3'RACE primers for MyLC2, spanned
intron sequences to exclude possible contamination of genomic DNA.
Primer sequences
Statistics
Significant differences (P<0.05) between treatments in somite
recruitment rate and number of somites, standard length and myotome height at
hatching were determined by a one-way ANOVA followed by a Tukey's B
post-hoc test.
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Halibut MyoD and myogenin exhibited the highest overall aa sequence
identity to gilthead seabream (Sparus aurata) MyoD (71%) and
pufferfish (Tetraodon nigroviridis) myogenin (82%), respectively. In
addition to the conserved bHLH domain and the juxtaposed motif CLP/LWACKA/LCK,
vertebrate MyoD and myogenin also showed homology in the C-terminal end
(Fujisawa-Sehara et al., 1990)
(Fig. 3). This part includes
the so-called helix III, which appears to have adopted distinct functions in
MyoD and myogenin mainly due to a negatively or positively charged residue in
the marked position in Fig. 3
(Bergstrom and Tapscott, 2001).
These authors demonstrated that the substitution of the Asp residue at this
position in mouse MyoD with His of mouse myogenin disrupted the function of
the MyoD helix III. The sequence alignment indicates that the negatively
charged Asp, or Glu in cod, is conserved in the vertebrate MyoDs, whereas the
positively charged His or Arg are found at this position in the myogenin of
mammals and teleosts, respectively (Fig.
3). The exception seems to be the two myogenins of
Xenopus both containing Asn at this position
(Charbonnier et al., 2002).
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Embryonic development
The embryos in all temperature groups were halfway through epiboly at 30
d° and the first movements of the embryos were observed around 70 d°.
Hatching occurred at 84, 88 and 77 d°, or 19, 15 and 9 days after
fertilisation at 4, 6 and 8°C, respectively. The first somites were formed
between half and complete epiboly in all temperature groups, and at blastopore
closure 8–11 somites were formed. Recruitment rate of new somites
increased significantly with increasing incubation temperature
(Table 1;
Fig. 4), and was
4.6±0.3, 5.4±0.1 and 10.5±0.3 somites per day at 4, 6 and
8°C, respectively. The overall number of somites at hatching was
52±2 and was not significantly different among treatments
(Table 1). The newly hatched
larvae from the 4°C group were significantly shorter than larvae from the
other temperature groups, whereas the myotome height of larvae from the
6°C group was significantly larger than that of larvae from the 4°C
and 8°C groups (Table
1).
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Spatiotemporal expression of muscle genes
WM-ISH with the RNA antisense probes enabled the visualisation of a
cranial-to-caudal progression in the expression of MyoD, myogenin and
MyHC (Fig. 5), whereas
sense probes produced no colour reaction. RT–PCR was used to verify the
temporal expression of MyoD and myogenin (not shown). Owing
to the difficulty of making specific ISH probes for the two highly similar
MyLC2 genes, their temporal expression was studied by 3'RACE
(Fig. 6), which amplified the
two cDNAs differing in the length of the 3'UTR. Expression patterns of
the muscle genes studied closely followed somite formation, and no significant
effects of egg incubation temperature on the timing and spatial expression of
MyoD, myogenin and MyHC were found when studied in relation
to somite stage. The WH-ISH results shown in
Fig. 5 are, therefore, a
representative selection from all three temperature groups.
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Initial MyoD expression was detected prior to somitogenesis at 50% epiboly only by RT–PCR, whereas the first detection of MyoD by WM-ISH was first observed very faintly at the 9-somite stage, and only in the lateral cells of somites 7–9 (Fig. 5A). This indicates that somites 1–6 expressed MyoD prior to the 9-somite stage, but this could not be detected by WM-ISH. Intriguingly, the MyoD signal was shown on only one side of the notochord, or in more somites on one side than on the other. This novel bilaterally asymmetric expression of MyoD persisted until 20–30 somites were formed (Fig. 5A–C). As MyoD expression progressed caudally in an increasing number of somites, the pattern appeared to become more symmetric. From the 25- to 45-somite stage the expression was downregulated in more and more cranial somites (Fig. 5D–F). MyoD signals were still present close to hatching (>50 somites), but the individual spatial expression varied from none at all to expression in the caudal half of the newly hatched larvae (Fig. 5G).
Myogenin mRNA was first detected at the 9- to 10-somite stage by the more sensitive RT–PCR method as a heavily stained band. The adaxial cells of somites 3–10 had a faintly bilaterally symmetrical myogenin signal at the 9- to 10-somite stage (Fig. 5H), but myogenin was not detected in lateral cells of somites 1 and 2. The myogenin expression became stronger at the 14- to 15-somite stage as it progressed caudally and was found in adaxial cells of somites 3–15 and in lateral cells of somites 7–15 (Fig. 5I). In 20-somite stage embryos (Fig. 5J) the whole of somites 3–20 expressed myogenin mRNA. All somites except number 1 and 2 had a myogenin signal in all somitic cells throughout the 25- to 30-somite stages (Fig. 5K,L). Between somite stages 30 to 40 myogenin expression was downregulated in more cranial somites so that only somites 25–40 had myogenin expression at the 40-somite stage (Fig. 5L,M). Finally, at the 40- to 50-somite stages only the most caudal somites expressed myogenin and the staining decreased in intensity (Fig. 5M,N). Myogenin expression preceded MyHC expression by approximately 5 somites.
Whereas no MyHC mRNA was found in the earliest embryonic stages (Fig. 5O), WM-ISH signals were identified adaxially in somites 4–10 at the 17-somite stage (Fig. 5P). By the 20-somite stage, MyHC mRNA was also found more laterally at the posterior end of somites 11–20, whereas somites 9–12 showed only adaxial MyHC expression (Fig. 5Q). From the 25-somite stage onwards MyHC expression gradually progressed caudally (Fig. 5R–T), and close to hatching (>50 somites) the MyHC signal was only found in somites 40–50 (Fig. 5U).
The expression of the MyLC2 isoforms was only studied by RT–PCR, hence no spatial information is available. Whereas MyLC2a mRNA was detected from the 40-somite stage onwards, expression of MyLC2b was initiated just before hatching from the 50-somite stage onwards (Fig. 6). By contrast to MyLC2a, the MyLC-2b mRNA level increased in intensity throughout embryogenesis, and was the only isoform identified in adult fast and slow skeletal muscle (not shown).
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). Hence, development of the halibut embryo seems to be a
fine-tuned process, and the natural temperature regime should be mimicked in
aquaculture to produce high-quality larvae with a high growth potential.
Temperature has been shown to affect the sequence of organogenesis
(Johnston et al., 1995), rate
of somitogenesis (Brooks and Johnston,
1994) (present study), number of somites
(Brooks and Johnston, 1994),
number and size of muscle fibres at hatching
(Vieira and Johnston, 1992;
Brooks and Johnston, 1993;
Usher et al., 1994;
Nathanailides et al., 1995;
Johnston and McLay, 1997;
Alami-Durante et al., 1997;
Galloway et al., 1998;
Galloway et al., 1999;
Hall and Johnston, 2003) and
the relative timing of myofibrillogenesis
(Johnston et al., 1995) in
different fish species. However, in agreement with our halibut study, relative
temporal and spatial expression of several muscle genes in Atlantic herring
Clupea harengus (Temple et al.,
2001), Atlantic cod Gadus morhua
(Hall et al., 2003) and common
carp Cyprinus carpio (Cole et al.,
2004) do not seem to be influenced by temperature. Temple et al.
concluded (Temple et al.,
2001) that temperature probably affects myofibril synthesis
downstream from MyHC transcription, at the translation or assembly stage. By
contrast, the relative expression of MRF genes was shown to be
delayed and prolonged at low egg incubation temperatures in other species such
as rainbow trout (Oncorhynchus mykiss) and gilthead seabream
(Wilkes et al., 2001;
Xie et al., 2001). These
authors suggested that the delayed muscle differentiation at lower
temperatures could extend the proliferation phase, enable more myoblast
hyperplasia and hence increase muscle fibre numbers at hatching, something
that is also seen in fish species with temperature-independent MRF
expression.
In most fish species where MyoD has been studied to date
[zebrafish Danio rerio (Weinberg
et al., 1996); Atlantic herring
(Temple et al., 2001);
gilthead seabream (Tan and Du,
2002); common carp (Cole et
al., 2004); rainbow trout
(Delalande and Rescan, 1999;
Xie et al., 2001); flounder
Paralichthys olivaceus (Zhang et
al., 2006)], the gene is expressed in presomitic and somitic
adaxial cells, which will later become the superficial slow red muscle fibres
(Devoto et al., 1996).
Subsequently, MyoD is expressed in lateral cells of the somites,
which will form the bulk of the fast white muscle mass. In Atlantic halibut,
MyoD mRNA was detected prior to somitogenesis, but was never observed
in adaxial cells of the presomitic or somitic mesoderm at any later
developmental stage. This absence of MyoD mRNA in the adaxial cells
has also been observed in Atlantic cod
(Hall et al., 2003).
Furthermore, MyoD was expressed bilaterally asymmetrically in the
earliest stages of halibut somitogenesis. These findings indicate that MyoD
may have a different function in halibut and cod than in other fish species,
and/or that halibut and cod may have more than one MyoD gene. Two
MyoD encoding genes have previously been reported in the tetraploid species
Xenopus (Scales et al.,
1990), rainbow trout (Rescan
and Gauvry, 1996), gilthead seabream
(Tan and Du, 2002) and
amphioxus Branchiostoma floridae
(Schubert et al., 2003). After
the present study was performed, another MyoD isoform, referred to as MyoD1,
was identified in halibut (Ø. Andersen, S. V. W. Dahle, E.
Kjørsvik, T. Bardal, H. Munck and T. F. Galloway, manuscript submitted
for publication). The two MyoD isoforms of both halibut and gilthead seabream
show about 72% sequence identity, indicating that the two paralogues are the
result of a common duplication event about 350 million years ago
(Robinson-Rechavi et al.,
2001). By contrast to the MyoD gene identified in the
present study (which should be referred to as MyoD2), the halibut
MyoD1 gene was symmetrically expressed in presomitic and somitic
adaxial cells from the 5-somite stage onwards, and subsequently spread to
lateral cells (Ø. Andersen, S. V. W. Dahle, E. Kjørsvik, T.
Bardal, H. Munck and T. F. Galloway, manuscript submitted for publication).
The MyoD gene isolated from flounder was also initially expressed
bilaterally symmetrically in adaxial cells and subsequently in lateral cells
(Zhang et al., 2006), but it
is not known whether the flounder has two MyoD isoforms. The functional
significance of the bilaterally asymmetrical expression pattern observed for
halibut MyoD2 is not known, but we hypothesise that this isoform may
have adopted a new function related to the development of external asymmetry
in this flatfish species. Alternatively, the asymmetric expression pattern may
be related to the development of a greater muscle mass on the ocular side than
on the abocular side, as observed in adult halibut
(Johnston, 2004). Asymmetry of
the muscle mass is not present in halibut yolk sac larvae
(Galloway et al., 1995;
Galloway et al., 1999),
indicating that the early asymmetrical MyoD2 expression has an effect
on gross muscle anatomy during the transformation from larva to juvenile and
onwards. During a sizeable part of early vertebrate embryogenesis the Wnt, FGF
and Notch signalling pathways convey both symmetric and asymmetric information
for bilaterally synchronised somitogenesis and left–right asymmetric
patterning of internal organs, respectively
(Kawakami et al., 2005). These
signals operate upstream from the MRFs, and have so far only been studied in
vertebrates with external bilateral symmetry
(Levin, 2005). Therefore,
there is a need to study more flatfish species with respect to muscle
development, in order to elucidate the possible functions of the asymmetrical
MyoD2 expression in embryos of halibut and possibly other
species.
Expression of the single halibut myogenin gene was initiated at
the 9- to 10-somite stage in adaxial cells, possibly correlating with the
initiation of myoblast differentiation and indicating that these red muscle
precursor cells differentiate prior to the lateral white muscle precursor
cells (Devoto et al., 1996;
Rescan, 2001). Subsequently,
the halibut myogenin signals spread to lateral somitic cells of newly
formed somites. When the halibut embryos contained between 30 and 40 somites,
the myogenin signals began to disappear from the cranial somites in
the same manner as the MyoD2 transcript did. This transient
expression pattern of myogenin resembles that observed in the
Atlantic herring and common carp (Temple
et al., 2001; Cole et al.,
2004), but is different from that observed in rainbow trout and
zebrafish (Delalande and Rescan,
1999; Weinberg et al.,
1996). This probably reflects differences in species, final size,
muscle fibre types and ecology.
Vertebrate muscle contains many myosin isoforms, depending on developmental
stage, muscle fibre type and environmental characteristics
(Goldspink et al., 2001). The
expression of the myofibrillar contractile proteins MyHC and MyLC has been
shown to follow expression of MRFs in all vertebrate species studied to date
(Watabe, 2001). The halibut
MyHC studied here was probably a transient embryonic isoform, and was
detected from the 15 somite stage, whereas MyLC2a mRNA was first
identified at the 40-somite stage. Neither MyHC nor MyLC2a
were expressed post-hatching (results not shown). Different MyLC2 isoforms
have been isolated from several teleosts, and a larval MyLC2 was shown to be
gradually replaced by an adult isoform in metamorphosing flatfish
(Brooks and Johnston, 1993;
Yamano et al., 1994;
Focant et al., 2000;
Focant et al., 2003). Whereas
the sequence of flatfish MyLC2 has so far been unknown, the similarity in the
expression patterns strongly indicates that the deduced halibut MyLC2a and
MyLC2b represent embryonic and larval/juvenile isoforms, respectively.
Factors regulating muscle determination, differentiation and development have so far mostly been studied in vertebrates with external bilateral symmetry. The present study reveals that a MyoD isoform, but not the other muscle genes studied, is expressed bilaterally asymmetrically during early somitogenesis of the halibut. This suggests that more such investigations of flatfish species could provide valuable information on how muscle-regulating mechanisms work in species with different anatomical, physiological and ecological traits. The present study also shows that many important events in the determination and differentiation of muscle in halibut occur within a very short time period (10 d° or approximately 2 days) between the formation of the 10th and 20th somite, a period that should be treated with special care in halibut hatcheries.
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Acknowledgments |
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References |
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