Effects of decreased muscle activity on developing axial musculature in nicb107 mutant zebrafish (Danio rerio)
Experimental Zoology Group, Wageningen Institute of Animal Sciences, Wageningen University, Marijkeweg 40, NL-6709 PG Wageningen, The Netherlands
* Author for correspondence (e-mail: talitha.vandermeulen{at}wur.nl)
Accepted 8 August 2005
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Summary |
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Key words: decreased activity, development, zebrafish, nicb107 mutant, muscle
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Introduction |
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Increasing muscle activity by forced swimming in fish stimulates red muscle
development, enhances muscle enzyme activity, increases blood oxygen carrying
capacity, increases mitochondrial density, improves swimming efficiency and
increases hypoxia tolerance (Bagatto et
al., 2001; Davison,
1989
,
1997
;
De Graaf et al., 1990
;
Kieffer, 2000
;
Pelster et al., 2003
).
Decreased muscle activity transiently downregulates the activity of the muscle
enzyme succinate dehydrogenase (De Graaf
et al., 1990
). Decreased activity by immobility in zebrafish and
Xenopus laevis embryos correlated with abnormal muscle fibre
distribution and axial musculature architecture in some
(Droin and Beauchemin, 1974
;
Granato et al., 1996
; Van
Raamsdonk et al., 1977
,
1982
) but not all cases
(Granato et al., 1996
). The
effects of altered muscle activity on gene expression levels during
development have been only rarely investigated.
The bulk of zebrafish axial muscle consists of fast white fibres. Slow red
fibres form a superficial layer, and intermediate pink fibres are located in
between (Waterman, 1969). The
white trunk axial musculature is arranged in a series of myomeres, which are
separated by collagenous sheets, the myosepta. Muscle fibres in adult fish run
between the myosepta in a pseudo-helical pattern
(Alexander, 1969
;
Ellerby and Altringham, 2001
;
Gemballa and Vogel, 2002
;
Johnston et al., 1995
;
Mos and Van Der Stelt, 1982
;
Van der Stelt, 1968
;
Van Leeuwen, 1999
). This
pseudo-helical arrangement is thought to be an optimisation for muscle work
output (Alexander, 1969
) and,
as such, may be influenced by mechanical loading. At 18 hours
post-fertilisation (18 hpf),zebrafish embryos start making their first feeble
movements (Westerfield, 1995
).
At this age, slow muscle fibres in the zebrafish embryo are adjacent and
parallel to the notochord, can be stained for heavy myosin chains and are
about to migrate laterally through the somite
(Devoto et al., 1996
). Fast
fibres, which already form the bulk of the muscle mass, do not stain for heavy
myosin chains until after 23 hpf (Devoto
et al., 1996
). Helical arrangements of zebrafish muscle fibres are
first observed at 4 days after hatching
(Van Raamsdonk et al., 1974
).
This timeline of development and use of the axial musculature also suggest
that early use of the axial musculature is crucial for its proper development
(Van Raamsdonk et al.,
1977
).
As the use of the musculature appears crucial for proper development of the
axial musculature, lack of use, i.e. immobility, is expected to hamper proper
development. We used the nicb107 mutant to study the
effect of immobility on axial muscle development. In this mutant, the
-subunit of the acetylcholine receptor is defective, which blocks
assembly of functional acetylcholine receptors on the muscle fibres
(Sepich et al., 1998
). As a
result of this lack of innervation, the muscle fibres fail to contract in
vivo. They are mechanically intact, however, as they are able to contract
upon electrical stimulation (Westerfield
et al., 1990
). In the present paper, we report several effects of
immobility on the morphogenetic development of axial musculature in early
zebrafish larvae by studying the expression levels of a selection of
structural as well as regulatory muscle genes and studying muscle structure at
different organisational levels.
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Materials and methods |
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Collecting and processing morphometric data
Embryos were positioned in sedative (1 g l-1 MS-222 and 1.5 g
l-1 Na2CO3. H2O) on a 1% agarose
gel. Lateral photographs were taken using an Olympus DP50 digital camera
mounted on a Zeiss Stemi SV11 microscope with AnalySIS software V3.1 (Soft
Imaging System GmbH, Münster, Germany). At 24, 48, 72, 96 and 120 hpf,
total body length and, at anus level, muscle height, notochord height, somite
size in anteriorposterior direction and angle of the somite were
measured from photos using AnalySIS software in both
nicb107 embryos and their wild-type siblings. Five to
eight animals were used per group per measurement. Statistical differences
between wild-type and nicb107 data were detected using the
MannWhitney U test and were considered significant when
P<0.05.
Transverse sections for fibre orientation
Embryos were fixed in 4% paraformaldehyde (PFA) in PBS overnight at
4°C, then stored in 1% PFA in PBS, postfixed in 10% PFA in PBS and
embedded in 15% gelatin in PBS and fixed overnight at 4°C in 4% PFA in
PBS. Transverse sections of 100 µm thickness in the area just behind the
anus were cut on a vibratome 1500 (Vibratome, St Louis, MO, USA) and stained
overnight at 4°C with propidium iodide (1 µg ml-1) in PBS.
They were incubated in 25% (1 h), 50% (1 h), 75% (overnight) and 90% glycerol
in PBS (3 h) and then embedded in 90% glycerol in PBS immediately prior to
examination.
Fibre tracking
A 15 µm-thick Z-stack of 1 µm-thick consecutive optical sections was
created from the 100 µm-thick transverse section using a laser scanning
microscope (ZEISS LSM-510). The Z-stack was exported as individual TIFF files
to AnalySIS software and calibrated. Each fibre that was present as a complete
cross section in 15 consecutive sections (i.e. over a distance of 15 µm
from anterior to posterior) was individually and manually tracked. This
implies that fibres close to myosepta were not digitised, due to tapering. For
each cross section, the centre of area (CA) in coordinates of the Z-stack was
computed with AnalySIS software. The fibre orientations in
XzYzZz coordinates
of the Z-stack were computed in Matlab 6.5 (The Mathworks, Inc., Gouda, The
Netherlands) from the CA in the first section with that of the nearest CA in
the second section. As a control of the validity, two such computations were
made per embryo, using different sections. For final analyses, optical
sections that were 5 µm apart were analysed. At this distance, individual
fibres can be easily identified and tracked, a straight vector is a relatively
accurate description of the local fibre orientation and the error in the
computed orientation was determined to be less than 5°.
In general, the computed orientation of the fibres in the
XzYzZz coordinates
of the Z-stack is not a fair representation of the fibres in a fish-bound
XfYfZf coordinate
system, because the sections in the
XzYz plane are not exactly parallel to
the XfYf plane (transverse plane) of
the fish (Fig. 1). Based on the
leftright symmetry of the fish, the computed fibre orientations were
rotated over three perpendicular axes to obtain a visually leftright
symmetrical vector field. The result is a series of vectors describing the
elevation and azimuth of each individual fibre in a fish-bound
XfYfZf coordinate
system. Elevation is the angle of the fibre with the horizontal plane (or
XfZf plane). Azimuth is the angle of
the projection of the fibre on the horizontal plane with the sagittal plane
(YfZf). For visualisation purposes,
these vectors were projected on the first section. The distance
between sections is 5 µm. From
and the length of the projections in
Xf and Yf directions, the size of the
azimuth and elevation of each vector can be calculated. Azimuth =
atan(Lx/
), and elevation =
atan[Ly/
(Lx2 +
2)], where Lx is the length of the
projection in the Xf direction and Ly
is the length of the projection in the Yf direction.
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Real-time quantitative PCR (RQ-PCR)
Primer Express software (Applied Biosystems, Nieuwerkerk a/d IJssel, The
Netherlands) was used to design primers for use in real-time quantitative PCR
(RQ-PCR). Primers are given in Table 1 in supplementary material. Primers were
tested for specificity and efficiency of template amplification by sequencing
the PCR product and using a known template dilution series in RQ-PCR,
respectively. For RQ-PCR, 5 µl cDNA and forward and reverse primers (1.5
µl of 5 µmol l-1 each) were added to 12.5 µl Quantitect
SYBR Green PCR Mastermix (Qiagen, Venlo, The Netherlands) and filled up with
4.5 µl demineralised water to a final volume of 25 µl. RQ-PCR (15 min at
95°C, 40 cycles of 15 s at 94°C, 30 s at 60°C, 30 s at 72°C)
and melt analysis (6090°C in 1° steps) was carried out on the
Rotor-Gene 200 Real Time cycler (Corbett Research, Mortlake, New South Wales,
Australia). Data were analysed using the Pfaffl method to include primer
efficiencies (Pfaffl, 2001).
This method requires the use of a designated `housekeeping gene', a gene whose
expression is not affected by the treatment, decreased muscle activity in this
case. Two such housekeeping genes were used to normalise the expression
between nicb107 and wild-type embryos: ribosomal protein
40S and ß-actin. Results were similar with either gene, and the results
for 40S are shown. Immobility might influence the process of muscle
development but it might also influence the final muscle structure. We
therefore chose to test several groups of genes: structural components of
sarcomeres (myosins, titin, troponin C), muscle activity genes [myoglobin,
phosphofructokinase for muscle (PFK-m), mitochondrial diaphorase (NADHd) and
succinate dehydrogenase complex subunit A (SDHa)] and muscle growth factors
(insulin-like growth factor and its receptors, myostatin/growth and
differentiation factor 8, myogenin). Two collagens were tested as they
represent two different tissue types that may be indirectly influenced by
muscle activity. Collagen type 1
2 is found mainly in skin in
embryonic stages and later in bone. Collagen type 2
1 is found around
the notochord in early development and later in cartilage. To determine
whether differences were statistically significant, analysis of variance
(ANOVA) contrasts were determined in SPSS v. 12.0.1 (SPSS Inc., Chicago, IL,
USA) between nicb107 and wild-type gene expression at the
same age, while taking into account whether or not variances were homogeneous.
Differences were considered significant when P<0.05.
Transverse sections and slow muscle antibody staining
Embryos were fixed in 4% PFA in PBS overnight at 4°C, washed in PBS,
incubated in 5% sucrose for 30 min, embedded in 1.5% agarose in 5% sucrose.
After overnight storage at 4°C in 25% sucrose, the agarose block was
incubated for 3 h in fresh 25% sucrose before being flash frozen in liquid
nitrogen. 10 µm-thick sections were cut on a cryostat and collected on
polysine slides (Menzel-Gläser, Braunschweig, Germany). The sections were
washed twice for 5 min in PBS, incubated for 10 min with blocking solution
[10% normal calf serum (NCS) in PBS], for 60 min with 1:10 F59 supernatant (a
generous gift of Dr Frank E. Stockdale;
Crow and Stockdale, 1986),
which stains slow muscle cells in zebrafish
(Devoto et al., 1996
), in 3%
NCS in PBS, washed three times for 5 min in PBS, incubated with 1:50
FITC-labelled goat anti-mouse antibody (DAKO, Hevelee, BE) in 3% NCS in PBS,
washed three times for 5 min in PBS and mounted in Vectashield with propidium
iodide (Vector Laboratories, Burlingame, CA, USA). Photographs were taken
using an Olympus DP50 digital camera mounted on a Nikon MicroPhot microscope
and AnalySIS software.
EM preparations
Embryos were fixed for at least one day in 4% PFA in PBS at 4°C,
postfixed for 60 min in a 0.1 mol l-1 cacodylate buffer (pH 7.2)
containing 1% osmium (OsO4), 2% glutaraldehyde and 1%
K2Cr2O7 on ice, rinsed twice with milliQ
water and stored in 70% ethanol until embedding in epon.
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Results |
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Muscle fibre arrangement
The muscle fibre directions at anus level for 48, 72 and 96 hpf are given
in Fig. 4. The projection of
the fibres is taken from anterior to posterior, with the anterior end of each
fibre as the centre of a circle. The nicb107 larvae
(Fig. 4DF) show a
topology very similar to the wild-type larvae
(Fig. 4AC). Overall, the
fibre orientations in wild-type and nicb107 are similar,
and helices can be discerned as early as 48 hpf. The
nicb107 fibre orientation patterns are somewhat more
irregular and dorso-ventrally flattened at 96 hpf, with fibres displaying
smaller elevation angles than in wild-type embryos, especially laterally
(Fig. 4F). The lack of data
near the myosepta and the different locations of the myosepta in different
sections prevent an absolute comparison of the elevation and azimuth data
between wild-type and nicb107 embryos or to follow changes
over time. For the fibres on the sections shown here, the mean absolute values
for the elevation and azimuth angles are both 8°.
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Ultrastructural muscle morphology
Sarcomere architecture results in a repeated banding pattern in striated
muscle, a result of stacking of sarcomeres on top of one another in a single
myofibril (Fig. 6A,B). In
wild-type embryos, myofibrils are positioned in parallel to one another over
some distance. The sarcomeres in adjacent myofibrils are juxtaposed regularly
and the banding pattern stretches out over multiple myofibrils. In the
nicb107 embryos, the myofibrils are arranged in parallel
over shorter distances than in the wild type. This can be inferred from the
tapering of the myofibrils (Fig.
6C,D). In addition, sarcomeres are present, but the stacking of
sarcomeres is less regular than in the wild-type embryos. The sarcomere bands
are not strictly juxtaposed, but a shift in banding between myofibrils in
nicb107 embryos is present
(Fig. 6C,D).
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Discussion |
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IMA and DMA have opposing effects on muscle, as is shown in
Fig. 8. IMA promotes muscle
growth amongst others through IGF (Adams,
1998) and myogenin signalling
(Hasty et al., 1993
;
Nabeshima et al., 1993
;
Rescan, 2001
), and DMA
inhibits muscle growth amongst others by stimulating gdf8 signalling
(Lee, 2004
;
McPherron et al., 1997
; Xu et
al., 2000
,
2003
). Gdf8 signalling acts in
part through downregulation of myogenin and IGF expression
(Amali et al., 2004
). In
addition to promoting growth, a high relative expression of myogenin over MyoD
promotes a shift towards a slow muscle phenotype
(Talmadge, 2000
). Overcrowding
stress acts contradictively as it inhibits gdf8 expression in zebrafish but it
also represses muscle growth (not shown in
Fig. 8). This was suggested to
result from a general depression of muscle protein synthesis that does not
spare myostatin (Vianello et al.,
2003
).
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The aim of the present study was to investigate the effects of DMA on the
early development of the axial musculature in zebrafish. It was hypothesised
that DMA would hamper proper muscle development. As a model for DMA, the
nicb107 mutant was used. Up to 18 hpf, wild-type and
nicb107 mutant embryos are indistinguishable by eye.
During the first 18 h of development, a great many processes involving
specification and differentiation of somite derivatives have already taken
place. In the early hours after fertilisation, involution and convergent
extension have created axial and paraxial mesoderm. The axial mesoderm induces
local differentiation of the paraxial mesoderm, and slow muscle precursors
have been specified. The paraxial mesoderm is segmented into somites from 10.5
hpf onwards, and, by 18 hpf, 18 somites have been formed
(Kimmel et al., 1995;
Stickney et al., 2000
). The
wild-type embryos now start making their first feeble coiling movements
(Westerfield, 1995
) by
contraction of the muscle pioneer cells through cholinergic signalling
(Melancon et al., 1997
). By 21
hpf, they become sensitive to touch and respond with vigorous coiling, and, at
1 day post-fertilisation, fast and slow muscle are both activated in coiling
behaviour (Buss and Drapeau,
2002
). Muscle action is conferred via cholinergic
signalling through the release of acetylcholine (ACh), which, after binding to
its receptor on the muscle cell, results in an action potential. After
subsequent release of calcium in the muscle cell, the sarcomeres, the smallest
functional units of muscle, contract. Sarcomeres are composed of thick
(myosins) and thin (actin, tropomyosin and the troponin complex) filaments,
titin and several other proteins (Marieb,
2004
). In the nicb107 mutant, cholinergic
signalling is defective because the acetylcholine receptor is not functional
(Sepich et al., 1994
,
1998
;
Westerfield et al., 1990
), no
action potentials are generated
(Westerfield et al., 1990
) and
thus no calcium is released. Consequently, the nicb107
mutant embryos do not perform any movement, not even when stimulated (Sepich
et al., 1994
,
1998
;
Westerfield et al., 1990
). The
lack of signalling and ensuing lack of muscle contraction possibly result in
DMA when tissues are still developing. This is expected to result in increased
gdf8 expression, decreased myogenin and IGF signalling, a decrease in muscle
growth and a slow-to-fast transition of muscle fibres, according to literature
for adult muscle (Fig. 8).
DMA effects on gene expression levels in muscle
The expression of myogenin in the nicb107 embryos is
higher than in wild-type embryos, which is not in line with the effects of DMA
in Fig. 8. A study addressing
the effects of innervation and blocking neuromuscular transmission in
developing and adult rats reported that developmental innervation, although
IMA-like, downregulates myogenin expression. Preventing developmental
innervation, although it is DMA-like, prevented this downregulation
(Witzemann and Sakmann, 1991).
Blocking neuromuscular transmission (DMA-like) using toxins that leave the
neuromuscular junction itself intact, and thus the possibility of
communication, increased myogenin expression
(Witzemann and Sakmann, 1991
).
In our study, developmental innervation takes place; only the neuromuscular
transmission is defective. The observed increase in myogenin could thus result
from a lack of downregulation. Along a similar line of reasoning, the
increased IGF signalling, which also promotes muscle growth
(Adams, 1998
), may perhaps be
viewed. IGF signalling is increased through increases in IGF-Rb expression.
IGF-1 and IGF-Ra expression is not affected, which is consistent with effects
of altered muscle activity by space flight
(Lalani et al., 2000
) and
resistance training (Walker et al.,
2004
) but not mechanical ventilation, which decreased IGF-1
expression (Gayan-Ramirez and Decramer,
2002
). We also observed a transient increase in gdf8 expression,
which is in line with Fig. 8,
followed by a decrease by 96 hpf. A (transient) increase fits with mammalian
literature (Carlson et al.,
1999
; Lalani et al.,
2000
). Since increases in opposing growth factor signalling are
detected, the question is whether growth of muscle is promoted or inhibited.
Titin expression, which is found in red and white muscle alike, is elevated
and suggests that growth is promoted.
High relative expression of myogenin has been implied in a fast-to-slow
transition in muscle fibres (Talmadge,
2000). The observed downregulation of fast muscle specific myosin
heavy chain 2 and troponin C expression is consistent with this idea. The
downregulation of slow troponin C is not. Troponin C is involved in binding
calcium that is released upon cholinergic signalling. In the absence of this
calcium release in the nicb107 mutants, there is no
functional demand for troponin C in either muscle type. If a direct
relationship between calcium release and troponin C expression exists, this
can explain the downregulation of both fast and slow troponin C in the
nicb107 mutant. This would also suggest a downregulation
of both types of troponin C in the relaxed mutant, which also lacks
calcium signalling, despite successfully generating an electrical signal
(Ono et al., 2001
).
In zebrafish, increased activity at early life stages increased the
mitochondrial content of red and intermediate muscle, suggesting an increased
demand for oxidative metabolism (Pelster
et al., 2003). Decreased activity resulted in a transient decrease
in the activity of SDHa, an enzyme of the oxidative phosphorylation pathway
(De Graaf et al., 1990
). The
nicb107 mutant does not require energy for movement, only
for basal cell metabolism, and therefore the energy-generating pathways may be
less active in nicb107 than in wild-type embryos. We
assayed the expression of genes that are involved in cellular energy
metabolism. Expression of myoglobin, which acts as a muscle oxygen carrier,
was not changed. Glycolysis is the first step to generate energy, and
muscle-specific phosphofructokinase is involved in this process. No
differences in expression level were found between wild-type and
nicb107 mutant embryos. End products of glycolysis are
oxidised in mitochondria in the oxidative phosphorylation pathway, a.o. by the
enzymes SDHa and NADHd. These genes are also not altered in expression. The
transient decrease in SDHa enzyme activity in adult zebrafish
(De Graaf et al., 1990
) may
have been the result of (post)-translational control of activity or may occur
only later in life. Together, these observations suggest that energy
metabolism in the nicb107 mutant is not grossly affected
by the lack of muscle activity. Overall, it appears that the composition of
developing muscle is altered at the molecular level, in line with DMA effects
on adult muscle. Contrary to literature for adult animals, however, muscle
growth and energy metabolism are not impeded by DMA during development
(Fig. 8).
DMA effects on gene expression levels outside muscle: skin and notochord
In addition to affecting muscle, muscle activity may also affect the
tissues peripheral (skin) and central (notochord) to muscle by exerting force.
Major force-resistant molecules in these tissues are collagens. DMA was
expected to result in lower collagen expression in these tissues since less
force is applied. The skin collagen fibrils consist mainly of collagen type 1
(Dubois et al., 2002;
Le Guellec et al., 2004
). In
fact, the bulk of expression of collagen type 1
2 is concentrated in
the skin, with minor expression in other organs
(Dubois et al., 2002
). The
expression of this gene is downregulated in nicb107
embryos, suggesting the skin to be less stiff in nicb107
than in wild-type embryos as a result of DMA. Consequently, the skin in the
nicb107 embryos may be less resistant to deformation. In
the nicb107 embryos, in the absence of muscle fibre
activity, deformation can still be generated by growth. For a direct effect of
muscle fibre activity, see below.
The notochord is a fluid-filled column that stiffens the body axis because
it is osmotically pressurised (Adams et
al., 1990). Collagen type 2
1 (col2
1) is a major
component of the notochordal sheet (Adams
et al., 1990
) during the first 24 h of development in the
zebrafish (Yan et al., 1995
)
and has been suggested to counteract the internal pressure
(Aszódi et al., 1998
).
The expression of col2
1 is not different between
nicb107 and wild-type embryos at 24 hpf, which suggests
unchanged stiffness between wild-type and nicb107
embryos.
Body shape
From 72 hpf onwards, about half of the mutant embryos show curvature of the
body with their tails curved up. Abnormal body curvature is a common feature
in mutants in the diverse mutagenesis screens. These mutants usually have
additional defects. Abnormal body curvature is especially common in mutants
with defects in notochord (Stemple et al.,
1996) and midline structures
(Brand et al., 1996
),
suggesting that these tissues normally stabilise a straight body shape or
induce tissues that do so. Searching the 2966 entries in the zfin database
(Sprague et al., 2001
) for
body curvature (tail up or down and bent body) results in 209 hits. Only one
of these is immobile (Brand et al.,
1996
). Vice versa, out of 145 hits for `nonmotile' or
`reduced motility', only 10, representing three loci, show changes in body
curvature (Granato et al.,
1996
). This suggests that there is not a one-to-one relationship
between body curvature and mobility, which is also indicated by the fact that
the body curvature phenotype is not fully penetrant in the
nicb107 mutants.
Somite size, measured ventrally in the anal somite, is larger in
nicb107 embryos than in wild-type embryos at 120 hpf, a
possible result of lower gdf8 expression in nicb107
embryos (Amali et al., 2004).
Total length measured along the notochord did not differ. This incongruity
correlates with the presence of progressive dorsad curvature of the tail in
half of the nicb107 embryos
(Fig. 2B). Despite this total
body curvature, the somite angle did not differ significantly between groups.
Other authors found larger somite angles and smaller somite sizes at 72 hpf
for immobile zebrafish embryos (Van
Raamsdonk et al., 1977
). They looked at spontaneously immobile
embryos and also induced immobility by removal of the brain or making a
midbody lesion. Both types of immobile embryos in these experiments were
morphologically different from the untreated embryos, e.g. they had shorter,
less developed tails (Van Raamsdonk et
al., 1977
). These results are therefore difficult to compare with
ours, in which development was not altered by surgical procedures.
Morphometric measurements are not part of the large genetic screens and
therefore this type of data is scarce. Experiments with spontaneously immobile
tadpole larvae show body curvature with the tail pointing dorsally and larger
somite size (Droin and Beauchemin,
1974
), similar to our findings. Somite angle became smaller in
these embryos, whereas total length increased
(Droin and Beauchemin, 1974
),
which contrasts with our results. There appears to be a correlation between
the lengthening of the embryonic body and the angle of the somites. Shorter
bodies correlate with larger angles (Van
Raamsdonk et al., 1977
), longer bodies correlate with smaller
somite angles (Droin and Beauchemin,
1974
) and, in our experiments, neither of the two changed. The
larger somite size in nicb107 embryos can be a result of
activity of the muscle fibres. Muscle fibres shorten when active and pull
adjacent myosepta together, effectively decreasing somite size. The inactive
muscle fibres in the nicb107 embryos will not pull
adjacent myosepta together, resulting in larger somite sizes.
No indications for notochord differences between wild-type and
nicb107 embryos were found at the molecular level (see
above), but in wild-type embryos extra pressure may be generated by the muscle
fibre contractions that exert a compressive stress on the notochord in a
longitudinal direction. This activity of the muscle fibres acts to raise
internal pressure, which in turn results in greater mechanical stiffness of
the notochord (Adams et al.,
1990). A more circular transverse circumference is then adopted
automatically in the pressurised notochord, as is seen in wild-type embryos.
In addition, in a simulation of muscle fibre activity in adult fish, Van
Leeuwen (1999
) proposed that
the external shape of a fish body could be affected by skin stiffness. In
simulations with reduced skin stiffness, muscle tissue moved inwards close to
the mid-horizontal plane and outwards in the dorsal and ventral regions upon
activation of the muscle fibres, leading to two concavities near the
mid-horizontal plane (Van Leeuwen,
1999
). The embryonic skin consists of only two layers of cells
with few collagen fibrils (Le Guellec et
al., 2004
) and can therefore be expected to be weak. This holds
for both the wild-type and the nicb107 embryos, but muscle
fibre activity occurs only in the wild-type embryos. Indeed, the predicted
concavities are found in the wild-type embryos, especially at 48 and 72 hpf,
but are less prominent in nicb107 embryos, despite the
fact that they have a potentially weaker skin, suggesting a direct effect of
DMA on cross-sectional body shape.
Muscle fibre morphology
Wild-type zebrafish embryos start to make movements around 18 hpf, and this
is also the time that slow muscle fibres migrate laterally through the somite
towards the muscle periphery (Devoto et
al., 1996). Agar immobilisation of zebrafish embryos resulted in a
mosaic pattern of slow and fast fibres in the bulk of fast muscle
(Van Raamsdonk et al., 1982
),
which may be the result of altered migration of slow muscle fibres or
phenotypic switching of fast muscle fibres. In any case, it implies altered
ratios of slow and fast fibres, i.e. of slow and fast myosins. The expression
of a fast-twitch myosin heavy chain is indeed affected
(Fig. 7), which is in line with
this idea. We therefore stained slow fibres in zebrafish to check for possible
mosaic formation of slow fibres in the bulk of fast fibres in situ.
The muscle fibres in nicb107 embryos are normally
segregated as slow and fast fibres and no mosaic is found
(Fig. 5). Apparently, genetic
immobilisation is different from agar immobilisation. In 5% agar, growth
defects become apparent; 0.5% agar allows for growth but also for body bending
(Van Raamsdonk et al., 1979
).
Firing of neurones is not inhibited by embedding. Indeed, in 1% agar, the
embryos are able to contract their muscles and they will make shrugging
movements (T.v.d.M., unpublished), even when the action does not result in
body deformation and embryos appear immobile
(Van Raamsdonk et al., 1979
).
It is not implausible that embedded embryos will try to escape the agar
confinement or try to refresh the oxygen-poor stagnant agar boundary layer. In
their efforts to do so, they may even show IMA. IMA leads to a fast-to-slow
transition in muscle fibres (Fig.
8) and could explain the mosaic formation. The difference
therefore lies in the activity of muscles, i.e. whether the immobility is
intrinsic (in the nicb107 mutant) or extrinsic (in agarose
embedding). Our results showed that in the nicb107 mutant,
fast and slow muscle fibres segregate correctly. Muscle fibre type is
determined largely by sarcomere components
(Fig. 8), indicating that these
are assimilated correctly.
Another feature of muscle development is the development of a
pseudo-helical arrangement of white muscle fibres. This pseudo-helical
arrangement in adult fish is thought to allow equal amounts of work by each
individual fibre of a single myomere and can thus be viewed as an optimisation
of the fibre architecture to activity
(Alexander, 1969). The
development of this pattern occurs only after the embryos make their first
movements. We found helical arrangements as early as 48 hpf, which is four
days earlier than previously reported (Van
Raamsdonk et al., 1974
). As both wild-type and
nicb107 embryos arranged their muscle fibres in helical
patterns, it appears that the mechanical loading of the early parallel fibres
is not necessary for the development of a helical pattern. Loading may be
necessary for further development of muscle fibre architecture, as helices in
nicb107 mutants appear less organised and the change in
body shape concomitantly affects the shape of the helix. As a result, at 96
hpf, the nicb107 lateral muscle fibres have a smaller
elevation angle. This can be seen in Fig.
4C vs Fig.
4F: the line segments indicating fibre direction have a greater
vertical component in lateral wild-type fibres than in lateral
nicb107 mutant fibres. The larger elevation angles in the
lateral wild-type fibres are necessary for equalising the strains over the
fibres in a single myotome, which forms the basis of the optimised fibre
architecture (Alexander, 1969
;
J.L.v.L., unpublished). In conclusion, the pseudo-helical arrangement of
muscle fibres develops in the case of DMA but is affected by it.
Within muscle fibres, stacks of sarcomeres are arranged in myofibrils. DMA
in the nicb107 mutant, results in apparently normal
sarcomeres, but the stacking of sarcomeres on top of one another and the
myofibril organisation are less regular
(Fig. 6). A similar phenotype
is observed in the twister mutant. The IMA in this mutant results
from increased synaptic decay times, due to a dominant mutation in the
subunit of the acetylcholine receptor. Contractile filaments appear
disorganised and myofibril organisation is severely impaired
(Lefebvre et al., 2004
). This
shows that basal components, such as the sarcomeres, develop more or less
normally, but the integration of multiple basal components into a higher-level
architecture goes astray when muscle activity deviates from the normal.
Conclusion
Despite the lack of muscle fibre use, slow and fast muscle fibres develop,
the intricate sarcomere architecture is built and the necessary genes are
transcribed. It appears therefore that the information that is needed to
develop and differentiate the `basal components' of muscle does not depend on
external signalling from muscle activity. The DMA that results from a lack of
cholinergic signalling during development distinguishes
nicb107 embryos from wild-type embryos, however, as body
shape, gene expression levels, sarcomere stacking and myofibril arrangement
are altered. In conclusion, DMA affects morphological as well as genetic
parameters during early development, influencing differentiation of basal
components.
Recommendations for future research
When muscle activity is viewed as an, as yet, unknown pathway that affects
muscle development and differentiation, it can be interfered with at several
steps that result in DMA or IMA. The nicb107 mutant, a
model for DMA, has defective electrical signalling, and downstream muscle
activities are affected as well. These include the release of calcium and
force production by muscle contraction. In the relaxed mutant,
electrical signalling is intact, but a defect in dihydropyridine
receptor-mediated release of internal calcium renders them motionless
(Ono et al., 2001). Comparing
these two mutants will provide information on the importance of the electrical
signal in the muscle activity pathway. The twister mutant provides a
model for IMA. It has a dominant mutation in the
subunit of the
acetylcholine receptor that increases synaptic decay times and causes
uncoordinated movements (Lefebvre et al.,
2004
). It has already been shown to have profound effects on
muscle development, but data on the levels of expression of genes that appear
affected morphologically are currently not available
(Lefebvre et al., 2004
).
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Acknowledgments |
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References |
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