Download The zebrafish as a model for muscular dystrophy and congenital

Human Molecular Genetics, 2003, Vol. 12, Review Issue 2
DOI: 10.1093/hmg/ddg279
R265–R270
The zebrafish as a model for muscular dystrophy
and congenital myopathy
David I. Bassett1,* and Peter D. Currie2
1
Comparative and Developmental Genetics Section, MRC Human Genetics Unit, Western General Hospital,
Crewe Road, Edinburgh EH4 2XU, UK and Institute of Human Genetics, University of Newcastle upon Tyne,
International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK and 2Victor Chang Cardiac
Research Institute, 384 Victoria St, Darlinghurst 2010, Sydney, Australia
Received July 10, 2003; Revised and Accepted August 8, 2003
The muscular dystrophies and congenital myopathies are inherited diseases of the skeletal muscle, which
lead to a loss of muscle function and are often fatal. While many of the loci involved are already known, these
conditions remain incurable, and genetic models are being developed in an effort to understand the
pathological mechanisms involved. Recently several papers have shown that the zebrafish, which is now
widely used in developmental genetic studies, will provide a useful addition to our toolkit in this regard. Here
we describe these studies, including a zebrafish model of what is potentially the novel pathological
mechanism of muscle attachment failure in Duchenne and other muscular dystrophies.
INTRODUCTION
Muscle tissue can be subject to a tremendous variety of
diseases, both inherited, such as the muscular dystrophies, in
which cycles of de- and re-generation destroy skeletal muscles,
and acquired, such as the cardiomyopathies in which abnormal
growth of cardiac muscle compromises heart function. In order
to develop the necessary preventative strategies and treatments
for conditions like these, it is likely to be of great benefit to
understand their pathology at the level of the genes and cellular
processes affected. Molecular genetic studies, focussing on
developmental processes are currently providing a torrent of
such information, and during the last decade the zebrafish,
Danio rerio, has come to rank alongside the nematode
Caenorhabditis elegans, the fruit fly, the frog Xenopus laevis,
and the mouse, as a model system of choice in the burgeoning
field of developmental genetics. It shares with invertebrate
models the advantage of quickly producing high numbers of
externally developing, diploid embryos, making large-scale
random mutagenesis screens a feasible, almost routine, undertaking. It also conforms to the same fundamental body plan as
humans, containing an equivalent to nearly every human organ,
cell and molecular pathway, allowing the examination of gene
function in a context likely to produce data applicable to human
development and disease. This formidable combination of
‘forward’ genetics, which trawls a genome for new gene
functions, wherever they may reside, and ‘reverse’ genetics,
which entails functional studies of known genes including
disease loci, is now being employed in the study of genetic
diseases of skeletal muscle. Here we concentrate on a recent
series of papers have begun to employ the zebrafish in the study
of muscular dystrophies of the types caused by disruption of
the dystrophin-associated protein complex (DAPC).
MUSCLE DEVELOPMENT IN THE ZEBRAFISH
Zebrafish embryos are particularly suited to the study of muscle
development for several reasons, most importantly they
develop externally, are transparent, somitic muscle comprises
a large proportion of the body and is accessible, and they begin
to move very soon after gastrulation. Both embryological and
genetic studies have taken advantage these qualities to examine
early stages of muscle development in the zebrafish with great
success, such as the specification of slow-twitch muscle fibres
(1). However, the later stages of development have been
relatively little studied beyond the initial identification of
mutations that affect muscle fibre differentiation, function and
integrity.
Somitic muscle in fish forms initially from segmented
paraxial mesoderm, with the posterior compartment of each
somite expressing myogenic basic–helix–loop–helix transcription factors from the end of gastrulation. Muscle fibres
differentiate to span an entire somite in the anterior–posterior
axis. The first somitic muscle cells to differentiate are slowtwitch fibres that are specified medially by midline-derived
hedgehog family signalling proteins, differentiate by 16 h
*To whom correspondence should be addressed. Email: david.bassett@hgu.mrc.ac.uk
Human Molecular Genetics, Vol. 12, Review Issue 2 # Oxford University Press 2003; all rights reserved
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Human Molecular Genetics, 2003, Vol. 12, Review Issue 2
post-fertilisation (hpf) and subsequently migrate to the lateral
periphery of the somites (reviewed in 2). The bulk of the somite
develops as fast-twitch muscle fibres once this migration has
occurred. The somite adopts its distinctive chevron shape early
on, by 24 hpf, with the dorsal and ventral halves being
separated by a sheet of matrix material called the horizontal
myoseptum and each pair of adjacent somites being separated
by a sheet of matrix called the vertical myoseptum (see Fig. 1
for a schematic representation). These myosepta, along with the
notochord (a primitive stiffening rod common to all chordates)
serve as the attachment sites for somitic muscle fibres. These
structures are essentially laminar tendons that transmit force to
the notochord, and later to the vertebral column. These muscle
attachment sites have now come under the spotlight with the
finding that their mechanical failure is the pathological
mechanism in a zebrafish mutation that provides the first
zebrafish model of an inherited disease of skeletal muscle (3).
THE DAPC IN HUMAN DISEASE AND
MOUSE MODELS
The DAPC is a multi-protein assembly that provides
a transmembrane link between the cytoskeleton and the
extracellular matrix. This complex also acts as an anchor for
certain signalling components such as nitric oxide synthase (4–6).
The complex consists of several sub-complexes of a few
proteins, with the main structural axis being provided by a
series of three proteins that link intracellular F-actin below the
sarcolemma to laminin outside the cell. The laminin receptor
within the DAPC is dystroglycan, which is formed by the
cleavage of a precursor protein into extracellular a and
transmembrane b subunits. Dystrophin is a large rod-like
protein related to the spectrin family, which binds to
b-dystroglycan at its C-terminus and to actin filaments via its
N-terminus. A second sub-complex of the DAPC comprises a
group of related transmembrane proteins called sarcoglycans;
a third is based on syntrophin proteins and nitric oxide synthase,
and several further proteins are known to associate with the
complex. The DAPC is found at the membrane of skeletal
muscle fibres and several other cell types in the body, while a
similar complex in which dystrophin is replaced by the related
protein utrophin is distributed widely throughout the body.
Dystrophin is the largest product of the DMD or Duchenne
muscular dystrophy gene, which is responsible for a spectrum
of X-linked conditions including Duchenne and the milder
Becker muscular dystrophies, cardiomyopathies and mental
retardation (7,8). In Duchenne muscular dystrophy the
sarcolemma is torn during muscle contraction, leading to a
cycle of death and replacement of muscle fibres which results
in an accumulation of scar tissue in the muscle and a gradual
loss of function and eventually death. Many other muscular
dystrophies and congenital myopathies are caused by mutations
affecting other components of the complex, such as a congenital
dystrophy linked to laminin-a2, and type-2 limb girdle
muscular dystrophies (LGMD2), some of which are linked to
sarcoglycans, calpain 3, caveolin 3 and dysferlin (9–13).
The distribution of dystrophin- and utrophin-containing
DAPC within muscle fibres and the consequences of loss of
dystrophin for the integrity of the sarcolemma and ultimately
Figure 1. The somitic muscle in the trunk of a fish forms a series of pairs
of bilaterally symmetrical chevron-shaped somites, shown here as alternating
yellow and blue segments. Each somite is divided from its anterior (left) and
posterior (right) neighbours by sheets of extracellular matrix, the vertical
myosepta (VM). These act as tendons, transducing force towards the notochord
(NC). The notochord is a stiff rod packed with vacuolated cells, and has a
vestigial equivalent in human embryos, which in turn gives rise to the intervertebral discs. The dorsal and ventral halves of each chevron somite are divided
by a further sheet of extracellular matrix, the horizontal myoseptum (HM),
which also attaches to the notochord medially and provides a guidance cue
for migrating neural crest derivatives and motor axons. The neural tube (NT)
runs between the dorsal somites. Horizontal and parasagittal planes of section
are shown for orientation, while the anterior (left) end of the diagram represents
the typical appearance of a transverse section. Individual muscle fibres run
parallel with the axis of the body in an anterior–posterior direction and span
across an entire somite between two vertical myosepta. Muscle fibres form
myotendinous junctions with the vertical myosepta, which provide their main
site of force transmission. These junctions are specialised areas of sarcolemma
at which the DAPC is localized in embryonic muscle, and which are subject to
failure in embryos homozygous for sap, a mutation in the zebrafish orthologue
of the Duchenne muscular dystrophy gene.
the survival of the muscle fibre have been well documented in
studies of both patient biopsies and the dystrophin-deficient
mdx mouse. It is thought that, in Duchenne patients, remaining
structural proteins in the sarcolemma such as spectrin and
integrin complexes are insufficient to protect non-specialized
areas of the membrane from forces generated during exercise.
This leads to the appearance of tears along the length of the
fibre, and eventually to the replacement of dead muscle fibres
with scar tissue (14). The specialized neuromuscular junction is
unaffected because it contains utrophin, which despite some
up-regulation is insufficient to compensate for dystrophin loss
over the whole fibre (15–17). By contrast to Duchenne patients,
the mdx mouse is only mildly affected, perhaps (18–20)
because of efficient regeneration and effective compensation by
Human Molecular Genetics, 2003, Vol. 12, Review Issue 2
utrophin (21,22). This last observation has lead to attempts to
identify ways to increase utrophin production in the hope of
allowing a therapeutic level of compensation (23).
DAPC IN ZEBRAFISH
Homologues of dystrophin and other DAPC components have
now been identified in all of the common genetic
model organisms, and have been shown to have a necessary
function in muscle in animals as diverged from humans as the
nematode and Drosophila, indicating that the complex is
evolutionarily ancient (24–30). Given that muscle fibres are
similar in animals and essentially the same in all vertebrates, gene
functions can be studied in muscle using a range of organisms.
Taking the gene-first route, several groups have therefore begun
to address the function of known DAPC components and
muscular dystrophy loci in zebrafish. Their work has shown that
orthologues of dystrophin, dystroglycan and the sarcoglycans
show conserved sequence and expression between humans and
zebrafish, with the additional discovery that in embryonic
zebrafish the DAPC is localized initially at a distinct specialization of the sarcolemma, the end attachment point.
Bolanos-Jimenez et al. (31,32) first described the embryonic
expression of mRNA produced from the zebrafish DMD gene
orthologue. They found that muscle cells contain mRNA
dystrophin and a shorter product Dp71 that is localized to the
ends of fibres. The possible conservation of this mechanism
and its functional significance remain to be investigated.
A complementary set of findings was made by Parsons et al.
(33), who investigated the distribution and role in zebrafish of
the laminin receptor dystroglycan, to which dystrophin binds
directly. Having identified laminin mutations that disrupt
notochord development, they were seeking to identify the
laminin receptors important in this process when they described
the localization of laminin and dystrophin proteins at the ends
of embryonic muscle fibres. Furthermore, they used morpholinos, modified antisense oligonucleotides, to knock down
levels of dystroglycan protein in wild-type embryos. Using this
method they found that, as in the mouse, dystroglycan is
essential for the normal differentiation of muscle fibres, too
early to cause muscular dystrophy. Dystroglycan itself has not
been demonstrated to be the mutated gene in any muscular
dystrophy as yet, but several diseases including Fukuyama type
muscular dystrophy result from defects in proteins responsible
for modifying it (34–36). Dystroglycan removal from zebrafish
results in muscle fibres lacking the ability to form the regular
array of the sarcomeric proteins that are the engine of
contraction. This phenotype and the large number of mutations
in unidentified zebrafish genes that cause failure of muscle
differentiation, may represent models of congenital myopathies
(37–39).
The similarity between human and zebrafish muscle was
further emphasized by Donald Love’s group, who have carried
out an immunohistochemical analysis of dystrophin in adult
zebrafish muscle that was designed to reproduce the type of
data that is familiar to physicians and those working with
mouse models (40,41). By labelling transverse sections of adult
skeletal muscle with anti-dystrophin, they observed characteristic rings of signal, indicating that the expected localization in
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non-specialized sarcolemma is conserved as a later phase of
expression. They also further confirmed the conservation of
dystrophin gene structure and expression by showing in
western blots, as did Bolanos-Jimenez et al., that the same
range of isoforms are present as in mammalian muscle. This
group has also identified zebrafish orthologues of the
sarcoglycan family of transmembrane proteins, which form a
sub-complex of the DAPC. Mutations in these loci lead to
human type-2 limb girdle muscular dystrophies, and Chambers
et al. (41) have shown that these proteins localize to the
sarcolemma and to the ends of muscle fibres in zebrafish. They
are currently applying gene-silencing RNAs to DAPC
components in the zebrafish with the aim of developing this
technology for the rapid creation of genetic models for a variety
of diseases. This approach may well eventually deliver the
ability to create stable inducible transgenic lines in which a
gene of interest can be down-regulated with both spatial and
temporal control, hugely increasing the scope of the zebrafish
as a system for studying monogenic disorders.
Most recently, Guyon et al. (42) have used similar techniques
to identify zebrafish sarcoglycan homologues, and have used
the morpholino knockdown technique to begin to address the
essential issue of how far the function of the DAPC is
conserved in the zebrafish. They used morpholinos to the
reduce levels of dystrophin protein in embryos and showed that
this in turn affected the levels of other co-localized DAPC
components, a good indication that the composition of the
complex is conserved, and therefore that the function may well
be too. However, this did not reveal any insights to the role the
DAPC plays in zebrafish in maintaining muscle cell integrity.
In combination, these data show us that there is a highlyconserved dystrophin-based form of the DAPC present in
zebrafish muscle and that it shifts from being localized at fibre
ends to the more familiar pattern of sarcolemmal distribution as
development proceeds. However, the question of whether
loss of the DAPC causes a muscular dystrophy in the zebrafish
has recently been answered using a mutation that causes exactly
the right kind of phenotype (3).
A NOVEL MECHANISM OF PATHOLOGY IN
A MODEL OF MUSCULAR DYSTROPHY
Large-scale genetic screens in zebrafish have identified a large
number of mutants that affect muscle formation, with one class
showing a very specific degeneration of skeletal muscle.
Preliminary investigation during the screening process revealed
that mutations at several independent loci share the broad
phenotype of developing visible lesions in the somitic trunk
muscle during the second day of development once movement
begins, which gradually accumulate until the animals die
before reaching adulthood, a phenotype superficially similar to
muscular dystrophy in humans. This class of mutations is now
being examined by the authors, and, in particular, one specific
locus, sapje (sap), has been identified as a model of a human
muscular dystrophy that reveals a novel pathological mechanism that we suggest may be of clinical relevance (3).
We have found that dystrophin is lost from the end of
muscle fibres in sap mutant zebrafish embryos, confirming the
class of muscle degeneration mutants may represent muscular
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dystrophy phenotypes (Fig. 2A and B). Histological examination and confocal imaging of skeletal muscle expressing green
fluorescent protein (GFP) showed that the lesions occur where
the ends of sap mutant muscle fibres become separated from
their attachment sites (Fig. 2C–F). Many fibres are seen to
detach at one end and contract to a fraction of their original
length, showing compression or even collapse of the sarcomeric banding. Furthermore, these cells show nuclear condensations under electron microscopy that indicate cell death
that are not seen in intact neighbouring cells. A subset of these
detached fibres takes up the vital dye Evans Blue, which only
enters cells with compromised plasma membranes, indicating
that some tearing of the membrane does occur (Fig. 2G and H).
Thus, despite sharing the phenotype of muscle degeneration at
the whole organism level at the cellular level, the pathology of
sap mutant zebrafish is apparently different to that of human
muscular dystrophies, where membrane damage occurs along
the length of the fibre. The DAPC is localized embryonically in
zebrafish to the ends of muscle fibres before it becomes detectable at the sarcolemma, suggesting that loss of the complex
might compromise muscle attachments and possibly allowing
detachment of the kind seen in sap. This is a surprising finding
because this has not been reported in any human muscular
dystrophy and, even in mouse mutants that show ultrastructural abnormalities of the cytoskeleton at the MTJ, there
have been no reports of actual failure occurring in vivo (43–47).
We have now shown that sap corresponds to the dmd gene,
the zebrafish orthologue of the human Duchenne muscular
dystrophy locus, and thus revealed a novel functional
requirement for the DAPC in the stability of muscle
attachments. We have identified a nonsense mutation towards
in the N-terminal actin-binding domain of dystrophin that
removes the large muscle-specific isoform and causes a
progressive fatal muscle degeneration. sap is a far more severe
phenotype than the mouse dystrophin mutant mdx, showing
the same progression to early lethality as the human disease,
perhaps because both human and zebrafish lack the regenerative capacity and compensatory levels of utrophin that are
thought to protect mdx mice. Utrophin is not found at
embryonic muscle attachments in zebrafish, and is absent from
the non-specialized sarcolemma along the length of muscle
fibres during development, but is present in the skin and
pronephros. This lack of either dystrophin or utrophin in
muscle perhaps makes sap mutant embryos most similar to
mouse models thought to lack any functional DAPC link,
notably the laminin-a2 (dy), dystroglycan and mdx/utrophin
double mutants (21,22,48–50). In these animals, the MTJ lacks
folding almost completely and may be weaker than in mdx
animals, but it is unclear whether it ever fails completely. Only
very slight folding of the sarcolemma is present in wild-type
zebrafish muscle (51), indicating that sap resembles these
mouse models in its anatomical details, and that the complex
involutions of the mammalian MTJ may have evolved to
withstand the rigours of life on land. A similarly severe loss of
the mechanical function might occur in the merosin (laminina2) deficient congenital muscular dystrophies (6), a group of
very severe early muscle disorders, raising the possibility that
such diseases might involve MTJ defects in human muscle.
Within some mammalian muscles, dystrophin is also enriched
at specialized myomuscular junctions (MMJs) that also
Figure 2. sap is a mutation of zebrafish dmd that causes fibre detachment.
Dystrophin C-terminal immunoreactivity is localized to somite boundaries in
wild-type (in A, 27 h post-fertilisation, lateral views) but lacking in sap mutant
embryos (B). Toluidene blue histology reveals detached, retracted fibres in
association with lesions in sap (arrowhead in D; parasagittal sections, 72 h
post-fertilisation) but not in wild-type (C) embryos. Surface reconstructions
of somites made using confocal microscopy to image fluorescence from a
transgene expressing EGFP in skeletal muscle under the control of an alphaactin promoter reveals extensive fibre loss in sap mutant (arrowhead in F),
but not in wild-type embryos (E), and in particular the apparently random
nature of the damage sites. Evans blue is a vital dye (EBD, red fluorescence)
that does not accumulate in wild-type somites (G), but labels fibres with
compromised sarcolemmal membranes in sap (H). Labelled fibres are visible
that have both detached and retracted (arrowheads). Reproduced from Bassett
et al. (3) by permission of The Company of Biologists Ltd.
transmit force between the ends of muscle fibres (52). These
occur as either intrafascicular fibre terminations, connecting
single fibres into networks both end-to-end and end-to-side
(53), or as fibrous sheets called tendinous intersections that
separate segmented blocks of non-overlapping fibres. These in
particular bear a striking structural resemblance to the
dystrophin-dependent attachments between somites in zebrafish (53,54). If the failure of MMJs failure were a significant
factor in mammalian muscle disease, their differential deployment might contribute to the observed variations in pathology
between individual muscles, and between humans and the
different dystrophic animal models.
The sap mutation may only affect a subset of the products of
the dmd gene, as do many of the known human disease-causing
Human Molecular Genetics, 2003, Vol. 12, Review Issue 2
mutations. The phenotype of sap mutant embryos may be less
severe than that of embryos injected with a morpholino
designed to block dystrophin translation (42), as it does not
entail the same body curvature, perhaps because these two
situations disrupt different exons and therefore different
isoforms of dystrophin. To date no other mutations are known
in the zebrafish dmd gene, but it will be of great interest to
examine the effects of mutations at different points in the gene
that affect different gene products. This type of information is,
however, more likely to be provided in the near future by the
use of silencing RNAs and morpholinos directed to different
exons, which will hopefully begin to model the full spectrum of
dystrophinopathies.
As well as accurately representing the progressive nature and
fatality of DMD, sap closely resembles known Duchennecausing nonsense mutations in the N-terminal, making sap a
new model of the disease and raising the possibility that muscle
attachment failure contributes to the pathology of either
Duchenne or other muscular dystrophies. The known localization of the DAPC to this site is consistent with this, but it
seems possible that such pathology might so far have been
overlooked, as muscle biopsies are often deliberately taken
from sites at a distance from the tendon in order to simplify
histological examinations. It remains to be seen to what extent
this novel requirement for the DAPC for the stability of muscle
attachments might contribute to human muscle diseases, but it
might be prudent to re-examine these structures, especially
myomuscular junctions.
THE FUTURE OF ZEBRAFISH MUSCLE
DISEASE RESEARCH
The zebrafish is rapidly being developed as a system for the
study of muscle disease, with exciting results, and it is to
be hoped that this trend continues. In future there will be
more laboratories beginning to dedicate a proportion of their
time to this approach; both existing muscular dystrophy and
zebrafish groups may be converted, hopefully to the benefit of
the field and of patients. The zebrafish as a model system will
provide for both genetic and embryological manipulations,
and thus may provide insights into both the protein
interactions and cellular functions that underlie muscular
dystrophy and other muscle diseases, as it has begun to with
great effect in the field of cardiovascular development and
disease.
ACKNOWLEDGEMENTS
We would like to thank the members of the Currie group and
the staff of the MRC Human Genetics Unit. Our work has been
funded by the Muscular Dystrophy Campaign.
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