A class of recessive lethal zebrafish mutations has been identified in which normal skeletal muscle differentiation is followed by a tissue-specific degeneration that is reminiscent of the human muscular dystrophies. Here, we show that one of these mutations, sapje, disrupts the zebrafish orthologue of the X-linked human Duchenne muscular dystrophy (DMD) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology through disconnecting the cytoskeleton from the extracellular matrix in skeletal muscle by reducing the level of dystrophin protein at the sarcolemma. This is thought to allow tearing of this membrane, which in turn leads to cell death. Surprisingly, we have found that the progressive muscle degeneration phenotype of sapje mutant zebrafish embryos is caused by the failure of embryonic muscle end attachments. Although a role for dystrophin in maintaining vertebrate myotendinous junctions (MTJs) has been postulated previously and MTJ structural abnormalities have been identified in the Dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure has thus far been lacking. This zebrafish mutation may therefore provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases
Parasitic infections by the salmon louse, Lepeophtheirus salmonis (Krøyer), cause huge economic damage in salmon farming in the northern hemisphere, with combined treatment costs and production losses in 2014 having been estimated at US$ 350 million for Norway (annual production 1.25 million tonnes). The control of L. salmonis relies significantly on medicinal treatments, supplemented by non-pharmacological approaches. However, efficacy losses have been reported for several delousing agents, including the pyrethroid deltamethrin. The aim of the present study was to analyse the genetic basis of deltamethrin resistance in L. salmonis. Deltamethrin median effective concentrations (EC50) were 0.28 μg L-1 in the drug susceptible L. salmonis strain IoA-00 and 40.1 μg L-1 in the pyrethroid resistant strain IoA-02. IoA-00 and IoA-02 were crossed to produce families spanning one parental and three filial generations (P0, F1-F3). In three families derived from P0 crosses between an IoA-00 sire and an IoA-02 dam, 98.8% of F2 parasites (n = 173) were resistant, i.e. remained unaffected after exposure to 2.0 μg L-1 deltamethrin. F3 parasites from these crosses showed a deltamethrin EC50 of 9.66 μg L-1. In two families of the inverse orientation at P0 (IoA-02 sire x IoA-00 dam), 16.7% of F2 parasites were resistant (n = 84), while the deltamethrin EC50 in F3 animals was 0.26 μg L-1. The results revealed a predominantly maternal inheritance of deltamethrin resistance. The 15,947-nt mitochondrial genome was sequenced and compared among six unrelated L. salmonis strains and parasites sampled from wild salmon in 2010. IoA-02 and three further deltamethrin resistant strains, established from isolates originating from different regions of Scotland, showed almost identical mitochondrial haplotypes. In contrast, the mitochondrial genome was variable among susceptible strains and L. salmonis from wild hosts. Deltamethrin caused toxicity and depletion of whole body ATP levels in IoA-00 but not IoA-02 parasites. The maternal inheritance of deltamethrin resistance and its association with mitochondrial haplotypes suggests that pyrethroid toxicity in L. salmonis may involve molecular targets encoded by mitochondrial genes.
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.
The salmon louse (Lepeophtheirus salmonis (Krøyer, 1837)) is a parasitic copepod that can, if untreated, cause considerable damage to Atlantic salmon (Salmo salar Linnaeus, 1758) and incurs significant costs to the Atlantic salmon mariculture industry. Salmon lice are gonochoristic and normally show sex ratios close to 1:1. While this observation suggests that sex determination in salmon lice is genetic, with only minor environmental influences, the mechanism of sex determination in the salmon louse is unknown. This paper describes the identification of a sex-linked Single Nucleotide Polymorphism (SNP) marker, providing the first evidence for a genetic mechanism of sex determination in the salmon louse. Restriction site-associated DNA sequencing (RAD-seq) was used to isolate SNP markers in a laboratory-maintained salmon louse strain. A total of 85 million raw Illumina 100 base paired-end reads produced 281,838 unique RAD-tags across 24 unrelated individuals. RAD marker Lsa101901 showed complete association with phenotypic sex for all individuals analysed, being heterozygous in females and homozygous in males. Using an allele-specific PCR assay for genotyping, this SNP association pattern was further confirmed for three unrelated salmon louse strains, displaying complete association with phenotypic sex in a total of 96 genotyped individuals. The marker Lsa101901 was located in the coding region of the prohibitin-2 gene, which showed a sex-dependent differential expression, with mRNA levels determined by RT-qPCR about 1.8-fold higher in adult female than adult male salmon lice. This study’s observations of a novel sex-linked SNP marker are consistent with sex determination in the salmon louse being genetic and following a female heterozygous system. Marker Lsa101901 provides a tool to determine the genetic sex of salmon lice, and could be useful in the development of control strategies.
1. Large-scale mutagenic screens of the zebrafish genome have identified a number of different classes of mutations that disrupt skeletal muscle formation. Of particular interest and relevance to human health is a class of recessive lethal mutations in which muscle differentiation occurs normally, but is followed by tissue-specific degeneration reminiscent of human muscular dystrophies. 2. We have shown that one member of this class of mutations, sapje (sap), results from mutations within the zebrafish orthologue of the human Duchenne muscular dystrophy (DMD) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology by disrupting the link between the actin cytoskeleton and the extracellular matrix in skeletal muscle cells. 3. We have found that the progressive muscle degeneration phenotype of sapje-mutant zebrafish embryos is caused by the failure of somitic muscle attachments at the embryonic myotendinous junction (MTJ). 4. Although a role for dystrophin at the MTJ has been postulated previously and MTJ structural abnormalities have been identified in the dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure is thus far lacking. Therefore, the sapjre mutation may provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases. In the present review, we discuss this finding in light of previously postulated models of dystrophin function.
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