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 Molecular characterization of the sloth locus showed the phenotype to be due to Zebrafish genetic screens and gne myopathy loss-of-function mutation in the hsp90A gene, implicating the chaperone protein that it encodes in myofibril formation Hawkins et al.
Muscular dystrophies are characterized by progressive muscle degeneration and progressive loss of muscle function. Developmental stages are annotated as hours postfertilization hpf or days postfertilization dpf.
To visualize incorporated sialic acid in live zebrafish embryos, we used Ac4-ManNAz, a nontoxic sialic acid precursor with an azido group. Petcharat has nothing to disclose. The Gne splice site mutation results in fish that generally do not survive into adulthood, whereas the missense mutation generally results in fish surviving into adulthood.
GFP i transgenic line has been previously described Elworthy et al. The zebrafish animal model, in particular, offers many advantages for drug discovery and for understanding drug mechanisms of action not only for DMD therapies but for therapies for other muscle diseases as well.
Analyses of Myo18B expression in the mouse are consistent with a role in the development and maintenance of both cardiac and skeletal muscle. 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.
Myosin heavy chain protein accumulated throughout the fast-twitch fibers of froto27c mutant embryos, in contrast to the ordered myofibrillar distribution typical of wild-type fibers Figure 1, F and H. Data availability The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article.
Previous work has demonstrated that sialylation is critical for normal muscle function.
These controversial data illustrate that the process by which GNE mutations lead to myopathy is certainly not well understood. Since the GNEMTin vivo mouse model and in vitro cell culture model are less informative than expected 24—27it is critical to develop novel model systems to comprehensively examine the functions of the GNE protein in muscle, which could eventually explain the pathophysiological downstream defects caused by GNE mutations.
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.
Here, I review how the zebrafish animal model has been employed to both identify, and study the mechanisms of, pharmacological approaches to muscle disease therapies. These occur as either intrafascicular fibre terminations, connecting single fibres into networks both end-to-end and end-to-side 53or as fibrous sheets called tendinous intersections that separate segmented blocks of non-overlapping fibres.
Actin was visualized using Phalloidin Tetramethylrhodamine B isothiocyanate Sigma 1: 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.
No cures are currently available for inherited myopathies. Among the latter type, members of six classes have been found to be expressed in muscle Redowicz 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.
Two different types of myosin can be distinguished: Marked GNE deficiency has not been observed in the patients; in fact, GNE protein is expressed at equal levels in patients and normal control subjects Enter and update disclosures at http: 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.
Materials and Methods Zebrafish and husbandry Zebrafish were raised, bred, and staged following standard methods Kimmel et al.
The sap mutation may only affect a subset of the products of the dmd gene, as do many of the known human disease-causing mutations. Furthermore, they used morpholinos, modified antisense oligonucleotides, to knock down levels of dystroglycan protein in wild-type embryos.
These results demonstrate a critical novel role for gne in embryonic development and particularly in myofiber development, muscle integrity and activity. Read any comments already posted on the article prior to submission. EGFP expression was observed throughout larval development, in juveniles and persisted in adult fish, where it appeared strong in eyes and pineal gland and lower in skeletal muscles Fig.
Zebrafish is recognized as a very potent vertebrate model for the study of development and of various human conditions, in particular neuromuscular disease.
These structures are essentially laminar tendons that transmit force to the notochord, and later to the vertebral column. Our work has been funded by the Muscular Dystrophy Campaign. Areas covered With this article, the author provides the reader with a description of the zebrafish animal model, which has been employed to identify and study pharmacological approaches to muscle disease.
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. Chaiyasap has nothing to disclose.
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.
Disruption of excitation—contraction coupling appears to be a common pathological mechanism in the congenital myopathies [ 35 ].Forward genetic screens for mutations affecting muscle development and function in the zebrafish yielded a number of mutants including frozen (fro) and sloth, originally identified on the basis of their complete lack of motility (Granato et al.
). GNE myopathy is an ultra-rare autosomal recessive disease, which starts as a distal muscle weakness and ultimately leads to a wheelchair bound state. Molecular research and animal modelling significantly moved forward understanding of GNE myopathy mechanisms. The rapid development of zebrafish and the ability to readily obtain large numbers of animals have facilitated forward genetic screens that have significantly contributed to the use of zebrafish as an animal model for human muscle disease.
Forward genetic screens have proven to be an excellent source of zebrafish mutants that model human disease. The first muscle mutants emerged from the original ENU mutagenesis-based screen by Nüsslein-Vollhard and colleagues, and include the first identified zebrafish mutant strains for dystrophin (sapje), titin (runzel), and laminin α2.
GNE Myopathy is a rare recessively inherited neuromuscular disorder caused by mutations in the GNE gene, which codes for the key enzyme in the metabolic pathway of sialic acid synthesis. We began by using TALE nucleases to generate germ line mutations in the zebrafish selenoprotein N (sepn1) gene, and in doing so created the first vertebrate to accurately model human SEPN1-related myopathy (SEPN1-RM).Download