Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays
Summary
The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.
Abstract
Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo's swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.
Protocol
Representative Results
Discussion
Primary neuromuscular disorders are traditionally classified as dystrophic or nondystrophic processes. Muscular dystrophies are characterized by myofiber degeneration followed by repeated rounds of regeneration, which ultimately leads to an end stage process typified by fibrosis and replacement by adipose tissue14. Nondystrophic myopathies, in contrast, are not associated with necrosis or regenerative changes, but do result in disorganized myofibers and overall skeletal muscle weakness.
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The authors have nothing to disclose.
Acknowledgements
We thank Behzad Moghadaszadeh for his wonderful help with quantification of birefringence images. This work was funded by the Muscular Dystrophy Association USA (MDA201302) and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01 AR044345), as well as generous support from A Foundation Building Strength, Cure CMD, and the AUism Charitable Foundation. VAG is supported by K01 AR062601 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, and LLS is supported by F31 NS081928 from the National Institute of Neurological Disorders and Stroke.
Materials
| Tricaine | Sigma | A5040 |
| Petri dishes | Fischer Scientific | 0875711Z |
| Forceps | Fischer Scientific | 100189-588 |
| Insect pin | Fischer Scientific | S67375 |
| Polarized lenses | Ritz Camera | Quantaray Tristar Optics C-PL 72mm |
| Incubator | Fischer Scientific | Isotemp Incubator Model 630D |
| Microscope | Nikon Instruments Inc. | SMZ 1500 |
| Camera | Diagnostic Instruments Inc. | SPOT RT3 |
| Imaging software | Diagnostic Instruments Inc. | SPOT 5.1 Advanced |
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