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.

1. In vivo Analysis of Skeletal Muscle Structure by Birefringence
<ol>
	<li>Prepare mating cages separating the male(s) from the female(s) of the desired zebrafish line(s) late in the afternoon after feeding.
	<ol>
		<li>Distinguish females by their bigger underbelly and slight blue/white coloration, and males by their slender body shape and pink/yellow hues.</li>
		<li>Success with pair-wise crosses (one male and one female) may only be about 50%. For a higher rate of success, mate one male with 2-3 females.<...

<ol>
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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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		<tr height="22">
			<td>Tricaine </td>
			<td>Sigma</td>
			<td>A5040</td>
		</tr>
		<tr height="22">
			<td>Petri dishes</td>
			<td>Fischer Scientific</td>
			<td>0875711Z</td>
		</tr>
		<tr height="22">
			<td>Forceps</td>
			<td>Fischer Scientific</td>
			<td>100189-588</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Insect pin</td>
			<td style="width:248px;">Fischer Scientific</td>
			<td style="width:259px;">S67375</td>
		</tr>
		<tr height="22">
			<td>Polarized lenses</td>
			<td>Ritz Camera</td>
			<td>Quantaray Tristar Optics C-PL 72mm</td>
		</tr>
		<tr height="22">
			<td>Incubator</td>
			<td>Fischer Scientific </td>
			<td>Isotemp Incubator Model 630D</td>
		</tr>
		<tr height="22">
			<td>Microscope</td>
			<td>Nikon Instruments Inc.</td>
			<td>SMZ 1500</td>
		</tr>
		<tr height="22">
			<td>Camera</td>
			<td>Diagnostic Instruments Inc.</td>
			<td>SPOT RT3</td>
		</tr>
		<tr height="22">
			<td>Imaging software</td>
			<td>Diagnostic Instruments Inc.</td>
			<td>SPOT 5.1 Advanced</td>
		</tr>
	</tbody>
</table>

analysis of skeletal muscle defects in larval zebrafish by birefringence and touch-evoke escape response assays

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&#39;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.

Birefringence can be used as an efficient, noninvasive assay to shed light on the state of myofibrillar organization in living zebrafish embryos. Examples of wild-type zebrafish as well as zebrafish with decreased expression of genes critical to skeletal muscle development and function are presented. Wild-type zebrafish at 5 dpf display highly birefringent skeletal muscles under polarized light due to the ordered array of myofilaments (Figures 2A-B). In contrast, an age-matched embryo homozygous for a pa...

Watch this Scientific Journal Video about Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays at JoVE.com

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

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.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

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7.7K Views.  Boston Children's Hospital, Harvard Medical School. 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.