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.</li>
	</ol>
	</li>
	<li>Remove cage dividers the next morning shortly after the onset of light and allow the fish to spawn undisturbed.</li>
	<li>Collect the eggs using a strainer when sufficient numbers of fertilized eggs are laid at the bottom of the tank.</li>
	<li>Transfer the embryos into a deep Petri dish by rinsing the strainer with fish water and return the parent fish to their tanks. Place dishes of embryos into a 28.5 &#176;C incubator.</li>
	<li>Clean out the inviable eggs and debris later that day or the following morning, and return the embryos to the incubator for further growth.</li>
	<li>Embryos/larvae reach the appropriate age to observe birefringence at 3-7 days post-fertilization (dpf), as results prior to 3 dpf may potentially suffer from limited contrast. Wild-type embryos hatch from their chorions between 48-60 hr post-fertilization (hpf), followed by a straightening of the body axis. Late-hatching wild-type or mutant embryos may require manual dechorionation.
	<ol>
		<li>Gently make a tear in the chorion with sharp forceps and turn it upside down so that the embryo falls out.</li>
		<li>Allow dechorionated embryos to straighten prior to beginning the assay.</li>
		<li>Anesthetize embryo(s) with tricaine (0.04% in fish water) to aid in their correct positioning.</li>
	</ol>
	</li>
	<li>Fit a dissecting microscope with a polarized lens that can be rotated to adjust the angle of polarized light (Figure 1A).</li>
	<li>Place dechorionated, anesthetized embryo(s) directly on top of a second polarized lens along the lateral axis of the body. The second lens should be positioned below the top lens, on the microscope stage (Figure 1B). Do not use plastic Petri dishes at any point during the birefringence assay, as plastic is not an appropriate medium for the transmission of refracted light.</li>
	<li>Rotate the top lens with the embryo of interest in view until the axes of polarization of the two lenses are oriented at 90&#176; from one another and the background is completely dark.</li>
	<li>The optimum output of the assay is dependent on the orientation of fish between two polarized lenses. Move the fish around to make sure that they are lying as flat as possible.
	<ol>
		<li>If the fish is curved, only the segments with this flat orientation will pass the polarized light and exhibit birefringence. In such cases, measure the birefringence in the flat areas and record the birefringence of the corresponding area in the wild-type fish (e.g.&#160;somites 1-10 in both wild-type and mutant fish).</li>
	</ol>
	</li>
	<li>Observe the birefringent phenotype of the fish.
	<ol>
		<li>Wild-type fish with highly organized skeletal muscle show bright birefringence, as the refractive index for the light parallel to the myofilaments is higher than the polarized light perpendicular to these structures.</li>
		<li>Fish with disorganized skeletal muscle suggestive of a nondegenerative myopathy or a developmental defect typically show an overall reduction in birefringence.</li>
		<li>Fish with disorganized skeletal muscle characteristic of a muscular dystrophy commonly exhibit a patch-like pattern of birefringence, with dark areas representing muscle degeneration among bright areas of normal muscle architecture.</li>
	</ol>
	</li>
	<li>Quantify birefringence by taking images of wild-type and mutant fish under polarized light at the same exposure settings and magnification. Save images as .tiff files.
	<ol>
		<li>Open birefringence images in ImageJ software (<a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a>).</li>
		<li>For each image, select the area of zebrafish muscle by drawing a line around the body using the &#8220;Polygon Selections&#8221; option from the toolbar.</li>
		<li>Use &#8220;Set Measurements&#8221; under the &#8220;Analyze&#8221; drop-down menu to select the required statistics for the image.
		<ol>
			<li>Minimum selection requires that boxes for &#8220;Area,&#8221; &#8220;Mean gray value,&#8221; and &#8220;Min &#38; max gray value&#8221; are checked.</li>
			<li>A maximum gray value of &#62;255 will indicate pixel saturation. Therefore, use images with maximum gray values less than 255.</li>
		</ol>
		</li>
		<li>Normalize the mean intensity with the selected area and repeat this same quantification procedure for each birefringence image.</li>
	</ol>
	</li>
</ol>
2. Touch-evoked Escape Behavior Assay
<ol>
	<li>Set up mating pairs of the appropriate zebrafish line(s) and collect embryos as described above in steps 1.1-1.5.</li>
	<li>At 2-7 days post-fertilization, place dechorionated embryos/larvae individually into a deep Petri dish or the chambers of multiwell plates.</li>
	<li>Working with only one embryo at a time, center the embryo in the field of view of a Nikon SMZ1500 stereomicroscope&#160;with a SPOT RT3 digital camera system&#160;or similar, and begin sequential imaging.
	<ol>
		<li>The same frequency of imaging should be used for both wild-type and mutant fish. Frame rates &#62;30 Hz are recommended, as slower rates will typically not be sufficient to capture the fast swimming of wild-type fish.</li>
	</ol>
	</li>
	<li>Deliver mechanosensory stimuli to the embryo by touching the tail with an insect pin.</li>
	<li>Stop imaging when the embryo has stopped swimming or has swam out of the field of view.</li>
	<li>Convert sequential images into a video file, or analyze individual frames of time-lapse images off-line, using SPOT 5.1 Advanced&#160;software. Similar software packages include Open Lab, NIS Elements, or freely available ImageJ (<a href="http://rsbweb.nih.gov/ij/" title="">http://rsbweb.nih.gov/ij/</a>).</li>
	<li>Quantify swimming behavior by performing the touch-evoked response in multiple embryos.
	<ol>
		<li>This can be achieved by averaging the distance embryos are able to swim within a fixed time interval using software or a metric ruler to mark the start and end points of the swimming bout.</li>
		<li>Avoid embryos becoming habituated to the touch stimulus over time by performing the assay with a new embryo for each experimental repeat. Behaviors in wild-type and reliable mutant models tend to be highly reproducible.</li>
	</ol>
	</li>
</ol>

<ol>
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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+candyfloss+mutant+implicates+extracellular+matrix+adhesion+failure+in+laminin+&#945;2-deficient+congenital+muscular+dystrophy.">The zebrafish candyfloss mutant implicates extracellular matrix adhesion failure in laminin &#945;2-deficient congenital muscular dystrophy.</a> Proc. Natl. Acad. Sci. U.S.A. 104 (17), 7092-7097 (2007).</li><li>Dowling, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+myotubularin+function+results+in+T-tubule+disorganization+in+zebrafish+and+human+myotubular+myopathy.">Loss of myotubularin function results in T-tubule disorganization in zebrafish and human myotubular myopathy.</a> PLoS Genet. 5 (2), e1000372 (2009).</li><li>Telfer, W. R., Nelson, D. D., Waugh, T., Brooks, S. V., Dowling, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neb:+a+zebrafish+model+of+nemaline+myopathy+due+to+nebulin+mutation.">Neb: a zebrafish model of nemaline myopathy due to nebulin mutation.</a> Dis. Model Mech. 5 (3), 389-396 (2012).</li><li>Ferrante, M. I., Kiff, R. M., Goulding, D. A., Stemple, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troponin+T+is+essential+for+sarcomere+assembly+in+zebrafish+skeletal+muscle.">Troponin T is essential for sarcomere assembly in zebrafish skeletal muscle.</a> J. Cell Sci. 124 (4), 565-577 (2011).</li><li>Hawkins, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ATPase-dependent+chaperoning+activity+of+Hsp90a+regulates+thick+filament+formation+and+integration+during+skeletal+muscle+myofibrillogenesis.">The ATPase-dependent chaperoning activity of Hsp90a regulates thick filament formation and integration during skeletal muscle myofibrillogenesis.</a> Development. 135 (6), 1147-1156 (2008).</li><li>Barbazuk, W. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+syntenic+relationship+of+the+zebrafish+and+human+genomes.">The syntenic relationship of the zebrafish and human genomes.</a> Genome Res. 10 (9), 1351-1358 (2000).</li><li>Schapira, G., Dreyfus, J. C., Joly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+flow+birefringence+of+myosin+as+a+result+of+muscular+atrophy.">Changes in the flow birefringence of myosin as a result of muscular atrophy.</a> Nature. 170 (4325), 494-495 (1952).</li><li>Kimmel, C. B., Patterson, J., Kimmel, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+behavioral+characteristics+of+the+startle+response+in+zebrafish.">The development and behavioral characteristics of the startle response in zebrafish.</a> Dev. Psychobiol. 7, 47-60 (1974).</li><li>Eaton, R. C., Bombardieri, R. A., Meyer, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mauthner-initiated+startle+response+in+teleost+fish.">The Mauthner-initiated startle response in teleost fish.</a> J. Exp. Biol. 66, 65-81 (1977).</li><li>Saint-Amant, L., Drapeau, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+the+development+of+motor+behaviors+in+the+zebrafish+embryo.">Time course of the development of motor behaviors in the zebrafish embryo.</a> J. Neurobiol. 37 (4), 622-632 (1998).</li><li>Hirata, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+relatively+relaxed+mutants+have+a+ryanodine+receptor+defect,+show+slow+swimming+and+provide+a+model+of+multi-minicore+disease.">Zebrafish relatively relaxed mutants have a ryanodine receptor defect, show slow swimming and provide a model of multi-minicore disease.</a> Development. 134 (15), 2771-2781 (2007).</li><li>Wallace, G. Q., McNally, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+muscle+degeneration,+regeneration,+and+repair+in+the+muscular+dystrophies.">Mechanisms of muscle degeneration, regeneration, and repair in the muscular dystrophies.</a> Annu. Rev. Physiol. 71, 37-57 (2009).</li><li>Granato, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+controlling+and+mediating+locomotion+behavior+of+the+zebrafish+embryo+and+larva.">Genes controlling and mediating locomotion behavior of the zebrafish embryo and larva.</a> Development. 123, 399-413 (1996).</li><li>Berger, J., Sztal, T., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+birefringence+readily+measures+the+level+of+muscle+damage+in+zebrafish.">Quantification of birefringence readily measures the level of muscle damage in zebrafish.</a> Biochem. Biophys. Res. Commun. 423 (4), 785-788 (2012).</li><li>Low, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPM7+is+required+within+zebrafish+sensory+neurons+for+the+activation+of+touch-evoked+escape+behaviors.">TRPM7 is required within zebrafish sensory neurons for the activation of touch-evoked escape behaviors.</a> J. Neurosci. 31 (32), 11633-11644 (2011).</li><li>Liu, D. W., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+identified+motoneurones+and+co-ordination+of+primary+and+secondary+motor+systems+during+zebrafish+swimming.">Function of identified motoneurones and co-ordination of primary and secondary motor systems during zebrafish swimming.</a> J. Physiol. 403, 73-89 (1988).</li></ol>

No conflicts of interest declared.

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.
Zebrafish models have emerged in recent years as a tremendously valuable resource for better understanding the molecular pathways and pathogenesis of different neuromuscular diseases. In addition to the general advantages of this model system, including rapid ex utero development, translucent embryos, high reproductive capacity, and a genome closely related to that of human, the zebrafish disease model often closely mimics the human disease. Although many zebrafish with skeletal muscle defects have morphological abnormalities, some mutant phenotypes are grossly normal and require finer characterizations. For these and all cases, the extent of muscle damage and weakness can quickly and easily be evaluated in zebrafish using noninvasive in vivo assays for birefringence and touch-evoked escape behavior. Birefringence is a sensitive indicator of muscle integrity in translucent zebrafish embryos that has been used to noninvasively identify skeletal muscle mutants since early in the establishment of the zebrafish model system15. The assay itself involves placing a zebrafish embryo between two polarizing filters and visualizing the fish in an otherwise dark background16. Wild-type fish with highly organized sarcomeric arrays appear bright, whereas muscle mutants usually present with dark patches or an overall reduction in birefringence, suggesting myofibrillar disorganization within the major axial skeletal muscles. As described above, birefringent images may be readily quantified using publically available ImageJ&#160;software.
Touch-evoked escape behaviors in zebrafish involve many steps, beginning with the sensing of tactile stimuli and culminating in the contraction of muscles. Zebrafish embryos swim in response to touch by 26 hpf, but the frequency of muscle contractions during swimming does not increase to a value comparable to that of adult zebrafish until 36 hpf12,18. The touch-evoked response is optimally captured between 2-7 dpf. The time at which the touch stimulus is applied to the tail is set as the experimental &#8220;start&#8221; point. Whereas both wild-type and mutant larvae begin stationary, video microscopy is able to capture that wild-type larvae respond to mechanosensory stimuli with rapid and vigorous swimming in a straight line. Muscular mutants instead tend to weakly flex, swim in circles, or otherwise move for only a short distance. It should be noted that while there is an inherent degree of variability in the timing and intensity of manual touch stimuli, quantitative measurements of touch-evoke behaviors and swimming episodes tend to be highly reproducible within wild-type and reliable fish models of skeletal muscle disease.
Both birefringence and touch-evoked escape assays provide a fast and noninvasive way to analyze defects in skeletal muscles, and are therefore appropriate for analyzing a large number of embryos when performing high-throughput mutagenesis or chemical screens.

<table border="0" cellpadding="0" cellspacing="0" width="722">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<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 pathogenic mutation in the dystroglycan gene (dag1) displays patchy birefringence, indicative of areas of muscle degeneration (Figures 2C-D)2. This patch-like pattern and rapid progression of muscle degeneration throughout early development is consistent with other dystrophic fish models1,3. Quantifying the birefringence of 5 dpf dystrophic dag1 mutants identifies a significant reduction in brightness to 27.9%&#177;5.3&#160;of the maximal birefringence seen in their wild-type siblings. This reduction is more severe than the reduction to 52.4%&#177;5.5&#160;observed in 3 dpf myopathic mtm1 morphants (wild-type/dag1/mtm1: n = 3; Student&#39;s t-test, P &#60; 0.001) (Figure 4). Myopathic fish models (Figures 3C-D) instead tend to show an overall reduction in birefringence compared to wild-type (Figures 3A-B), as there is often myofibrillar disorganization in the major axial skeletal muscles but no evidence of degenerative changes.
Touch-evoked escape behaviors can be used to demonstrate that zebrafish models of skeletal muscle disease display locomotive defects and swimming difficulties. The live swimming phenotypes of wild-type and dag1 fish were examined by video microscopy with a touch-evoked escape behavior assay (Videos 1 and 2)2. Videos were then analyzed frame by frame to confirm that dag1 mutants exhibit impaired movement compared to wild-type controls in response to tactile stimuli (Figure 5). The distances both types of embryos were able to move within a fixed time interval were averaged (wild-type: 6.2&#177;0.4 cm/0.1 sec, n = 10; mutant: 0.75&#177;0.08 cm/0.1 sec, n = 10; Student&#39;s t-test, P &#60; 0.001) and indicate diminished motor function in dag1 mutants. However, reduced touch-evoke responses in neuromuscular mutants could also be attributed to defects in the central nervous system and/or the neuromuscular junction. Therefore, electrophysiological measurement of the voltage response in muscles evoked by tactile stimulation may help to distinguish whether touch-evoke impairments are due to a sensory deficit or to a primary defect in skeletal muscle13.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/50925/50925fig1.jpg" width="600px" /> 
Figure 1. Diagram of a standard dissecting microscope fitted with a camera and polarized lenses (A). Sample set-up with polarized lenses and anesthetized zebrafish embryos is presented larger in (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50925/50925fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/50925/50925fig2.jpg" width="600px" /> 
Figure 2. Wild-type fish show bright birefringence indicative of highly organized muscles at 5 dpf (B). Age-matched dag1 mutant fish appear grossly normal in normal light whereas extensive muscle degeneration in most somites is demonstrated by the loss of birefringence under polarized light and is consistent with a dystrophic phenotype (D). Brightfield images of the wild-type fish (A) and the dag1 mutant (C) are shown for reference. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50925/50925fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/50925/50925fig3.jpg" width="600px" /> 
Figure 3. Morpholino-based knockdown of myotubularin (mtm1) in zebrafish results in defects in skeletal muscle at 3 dpf. An overall reduction in birefringence is observed in mtm1 morphant fish (D) in comparison to wild-type fish (B), consistent with a myopathic phenotype. Brightfield images of the wild-type fish (A) and the mtm1 morphant (C) are shown for reference. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50925/50925fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/50925/50925fig4.jpg" width="600px" /> 
Figure 4. Zebrafish embryos with skeletal muscle defects show a highly significant reduction in birefringence compared to wild-type controls. Quantification data are calculated by dividing the mean intensity by the selected area of the fish (ImageJ), and normalizing the percentage values to wild-type. Data are presented as means&#177;SEM and ** indicates P &#60; 0.001 by Student&#39;s t-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50925/50925fig4highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 5" fo:content-width="6in" src="/files/ftp_upload/50925/50925fig5.jpg" width="600px" /> 
Figure 5. Dag1 mutant embryos exhibit reduced touch-evoked response at 5 dpf (E-K) while mechanosensory stimulation causes 5 dpf wild-type embryos to swim away rapidly and fully exit the field of view (A-D). Scale bar = 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50925/50925fig5highres.jpg" target="_blank">Click here to view larger image</a>.
Video 1. Touch-evoked escape behavior assay on wild-type zebrafish. Normal fry (5 dpf) swim away very rapidly in response to touch. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50925/wtteeb(1).mp4" target="_blank">Click here to watch video.</a>
Video 2. Touch-evoked escape behavior assay on dag1 mutant zebrafish. Dag1 fry (5 dpf) show impaired response to touch and fail to exit the field of view. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50925/dag1teeb3(1).mp4" target="_blank">Click here to watch video</a>

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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Nanyang Technological University

Research

JoVE Journal

Biology

7.8K 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.

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

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.