We would like to thank Dr. Alex Fulcher, and Monash Micro Imaging for assistance with microscope maintenance and setup. The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government. This work was funded by a Muscular Dystrophy Association (USA) project grant to P.D.C (628882).

Zebrafish maintenance was carried out as per the standard operating procedures approved by the Monash University Animal Ethics Committee under breeding colony license ERM14481.
1. Determination of the genotype of live embryos using an embryo genotyping platform.
<ol>
	<li>Anesthetize 3 days post fertilization (dpf) zebrafish embryos by adding tricaine methanesulfonate to a final concentration of 0.016% (v/v) in embryo medium (5 mM NaCl, 0.17mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO...

<ol>
	<li>Egan, B., Zierath, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+Metabolism+and+the+Molecular+Regulation+of+Skeletal+Muscle+Adaptation.">Exercise Metabolism and the Molecular Regulation of Skeletal Muscle Adaptation.</a> Cell Metabolism. 17 (2), 162-184 (2013).</li><li>Seale, P., Sabourin, L. A., Girgis-Gabardo, A., Mansouri, A., Gruss, P., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax7+is+required+for+the+specification+of+myogenic+satellite+cells.">Pax7 is required for the specification of myogenic satellite cells.</a> Cell. 102 (6), 777-786 (2000).</li><li>Relaix, F., Rocancourt, D., Mansouri, A., Buckingham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pax3/Pax7-dependent+population+of+skeletal+muscle+progenitor+cells.">A Pax3/Pax7-dependent population of skeletal muscle progenitor cells.</a> Nature. 435 (7044), 948-953 (2005).</li><li>Sousa-Victor, P., 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=Geriatric+muscle+stem+cells+switch+reversible+quiescence+into+senescence.">Geriatric muscle stem cells switch reversible quiescence into senescence.</a> Nature. 506 (7488), 316-321 (2014).</li><li>Egerman, M. 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=GDF11+Increases+with+Age+and+Inhibits+Skeletal+Muscle+Regeneration.">GDF11 Increases with Age and Inhibits Skeletal Muscle Regeneration.</a> Cell Metabolism. 22 (1), 164-174 (2015).</li><li>Emery, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+muscular+dystrophies.">The muscular dystrophies.</a> The Lancet. 359 (9307), 687-695 (2002).</li><li>Emery, A. E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Duchenne muscular dystrophy. , (1993).</li><li>Anne Helbling-Leclerc, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+the+laminin+&#945;2-chain+gene+(LAMA2)+cause+merosin-deficient+congenital+muscular+dystrophy.">Mutations in the laminin &#945;2-chain gene (LAMA2) cause merosin-deficient congenital muscular dystrophy.</a> Nature Genetics. (11), 216-218 (1995).</li><li>Campbell, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+muscular+dystrophies:+loss+of+cytoskeleton-extracellular+matrix+linkage.">Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage.</a> Cell. 80 (5), 675-679 (1995).</li><li>Cerletti, 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=Highly+efficient,+functional+engraftment+of+skeletal+muscle+stem+cells+in+dystrophic+muscles.">Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles.</a> Cell. 134 (1), 37-47 (2008).</li><li>Dumont, N. 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=Dystrophin+expression+in+muscle+stem+cells+regulates+their+polarity+and+asymmetric+division.">Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division.</a> Nature Medicine. 21 (12), 1455-1463 (2015).</li><li>Lieschke, G. J., 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=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nature Reviews. Genetics. 8 (5), 353-367 (2007).</li><li>Gurevich, D. 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=Asymmetric+division+of+clonal+muscle+stem+cells+coordinates+muscle+regeneration+in+vivo.">Asymmetric division of clonal muscle stem cells coordinates muscle regeneration in vivo.</a> Science. 353 (6295), (2016).</li><li>Lambert, C. 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=An+automated+system+for+rapid+cellular+extraction+from+live+zebrafish+embryos+and+larvae:+Development+and+application+to+genotyping.">An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping.</a> PloS One. 13 (3), 0193180 (2018).</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> Biochemical and Biophysical Research Communications. 423 (4), 785-788 (2012).</li><li>Hall, T. 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+alpha2-deficient+congenital+muscular+dystrophy.">The zebrafish candyfloss mutant implicates extracellular matrix adhesion failure in laminin alpha2-deficient congenital muscular dystrophy.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (17), 7092-7097 (2007).</li><li>Otten, C., 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=Xirp+Proteins+Mark+Injured+Skeletal+Muscle+in+Zebrafish.">Xirp Proteins Mark Injured Skeletal Muscle in Zebrafish.</a> PLOS ONE. 7 (2), 31041 (2012).</li><li>Otten, C., Abdelilah-Seyfried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-inflicted+Injury+of+Zebrafish+Embryonic+Skeletal+Muscle.">Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle.</a> Journal of Visualized Experiments JoVE. (71), e4351 (2013).</li><li>Nguyen, P. D., 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=Muscle+Stem+Cells+Undergo+Extensive+Clonal+Drift+during+Tissue+Growth+via+Meox1-Mediated+Induction+of+G2+Cell-Cycle+Arrest.">Muscle Stem Cells Undergo Extensive Clonal Drift during Tissue Growth via Meox1-Mediated Induction of G2 Cell-Cycle Arrest.</a> Cell Stem Cell. 21 (1), 107-119 (2017).</li><li>Ruparelia, A. A., Ratnayake, D., 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=Stem+cells+in+skeletal+muscle+growth+and+regeneration+in+amniotes+and+teleosts:+Emerging+themes.">Stem cells in skeletal muscle growth and regeneration in amniotes and teleosts: Emerging themes.</a> Wiley Interdisciplinary Reviews. Developmental Biology. 9 (2), 365 (2020).</li></ol>

Skeletal muscle regeneration is driven by obligate tissue resident muscle stem cells, whose function is altered in many muscle diseases such as muscular dystrophy, subsequently impeding the process of muscle regeneration. Here, we describe a high throughput protocol to examine muscle regeneration in live zebrafish models of muscle disease. The first step of the pipeline utilizes a embryo genotyping platform14, which is a user-friendly and accurate method to determine the genotype of live larvae, b...

Skeletal muscle accounts for 30-50% of human body mass, and is not only indispensable for locomotion, but it also serves as a critical metabolic and storage organ1. Despite being postmitotic, skeletal muscle is highly dynamic and retains a tremendous regenerative capacity following injury. This is attributed to the presence of tissue resident stem cells (also called satellite cells), located under the basal lamina of myofibers and marked by the transcription factors paired box protein 7 (pax7) and/or paired box protein 3 (pax3), among others2,3. Following injury, the satellite cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. The highly conserved cascade of pro-regenerative signals regulating satellite cell activation and robust muscle repair is&#160;affected in various conditions such as myopathies and homeostatic ageing4,5.
One such diverse group of myopathies is muscular dystrophy, characterized by progressive muscle wasting and degeneration6. These diseases are the consequence of genetic mutations in key proteins, including dystrophin and laminin-&#945;2 (LAMA2), responsible for the attachment of muscle fibers to the extracellular matrix7,8. Given that proteins implicated in muscular dystrophy play such a central role in maintaining muscle structure, for many years it was believed that a failure in this process was the mechanism responsible for disease pathogenesis9. However, recent studies have identified defects in the regulation of muscle stem cells and subsequent impairment in muscle regeneration as a second possible basis for the muscle pathology observed in muscular dystrophy10,11. As such, further studies are needed to investigate how an impairment in muscle stem cell function and associated niche elements contributes to muscular dystrophy.
Over the past decade, zebrafish (Danio rerio) has emerged as an important vertebrate model for disease modeling12. This is attributed to the rapid external development of the zebrafish embryo, coupled with its optical clarity, which allows the direct visualization of muscle formation, growth, and function. Additionally, not only is the development and structure of muscle highly conserved in zebrafish, they also display a highly conserved process of muscle regeneration13. Consequently, zebrafish represent an excellent system to study the pathobiology of muscle diseases, and explore how muscle regeneration is affected in it. To this end, we have developed a method that enables the timely study of skeletal muscle regeneration in zebrafish models of muscle disease. This high throughput pipeline involves a method to genotype live embryos14, following which a needle-stab injury is performed and the extent of muscle regeneration is imaged using polarizing light microscopy. The utilization of this technique will therefore reveal the regenerative capacity of muscle in zebrafish models of muscle disease.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24 well plates</td>
			<td>Thermo Fischer</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>30 gauge needles</td>
			<td>Terumo</td>
			<td>NN-3013R</td>
			<td></td>
		</tr>
		<tr>
			<td>90 mm Petri Dishes</td>
			<td>Pacific Laboratory Products PT</td>
			<td>S9014S20</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA extraction chips</td>
			<td>wFluidx</td>
			<td>ZEG chips</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryo genotyping platform</td>
			<td>wFluidx</td>
			<td>ZEG base unit</td>
			<td>Zebrafish Embryo Genotyper</td>
		</tr>
		<tr>
			<td>Glass pipette</td>
			<td>Hirschmann</td>
			<td>9260101</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass plate dish</td>
			<td>WPI</td>
			<td>FD35-100</td>
			<td>Commonly referred to as FluoroDish</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermoline Scientific</td>
			<td>TEI-43L</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic pipette</td>
			<td>Livingstone</td>
			<td>PTP03-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Polarizing microscope</td>
			<td>Abrio</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

examining muscle regeneration in zebrafish models of muscle disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

The ability to quantify birefringence of skeletal muscle provides a non-invasive but highly reproducible method to examine and compare levels of muscle damage, and examine muscle regeneration in vivo. Birefringence results from the diffraction of polarised light through the pseudo-crystalline array of the muscle sarcomeres15, and following injury or damage to the muscle, a reduction in birefringence is evident. Likewise, the activation and differentiation ...

Watch this Scientific Journal Video about Examining Muscle Regeneration in Zebrafish Models of Muscle Disease at JoVE.com

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

The overall goal of this protocol is to examine and quantify muscle regeneration in a zebrafish model of muscle disease. This method provides a high-throughput pipeline that is not only cost-effective, but also highly reproducible in scoring the regenerative potential of a diseased muscle in an in vivo setting. Demonstrating the procedure with me will be Avnika Ruparelia, a research fellow at the Australian Regenerative Medicine Institute.For live embryo genotyping, peel the clear protective film from the top surface of a 24-chamber DNA extraction chip and use a 20 microliter filter tip with a cut tip to transfer a single anesthetized embryo in 13 microliters of embryo medium into each chamber of the chip. When all of the embryos have been loaded, gently mount the chip onto the zebrafish embryo genotyping platform by placing one side in first, followed by the rest of the chip. Affix the magnetic platform lid over the chip to prevent the evaporation of embryo medium during the DNA extraction protocol and close the lid.Set the base unit to 2.4 volts, 0.051 amps, and 0.12 watts and start the DNA extraction protocol. The platform should start vibrating, which can be assessed by gently touching the lid. While the program is running, prepare a 24-well plate by adding one milliliter of embryo medium to each well and label eight-well strip tubes to be used for the collection of DNA material from each embryo.After eight minutes of extraction, press the on/off button to stop the vibration of the platform, gently remove the magnetic lid and lift the chip from the platform. Transfer 10 microliters of embryo medium from one chamber into the appropriate well of the eight-well strip tube and immediately add two drops of fresh embryo medium to the chambers. Transfer the embryo from the chamber to an appropriate well of the previously prepared 24-well plate.When all of the embryos have been transferred, place the plate in a 28 degree Celsius incubator. Perform the appropriate downstream genotyping assays on the genetic material collected in the eight-well strip tubes to determine the genotype of each embryo. Once the genotype of each embryo has been identified, transfer the embryos to 90 millimeter Petri dishes containing 25 milliliters of medium water and incubate the plates at 28 degrees Celsius until muscle injury.At four days post-fertilization, use a pipette to transfer one anesthetized larva into a new Petri dish and carefully remove any excess medium from the dish under a dissecting microscope. Orient the fish such that the head is on the left, the tail is on the right, the dorsal region is up, and the ventral region is down, and use a 30 gauge needle to make a quick precise stab into one to two somites in the epaxial muscle above the anal pore and horizontal to the myoseptum. Apply a drop of embryo medium to the injured zebrafish and carefully transfer the larva into one well of a 24-well plate containing one milliliter of fresh embryo medium per well.When all of the larvae have been injured, place the plate in the 28 degree Celsius incubator until the zebrafish are imaged. To quantify the muscle regeneration, open a one-day post-injury image in an appropriate image analysis software program and use the polygon tool to draw a shape around the wound site. Use the software to measure the area and mean birefringence intensity of the region and copy these values to cells D3 and E3 of the provided template.Draw two additional regions each spanning one to two uninjured somites and measure the area and mean birefringence intensities of each of these regions. Copy these values to cells D4 and D5 and E4 and E5 in the template. Then repeat the measurement for same regions in the three days post-injury image.When the birefringence has been measured in the same manner in all of the images, calculate the normalized birefringence for each region by dividing the mean birefringence intensity of each region by its area. For each time point, calculate the average normalized birefringence of the two uninjured regions to provide a reference point for the uninjured muscle. To determine the extent of muscle injury at day one post-injury, divide the normalized birefringence of the injury region by the average normalized birefringence of the uninjured regions.To determine the extent of muscle regeneration, divide the normalized birefringence of the injury region in the three days post-injury image by the average normalized intensity of the uninjured regions at this stage. Finally, calculate the regenerative index by dividing the value for the extent of the muscle regeneration at day three post-injury by the value for the extent of the muscle injury at day one post-injury. In this representative analysis, a clutch of embryos from a cross between two Lama2 heterozygous zebrafish was transferred to a DNA extraction chip and subsequently genotyped.While muscle injury results in a reduction in birefringence intensity at day one post-injury, the successful regeneration of muscle results in an increased birefringence within the same region. It is also noteworthy that while wild type larvae display a uniform birefringence intensity due to a normal muscle patterning, the birefringence intensity in the muscle of Lama2 knockout larvae is uneven and highly sporadic likely due to a reduced muscle integrity. As observed, both wild type and Lama2-deficient larvae demonstrate a significantly increased birefringence intensity in the wound site at three days post-injury compared to at one day post-injury, indicating that the muscle has regenerated.Determination of the regenerative index reveals that Lama2-deficient larvae display a striking increase in muscle regeneration compared to wild type animals. While this protocol will allow us to identify any alterations in the ability of muscle to regenerate in models of muscle disease, downstream cellular and molecular analysis need to be performed to identify the mechanisms that are responsible for the observed changes. This method has allowed us to identify how impaired muscle stem cell function and an impairment in associated niche elements contributes to muscle disease pathogenesis, therefore allowing us to identify novel disease mechanisms.

examining-muscle-regeneration-in-zebrafish-models-of-muscle-disease

Research

JoVE Journal

Developmental Biology

4.7K vistas.  Monash University. El objetivo general de este protocolo es examinar y cuantificar la regeneración muscular en un modelo de pez cebra de enfermedad muscular. Este método proporciona una tubería de alto rendimiento que no solo es rentable, sino también altamente reproducible para calificar el potencial regenerativo de un músculo enfermo en un entorno in vivo. Demostrando el procedimiento conmigo estará Avnika Ruparelia, investigadora del Instituto Australiano de Medicina Regenerativa.Para el genotip...