Recent advances using zebrafish animal models for muscle disease drug discovery

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  • 1. Introduction

    2. Zebrafish as a model for muscle

    development and disease

    3. Pharmacological therapies for

    DMD studied in zebrafish

    4. Additional zebrafish muscle

    disease models and

    pharmacological studies

    5. Analyses of zebrafish muscle

    disease and assessment of

    pharmacological rescue

    6. Using zebrafish to study

    pharmacological approaches

    to muscle disease gene

    modification

    7. Conclusion

    8. Expert opinion

    Review

    Recent advances using zebrafishanimal models for muscle diseasedrug discoveryLisa MavesCenter for Developmental Biology and Regenerative Medicine, Seattle Childrens Research Institute,

    and Department of Pediatrics, University of Washington, Seattle, WA, USA

    Introduction: Animal models have enabled great progress in the discovery

    and understanding of pharmacological approaches for treating muscle

    diseases like Duchenne muscular dystrophy.

    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. In particu-

    lar, the author focuses on how both large-scale chemical screens and targeted

    drug treatment studies have established zebrafish as an important model for

    muscle disease drug discovery.

    Expert opinion: There are a number of opportunities arising for the use of

    zebrafish models for further developing pharmacological approaches to

    muscle diseases, including studying drug combination therapies and utilizing

    genome editing to engineer zebrafish muscle disease models. It is the authors

    particular belief that the availability of a wide range of zebrafish transgenic

    strains for labeling immune cell types, combined with live imaging and

    drug treatment of muscle disease models, should allow for new elegant

    studies demonstrating how pharmacological approaches might influence

    inflammation and the immune response in muscle disease.

    Keywords: birefringence, drug screen, Duchenne muscular dystrophy, muscle function,

    muscle structure, myopathy, zebrafish

    Expert Opin. Drug Discov. (2014) 9(9):1033-1045

    1. Introduction

    Myopathies are muscle diseases in which dysfunction of muscle fibers results inmuscle weakness. The diversity of inherited myopathies, which include the muscu-lar dystrophies, is reflected in the diversity of genetic mutations that are implicatedin these diseases [1]. No cures are currently available for inherited myopathies.Gaining increased understanding of the cellular and molecular mechanisms under-lying the muscle defects seen in inherited myopathies will help provide insight intopotential therapies [2,3]. Here, I review how the zebrafish animal model hasbeen employed to both identify, and study the mechanisms of, pharmacologicalapproaches to muscle disease therapies.

    Many animal models, including mice, dogs, pigs, fruit flies, worms and zebra-fish, have contributed to our understanding of the genetic basis of, and molecularand cellular mechanisms behind, inherited muscle diseases [4-9]. In particular, onemuscle disease that is well characterized in animal models is Duchenne musculardystrophy (DMD). DMD is the most common and severe form of musculardystrophy, affecting about 1 in 3500 males, and is caused by mutations in theX-linked DMD gene, which encodes dystrophin [10]. Dystrophin contributes to

    10.1517/17460441.2014.927435 2014 Informa UK, Ltd. ISSN 1746-0441, e-ISSN 1746-045X 1033All rights reserved: reproduction in whole or in part not permitted

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  • the multi-protein dystrophin--glycoprotein complex (DGC),also known as the dystrophin-associated protein complex,which links the extracellular matrix with the cytoskeletonand is critical for muscle cell membrane stability and cellsignaling [2,11]. Many types of muscular dystrophy are causedby mutations in genes that encode components of theDGC [2,11]. Muscular dystrophies are characterized by pro-gressive muscle degeneration and progressive loss of musclefunction. Mechanisms that contribute to muscle degenera-tion in the muscular dystrophies include muscle membraneinstability, disrupted calcium homeostasis and oxidativestress [2,11]. The congenital myopathies (sometimes alsoreferred to as inherited myopathies), such as nemalinemyopathy, are characterized by muscle weakness but do notusually show progressive muscle degeneration. Congenitalmyopathies typically show characteristic structural defects inthe contractile apparatus that lead to reduced myofiber con-tractile function [5]. Disruption of excitation--contractioncoupling appears to be a common pathological mechanismin the congenital myopathies [3,5].Animal models have also contributed to the development of

    therapeutic approaches for muscle diseases. For DMD inparticular, gene therapy and stem cell-mediated therapeuticstrategies hold tremendous promise, but these approaches stillface many obstacles [12,13]. Therefore, many different pharma-cological therapies are currently being pursued [14-17]. Oneadvantage of pharmacological therapy is that systemicallydelivered drugs could reach all muscle groups, including theheart, which undergoes cardiomyopathy in DMD patients[13-15]. Animal models from fruit flies [18] to dogs [19] havehelped advance our knowledge of potential pharmacologicaltherapies for DMD. The zebrafish animal model, in particular,offers many advantages for drug discovery and for understand-ing drug mechanisms of action not only for DMD therapiesbut for therapies for other muscle diseases as well.

    2. Zebrafish as a model for muscledevelopment and disease

    2.1 Zebrafish model organism advantagesThe zebrafish, Danio rerio, offers several advantages as amodel organism for human disease and drug discovery [20].Zebrafish can be produced readily in large numbers suchthat hundreds of embryos can be obtained in a single day.The embryos are transparent and develop rapidly outside ofthe mother, allowing the earliest stages of development to beexamined. Also, zebrafish are readily manipulated by bothgenetic and chemical approaches (Figure 1). I will discussgenetic manipulations below (Sections 2.3 and 2.4). Forchemical approaches, zebrafish embryos raised in a petri dishcan readily absorb drugs that are simply added to the embryobath (Figure 2) [21]. Embryos can be raised in 96- or 384-wellplates, allowing for high-throughput chemical screening (Fig-ure 1) [21]. Significantly, drugs identified through screening inthe zebrafish have led to clinical trials [22,23], underscoring therelevance of pharmacological studies in zebrafish to humandisease. In addition to these general advantages, there are sev-eral additional characteristics that have made zebrafish an out-standing model for human muscle disease, as I describe below.

    2.2 Zebrafish skeletal muscle features and motor

    behaviorsOne advantage of studying zebrafish muscle is that develop-ment of the main body musculature is rapid, such that by24 h post-fertilization, all 30 segmental blocks of muscle, ormyotomes, are present and easily visible along the trunk andtail of the embryo. The transparency of the zebrafish embryohas facilitated cellular observations of early muscle cell line-ages, cellular migrations and muscle fiber morphogenesis[24-26]. Many of the molecular events of skeletal muscle devel-opment and differentiation are conserved between zebrafishand mammals [25,26]. Structurally, zebrafish skeletal muscle isvery similar to human muscle, and zebrafish share the samemuscle disease genes with humans, including genes encodingcomponents of the sarcomere, the DGC and excitation--contraction coupling [6,27-32]; (see Section 2.4 Zebrafish muscledisease models).

    The mechanisms of skeletal muscle repair and regenerationare similar in zebrafish and mammals. Defective muscle mem-brane repair can lead to myopathy, and both zebrafish andmammals share requirements for dysferlin and associatedproteins such as annexins for sarcolemmal repair [33,34].Pax7-expressing skeletal muscle stem cells, or satellite cells,are required for mammalian skeletal muscle regeneration [35].Zebrafish have Pax7-expressing cells that migrate into areas ofmuscle damage [36]. Whether these Pax7-expressing cells func-tion as true satellite cells and contribute to new muscle fibersduring zebrafish muscle regeneration has not yet been defini-tively shown [37]. However, zebrafish muscle does appear toregenerate from proliferative stem cell-like cells and not

    Article highlights.

    . The zebrafish animal model offers many advantages fordrug discovery and for understanding drug mechanismsof action for muscle disease therapies.

    . Zebrafish has served as a vertebrate high-throughputscreening platform for new drug therapies for Duchennemuscular dystrophy.

    . Pharmacological studies in zebrafish muscle diseasemodels have helped provide new insights intomechanisms of drug action, therapeutic targets anddisease pathogenesis.

    . Zebrafish is an exceptional animal model for the rangeof extremely simple to sophisticated approaches forassessing muscle structure and function.

    . The use of new approaches, such as genome editingtechnologies, will further enhance the use of zebrafishfor muscle disease drug discovery.

    This box summarizes key points contained in the article.

    L. Maves

    1034 Expert Opin. Drug Discov. (2014) 9(9)

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  • from de-differentiated fibers [37]. These studies help supportthe idea that investigating the mechanisms of zebrafish muscleresponse to injury and disease will inform our understandingof human muscle.

    Early motor behaviors during zebrafish embryonic andlarval stages have been well characterized and can be easilyobserved and measured [38-41]. The first movements begin atabout 17 h post-fertilization as spontaneous contractions.

    Forward geneticscreening

    Injectmorpholinos

    InjectCRISPR/Cas9

    mRNAs

    Gene knock-down

    dmd+/+

    dmd-/-

    Genome editing

    Zebrafishmuscle disease

    models

    In vivo imaging

    High-throughputdrug screening

    Time-lapse larval movement/muscle functions studies

    Figure 1. Zebrafish offer many advantages as an animal model for muscle diseases. There are several ways to generate

    zebrafish muscle disease models, including using forward genetic screens and birefringence, injecting antisense morpholinos

    into zebrafish one-cell embryos to cause targeted gene knock-down, and injecting CRISPR/Cas9 mRNAs to induce targeted

    gene editing. Zebrafish muscle disease models, such as dmd mutants, can show disruptions in the muscle birefringence

    pattern (center figure). Zebrafish muscle disease models can be used in high-throughput drug screens to identify compounds

    that ameliorate the muscle disease phenotype, for in vivo muscle fiber imaging for muscle structure analyses, and for muscle

    function analyses, such as time-lapse tracking of larval movements in petri dishes.DMD: Duchenne muscular dystrophy.

    4 dayspost fertilization

    1 day post fertilization

    +DMSO

    Birefringence

    Phalloidin

    +drug

    Zebrafish embryosfrom dmd+/-X dmd+/-cross

    Zebrafish embryosfrom dmd+/-X dmd+/-cross

    3 days incubation

    dmd+/+ or dmd+/- dmd+/+ or dmd+/-dmd-/- dmd-/-

    Figure 2. Example of a strategy for testingpharmacological rescue of a zebrafishmuscle diseasemodel. In this example, embryos

    are collected from dmd+/- parents and raised in awater bath in petri dishes. Drugs, and vehicle control such as dimethyl sulfoxide

    (DMSO) can be added to the water bath at any time, typically at 1 day post-fertilization [54,55,59,62]. Following incubation, larvae

    can be scored formuscle lesions using birefringence (shown in lowmagnification views; [39,54]) ormuscle stains such as phalloidin

    (larval trunkmyotomes shown inhighermagnificationviews; [62,73]).At4dayspost-fertilization, about25%of larvae fromDMSO-

    control treated dmd+/-crosses show disruptions in the muscle fiber pattern, whereas lower frequencies of larvae with muscle

    defects appear upon treatment with drugs that improve the dmd phenotype (Table 1). Arrows point to larvae with disrupted

    birefringence pattern. Phalloidin images were previously published in [62] and appear with permission of PLOS Currents.DMD: Duchenne muscular dystrophy.

    Recent advances using zebrafish animal models for muscle disease drug discovery

    Expert Opin. Drug Discov. (2014) 9(9) 1035

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  • At about 24 h, embryos will coil in response to touch. Atabout 26 h, embryos exhibit swimming movements inresponse to touch, and by 96 h the larva is freely swimming.These stereotyped movements have formed the basis forgenetic screens for zebrafish mutant strains with muscledefects, as I describe next, and for characterization of zebrafishmuscle disease models (see Section 5.2 Assessing musclefunction).

    2.3 Zebrafish genetic screens for muscle defectsThe rapid development of zebrafish and the ability to readilyobtain large numbers of animals have facilitated forwardgenetic screens that have significantly contributed to the useof zebrafish as an animal model for human muscle disease.Some of the earliest genetic screens in zebrafish usedembryonic motility to identify loci required for muscle func-tion [39,42]. The stereotypic touch-evoked escape response inzebrafish larvae has formed the basis of several genetic screensin an effort to identify genes involved in motor control ofbehavior [39,40,43,44]. Additional zebrafish forward geneticscreens have utilized muscle birefringence, a measure of skele-tal muscle structural integrity (see Section 5.1 Assessing mus-cle structure; Figure 1), to identify genes relevant to skeletalmuscle disease [40,45]. Many of the genes responsible for thephenotypes in the mutant strains recovered from these screenshave been identified, and most are orthologs of humanmuscular dystrophy and myopathy genes [28,30,45-48].

    2.4 Zebrafish muscle disease modelsThese genetic screens and the resulting mutant zebrafish strainshave thus provided a wealth of zebrafish models of humanmuscle diseases. Additional approaches to generating zebrafishmodels of humanmuscle diseases have been the use of antisensemorpholinos (Figure 1) or, in cases where the human diseaseresults from a dominant mutation or misexpression, transgeniczebrafish or transient overexpression in zebrafish embryos.Recent reviews provide tables with comprehensive lists ofknown zebrafish models of human muscle diseases [6,32,49].Genome editing technology now opens up new approaches togenerating zebrafish muscle disease models, as I will discussfurther below (Section 8 Expert opinion; Figur...

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