Recent advances using zebrafish animal models for muscle disease drug discovery

Abstract

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 particular, 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 author's 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: Duchenne muscular dystrophy; birefringence; drug screen; muscle function; muscle structure; myopathy; zebrafish.

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.

Figure 2. Example of a strategy for testing pharmacological rescue of a zebrafish muscle disease model

In this example, embryos are collected from dmd+/− parents and raised in a water 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 for muscle lesions using birefringence (shown in low magnification views; [39,54]) or muscle stains such as phalloidin (larval trunk myotomes shown in higher magnification views; [62,73]). At 4 days post-fertilization, about 25% of larvae from DMSO-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.