Joachim Berger

The zebrafish candyfloss mutant implicates extracellular matrix adhesion failure in laminin α2-deficient congenital muscular dystrophy

Mutations in the human laminin α2 (LAMA2) gene result in the most common form of congenital muscular dystrophy (MDC1A). There are currently three models for the molecular basis of cellular pathology in MDC1A: (i) lack of LAMA2 leads to sarcolemmal weakness and failure, followed by cellular necrosis, as is the case in Duchenne muscular dystrophy (DMD); (ii) loss of LAMA2-mediated signaling during the development and maintenance of muscle tissue results in myoblast proliferation and fusion defects; (iii) loss of LAMA2 from the basement membrane of the Schwann cells surrounding the peripheral nerves results in a lack of motor stimulation, leading to effective denervation atrophy. Here we show that the degenerative muscle phenotype in the zebrafish dystrophic mutant, candyfloss (caf) results from mutations in the laminin α2 (lama2) gene. In vivo time-lapse analysis of mechanically loaded fibers and membrane permeability assays suggest that, unlike DMD, fiber detachment is not initially associated with sarcolemmal rupture. Early muscle formation and myoblast fusion are normal, indicating that any deficiency in early Lama2 signaling does not lead to muscle pathology. In addition, innervation by the primary motor neurons is unaffected, and fiber detachment stems from muscle contraction, demonstrating that muscle atrophy through lack of motor neuron activity does not contribute to pathology in this system. Using these and other analyses, we present a model of lama2 function where fiber detachment external to the sarcolemma is mechanically induced, and retracted fibers with uncompromised membranes undergo subsequent apoptosis.

Dystrophin-deficient zebrafish feature aspects of the Duchenne muscular dystrophy pathology

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene. As in humans, zebrafish dystrophin is initially expressed at the peripheral ends of the myofibres adjacent to the myotendinous junction and gradually shifts to non-junctional sites. Dystrophin-deficient zebrafish larvae are characterised by abundant necrotic fibres being replaced by mono-nucleated infiltrates, extensive fibrosis accompanied by inflammation, and a broader variation in muscle fibre cross-sectional areas. Muscle progenitor proliferation cannot compensate for the extensive skeletal muscle loss. Live imaging of dystrophin-deficient zebrafish larvae documents detaching myofibres elicited by muscle contraction. Correspondingly, the progressive phenotype of dystrophin-deficient zebrafish resembles many aspects of the human disease, suggesting that specific advantages of the zebrafish model system, such as the ability to undertake in vivo drug screens and real time analysis of muscle fibre loss, could be used to make novel insights relevant to understanding and treating the pathological basis of dystrophin-deficient muscular dystrophy.

The zebrafish dystrophic mutant softy maintains muscle fibre viability despite basement membrane rupture and muscle detachment

The skeletal muscle basement membrane fulfils several crucial functions during development and in the mature myotome and defects in its composition underlie certain forms of muscular dystrophy. A major component of this extracellular structure is the laminin polymer, which assembles into a resilient meshwork that protects the sarcolemma during contraction. Here we describe a zebrafish mutant, softy, which displays severe embryonic muscle degeneration as a result of initial basement membrane failure. The softy phenotype is caused by a mutation in the lamb2 gene, identifying laminin β2 as an essential component of this basement membrane. Uniquely, softy homozygotes are able to recover and survive to adulthood despite the loss of myofibre adhesion. We identify the formation of ectopic, stable basement membrane attachments as a novel means by which detached fibres are able to maintain viability. This demonstration of a muscular dystrophy model possessing innate fibre viability following muscle detachment suggests basement membrane augmentation as a therapeutic strategy to inhibit myofibre loss.

Zebrafish models flex their muscles to shed light on muscular dystrophies

Muscular dystrophies are a group of genetic disorders that specifically affect skeletal muscle and are characterized by progressive muscle degeneration and weakening. To develop therapies and treatments for these diseases, a better understanding of the molecular basis of muscular dystrophies is required. Thus, identification of causative genes mutated in specific disorders and the study of relevant animal models are imperative. Zebrafish genetic models of human muscle disorders often closely resemble disease pathogenesis, and the optical clarity of zebrafish embryos and larvae enables visualization of dynamic molecular processes in vivo. As an adjunct tool, morpholino studies provide insight into the molecular function of genes and allow rapid assessment of candidate genes for human muscular dystrophies. This unique set of attributes makes the zebrafish model system particularly valuable for the study of muscle diseases. This review discusses how recent research using zebrafish has shed light on the pathological basis of muscular dystrophies, with particular focus on the muscle cell membrane and the linkage between the myofibre cytoskeleton and the extracellular matrix.

Quantification of birefringence readily measures the level of muscle damage in zebrafish.

Muscular dystrophies are a group of genetic disorders that progressively weaken and degenerate muscle. Many zebrafish models for human muscular dystrophies have been generated and analysed, including dystrophin-deficient zebrafish mutants dmd that model Duchenne Muscular Dystrophy. Under polarised light the zebrafish muscle can be detected as a bright area in an otherwise dark background. This light effect, called birefringence, results from the diffraction of polarised light through the pseudo-crystalline array of the muscle sarcomeres. Muscle damage, as seen in zebrafish models for muscular dystrophies, can readily be detected by a reduction in the birefringence. Therefore, birefringence is a very sensitive indicator of overall muscle integrity within larval zebrafish. Unbiased documentation of the birefringence followed by densitometric measurement enables the quantification of the birefringence of zebrafish larvae. Thereby, the overall level of muscle integrity can be detected, allowing the identification and categorisation of zebrafish muscle mutants. In addition, we propose that the establish protocol can be used to analyse treatments aimed at ameliorating dystrophic zebrafish models.

Asymmetric division of clonal muscle stem cells coordinates muscle regeneration in vivo

Dividing asymmetrically to fix muscle Resident tissue stem cells called satellite cells repair muscle after injury. However, how satellite cells operate inside living tissue is unclear. Gurevich et al. exploited the optical clarity of zebrafish larvae and used a series of genetic approaches to study muscle injury. After injury, satellite cells divide asymmetrically to generate a progenitor pool for muscle replacement and at the same time “self-renew” the satellite stem cell. This results in regeneration that is highly clonal in nature, validating many decades of in vitro analyses examining the regenerative capacity of skeletal muscle. Science, this issue p. 136 The visualization of myogenic repair in zebrafish muscle reveals a dynamic regeneration process in living animals. INTRODUCTION Mammalian skeletal muscle harbors tissue-specific stem cells that are triggered to replace damaged fibers after injury. Genetic ablation of satellite cells in the mouse results in a failure to regenerate muscle, which indicates that these cells are the major (and possibly only) mediators for repair of skeletal muscle. Further evidence for the central role of satellite cells in muscle regeneration comes from transplantation experiments with genetically marked cells, which demonstrate that satellite cells are highly proliferative myogenic precursors capable of self‐renewal and the resumption of quiescence, properties deemed important in a cell population responsible for muscle repair. Considerable in vitro evidence, derived from cultured fibers and myoblasts, is suggestive of a role for asymmetric division in generating both a self-renewing “immortal” stem cell and a differentiation-competent progenitor cell that proliferates and ultimately replaces damaged muscle. However, asymmetric division of satellite cells has not been documented in vivo. Furthermore, considerable doubt remains over how accurately in vitro studies can model satellite cell behavior, given that the isolation and culture of individual muscle fibers and cells stimulates satellite cell proliferation. Finally, it is not clear whether the environment an activated satellite cell encounters in a single fiber explant, or in culture, mimics the molecular and biophysical architecture of a regenerating muscle injury in vivo. Consequently, what role, if any, the wound environment itself plays in regeneration and self-renewal is difficult to address in these systems. RATIONALE Using the optical clarity and genetic tractability of the zebrafish system, we developed tools to track and image the regeneration of living muscle tissue after injury. Marking muscle stem and progenitor cells with transgenes and using long-term imaging and lineage-tracing modalities enabled us to visualize cell movements and behaviors during regeneration in vivo. RESULTS In vivo cell tracking permitted high-resolution imaging of the entire process of muscle regeneration, from injury to fiber replacement. Using this approach, we were able to determine the morphological, cellular, and genetic basis for zebrafish muscle regeneration. Our analysis identified a stem cell niche in the zebrafish myotome that is equivalent to the mammalian satellite cell system, revealing that this evolutionarily ancient stem cell is probably present throughout the vertebrate phylogeny. Complex interactions were observed between satellite cells and both injured and uninjured fibers within the wound environment. Among the most notable of these was the identification of filopodia-like projections, emanating from uninjured fibers, which adhere to and “lasso” the activated satellite cell to guide it to the wound edge. Furthermore, we documented the in vivo occurrence of asymmetric satellite cell division, a process that drives both self-renewal and regeneration via a clonally restricted progenitor pool. CONCLUSION Asymmetric divisions occur during in vivo muscle regeneration to generate clonally related progenitors required for muscle repair. This finding resolves a long-term debate surrounding the existence of this mechanism of stem cell self-renewal and muscle repair in vivo. Our results also reveal the highly dynamic nature of the wound environment, where uninjured fibers at the wound edge play a crucial role in directing differentiating progenitors to regions of the wound that are most in need of new fiber addition. Mechanism of in vivo muscle repair. (A to C) Muscle regeneration is clonal. Regenerating fibers (outlined in white) express the same color after fluorescent lineage tracing, indicating clonal derivation from a single stem cell. Sagittal, transverse, and coronal sections are shown in (A) to (C), respectively. (D) Regeneration dynamics in vivo. Quiescent satellite cells, activated upon injury, undergo asymmetric division, which results in self-renewing or proliferating cells. Proliferative cells undergo myogenesis to generate de novo immature fibers. Skeletal muscle is an example of a tissue that deploys a self-renewing stem cell, the satellite cell, to effect regeneration. Recent in vitro studies have highlighted a role for asymmetric divisions in renewing rare “immortal” stem cells and generating a clonal population of differentiation-competent myoblasts. However, this model currently lacks in vivo validation. We define a zebrafish muscle stem cell population analogous to the mammalian satellite cell and image the entire process of muscle regeneration from injury to fiber replacement in vivo. This analysis reveals complex interactions between satellite cells and both injured and uninjured fibers and provides in vivo evidence for the asymmetric division of satellite cells driving both self-renewal and regeneration via a clonally restricted progenitor pool.

Evaluation of exon‐skipping strategies for Duchenne muscular dystrophy utilizing dystrophin‐deficient zebrafish

Duchenne muscular dystophy (DMD) is a severe muscle wasting disease caused by mutations in the dystrophin gene. By utilizing antisense oligonucleotides, splicing of the dystrophin transcript can be altered so that exons harbouring a mutation are excluded from the mature mRNA. Although this approach has been shown to be effective to restore partially functional dystrophin protein, the level of dystrophin protein that is necessary to rescue a severe muscle pathology has not been addressed. As zebrafish dystrophin mutants (dmd) resemble the severe muscle pathology of human patients, we have utilized this model to evaluate exon skipping. Novelu2002dmdu2002mutations were identified to enable the design of phenotype rescue studiesu2002viau2002morpholino administration. Correlation of induced exon‐skipping efficiency and the level of phenotype rescue suggest that relatively robust levels of exon skipping are required to achieve significant therapeutic ameliorations and that pre‐screening analysis of exon‐skipping drugs in zebrafish may help to more accurately predict clinical trials for therapies of DMD.

The role of zebrafish in chemical genetics

The identification and exploration of new drug candidates to fight diseases is a major imperative for improving human health. The traditional mechanism utilised to identify new compounds with therapeutic potential has been to systematically analyse large libraries of small molecules for lead compounds with a desired bioactivity in protein or cell based assays. Identified lead compounds were subsequently assessed for their potential as lead drugs. In the last few years, small molecule screens were also carried out in vivo, on whole organisms such as the zebrafish. Cost efficient maintenance together with abundant manipulatory techniques and molecular tools have made the zebrafish a preferred system in which to perform large-scale screens. Numerous studies have revealed that zebrafish mutants can accurately model many aspects of human diseases. Therefore, small molecules identified in zebrafish-based screens can be particularly valuable in identifying lead compounds with direct therapeutic relevance to specific human disease states. Here, we review the role of zebrafish-based screens in the emerging field of chemical genetics.

Chemical genetics unveils a key role of mitochondrial dynamics, cytochrome c release and IP3R activity in muscular dystrophy

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the dystrophin gene. The subcellular mechanisms of DMD remain poorly understood and there is currently no curative treatment available. Using a Caenorhabditis elegans model for DMD as a pharmacologic and genetic tool, we found that cyclosporine A (CsA) reduces muscle degeneration at low dose and acts, at least in part, through a mitochondrial cyclophilin D, CYN-1. We thus hypothesized that CsA acts on mitochondrial permeability modulation through cyclophilin D inhibition. Mitochondrial patterns and dynamics were analyzed, which revealed dramatic mitochondrial fragmentation not only in dystrophic nematodes, but also in a zebrafish model for DMD. This abnormal mitochondrial fragmentation occurs before any obvious sign of degeneration can be detected. Moreover, we demonstrate that blocking/delaying mitochondrial fragmentation by knocking down the fission-promoting gene drp-1 reduces muscle degeneration and improves locomotion abilities of dystrophic nematodes. Further experiments revealed that cytochrome c is involved in muscle degeneration in C. elegans and seems to act, at least in part, through an interaction with the inositol trisphosphate receptor calcium channel, ITR-1. Altogether, our findings reveal that mitochondria play a key role in the early process of muscle degeneration and may be a target of choice for the design of novel therapeutics for DMD. In addition, our results provide the first indication in the nematode that (i) mitochondrial permeability transition can occur and (ii) cytochrome c can act in cell death.

503unc, a small and muscle-specific zebrafish promoter

The muscle‐specific UNC‐45b assists in the folding of sarcomeric myosin. Analysis of the zebrafish unc‐45b upstream region revealed that unc‐45b promoter fragments reliably drive GFP expression after germline transmission. The muscle‐specific 503‐bp minimal promoter 503unc was identified to drive gene expression in the zebrafish musculature. In transgenic Tg(−503unc:GFP) zebrafish, GFP fluorescence was detected in the adaxial cells, their slow fiber descendants, and the fast muscle. At later stages, robust GFP fluorescence is eminent in the cardiac, cranial, fin, and trunk muscle, thereby recapitulating the unc‐45b expression pattern. We propose that the 503unc promoter is a small and muscle‐specific promoter that drives robust gene expression throughout the zebrafish musculature, making it a valuable tool for the exploration of zebrafish muscle. genesis 51:443–447.