Robert J. Bryson-Richardson

Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo

A class of recessive lethal zebrafish mutations has been identified in which normal skeletal muscle differentiation is followed by a tissue-specific degeneration that is reminiscent of the human muscular dystrophies. Here, we show that one of these mutations, sapje, disrupts the zebrafish orthologue of the X-linked human Duchenne muscular dystrophy (DMD) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology through disconnecting the cytoskeleton from the extracellular matrix in skeletal muscle by reducing the level of dystrophin protein at the sarcolemma. This is thought to allow tearing of this membrane, which in turn leads to cell death. Surprisingly, we have found that the progressive muscle degeneration phenotype of sapje mutant zebrafish embryos is caused by the failure of embryonic muscle end attachments. Although a role for dystrophin in maintaining vertebrate myotendinous junctions (MTJs) has been postulated previously and MTJ structural abnormalities have been identified in the Dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure has thus far been lacking. This zebrafish mutation may therefore provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases.

The genetics of vertebrate myogenesis

The molecular, genetic and cellular bases for skeletal muscle growth and regeneration have been recently documented in a number of vertebrate species. These studies highlight the role of transient subcompartments of the early somite as a source of distinct waves of myogenic precursors. Individual myogenic progenitor populations undergo a complex series of cell rearrangements and specification events in different regions of the body, all of which are controlled by distinct gene regulatory networks. Collectively, these studies have opened a window into the morphogenetic and molecular bases of the different phases of vertebrate myogenesis, from embryo to adult.

Analysis of protein sequence and interaction data for candidate disease gene prediction

Linkage analysis is a successful procedure to associate diseases with specific genomic regions. These regions are often large, containing hundreds of genes, which make experimental methods employed to identify the disease gene arduous and expensive. We present two methods to prioritize candidates for further experimental study: Common Pathway Scanning (CPS) and Common Module Profiling (CMP). CPS is based on the assumption that common phenotypes are associated with dysfunction in proteins that participate in the same complex or pathway. CPS applies network data derived from protein–protein interaction (PPI) and pathway databases to identify relationships between genes. CMP identifies likely candidates using a domain-dependent sequence similarity approach, based on the hypothesis that disruption of genes of similar function will lead to the same phenotype. Both algorithms use two forms of input data: known disease genes or multiple disease loci. When using known disease genes as input, our combined methods have a sensitivity of 0.52 and a specificity of 0.97 and reduce the candidate list by 13-fold. Using multiple loci, our methods successfully identify disease genes for all benchmark diseases with a sensitivity of 0.84 and a specificity of 0.63. Our combined approach prioritizes good candidates and will accelerate the disease gene discovery process.

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.

The structure and evolution of the melanocortin and MCH receptors in fish and mammals

Zebrafish are an excellent genetic model system for studying developmental and physiological processes. Pigment patterns in zebrafish are affected by mutations in three types of chromatophores. The behavior of these cells is influenced by alpha-melanocyte-stimulating hormone (alphaMSH) and melanin-concentrating hormone (MCH). Mammals have five alphaMSH receptors (melanocortin receptors) and one or two MCH receptors. We have identified the full complement of melanocortin and MCH receptors in both zebrafish and the pufferfish, Fugu. Zebrafish have six melanocortin receptors, including two MC5R orthologues, while Fugu, lacking MC3R, has only four. We also demonstrate that Fugu and zebrafish have two and three MCHR genes, respectively. MC2R and MC5R are physically linked in all species examined. Unlike other species, we find the Fugu genes contain introns, one of which is in a conserved location and is probably ancestral. We also detail the differential expression of the zebrafish genes throughout development.

Mutations in KLHL40 Are a Frequent Cause of Severe Autosomal-Recessive Nemaline Myopathy

Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.

Cadherin-Mediated Differential Cell Adhesion Controls Slow Muscle Cell Migration in the Developing Zebrafish Myotome

Slow-twitch muscle fibers of the zebrafish myotome undergo a unique set of morphogenetic cell movements. During embryogenesis, slow-twitch muscle derives from the adaxial cells, a layer of paraxial mesoderm that differentiates medially within the myotome, immediately adjacent to the notochord. Subsequently, slow-twitch muscle cells migrate through the entire myotome, coming to lie at its most lateral surface. Here we examine the cellular and molecular basis for slow-twitch muscle cell migration. We show that slow-twitch muscle cell morphogenesis is marked by behaviors typical of cells influenced by differential cell adhesion. Dynamic and reciprocal waves of N-cadherin and M-cadherin expression within the myotome, which correlate precisely with cell migration, generate differential adhesive environments that drive slow-twitch muscle cell migration through the myotome. Removing or altering the expression of either protein within the myotome perturbs migration. These results provide a definitive example of homophilic cell adhesion shaping cellular behavior during vertebrate development.

Myosin heavy chain expression in zebrafish and slow muscle composition

In the zebrafish embryo, two distinct classes of muscle fibers have been described in the forming myotome that arise from topographically separable precursor populations. Based entirely on cross‐reactivity with antibodies raised against mammalian and chick myosin heavy chain isoforms slow twitch muscle has been shown to arise exclusively from “adaxial” myoblasts, which migrate from their origin flanking the notochord to form a single layer of subcutaneous differentiated muscle cells. The remainder of the myotome differentiates behind this migration as muscle fibers recognized by anti‐fast myosin heavy chain (MyHC) antibodies. To identify unambiguous molecular markers of cell fate in the myotome, we have characterized genes encoding zebrafish fast and slow MyHC. Using phylogenetic and expression analysis, we demonstrate that these genes are definitive molecular markers of slow and fast twitch fates. We also demonstrate that zebrafish embryonic slow twitch muscle co‐expresses both slow and fast twitch MyHC isoforms, a property that they share with primary fibers of the amniote myotome. Developmental Dynamics 233:1018–1022, 2005.

Met and Hgf signaling controls hypaxial muscle and lateral line development in the zebrafish

Somites give rise to a number of different embryonic cell types, including the precursors of skeletal muscle populations. The lateral aspect of amniote and fish somites have been shown to give rise specifically to hypaxial muscle, including the appendicular muscle that populates fins and limbs. We have investigated the morphogenetic basis for formation of specific hypaxial muscles within the zebrafish embryo and larvae. Transplantation experiments have revealed a developmentally precocious commitment of cells derived from pectoral fin level somites to forming hypaxial and specifically appendicular muscle. The fate of transplanted somites cannot be over-ridden by local inductive signals, suggesting that somitic tissue may be fixed at an early point in their developmental history to produce appendicular muscle. We further show that this restriction in competence is mirrored at the molecular level, with the exclusive expression of the receptor tyrosine kinase met within somitic regions fated to give rise to appendicular muscle. Loss-of-function experiments reveal that Met and its ligand, hepatocyte growth factor, are required for the correct morphogenesis of the hypaxial muscles in which met is expressed. Furthermore, we demonstrate a requirement for Met signaling in the process of proneuromast deposition from the posterior lateral line primordia.

FishNet: an online database of zebrafish anatomy

BackgroundOver the last two decades, zebrafish have been established as a genetically versatile model system for investigating many different aspects of vertebrate developmental biology. With the credentials of zebrafish as a developmental model now well recognized, the emerging new opportunity is the wider application of zebrafish biology to aspects of human disease modelling. This rapidly increasing use of zebrafish as a model for human disease has necessarily generated interest in the anatomy of later developmental phases such as the larval, juvenile, and adult stages, during which many of the key aspects of organ morphogenesis and maturation take place. Anatomical resources and references that encompass these stages are non-existent in zebrafish and there is therefore an urgent need to understand how different organ systems and anatomical structures develop throughout the life of the fish.ResultsTo overcome this deficit we have utilized the technique of optical projection tomography to produce three-dimensional (3D) models of larval fish. In order to view and display these models we have created FishNet http://www.fishnet.org.au, an interactive reference of zebrafish anatomy spanning the range of zebrafish development from 24 h until adulthood.ConclusionFishNet contains more than 36 000 images of larval zebrafish, with more than 1 500 of these being annotated. The 3D models can be manipulated on screen or virtually sectioned. This resource represents the first complete embryo to adult atlas for any species in 3D.