FKRP-dependent glycosylation of fibronectin regulates muscle pathology in muscular dystrophy.

The muscular dystrophies encompass a broad range of pathologies with varied clinical outcomes. In the case of patients carrying defects in fukutin-related protein (FKRP), these diverse pathologies arise from mutations within the same gene. This is surprising as FKRP is a glycosyltransferase, whose only identified function is to transfer ribitol-5-phosphate to α-dystroglycan (α-DG). Although this modification is critical for extracellular matrix attachment, α-DG's glycosylation status relates poorly to disease severity, suggesting the existence of unidentified FKRP targets. Here we reveal that FKRP directs sialylation of fibronectin, a process essential for collagen recruitment to the muscle basement membrane. Thus, our results reveal that FKRP simultaneously regulates the two major muscle-ECM linkages essential for fibre survival, and establishes a new disease axis for the muscular dystrophies.

Cellular rescue in a zebrafish model of congenital muscular dystrophy type 1A.

Laminins comprise structural components of basement membranes, critical in the regulation of differentiation, survival and migration of a diverse range of cell types, including skeletal muscle. Mutations in one muscle enriched Laminin isoform, Laminin alpha2 (Lama2), results in the most common form of congenital muscular dystrophy, congenital muscular dystrophy type 1A (MDC1A). However, the exact cellular mechanism by which Laminin loss results in the pathological spectrum associated with MDC1A remains elusive. Here we show, via live tracking of individual muscle fibres, that dystrophic myofibres in the zebrafish model of MDC1A maintain sarcolemmal integrity and undergo dynamic remodelling behaviours post detachment, including focal sarcolemmal reattachment, cell extension and hyper-fusion with surrounding myoblasts. These observations imply the existence of a window of therapeutic opportunity, where detached cells may be "re-functionalised" prior to their delayed entry into the cell death program, a process we show can be achieved by muscle specific or systemic Laminin delivery. We further reveal that Laminin also acts as a pro-regenerative factor that stimulates muscle stem cell-mediated repair in lama2-deficient animals in vivo. The potential multi-mode of action of Laminin replacement therapy suggests it may provide a potent therapeutic axis for the treatment for MDC1A.

Muscular dystrophy modeling in zebrafish.

Skeletal muscle performs an essential function in human physiology with defects in genes encoding a variety of cellular components resulting in various types of inherited muscle disorders. Muscular dystrophies (MDs) are a severe and heterogeneous type of human muscle disease, manifested by progressive muscle wasting and degeneration. The disease pathogenesis and therapeutic options for MDs have been investigated for decades using rodent models, and considerable knowledge has been accumulated on the cause and pathogenetic mechanisms of this group of human disorders. However, due to some differences between disease severity and progression, what is learned in mammalian models does not always transfer to humans, prompting the desire for additional and alternative models. More recently, zebrafish have emerged as a novel and robust animal model for the study of human muscle disease. Zebrafish MD models possess a number of distinct advantages for modeling human muscle disorders, including the availability and ease of generating mutations in homologous disease-causing genes, the ability to image living muscle tissue in an intact animal, and the suitability of zebrafish larvae for large-scale chemical screens. In this chapter, we review the current understanding of molecular and cellular mechanisms involved in MDs, the process of myogenesis in zebrafish, and the structural and functional characteristics of zebrafish larval muscles. We further discuss the insights gained from the key zebrafish MD models that have been so far generated, and we summarize the attempts that have been made to screen for small molecules inhibitors of the dystrophic phenotypes using these models. Overall, these studies demonstrate that zebrafish is a useful in vivo system for modeling aspects of human skeletal muscle disorders. Studies using these models have contributed both to the understanding of the pathogenesis of muscle wasting disorders and demonstrated their utility as highly relevant models to implement therapeutic screening regimens.

Decreased anti-regenerative effects after spinal cord injury in spry4-/- mice.

Previously, we have demonstrated a role for fibroblast growth factor (Fgf) in spinal cord regeneration in both zebrafish and mouse. We have shown that exogenous Fgf2 treatment attenuates astrocytic gliosis and induces glia cells to become progenitors that undergo neurogenesis as well as differentiating into bipolar astrocytes that support axonal regeneration (Goldshmit et al., 2012, 2014). One of the downstream signaling target genes of Fgf is spry4, which acts as a feedback inhibitor for Fgf signaling. In this study we examined the effects of increased endogenous Fgf signaling, in spry4-/- mice, on the early events that occur after spinal cord injury (SCI). We demonstrate that in spry4-/- mice inflammatory responses, such as tumor necrosis factor α (TNFα) secretion and macrophage/neutrophil invasion into the lesion site are reduced. In addition, astrocytic gliosis is attenuated and neuronal survival is increased. These results further support a pro-regenerative role of Fgf after SCI, and suggest that increased endogenous Fgf signaling after SCI may contribute to functional recovery and therefore presents this pathway as a target for new therapy development.

Analysing regenerative potential in zebrafish models of congenital muscular dystrophy.

The congenital muscular dystrophies (CMDs) are a clinically and genetically heterogeneous group of muscle disorders. Clinically hypotonia is present from birth, with progressive muscle weakness and wasting through development. For the most part, CMDs can mechanistically be attributed to failure of basement membrane protein laminin-α2 sufficiently binding with correctly glycosylated α-dystroglycan. The majority of CMDs therefore arise as the result of either a deficiency of laminin-α2 (MDC1A) or hypoglycosylation of α-dystroglycan (dystroglycanopathy). Here we consider whether by filling a regenerative medicine niche, the zebrafish model can address the present challenge of delivering novel therapeutic solutions for CMD. In the first instance the readiness and appropriateness of the zebrafish as a model organism for pioneering regenerative medicine therapies in CMD is analysed, in particular for MDC1A and the dystroglycanopathies. Despite the recent rapid progress made in gene editing technology, these approaches have yet to yield any novel zebrafish models of CMD. Currently the most genetically relevant zebrafish models to the field of CMD, have all been created by N-ethyl-N-nitrosourea (ENU) mutagenesis. Once genetically relevant models have been established the zebrafish has several important facets for investigating the mechanistic cause of CMD, including rapid ex vivo development, optical transparency up to the larval stages of development and relative ease in creating transgenic reporter lines. Together, these tools are well suited for use in live-imaging studies such as in vivo modelling of muscle fibre detachment. Secondly, the zebrafish's contribution to progress in effective treatment of CMD was analysed. Two approaches were identified in which zebrafish could potentially contribute to effective therapies. The first hinges on the augmentation of functional redundancy within the system, such as upregulating alternative laminin chains in the candyfloss fish, a model of MDC1A. Secondly high-throughput small molecule screens not only provide effective therapies, but also an alternative strategy for investigating CMD in zebrafish. In this instance insight into disease mechanism is derived in reverse. Zebrafish models are therefore clearly of critical importance in the advancement of regenerative medicine strategies in CMD. This article is part of a Directed Issue entitled: Regenerative Medicine: The challenge of translation.

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.

Morphogenesis and evolution of vertebrate appendicular muscle.

Two different modes are utilised by vertebrate species to generate the appendicular muscle present within fins and limbs. Primitive Chondricthyan or cartilaginous fishes use a primitive mode of muscle formation to generate the muscle of the fins. Direct epithelial myotomal extensions invade the fin and generate the fin muscles while remaining in contact with the myotome. Embryos of amniotes such as chick and mouse use a similar mechanism to that deployed in the bony teleost species, zebrafish. Migratory mesenchymal myoblasts delaminate from fin/limb level somites, migrate to the fin/limb field and differentiate entirely within the context of the fin/limb bud. Migratory fin and limb myoblasts express identical genes suggesting that they possess both morphogenetic and molecular identity. We conclude that the mechanisms controlling tetrapod limb muscle formation arose prior to the Sarcopterygian or tetrapod radiation.

Evolutionary origins of vertebrate appendicular muscle.

The evolution of terrestrial tetrapod species heralded a transition in locomotor strategies. While most fish species use the undulating contractions of the axial musculature to generate propulsive force, tetrapods also rely on the appendicular muscles of the limbs to generate movement. Despite the fossil record generating an understanding of the way in which the appendicular skeleton has evolved to provide the scaffold for tetrapod limb musculature, there is, by contrast, almost no information as to how this musculature arose. Here we examine fin muscle formation within two extant classes of fish. We find that in the teleost, zebrafish, fin muscles arise from migratory mesenchymal precursor cells that possess molecular and morphogenetic identity with the limb muscle precursors of tetrapod species. Chondrichthyan dogfish embryos, however, use the primitive mechanism of direct epithelial somitic extensions to derive the muscles of the fin. We conclude that the genetic mechanism controlling formation of tetrapod limb muscles evolved before the Sarcopterygian radiation.

Slow muscle induction by Hedgehog signalling in vitro.

Muscles are composed of several fibre types, the precise combination of which determines muscle function. Whereas neonatal and adult fibre type is influenced by a number of extrinsic factors, such as neural input and muscle load, there is little knowledge of how muscle cells are initially determined in the early embryo. In the zebrafish, fibres of the slow twitch class arise from precociously specified myoblasts that lie close to the midline whereas the remainder of the myotome differentiates as fast myosin expressing muscle. In vivo evidence has suggested the Sonic Hedgehog glycoprotein, secreted from the notochord, controls the formation of slow twitch and fast twitch muscle fates. Here we describe an in vitro culture system that we have developed to test directly the ability of zebrafish myoblasts to respond to exogenous Sonic Hedgehog peptide. We find that Sonic Hedgehog peptide can control the binary cell fate choice of embryonic zebrafish myoblasts in vitro. We have also used this culture system to assay the relative activities of different Hedgehog-family proteins and to investigate the possible involvement of heterotrimeric G-proteins in Hedgehog signal transduction.

Control of muscle cell-type specification in the zebrafish embryo by Hedgehog signalling.

The specification of different muscle cell types in the zebrafish embryo requires signals that emanate from the axial mesoderm. In previous studies we and others have shown that overexpression of different members of the Hedgehog protein family can induce the differentiation of two types of slow-twitch muscles, the superficially located slow-twitch fibres and the medially located muscle pioneer cells. Here we have investigated the requirement for Hedgehog signalling in the specification of these distinct muscle cell types in two ways: first, by characterising the effects on target gene expression and muscle cell differentiation of the u-type mutants, members of a phenotypic group previously implicated in Hedgehog signalling, and second, by analysing the effects of overexpression of the Patched1 protein, a negative regulator of Hedgehog signalling. Our results support the idea that most u-type genes are required for Hedgehog signalling and indicate that while such signalling is essential for slow myocyte differentiation, the loss of activity of one signal, Sonic hedgehog, can be partially compensated for by other Hedgehog family proteins.

Hedgehog's escape from Pandora's box.

The Hedgehog family of secreted glycoproteins proteins plays multifarious roles during vertebrate embryogenesis. In both the Drosophila and vertebrate embryo correct deployment of Hedgehog-like proteins is critical for the generation of pattern in many tissues and organs. New evidence now reveals that genes involved in hedgehog signalling are mutated in a number of common human genetic disorders, including skin cancer and craniofacial defects. The understanding of how cells generate, receive and transduce the Hedgehog signal during development has led to the establishment of molecular paradigms for the pathogenesis of these diseases. These studies clearly illustrate that knowledge of the normal role of a gene during development is critical for generating an understanding of the disease state in which it is mutated.

The generation and interpretation of positional information within the vertebrate myotome.

How somitic cells become restricted to the muscle fate has been investigated on a number of levels. Classical embryological manipulations have attempted to define the source of inductive signals that control the formation of the myotome. Recently, these studies have converged with others dissecting the role of secreted proteins in embryonic patterning to demonstrate a role for specific peptides in inducing individual cell types of the myotome. Collectively, these investigations have implicated the products of the Wnt, Hedgehog (Hh) and Bone morphogenetic protein (Bmp) gene families as key myogenic regulators; simultaneously controlling both the initiation of myogenesis and the fate of individual myoblasts.

Notochord induction of zebrafish slow muscle mediated by Sonic hedgehog.

The patterning of vertebrate somitic muscle is regulated by signals from neighboring tissues. We examined the generation of slow and fast muscle in zebrafish embryos and show that Sonic hedgehog (Shh) secreted from the notochord can induce slow muscle from medial cells of the somite. Slow muscle derives from medial adaxial myoblasts that differentiate early, whereas fast muscle arises later from a separate myoblast pool. Mutant fish lacking shh expression fail to form slow muscle but do form fast muscle. Ectopic expression of shh, either in wild-type or mutant embryos, leads to ectopic slow muscle at the expense of fast. We suggest that Shh acts to induce myoblasts committed to slow muscle differentiation from uncommitted presomitic mesoderm.

Zebrafish genetics: mutant cornucopia.

The initial characterization of mutations from the large-scale mutagenesis of the zebrafish genome has been reported. What new insights will we gain about vertebrate development from these studies?

Induction of a specific muscle cell type by a hedgehog-like protein in zebrafish.

The notochord plays a central role in vertebrate development, acting as a signalling source that patterns the neural tube and somites. In in vitro assays, the secreted protein Sonic hedgehog mimics the inducing effects of notochord on both presomitic mesoderm and neural plate explants of amniote embryos, suggesting that both patterning activities of the notochord may be mediated by this protein in vivo. In zebrafish, however, mutants with disrupted notochord development lack a specific muscle cell type, the muscle pioneers, although they retain the ability to induce neural differentiation, raising the possibility that neural tube and somite patterning may be mediated by distinct signals. Here we describe a new member of the hedgehog family, echidna hedgehog, that is expressed exclusively in the notochord and has the ability to rescue the differentiation of muscle pioneer cells in mutants with no notochord. Moreover, we show that a combination of ectopic echidna hedgehog and sonic hedgehog expression induces supernumary muscle pioneers in wild-type embryos, suggesting that both signals act sequentially to pattern the developing somites.

Structure, expression and duplication of genes which encode phosphoglyceromutase of Drosophila melanogaster.

We report here the isolation and characterization of genes from Drosophila that encode the glycolytic enzyme phosphoglyceromutase (PGLYM). Two genomic regions have been isolated that have potential to encode PGLYM. Their cytogenetic localizations have been determined by in situ hybridization to salivary gland chromosomes. One gene, Pglym78, is found at 78A/B and the other, Pglym87, at 87B4,5 of the Drosophila polytene map. Pglym78 transcription follows a developmental pattern similar to other glycolytic genes in Drosophila, i.e., substantial maternal transcript deposited during oogenesis; a decline in abundance in the first half of embryogenesis; a subsequent increase in the second half of embryogenesis which continues throughout larval life; a decline in pupae and a second increase to a plateau in adults. This transcript has been mapped by cDNA and genomic sequence comparison, RNase protection, and primer extension. Using similar analyses transcripts of Pglym87 could not be detected. Pglym78 has two introns which interrupt the coding region, while the Pglym87 gene lacks introns. This and other features support a model of retrotransposition mediated gene duplication for the origin of Pglym87. The apparent absence of a complete, intact coding frame and transcript suggest that Pglym87 is a pseudogene. However, retention of reading frame and codon bias suggests that Pglym87 may retain coding function, or may have been inactivated recently, substantially after the time of duplication, or that the molecular evolution of Pglym87 is unusual. Similarities of the unusual molecular evolution of Pglym87 and other proposed pseudogenes are discussed.

Structure and expression of the gene encoding phosphofructokinase (PFK) in Drosophila melanogaster.

The gene from Drosophila melanogaster that encodes phosphofructokinase has been isolated and its structure and expression characterized. Southern blots of genomic DNA, comparison of multiple clones, and in situ hybridization to salivary gland chromosomes indicate that the Drosophila genome contains a single copy of the gene which encodes phosphofructokinase (PFK) that is located at cytogenetic position 46D/E on the polytene map. Analysis of the gene structure reveals a single coding region that spans 6.5 kilobase pairs and is composed of eight exons and seven introns. Three developmentally regulated transcripts are generated through the use of alternative polyadenylation sites. Transcription initiation occurs at a single site. The pattern of transcript accumulation during development is similar to other genes of Drosophila which encode enzymes of the glycolytic pathway. Deficiency analysis of the 46D/E region has identified a lethal complementation group associated with reduced PFK activity, thereby establishing the vital role of PFK function in Drosophila.

Intragenic dominant suppressors of glp-1, a gene essential for cell-signaling in Caenorhabditis elegans, support a role for cdc10/SWI6/ankyrin motifs in GLP-1 function.

The glp-1 gene product mediates cell-cell interactions required for cell fate specification during development in Caenorhabditis elegans. To identify genes that interact with glp-1, we screened for dominant suppressors of two temperature-sensitive glp-1 alleles and recovered 18 mutations that suppress both germline and embryonic glp-1 phenotypes. These dominant suppressors are tightly linked to glp-1 and do not bypass the requirement for a distal tip cell, which is thought to be the source of a signal that is received and transduced by the GLP-1 protein. Using single-strand conformation polymorphism (SSCP) analysis and DNA sequencing, we found that at least 17 suppressors are second-site intragenic revertants. The suppressors, like the original glp-1(ts) mutations, are all located in the cdc10/SWI6/ankyrin domain of GLP-1. cdc10/SWI6/ankyrin motifs have been shown to mediate specific protein-protein interactions in other polypeptides. We propose that the glp-1(ts) mutations disrupt contact between GLP-1 and an as yet unidentified target protein(s) and that the dominant suppressor mutations restore appropriate protein-protein interactions.