• BioID2
• cell migration
• centrosome
• focal adhesions
• protein–protein interactions
• septin

• Paxillin/metabolism
• Humans
• LIM Domain Proteins/metabolism
• Focal Adhesions/metabolism
• Cell Line, Tumor
• Centrosome/metabolism
• Vinculin/metabolism
• Intracellular Signaling Peptides and Proteins

• R35 GM131709 NIGMS NIH HHS
• S10 OD023617 NIH HHS
• S10 1S10OD023617-01A1 NIH HHS
• R35 GM131709 NIH HHS
• R35 GM131709 NIH HHS
• S10 1S10OD023617-01A1 NIH HHS

• Extracellular matrix
• Fibronectin
• Integrin
• Organogenesis
• Zebrafish

• Animals
• Zebrafish/genetics
• Zebrafish/metabolism
• Fibronectins/genetics
• Fibronectins/metabolism
• Alleles
• Integrins/genetics
• Embryonic Structures
• Portal System/embryology

• R35 GM148348 NIGMS NIH HHS

• elastic modulus
• finite element method
• force sensor
• optical elastography
• somite
• zebrafish embryo

• Animals
• Zebrafish
• Elasticity Imaging Techniques/methods
• Microscopy
• Embryonic Development
• Elastic Modulus

• ECM
• EMT
• Hox
• Integrin
• Neural crest

• Animals
• Neural Crest/metabolism
• DNA-Binding Proteins
• Neural Tube
• Epithelial-Mesenchymal Transition/genetics
• Extracellular Matrix/genetics
• Cell Movement/genetics

• R01 DK124804 NIDDK NIH HHS

• Collagen
• Collagen hybridizing peptide
• EMMA assay
• Extracellular matrix
• Gelatinase a
• MMP-2
• Morphogenesis
• Post-translational regulation
• Proteolytic activation
• Tissue remodelling

• Cell mobility
• Matrix stiffness
• Mechanosensing
• Metastatic breast cancer
• YAP

Focal adhesion kinase (FAK) mediates vital cellular pathways during development. Despite its necessity, how FAK regulates and integrates with other signals during early embryogenesis remains poorly understood. We found that the loss of Fak1a impaired epiboly, convergent extension and hypoblast cell migration in zebrafish embryos. We also observed a clear disturbance in cortical actin at the blastoderm margin and distribution of yolk syncytial nuclei. In addition, we investigated a possible link between Fak1a and a well-known gastrulation regulator, Wnt5b, and revealed that the overexpression of fak1a or wnt5b could cross-rescue convergence defects induced by a wnt5b or fak1a antisense morpholino (MO), respectively. Wnt5b and Fak1a were shown to converge in regulating Rac1 and Cdc42, which could synergistically rescue wnt5b and fak1a morphant phenotypes. Furthermore, we generated several alleles of fak1a mutants using CRISPR/Cas9, but those mutants only revealed mild gastrulation defects. However, injection of a subthreshold level of the wnt5b MO induced severe gastrulation defects in fak1a mutants, which suggested that the upregulated expression of wnt5b might complement the loss of Fak1a. Collectively, we demonstrated that a functional interaction between Wnt and FAK signalling mediates gastrulation cell movements via the possible regulation of Rac1 and Cdc42 and subsequent actin dynamics.

• FAK
• Wnt
• actin
• cell migration
• convergent extension
• gastrulation

Secondary dystroglycanopathies are muscular dystrophies that result from mutations in genes that participate in Dystroglycan glycosylation. Glycosylation of Dystroglycan is essential for muscle fibers to adhere to the muscle extracellular matrix (myomatrix). Although the myomatrix is disrupted in a number of secondary dystroglycanopathies, it is unknown whether improving the myomatrix is beneficial for these conditions. We previously determined that either NAD+ supplementation or overexpression of Paxillin are sufficient to improve muscle structure and the myomatrix in a zebrafish model of primary dystroglycanopathy. Here, we investigate how these modulations affect neuromuscular phenotypes in zebrafish fukutin-related protein (fkrp) morphants modeling FKRP-associated secondary dystroglycanopathy.

We found that NAD+ supplementation prior to muscle development improved muscle structure, myotendinous junction structure, and muscle function in fkrp morphants. However, Paxillin overexpression did not improve any of these parameters in fkrp morphants. As movement also requires neuromuscular junction formation, we examined early neuromuscular junction development in fkrp morphants. The length of neuromuscular junctions was disrupted in fkrp morphants. NAD+ supplementation prior to neuromuscular junction development improved length. We investigated NMJ formation in dystroglycan (dag1) morphants and found that although NMJ morphology is disrupted in dag1 morphants, NAD+ is not sufficient to improve NMJ morphology in dag1 morphants. Ubiquitous overexpression of Fkrp rescued the fkrp morphant phenotype but muscle-specific overexpression only improved myotendinous junction structure.

These data indicate that Fkrp plays an early and essential role in muscle, myotendinous junction, and neuromuscular junction development. These data also indicate that, at least in the zebrafish model, FKRP-associated dystroglycanopathy does not exactly phenocopy DG-deficiency. Paxillin overexpression improves muscle structure in dag1 morphants but not fkrp morphants. In contrast, NAD+ supplementation improves NMJ morphology in fkrp morphants but not dag1 morphants. Finally, these data show that muscle-specific expression of Fkrp is insufficient to rescue muscle development and homeostasis.

The online version of this article (10.1186/s13395-019-0206-1) contains supplementary material, which is available to authorized users.

• Dystroglycanopathy
• FKRP
• NAD+
• Neuromuscular junction
• Zebrafish

• Cadherin
• Cell adhesion
• Embryogenesis
• Extracellular matrix
• Integrin
• Mechanotransduction
• Morphogenesis

• Intramuscular connective tissue
• Muscle hyperplasia
• Myogenesis
• Myoseptal cells
• Somite
• Teleosts

• CRM1
• FAK
• Mechanotransduction
• Nanog
• Oct4
• cytoplasmic translocation
• hESCs
• stem cells

• R01 DE016530 NIDCR NIH HHS
• R01 EB019436 NIBIB NIH HHS
• U19 AI116482 NIAID NIH HHS

• cxcr4a
• miR-206
• rtn4a
• somite boundary formation
• thbs3a
• zebrafish

• EMMA assay
• Gelatinase A
• Matrix metalloproteinase
• Post-translational regulation
• Proteolytic activation
• Zebrafish
• Zymogen

Cranial neural crest cells are a multipotent cell population that generate all the elements of the pharyngeal cartilage with differentiation into chondrocytes tightly regulated by temporal intracellular and extracellular cues. Here, we demonstrate a novel role for miR-27, a highly enriched microRNA in the pharyngeal arches, as a positive regulator of chondrogenesis. Knock down of miR-27 led to nearly complete loss of pharyngeal cartilage by attenuating proliferation and blocking differentiation of pre-chondrogenic cells. Focal adhesion kinase (FAK) is a key regulator in integrin-mediated extracellular matrix (ECM) adhesion and has been proposed to function as a negative regulator of chondrogenesis. We show that FAK is downregulated in the pharyngeal arches during chondrogenesis and is a direct target of miR-27. Suppressing the accumulation of FAK in miR-27 morphants partially rescued the severe pharyngeal cartilage defects observed upon knock down of miR-27. These data support a crucial role for miR-27 in promoting chondrogenic differentiation in the pharyngeal arches through regulation of FAK.

• Chondrogenesis
• Focal adhesion kinase
• Pharyngeal arches
• Zebrafish
• miR-27

• Cell adhesion
• Extracellular matrix
• Genome editing
• Myotendinous junction
• Somite

Paxillin family proteins regulate intracellular signaling downstream of extracellular matrix adhesion. Tissue expression patterns and cellular functions of Paxillin proteins during embryo development remain poorly understood. Additionally, the evolution of this gene family has not been thoroughly investigated.

This report characterizes the evolution and expression of a novel Paxillin gene, called Paxillin-b, in Teleosts. Alignments indicate that Teleost Paxillin-a and Paxillin-b proteins are highly homologous to each other and to human Paxillin. Phylogenetic and synteny analyses suggest that these genes originated from the duplication of an ancestral Paxillin gene that was in a common ancestor of Teleosts and Tetrapods. Analysis of the spatiotemporal expression profiles of Paxillin-a and Paxillin-b using zebrafish revealed both overlapping and distinct domains for Paxillin-a and Paxillin-b during embryo development. Localization of zebrafish Paxillin orthologs expressed in mammalian cells demonstrated that both proteins localize to focal adhesions, similar to mammalian Paxillin. This suggests these proteins regulate adhesion-dependent processes in their endogenous tissues.

Paxillin-a and Paxillin-b were generated by duplication in Teleosts. These genes likely play similar roles as Paxillin genes in other organisms. This work provides a framework for functional investigation of Paxillin family members during development using the zebrafish as an in vivo model system.

• Animals
• Evolution, Molecular
• Fish Proteins/genetics
• Fish Proteins/metabolism
• Fishes/embryology
• Fishes/genetics
• Fishes/metabolism
• Focal Adhesions/metabolism
• Gene Duplication
• Gene Expression Regulation, Developmental
• Humans
• Paxillin/genetics
• Paxillin/metabolism
• Phylogeny
• Synteny
• Zebrafish/embryology
• Zebrafish/genetics
• Zebrafish Proteins/genetics

• R01 GM047607 NIGMS NIH HHS
• R01 HL095690 NHLBI NIH HHS
• National Institutes of Health

• barrier function
• endothelium
• kinases
• lung
• permeability

• Cardiac fusion
• Extracellular matrix
• Muscle morphogenesis
• Zebrafish

• EphA4
• boundary formation
• cadherin 2
• cytoskeletal contractility
• differential adhesion
• ephrinB2a
• fibronectin
• integrin α5
• morphogenesis
• segmentation
• somite
• zebrafish

The tumour suppressor Merlin, encoded by the gene NF2, is frequently mutated in the autosomal dominant disorder neurofibromatosis type II, characterised primarily by the development of schwannoma and other glial cell tumours. However, NF2 is expressed in virtually all analysed human and rodent organs, and its deletion in mice causes early embryonic lethality. Additionally, NF2 encodes for two major isoforms of Merlin of unknown functionality. Specifically, the tumour suppressor potential of isoform 2 remains controversial. In this study, we used Nf2 isoform-specific knockout mouse models to analyse the function of each isoform during development and organ homeostasis. We found that both isoforms carry full tumour suppressor functionality and can completely compensate the loss of the other isoform during development and in most adult organs. Surprisingly, we discovered that spermatogenesis is strictly dependent on the presence of both isoforms. While the testis primarily expresses isoform 1, we noticed an enrichment of isoform 2 in spermatogonial stem cells. Deletion of either isoform was found to cause decreased sperm quality as observed by maturation defects and head/midpiece abnormalities. These defects led to impaired sperm functionality as assessed by decreased sperm capacitation. Thus, we describe spermatogenesis as a new Nf2-dependent process. Additionally, we provide for the first time in vivo evidence for equal tumour suppressor potentials of Merlin isoform 1 and isoform 2.

• Development
• EMT
• Fibrosis
• Metastasis
• TM4SF5

• Cell adhesion
• Cellular adaptation
• Extracellular matrix
• Morphogenesis
• Muscular dystrophy
• Skeletal muscle

• Cadherin
• Cell migration
• Focal adhesion kinase
• Gastrulation
• Integrin
• Keratin

• Animals
• Biomechanical Phenomena
• Blotting, Western
• Cadherins/metabolism
• Cell Movement/physiology
• Cell Polarity/physiology
• Focal Adhesion Kinase 1/genetics
• Focal Adhesion Kinase 1/metabolism
• Gastrulation/physiology
• Gene Knockdown Techniques
• Germ Layers/embryology
• Immunoprecipitation
• Keratins/metabolism
• Microscopy, Fluorescence
• Morpholinos/genetics
• Statistics, Nonparametric
• Xenopus/embryology

• R01-HD26402 NICHD NIH HHS
• R01 GM094793 NIGMS NIH HHS
• R21-HD071136 NICHD NIH HHS
• GM094793 NIGMS NIH HHS
• R01 HD026402 NICHD NIH HHS
• T32 GM008136 NIGMS NIH HHS
• R21 HD071136 NICHD NIH HHS
• T32-GM08136 NIGMS NIH HHS

Tendons are extracellular matrix (ECM)-rich structures that mediate muscle attachments with the skeleton, but surprisingly little is known about molecular mechanisms of attachment. Individual myofibers and tenocytes in Drosophila interact through integrin (Itg) ligands such as Thrombospondin (Tsp), while vertebrate muscles attach to complex ECM fibrils embedded with tenocytes. We show for the first time that a vertebrate thrombospondin, Tsp4b, is essential for muscle attachment and ECM assembly at myotendinous junctions (MTJs). Tsp4b depletion in zebrafish causes muscle detachment upon contraction due to defects in laminin localization and reduced Itg signaling at MTJs. Mutation of its oligomerization domain renders Tsp4b unable to rescue these defects, demonstrating that pentamerization is required for ECM assembly. Furthermore, injected human TSP4 localizes to zebrafish MTJs and rescues muscle detachment and ECM assembly in Tsp4b-deficient embryos. Thus Tsp4 functions as an ECM scaffold at MTJs, with potential therapeutic uses in tendon strengthening and repair.

DOI:http://dx.doi.org/10.7554/eLife.02372.001

Tendons, the tough connective tissues that link muscles to bones, are essential for lifting, running and other movements in animals. A matrix of proteins, called the extracellular matrix, connects the cells in a tendon, giving it the strength it needs to prevent muscles from detaching from bones during strenuous activities.

To achieve this strength, extracellular matrix proteins bind to one another and to receptors on the muscle cell surface that are linked to its internal scaffolding, thereby organizing other proteins into a structure called a myotendinous junction. However, despite the essential roles of tendons, scientists do not fully understand how this organization occurs, or how it can go awry.

Subramanian and Schilling screened zebrafish for genes that are essential for proper muscle attachment, and zeroed in on a gene encoding a protein called Thrombospondin-4b (Tsp4b). A similar protein helps to connect muscle and tendon cells in fruit flies. Without Tsp4b, zebrafish are able to form connections between muscles and tendons, but the muscles detach easily during movement. This weakened connection is caused by disorganization of the proteins in the extracellular matrix, which results in reduced signaling from the muscle cell receptors.

When a human form of this protein was injected into zebrafish embryos lacking Tsp4b, it settled into the junctions between muscle and tendon cells. The human protein repaired the detached muscles and restored the proper organization of the matrix. This improved the strength of the muscle-tendon attachment in the treated fish embryos, suggesting that similar injections could also help to strengthen and repair muscles and tendons in people.

DOI:http://dx.doi.org/10.7554/eLife.02372.002

• ECM organization
• cell biology
• developmental biology
• muscle attachment
• myotendinous junctions
• stem cells
• tendon development
• zebrafish

Striated muscle has a highly ordered structure in which specialized domains of the cell membrane involved in force transmission (costameres) and excitation-contraction coupling (T tubules) as well as the internal membranes of the sarcoplasmic reticulum are organized over specific regions of the sarcomere. Optimal muscle function is dependent on this high level of organization but how it established and maintained is not well understood. Due to its ex utero development and transparency, the zebrafish embryo is an excellent vertebrate model for the study of dynamic relationships both within and between cells during development. Transgenic models have allowed the delineation of cellular migration and complex morphogenic rearrangements during the differentiation of skeletal myocytes and the assembly and organization of new myofibrils. Molecular targeting of genes and transcripts has allowed the identification of key requirements for myofibril assembly and organization. With the recent advances in gene editing approaches, the zebrafish will become an increasingly important model for the study of human myopathies and muscular dystrophies. Its high fecundity and small size make it well suited to high-throughput screenings to identify novel pharmacologic and molecular therapies for the treatment of a broad range of neuromuscular conditions. In this review, we examine the lessons learned from the zebrafish model regarding the complex interactions between the sarcomere and the sarcolemma that pattern the developing myocyte and discuss the potential for zebrafish as a model system to examine the pathophysiology of, and identify new treatments for, human myopathies and muscular dystrophies.

• T tubules
• myofibrils
• sarcolemma
• sarcoplasmic reticulum
• skeletal muscle
• zebrafish

• Animals
• Animals, Genetically Modified
• Embryo, Nonmammalian/embryology
• Embryo, Nonmammalian/metabolism
• Fibronectins/genetics
• Fibronectins/metabolism
• Focal Adhesion Kinase 1/genetics
• Focal Adhesion Kinase 1/metabolism
• Gene Expression Regulation, Developmental
• Gene Knockdown Techniques
• Immunohistochemistry
• In Situ Hybridization
• Integrin alpha5/genetics
• Integrin alpha5/metabolism
• Lens, Crystalline/embryology
• Lens, Crystalline/metabolism
• Lens, Crystalline/ultrastructure
• Luminescent Proteins/genetics
• Luminescent Proteins/metabolism
• Microscopy, Confocal
• Microscopy, Electron
• Models, Genetic
• Morphogenesis/genetics
• Mutation
• Zebrafish/embryology
• Zebrafish/genetics
• Zebrafish/metabolism
• Zebrafish Proteins/genetics
• Zebrafish Proteins/metabolism

• R01 EY018005 NEI NIH HHS
• EY18005-04S1 NEI NIH HHS
• R01 EY016060 NEI NIH HHS
• R01-EY016060 NEI NIH HHS
• R01-EY18005 NEI NIH HHS

NAD+ improves muscle tissue structure and function in dystrophic zebrafish by increasing basement membrane organization.

Muscular dystrophies are common, currently incurable diseases. A subset of dystrophies result from genetic disruptions in complexes that attach muscle fibers to their surrounding extracellular matrix microenvironment. Cell-matrix adhesions are exquisite sensors of physiological conditions and mediate responses that allow cells to adapt to changing conditions. Thus, one approach towards finding targets for future therapeutic applications is to identify cell adhesion pathways that mediate these dynamic, adaptive responses in vivo. We find that nicotinamide riboside kinase 2b-mediated NAD+ biosynthesis, which functions as a small molecule agonist of muscle fiber-extracellular matrix adhesion, corrects dystrophic phenotypes in zebrafish lacking either a primary component of the dystrophin-glycoprotein complex or integrin alpha7. Exogenous NAD+ or a vitamin precursor to NAD+ reduces muscle fiber degeneration and results in significantly faster escape responses in dystrophic embryos. Overexpression of paxillin, a cell adhesion protein downstream of NAD+ in this novel cell adhesion pathway, reduces muscle degeneration in zebrafish with intact integrin receptors but does not improve motility. Activation of this pathway significantly increases organization of laminin, a major component of the extracellular matrix basement membrane. Our results indicate that the primary protective effects of NAD+ result from changes to the basement membrane, as a wild-type basement membrane is sufficient to increase resilience of dystrophic muscle fibers to damage. The surprising result that NAD+ supplementation ameliorates dystrophy in dystrophin-glycoprotein complex– or integrin alpha7–deficient zebrafish suggests the existence of an additional laminin receptor complex that anchors muscle fibers to the basement membrane. We find that integrin alpha6 participates in this pathway, but either integrin alpha7 or the dystrophin-glycoprotein complex is required in conjunction with integrin alpha6 to reduce muscle degeneration. Taken together, these results define a novel cell adhesion pathway that may have future therapeutic relevance for a broad spectrum of muscular dystrophies.

A variety of diseases, both inherited and acquired, affect muscle tissues in humans. Critical to muscle homeostasis is the anchoring of muscle fibers to their surrounding microenvironment through cell adhesion complexes that help to resist the repeated stress experienced during muscle contraction. Genetic mutations in these complexes weaken this mechanical attachment, making fibers more susceptible to damage and death. The resulting increased fiber degeneration can eventually lead to progressive muscle-wasting diseases, known collectively as muscular dystrophies. Although clinical trials are ongoing, there is presently no way to cure the loss of muscle structure and function associated with these diseases. We identified a novel cell adhesion pathway involving integrin alpha6 that promotes adhesion of muscle cells to their microenvironment. Here, we show that activation of this pathway not only significantly reduces muscle degeneration but also improves the swimming ability of dystrophic zebrafish. We explore the likely benefits and limitations of this pathway in treating symptoms of congenital muscular dystrophies. Our findings suggest that activation of this pathway (for example, by boosting levels of NAD+) has the potential to ameliorate loss of muscle structure and function in multiple muscular dystrophies.