Le rôle de la matrice extracellulaire dans la régénération des nerfs moteurs

International audienceThe motor neurons (MN) form the ultimate route to convey the commands from the central nervous system to muscles. During development, MN extend axons that follow stereotyped trajectories to their muscle targets, guided by various attractive and repulsive molecular cues. Extracellular matrix (ECM) is a major source of guidance cues, but its role in axonal development and regeneration remains poorly documented. Regenerating axons are able to return to their synaptic target following their original trajectory. The same guidance cues could be thus involved in motor nerve regeneration. Zebrafish has become a popular model system in understanding the development of the peripheral nervous system. Thanks to the generation of fluorescent transgenic lines and the optical transparency of embryos and larvae, it allows direct visualization of axonogenesis. Additionally, and contrary to humans, its remarkable capacity to regenerate makes it well suited for the study of nerve regeneration. A laser method to ablate nerves in living zebrafish larvae has been developed in our laboratory that, combined with the use of the fluorescent mnx1:gfp zebrafish transgenic line, allows the follow up of the dynamics of the nerve regeneration process. To study the role of ECM proteins present in the axonal path, mutant lines for different ECM proteins (already available in our laboratory or generated in mnx1:gfp fish using CRISPR-Cas9 method) will be used to analyze their role during the regeneration process. These mutant lines for ECM will be crossed with existing fluorescent transgenic lines to visualize different cell types involved in the nerve regeneration, such as macrophages ( mfap4:mcherry ), neutrophils ( mpx:gfp ) or even Schwann cells ( sox10:mrfp ). Overall, this study will depict the role of ECM in nerve regeneration and will provide essential knowledge for the development of new biomaterials to promote the regeneration of injured motor nerves

Fishing for collagen function: About development, regeneration and disease

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Ion channels properties and intracellular Ca2+ handling of skeletal muscle fibers in a zebrafish model of Bethlem

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Étude physiopathologique de la myopathie de Bethlem à l’aide d’un modèle de poisson zèbre

Bethlem myopathy (BM) is a neuromuscular disease characterized by joint contractures and muscle weakness. BM is caused by mutations in one of the genes encoding one of the three alpha-chains of collagen VI (COLVI), a component of the skeletal muscle extracellular matrix. Nowadays, an unresolved question is to understand how alteration of COLVI located outside the muscle cells leads to functional modifications in muscle fibers. The zebrafish model col6a1(Delta ex14) is currently the unique animal model of the disease since it is the only model to reproduce a mutation that is the most frequently found in BM patients. In patient and col6a1(Delta ex14) zebrafish muscles, the structure of the sarcoplasmic reticulum has been found to be altered, thus suggesting dysfunction in intracellular Ca2+ handling and/or in ion channels that are known to control Ca2+ homeostasis and to play pivotal roles in muscle function and pathogenesis. Therefore, our project aims at exploring the properties of ion channels and intracellular Ca2+ regulation using electrophysiological approaches and intracellular Ca2+ measurement at rest and during activity in isolated muscle fibers from col6a1(Delta ex14) zebrafish. On one hand, this project should contribute to decipher how alteration in an extracellular matrix component transduces pathogenic signals within muscle fiber and should possibly lead to identify therapeutic targets for this currently incurable disease. On the other hand, because functional studies on zebrafish muscle cells are scarce, this project will provide a sound database on the electrophysiological properties of this cell model

Superfast excitation–contraction coupling in adult zebrafish skeletal muscle fibers

International audienceThe zebrafish has emerged as a very relevant animal model for probing the pathophysiology of human skeletal muscle disorders. This vertebrate animal model displays a startle response characterized by high-frequency swimming activity powered by contraction of fast skeletal muscle fibers excited at extremely high frequencies, critical for escaping predators and capturing prey. Such intense muscle performance requires extremely fast properties of the contractile machinery but also of excitation–contraction coupling, the process by which an action potential spreading along the sarcolemma induces a change in configuration of the dihydropyridine receptors, resulting in intramembrane charge movements, which in turn triggers the release of Ca2+ from the sarcoplasmic reticulum. However, thus far, the fastest Ca2+ transients evoked by vertebrate muscle fibers has been described in muscles used to produce sounds, such as those in the toadfish swim bladder, but not in muscles used for locomotion. By performing intracellular Ca2+ measurements under voltage control in isolated fast skeletal muscle fibers from adult zebrafish and mouse, we demonstrate that fish fast muscle fibers display superfast kinetics of action potentials, intramembrane charge movements, and action potential–evoked Ca2+ transient, allowing fusion and fused sustained Ca2+ transients at frequencies of excitation much higher than in mouse fast skeletal muscle fibers and comparable to those recorded in muscles producing sounds. The present study is the first demonstration of superfast kinetics of excitation–contraction coupling in skeletal muscle allowing superfast locomotor behaviors in a vertebrate

Spatio-temporal expression and distribution of collagen VI during zebrafish development

International audienceCollagen VI (ColVI) is an extracellular matrix (ECM) protein involved in a range of physiological and pathological conditions. Zebrafish (Danio rerio) is a powerful model organism for studying vertebrate development and for in vivo analysis of tissue patterning. Here, we performed a thorough characterization of ColVI gene and protein expression in zebrafish during development and adult life. Bioinformatics analyses confirmed that zebrafish genome contains single genes encoding for α1(VI), α2(VI) and α3(VI) ColVI chains and duplicated genes encoding for α4(VI) chains. At 1 day post-fertilization (dpf) ColVI transcripts are expressed in myotomes, pectoral fin buds and developing epidermis, while from 2 dpf abundant transcript levels are present in myosepta, pectoral fins, axial vasculature, gut and craniofacial cartilage elements. Using newly generated polyclonal antibodies against zebrafish α1(VI) protein, we found that ColVI deposition in adult fish delineates distinct domains in the ECM of several organs, including cartilage, eye, skin, spleen and skeletal muscle. Altogether, these data provide the first detailed characterization of ColVI expression and ECM deposition in zebrafish, thus paving the way for further functional studies in this species

Collagen XV, a multifaceted multiplexin present across tissues and species

Abstract Type XV collagen is a non-fibrillar collagen that is associated with basement membranes and belongs to the multiplexin subset of the collagen superfamily. Collagen XV was initially studied because of its sequence homology with collagen XVIII/endostatin whose anti-angiogenic and anti-tumorigenic properties were subjects of wide interest in the past years. But during the last fifteen years, collagen XV has gained growing attention with increasing number of studies that have attributed new functions to this widely distributed collagen/proteoglycan hybrid molecule. Despite the cumulative evidence of its functional pleiotropy and its evolutionary conserved function, no review compiling the current state of the art about collagen XV is currently available. Here, we thus provide the first comprehensive view of the knowledge gathered so far on the molecular structure, tissue distribution and functions of collagen XV in development, tissue homeostasis and disease with an evolutionary perspective. We hope that our review will open new roads for promising research on collagen XV in the coming years

Generation and characterization of alpha-1 type VI collagen zebrafish mutant line

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A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish.

International audiencePresently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders

A mechano- and heat-gated two-pore domain K + channel controls excitability in adult zebrafish skeletal muscle

TRAAK channels are mechano-gated two-pore-domain K + channels. Up to now, activity of these channels has been reported in neurons but not in skeletal muscle, yet an archetype of tissue challenged by mechanical stress. Using patch clamp methods on isolated skeletal muscle fibers from adult zebrafish, we show here that single channels sharing properties of TRAAK channels, i.e., selective to K + ions, of 56 pS unitary conductance in the presence of 5 mM external K + , activated by membrane stretch, heat, arachidonic acid, and internal alkaline pH, are present in enzymatically isolated fast skeletal muscle fibers from adult zebrafish. The kcnk4b transcript encoding for TRAAK channels was cloned and found, concomitantly with activity of mechano-gated K + channels, to be absent in zebrafish fast skeletal muscles at the larval stage but arising around 1 mo of age. The transfer of the kcnk4b gene in HEK cells and in the adult mouse muscle, that do not express functional TRAAK channels, led to expression and activity of mechano-gated K + channels displaying properties comparable to native zebrafish TRAAK channels. In whole-cell voltage-clamp and current-clamp conditions, membrane stretch and heat led to activation of macroscopic K + currents and to acceleration of the repolarization phase of action potentials respectively, suggesting that heat production and membrane deformation associated with skeletal muscle activity can control muscle excitability through TRAAK channel activation. TRAAK channels may represent a teleost-specific evolutionary product contributing to improve swimming performance for escaping predators and capturing prey at a critical stage of development