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  • Recently we have created CRISPR Cas mediated col a


    Recently, we have created CRISPR/Cas9-mediated col14a1a knockout fish to interrogate the function of col14a1a during regeneration using the caudal fin model. Using AFM, we showed that collagen XIV-A transiently acts as a molecular spacer responsible for BM biomechanics possibly by helping laminins integration within regenerative BM. These data indicated that 3D-organization of BM is fine-tunely regulated by minor components of the BM zone while the integral components of BM are involved in the assembly of its backbone [18]. In addition, our study unexpectedly provided new insights into the analysis of the epidermal BM topography and mechanics [18]. AFM and 3D reconstruction revealed that the epidermal BM is organized as a checkerboard structure made of alternation of soft and rigid regions. Interestingly, in absence of collagen XIV-A, the checkerboard organization is compromised leading to a more compact structure, suggesting that this collagen may act as a molecular organizer of the BM network [18].
    Zebrafish is a versatile model for collagenopathies The critical roles of collagens have been clearly illustrated by the wide spectrum of diseases that affect various tissues: osteogenesis imperfecta (OI, bone), Alport syndrome (kidney), Ehlers-Danlos syndrome (skin, joints and vessels), Bethlem myopathy and Ulrich congenital muscular dystrophy (UCMD) (skeletal muscle), epidermolysis bullosa (skin), Knoblock syndrome (retina)… [5,85]. These diseases are caused by more than 1,500 mutations identified in genes encoding more than 12 different collagen types. In common diseases, such as fibrotic disorders, tumor stroma, fibrosis, atherosclerosis inflammation, and neurodegenerative disease, the imbalance in the rates of collagen synthesis and breakdown is critical [85]. Mutations in collagen genes often led to intracellular assembly defects that induced ER stress if proteins are not properly degraded [85]. Glycine substitutions are the most common mutations found in collagens. They result in interruptions in the Gly-X-Y sequence that subsequently affect triple helix assembly. Frame knowing it skipping mutations are also a frequent cause of disease. They result in the synthesis of shorter collagenous domains that, in turn, affect proper assembly of the three α chains [86]. In the last 15 years, zebrafish has become an emerging model for studying human diseases (Table 1). Random mutagenesis has generated many mutants that can model a particular disease and even sometimes have help connecting a particular locus with a human disease [12]. In order to identify new genes involved in skeletal dysplasia, an adult fish skeletal defect screen of mutants generated by ENU mutagenesis was performed using X-ray radiography [87]. Over the 2,000 fish examined, only one mutant that was 20% shorter than wildtype fish was identified and named chihuahua. Mutation in col1a1a gene leading to a replacement of a glycine by an aspartate (G574D) in collagen I α1 chain was shown to be responsible for chihuahua phenotype. Collagen I is the most abundant protein in bones and mutations in any of the collagen I genes led to classical OI. This disorder is characterized by bone fragility and growth deficiency, as reported for the chihuahua mutant [88,89]. At the cell level, chihuahua mutant also recapitulated the human disease since ER stress caused by intracellular retention of misfolded collagen in ER [85] was observed in fin bones. Interestingly, this model opens new perspectives of investigation for OI treatment as the chihuahua phenotype could be improved in adults by treating embryos with the chemical chaperone 4PBA [88]. Zebrafish was also used to model collagen VI-related myopathies. The use of splicing MOs is a method particularly adapted to recapitulate type VI collagen disease as many exon skipping mutations in the three main collagen VI genes are present in the human genome of patients [86]. Depending on the exon deleted in the zebrafish col6a1 gene, Telfer and co-workers were able to induce either a severe or a mild muscle phenotype in agreement with the degree of severity observed in the two targeted myopathies, UCMD and Bethlem myopathy [90]. Zebrafish larvae showed the typical ultrastructural features of UCMD and Bethlem myopathy such as the abnormal structure of mitochondria and the swollen sarcoplasmic reticulum. The mitochondrial defects have been rescued after treatment with the mitochondrial permeability transition pore (mPTP) inhibitor cyclosporin A [90] as in the mouse model [91]. This drug has been successfully tested in clinical trials, but unfortunately it cannot be used for long term treatment due to its immunosuppressive properties [92]. In an attempt to find new treatment, another PTP inhibitor (NIM811) with non immunosuppressive properties was tested in zebrafish embryos and showed even stronger phenotype rescue than cyclosporin A [93]. Nevertheless, all these studies could be only performed during early development due to the transient efficiency of MOs. Using TALEN genome editing strategy, Radev and co-workers were able to create a stable line harboring a deletion in a splice donor site that led to the skipping of col6a1 exon14 that is the most frequent mutation found in Bethlem myopathy patients [48]. They also observed dilated sarcoplasmic reticulum and mitochondria defects and, most importantly, better recapitulated the Bethlem myopathy progressive disease. Such a stable line will be useful to dissect the pathophysiological mechanisms of the disease as well as to fully interrogate the function of collagen VI in muscle physiology and regeneration.