Abstract
Presently, 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.
Highlights
Collagen type VI proteins are found in the extracellular matrix; they are associated with interstitial type I–III collagen fibers, in connective tissues of skin [1] and skeletal muscle [2,3,4]
In patients, skipping of exon 14 of the col6a1 gene encoding the collagen type VI alpha-1 chain has been demonstrated to lead to the onset of Bethlem myopathy (BM), a case of mild Ullrich congenital muscular dystrophy (UCMD) caused by this mutation has been reported [42]
In order to reproduce the skipping of exon 14 in a stable zebrafish line, we targeted the splice donor site of intron 14 with a transcription activator-like effector nuclease (TALEN) directed against the exon/intron junction (Figs 1 and 2A)
Summary
Collagen type VI proteins are found in the extracellular matrix; they are associated with interstitial type I–III collagen fibers, in connective tissues of skin [1] and skeletal muscle [2,3,4]. Each alpha chain contains a short triple helical collagenous domain, flanked by N- and C-terminal globular domains of variable size [6]. In the functional collagen VI, the three alpha chains combine intracellularly into triple helical monomer that assembles into antiparallel dimer, and two dimers form a tetramer. The tetramers are secreted into the extracellular matrix where they assemble into beaded microfibrils, the functional unit of collagen VI [7,8,9,10]. Interstitial fibroblasts are the main source of collagen VI which is secreted in the endomysium surrounding the muscle fibers [3]
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