Collagen VI genes in zebrafish skeletal muscle: Implications for collagen VI-myopathies

Collagen VI (COLVI) is a heterotrimeric protein, ubiquitously expressed in connective tissues. COLVI plays different roles such as maintenance of structural integrity, cellular migration and survival. To date, six COLVI chains are recognized with two short (α1–2(VI)) and three long chains (α3–6(VI)). Little is known regarding the possible assembly of the newly characterized α4–6(VI) polypeptides with the short chains, and a putative compensation between the different chains. In humans, deficiency in α1–3(VI) due to mutations in the COL6A1–3 genes causes COLVI-myopathies a heterogeneous group of neuromuscular disorders. To study the development of muscular tissue as well as the early steps of the disease, we turned to the zebrafish model where we identified only two orthologs of the α4–6(VI) chains: col6a4a and col6a4b. We showed that the genes encoding the short chains are expressed at higher levels, and that only col6a4b was differentially expressed during development. We created COLVI deficient zebrafish embryos using morpholinos that block splicing of col6a2, col6a4a and col6a4b, thereby creating premature termination codons. By qPCR, we demonstrated that the targeted transcripts were degraded, likely by the nonsense mediated RNA decay. At the morphological level, all morphant embryos had a curved body and showed severely impaired motility. Birefringence analysis and whole mount immunohistochemistry showed alteration of the muscle structure, with disorganized fibers and U-shaped myosepta. These alterations were confirmed at the ultrastructural level by electron microscopy. In conclusion, col6a2 deficient embryos recapitulate the severe end of the COLVI-myopathy phenotypical spectrum, thereby confirming the importance of col6 genes in muscle development. Furthermore, the phenotypes associated with α4a and α4b deficiency may help orientate group(s) of patients to screen for mutations in the human genes.