Dissecting the Role of ZAK-beta in Skeletal Muscle Using Zebrafish as a Model Organism

Congenital myopathies are a group of inherited, heterogenous, rare muscle diseases, associated with progressive muscle wasting, chronic disability and a reduced quality of life. Inheritance of mutations in ZAK, a gene encoding a MAP triple kinase, has been identified as a novel cause of congenital myopathy in humans. This thesis utilises zebrafish to model the impacts of absence of ZAK on skeletal muscle, with investigations ranging from development studies to the analysis of the aging process of muscle in adult zebrafish. In contrast to other vertebrates, where the two isoforms are achieved through differential splicing of a single gene (to produce ZAK-alpha and ZAK-beta), in zebrafish the two isoforms exist as different genes on separate chromosomes, simplifying efforts to target each isoform. ZAK-beta is shown to be the isoform expressed in zebrafish skeletal muscle, and CRISPR-Cas9 gene editing was used to create mutations in each ZAK isoform and raise lines of zebrafish lacking either individual or both isoforms. Sequencing of the mRNA transcript and qRT-PCR confirmed mutations to each isoform result in a premature stop codon, and that there was a significant reduction in transcript levels. Breeding the ZAK-beta-/- line into transgenic reporter lines allowed the assessment of the structure of developing skeletal muscle, and the immune response of neutrophils following wounding in larval zebrafish, using confocal imaging. Individual muscle fibre area was significantly reduced in ZAK-beta-/- embryos at two and five days of development, compared to wild type, potentially indicating growth defects in the skeletal muscle. Aged ZAK-beta-/- zebrafish (35-months-old) show significantly decreased swimming capabilities, as well as abnormalities in the ultrastructure absent in age-matched wild type controls, suggesting a potential accelerated aging process in skeletal muscle with loss of ZAK. Thus, ZAK-beta signalling may represent a promising target for developing novel therapies for the treatment of sarcopenia.