A Splice Site Mutation in Laminin-α2 Results in a Severe Muscular Dystrophy and Growth Abnormalities in Zebrafish

Abstract

Congenital muscular dystrophy (CMD) is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A) is a form of CMD caused by a defect in the laminin-α2 gene (LAMA2). Laminin-α2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC) complex in membranes providing stability to muscle fibers. In an N-ethyl-N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2cl501/cl501, exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 8–15 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-α2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-α2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD.

Hanging on for the ride: Adhesion to the extracellular matrix mediates cellular responses in skeletal muscle morphogenesis and disease

“…Zebrafish mutants or morphants deficient for laminin alpha2, thrombospondin 4b or collagen 22a1 proteins have mild muscle defects; however, when muscle contraction is stimulated (either mechanically or electrically), the muscle phenotype becomes severe with many muscle fibers retracting from the MTJ (Charvet et al, 2013; Hall et al, 2007; Subramanian and Schilling, 2014). Unlike the case in mouse and zebrafish models with dystrophin mutations, the muscle fibers didn’t rupture, they were found to retract from MTJ intact (Charvet et al, 2013; Gupta et al, 2012; Hall et al, 2007; Subramanian and Schilling, 2014). Therefore, rather than stabilizing the sarcolemma, these ECM proteins play a role in muscle health by maintaining integrity of muscle cell adhesion to the ECM at the MTJ.…”

Influence of ovarian muscle contraction and oocyte growth on egg chamber elongation in Drosophila

“…Interestingly, some dystrophic muscles show hyper-contractility during early stages of their degeneration (Cullen and Fulthorpe, 1975;Gupta et al, 2012;Haines et al, 2007). We hypothesized, therefore, that the eggs with an increased aspect ratio might have developed within a hyper-contractile muscle sheath.…”

Satellite cell activation and populations on single muscle-fiber cultures from adult zebrafish (Danio rerio)

“…Experiments tested the hypotheses that SC activation on zebrafish muscle fibers is dependent on NO and HGF, that HGF responses are temperature-dependent and that stretch activation acts via NO release. Although zebrafish are a model organism in studying Duchenne and other muscular dystrophies (Lieschke and Currie, 2007;Berger et al, 2010;Guyon et al, 2007;Gupta et al, 2012;Guyon et al, 2003), this is the first physiological study on isolated zebrafish fibers to examine SC activation in vitro. …”