Proceedings of The Physiological Society
Durham University (2010) Proc Physiol Soc 21, PC25
Contraction-driven skeletal muscle differentiation in the embryo
R. Ashworth1, U. Karunarathna1, M. Lahne2, C. Krivcevska1
1. School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom. 2. Galvin Life Science Building, University of Notre Dame, Notre Dame, IN 46556, Indiana, United States.
Contraction drives not only movement but skeletal muscle differentiation within the embryo. Establishing muscle structure during development is critical for later function; however, the steps that connect contraction to differentiation remain unclear. Using the zebrafish (Danio rerio), we revealed that myofibril organization within the developing skeletal muscle is dependent on the excitation-contraction (E-C) pathway, namely actin-myosin interaction [1, 2]. Our current work is aimed at identifying the signaling components that control muscle differentiation. In mature muscle, adhesion complexes form an important route for force transmission between the extracellular matrix and the cytoskeleton. Focal adhesion kinase (FAK) is a component of the adhesion complex and its activity regulates mechanotransduction. In the zebrafish embryo FAK phosphorylation coincides with somite formation and may play a role in skeletal muscle morphogenesis . The action of FAK in embryonic skeletal muscle was assessed by treating embryos with inhibitors and examining the developmental consequences. Inhibitor 14 (Tocris) decreases FAK phosphorylation at tyrosine 397 (Tyr397), an autophosphorylation site that is critical for downstream signaling. Wild type zebrafish embryos were exposed to Inhibitor 14 (50 µM - 1mM) for 6 hrs starting at 18 hpf, a time that coincides with the initiation of embryonic movement. Drug treatment at 500µM and 1mM resulted in significant abnormalities and death, whereas development at the lower concentrations appeared normal. Inhibitor 14 treated (50-250 µM) and control embryos were fixed at 24 hpf and the myofibril structure and somite boundary formation analysed by immunocytochemistry using primary antibodies that recognise myosin and β-dystroglycan respectively. No differences in myofibril organisation or somite boundary formation between control and drug treated embryos were observed. Experiments were repeated with Okadaic acid (OA, Tocris), a phosphatase inhibitor reported to cause a loss of focal adhesions and dephosphorylation of FAK on Tyr397. OA drug uptake was facilitated by performing tail-cuts on the embryos and treatment (0.5µM and 1µM) appeared to cause a decrease in embryonic movements (tail-flipping and response to touch). However, there were no obvious defects in myofibril structure and somite boundary formation, as assessed by immunocytochemistry. In summary, application of FAK inhibitors to early embryos does not appear to disrupt skeletal muscle development, further work to determine the extent of FAK phosphorylation in the inhibitor-treated embryo will be necessary to confirm these initial observations. The mechanosensitive components of the contraction-driven skeletal muscle differentiation pathway in vivo remain to be established.
Where applicable, experiments conform with Society ethical requirements