PhDCalcium ions (Ca2+) are fundamental to the regulation of many cellular processes; however, the coordination of these signals during embryogenesis is not well understood. Ryanodine receptors (RyR) are a family of important intracellular ion channels that are responsible for the release of Ca2+ and they regulate the cytosolic Ca2+ concentration. Humans have three differentially expressed ryr genes (ryr1, ryr2 and ryr3) and mutations can cause both skeletal and cardiac diseases. Although the primary function of RyR is to mediate excitation-contraction coupling in muscle, they may also regulate Ca2+ signalling during developmental processes.
The project has addressed the role of RyR during embryonic development, using the zebrafish as an in vivo vertebrate model. Five zebrafish RyR genes (ryr1a, ryr1b, ryr2a, ryr2b and ryr3) were characterised and a comprehensive overview of their spatial and temporal expression in the embryo was determined. At 24 hours post-fertilisation (hpf), ryr1a, ryr1b and ryr3 are expressed in the skeletal muscle, ryr2a in specific neuronal populations and ryr2b in the cardiac muscle. Semi-quantitative PCR data and wholemount in situ hybridisation revealed strong maternal expression of ryr3 during the cleavage and blastula periods and into adulthood. The early expression of the ryr3 gene suggests that this receptor functions during the initial stages of development; a role that has not been described previously. The functional significance of RyR3 during early embryogenesis was investigated in a loss-of-
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function model using antisense morpholino oligonucleotides. The ryr3 specific knockdown experiments appeared to affect the establishment of embryonic axis prior to the segmentation periods (before 10 hpf). In addition, by 19 to 20 hpf ryr3 morphants failed to exhibit spontaneous muscle contractions and displayed a defect in neuromuscular development.
In conclusion, this study has characterised the ryr genes and provided an overview on their temporal and spatial expression. The work provides evidence that ryr3 expression provides the Ca2+ vital for myofibrils organisation and that is required for the spontaneous movements during zebrafish embryonic development. The knowledge of RyR tissue distribution in zebrafish has provided a strong foundation for loss-of-function studies aimed at addressing their role in development. In the long term, the work will also facilitate more focused studies on disease.School of Biological and Chemical Sciences Queen Mary University of London.
Central Research Fund and Physiological Society Travel Grant
