Application of complementary luminescent and fluorescent imaging techniques to visualize nuclear and cytoplasmic Ca 2+ signalling during the in vivo differentiation of slow muscle cells in zebrafish embryos under normal and dystrophic conditions

摘要

1. Evidence is accumulating for a role for Ca 2+ signalling in the differentiation and development of embryonic skeletal muscle. 2. Imaging of intact, normally developing transgenic zebrafish that express the protein component of the Ca 2+-sensitive complex aequorin, specifically in skeletal muscle, show that two distinct periods of spontaneous synchronised Ca 2+ transients occur in the trunk: one at approximately 17.5-19.5h post-fertilization (h.p.f.; termed signalling period SP1) and the other after approximately 23h.p.f. (termed SP2). These periods of intense Ca 2+ signalling activity are separated by a quiet period. 3. Higher-resolution confocal imaging of embryos loaded with the fluorescent Ca 2+ reporter calcium green-1 dextran shows that the Ca 2+ signals are generated almost exclusively in the slow muscle cells, the first muscle cells to differentiate, with distinct nuclear and cytoplasmic components. 4. Here, we show that coincidental with the SP1 Ca 2+ signals, dystrophin becomes localized to the vertical myoseptae of the myotome. Introduction of a dmd morpholino (dmd-MO) resulted in no dystrophin being expressed in the vertical myoseptae, as well as a disruption of myotome morphology and sarcomere organization. In addition, the Ca 2+ signalling signatures of dmd-MO-injected embryos or homozygous sapje mutant embryos were abnormal such that the frequency, amplitude and timing of the Ca 2+ signals were altered compared with controls. 5. Our new data suggest that, in addition to a structural role, dystrophin may function in the regulation of [Ca 2+] i during the early stages of slow muscle cell differentiation when the Ca 2+ signals generated in these cells coincide with the first spontaneous contractions of the trunk.