Application of complementary luminescent and fluorescent imaging techniques to visualize nuclear and cytoplasmic Ca²⁺ signalling during the in vivo differentiation of slow muscle cells in zebrafish embryos under normal and dystrophic conditions.

1. Evidence is accumulating for a role for Ca²⁺ 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 Ca²⁺-sensitive complex aequorin, specifically in skeletal muscle, show that two distinct periods of spontaneous synchronised Ca²⁺ transients occur in the trunk: one at approximately 17.5-19.5 h post-fertilization (h.p.f.; termed signalling period SP1) and the other after approximately 23 h.p.f. (termed SP2). These periods of intense Ca²⁺ signalling activity are separated by a quiet period. 3. Higher-resolution confocal imaging of embryos loaded with the fluorescent Ca²⁺ reporter calcium green-1 dextran shows that the Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ signalling signatures of dmd-MO-injected embryos or homozygous sapje mutant embryos were abnormal such that the frequency, amplitude and timing of the Ca²⁺ 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 [Ca²⁺](i) during the early stages of slow muscle cell differentiation when the Ca²⁺ signals generated in these cells coincide with the first spontaneous contractions of the trunk.