Programme Contents

The application of complementary luminescent and fluorescent imaging techniques to visualize nuclear and cytoplasmic Ca²⁺ signaling during in vivo differentiation of slow muscle cells in zebrafish embryos

Intact zebrafish embryos were used as an in vivo animal model to investigate the role of Ca²⁺ signaling during the differentiation of slow muscle cells (SMCs) within forming skeletal muscle. Transgenic zebrafish were generated using an α-actin promoter that targeted apoaequorin expression specifically to muscle cells. Two distinct Ca²⁺ signaling periods (CSPs) were visualized in the developing SMCs: between ∼17.5-19.5 hours post-fertilization (hpf) and after ∼23 hpf, separated by a ∼3.5 hour Ca²⁺ signaling quiet period. Further spatial characterization of these Ca²⁺ signals using confocal fluorescent microscopy and calcium green-1 dextran as a reporter, indicated that the earlier CSP displayed distinct nuclear and cytoplasmic components, whereas the later CSP was predominantly cytoplasmic. Both CSPs consisted of a series of oscillating Ca²⁺ waves generated at distinct frequencies, while the earlier CSP also displayed a slow rise then fall in the Ca²⁺ baseline-level. Imaging of cyclopamine- and forskolin-treated wild-type, or smo^-/- mutant embryos, where SMCs do not form, confirmed the specific cell population generating the signals. Treating embryos with antagonists indicated that both IP₃Rs and RyRs are responsible for generating the temporal characteristics of the Ca²⁺ signaling signature, and that the latter plays a necessary role in SMC differentiation and subsequent myotome patterning (Cheung, et al., 2010). Together, these data support and extend the proposition that specific spatiotemporal patterns of spontaneous Ca²⁺ signals might be used for different as well as combinatorial regulation of both nuclear and cytosolic signal transduction cascades, resulting in myofibrillogenesis in SMCs as well as myotome patterning (Webb & Miller, 2010).

Cheung, C.Y., Webb, S.E., Love, D.R., and Miller A.L. (2010) International Journal of Developmental Biology, In Press. DOI 10.1387/ijdb.103160cc

Webb, S.E., and Miller, A.L. (2010) In: Calcium Signaling, CHS Press (Eds. Berridge, M.J., Putney, J., Roderick, L., Bootman, M.D). In Press.