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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

C.Y. Cheung,1 S.E. Webb,2 D.R. Love3 and A.L. Miller,2 1Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, CUHK, Hong Kong, China, 2Section of Biochemistry and Cell Biology, Division of Life Science, HKUST, Hong Kong, China, and 3School of Biological Sciences, University of Auckland, Auckland, New Zealand. (Introduced by Grigori Rychkov)

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.