Int. J. Dev. Biol. 55: 153 - 174 (2011)

https://doi.org/10.1387/ijdb.103160cc

Vol 55, Issue 2

Visualization, characterization and modulation of calcium signaling during the development of slow muscle cells in intact zebrafish embryos

Original Article | Published: 23 February 2011

Chris Y. Cheung¹, Sarah E. Webb¹, Donald R. Love² and Andrew L. Miller*^,1,3

¹Division of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, PRC, ²School of Biological Sciences, University of Auckland, Auckland, New Zealand and ³MBL, Woods Hole, MA, USA

Abstract

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 a-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 h 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. 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.

Keywords

Ca²⁺ imaging, IP₃R, RyR, slow muscle cell, transgenic zebrafish