THESIS
2009
xviii, 150 p. : ill. (some col.) ; 30 cm
Abstract
Transgenic zebrafish that express apoaequorin (the protein part of bioluminescent Ca
2+ reporter, aequorin) specifically in the musculature were generated to study muscle Ca
2+ signals during late somitogenesis. Two distinct periods of spontaneous, synchronized Ca
2+ signals, with characteristic frequencies and durations, termed ‘Signaling Period 1’ (SP1) and ‘Signaling Period 2’ (SP2), were visualized in the trunk of intact embryos between ~17.5-19.5 hours post fertilization (hpf) and after ~23 hpf, respectively, separated by a quiet period. The individual Ca
2+ signals were further characterized with respect to their spatial signatures using the fluorescent Ca
2+ reporter, calcium green-1 dextran (10 kDa) in conjunction with confocal microscopy. These experiments revealed that the signals...[
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Transgenic zebrafish that express apoaequorin (the protein part of bioluminescent Ca
2+ reporter, aequorin) specifically in the musculature were generated to study muscle Ca
2+ signals during late somitogenesis. Two distinct periods of spontaneous, synchronized Ca
2+ signals, with characteristic frequencies and durations, termed ‘Signaling Period 1’ (SP1) and ‘Signaling Period 2’ (SP2), were visualized in the trunk of intact embryos between ~17.5-19.5 hours post fertilization (hpf) and after ~23 hpf, respectively, separated by a quiet period. The individual Ca
2+ signals were further characterized with respect to their spatial signatures using the fluorescent Ca
2+ reporter, calcium green-1 dextran (10 kDa) in conjunction with confocal microscopy. These experiments revealed that the signals appeared to be restricted to slow muscle cells (SMCs) and that the SP1 Ca
2+ signals had both a significant nuclear as well as cytoplasmic component, whereas the SP2 signals were predominantly cytoplasmic. These signals had, therefore, characteristic frequencies, durations and locations within developing SMCs. Aequorin-based imaging of cyclopamine- or forskolin-treated embryos, and of smu
-/- mutant embryos, in which SMCs do not form, confirmed the specific cellular location of the Ca
2+ signals. Furthermore, treating embryos with antagonists of the nicotinic acetylcholine receptor (nAChR; i.e., α-bungarotoxin), the dihydropyridine receptor (DHPR; i.e., nifedipine), the inositol 1,4,5-trisphosphate receptor (IP
3R; i.e., 2-APB) and the ryanodine receptor (RyR; i.e., ryanodine) suggested that the SP1 signals were mediated through endogenous neuronal activation of AChRs and then DHPRs to release Ca
2+ from intracellular stores via both IP
3Rs and RyRs. Immunohistochemistry showed that the differential expression and organization of IP
3Rs and RyRs in SMCs coincided with the initial generation of the SP1 Ca
2+ transients and their subsequent spatial nature. Furthermore, 2-APB and ryanodine were both shown to disrupt the organization of the SMCs, but in different ways. This suggests that different Ca
2+ signaling pathways may be responsible for different functions during development of SMCs.
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