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Dynamic changes in cytosolic and nuclear Ca2+ concentration are reported to play a critical regulatory role in different aspects of skeletal muscle development and differentiation. Here we review our current knowledge of the spatial dynamics of Ca2+ signals generated during muscle development in mouse, rat, and Xenopus myocytes in culture, in the exposed myotome of dissected Xenopus embryos, and in intact normally developing zebrafish. It is becoming clear that subcellular domains, either membrane-bound or otherwise, may have their own Ca2+ signaling signatures. Thus, to understand the roles played by myogenic Ca2+ signaling, we must consider: (1) the triggers and targets within these signaling domains; (2) interdomain signaling, and (3) how these Ca2+ signals integrate with other signaling networks involved in myogenesis. Imaging techniques that are currently available to provide direct visualization of these Ca2+ signals are also described.The recognition of Ca2+ as a key regulator of muscle contraction dates back to Sydney Ringer''s seminal observations in the latter part of the 19th Century (Ringer 1883; Ringer 1886; Ringer and Buxton 1887; see reviews by Martonosi 2000; Szent-Györgyi 2004). More recently, evidence is steadily accumulating to support the proposition that Ca2+ also plays a necessary and essential role in regulating embryonic muscle development and differentiation (Flucher and Andrews 1993; Ferrari et al. 1996; Lorenzon et al. 1997; Ferrari and Spitzer 1998, 1999; Wu et al. 2000; Powell et al. 2001; Jaimovich and Carrasco 2002; Li et al. 2004; Brennan et al. 2005; Harris et al. 2005; Campbell et al. 2006; Terry et al. 2006; Fujita et al. 2007; and see reviews by Berchtold et al. 2000; Ferrari et al. 2006; Al-Shanti and Stewart 2009). What is currently lacking, however, is extensive direct visualization of the spatial dynamics of the Ca2+ signals generated by developing and differentiating muscle cells. This is especially so concerning in situ studies. The object of this article, therefore, is to review and report the current state of our understanding concerning the spatial nature of Ca2+ signaling during embryonic muscle development, especially from an in vivo perspective, and to suggest possible directions for future research. The focus of our article is embryonic skeletal muscle development because of this being an area of significant current interest. Several of the basic observations reported, however, may also be common to cardiac muscle development and in some cases to smooth muscle development. What the recent development of reliable imaging techniques has most certainly done, is to add an extra dimension of complexity to understanding the roles played by Ca2+ signaling in skeletal muscle development. For example, it is clear that membrane-bound subcellular compartments, such as the nucleus (Jaimovich and Carrasco 2002), may have endogenous Ca2+ signaling activities, as do specific cytoplasmic domains, such as the subsarcolemmal space (Campbell et al. 2006). How these Ca2+ signals interact with specific down-stream targets within their particular domain, and how they might serve to communicate information among domains, will most certainly be one of the future challenges in elucidating the Ca2+-mediated regulation of muscle development.Any methodology used to study the properties of biological molecules and how they interact during development should ideally provide spatial information, because researchers increasingly need to integrate data about the interactions that underlie a biological process (such as differentiation) with information regarding the precise location within cells or an embryo where these interactions take place. Current Ca2+ imaging techniques are beginning to provide us with this spatial information, and are thus opening up exciting new avenues of investigation in our quest to understand the signaling pathways that regulate muscle development (Animal Intact animals/Cells in culture Ca2+ reporter Reporter Loading Protocol Reference Rat 1° cultures prepared from hind limb muscle of neonatal rat pups Fluo 3-AM Cells incubated in 5.4 µM reporter for 30 min at 25°C. Jaimovich et al. 2000 Mouse Myotubes grown from C2C12 subclone of the C2 mouse muscle cell line Fluo 3-AM Incubated in 5 µM reporter plus 0.1% pluronic F-127 for 1 h at r.t. Flucher and Andrews 1993 Myotubes isolated from the intercostal muscles of E18 wild-type and RyR type 3-null mice. Fluo 3-AM Cells incubated with 4 µM for 30 min at r.t. Conklin et al. 1999b Myotubes in culture prepared from newborn mice. Fluo 3-AM Cells incubated in 10 µM for 20 min. Shirokova et al. 1999 1° cultures prepared from hind limb muscle from newborn mice. Fluo 3-AM Cells incubated in 5.4 µM reporter for 30 min at 25°C. Powell et al. 2001 Embryonic day 18 (E18) isolated diaphragm muscle fibers Fluo 4-AM Incubated in 10 µM reporter for 30 min. Chun et al. 2003 Chick Myotubes prepared from leg or breast of 11-day chick embryos Fluo 3-AM Incubated in 5 µM reporter plus 0.1% pluronic F-127 for 1 h at r.t. Flucher and Andrews 1993 Myoblasts isolated from thigh muscle of E12 embryos. Fluo 3-AM 1 mM stock was diluted 1:200 with 0.2% pluronic F-127. Cells were incubated for 60 min at r.t. in the dark. Tabata et al. 2006 Xenopus Exposed myotome in dissected embryo Fluo-3 AM Incubated dissected tissue in 10 µM reporter for 30–60 min. Ferrari and Spitzer 1999 1° myocyte cultures prepared from stage 15 Xenopus embryos. Fluo-4 AM Cells incubated in 2 µM reporter plus 0.01% pluronic F-127 for 60 min. Campbell et al. 2006 Zebrafish Intact animals Calcium green-1 dextran (10S) Reporter at 20 mM was injected into a single blastomere between the 32- and 128-cell stage. Zimprich et al. 1998 Intact animals Oregon Green 488 BAPTA dextran Single blastomeres from 32-cell stage embryos injected with reporter (i.c. 100 µM) and tetramethylrhodamine dextran (i.c. 40 µM). Ashworth et al. 2001 Intact animals Oregon Green 488 BAPTA dextran Microinjected with rhodamine dextran to give an intracellular concentration of ∼40 µM. Ashworth 2004 Intact animals Aequorin aEmbryos injected with 700 pg aeq-mRNA at the 1-cell stage and then incubated with 50 µM f-coelenterazine from the 64-cell stage. Cheung et al. 2006 Intact animals Aequorin Transgenic fish that express apoaequorin in the skeletal muscles were incubated with 50 µM f-coelenterazine from the 8-cell stage. Cheung et al. 2010