Visualization of stochastic Ca²⁺ signals in the formed somites during the early segmentation period in intact, normally developing zebrafish embryos

Abstract

Localized Ca2+ signals were consistently visualized in the formed somites of intact zebrafish embryos during the early segmentation period. Unlike the regular process of somitogenesis, these signals were stochastic in nature with respect to time and location. They did, however, occur predominantly at the medial and lateral boundaries within the formed somites. Embryos were treated with modulators of [Ca 2+ ] i to explore the signal generation mechanism and possible developmental function of the stochastic transients. Blocking elements in the phosphoinositol pathway eliminated the stochastic signals but had no obvious effect, stochastic or otherwise, on the formed somites. Such treatments did, however, result in the subsequently formed somites being longer in the mediolateral dimension. Targeted uncaging of buffer (diazo-2) or Ca 2+ (NP-ethyleneglycoltetraacetic acid [EGTA]) in the presomitic mesoderm, resulted in a regular mediolateral lengthening and shortening, respectively, of subsequently formed somites. These data suggest a requirement for IP 3 receptor-mediated Ca 2+ release during convergence cell movements in the presomitic mesoderm, which appears to have a distinct function from that of the IP 3 receptor-mediated stochastic Ca 2+ signaling in the formed somites.

Expression and reconstitution of the bioluminescent Ca2+ reporter aequorin in human embryonic stem cells, and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes

“…The Triton X-100 was used to permeabilize the cardiosphere cells and thus expose any unspent holo-f-aequorin to extracellular Ca 2+ . This confirmed that the low level of aequorin-generated luminescence (and thus the [Ca 2+ ]) observed was real and not due to the Ca 2+ reporter being used up (Leung et al, 2009).…”

“…This is consistent with the expression of these two SR-based receptors during embryogenesis in animal models, where functional IP 3 Rs have been shown to be expressed in nearly every cell in mice and zebrafish embryos from the earliest stages of development and expression continues throughout development (Rosemblit et al, 1999;Lee et al, 2003;Leung et al, 2009;Cheung et al, 2011). On the other hand, functional RyRs are not expressed during the early stages of development, but they are expressed later on with the differentiation of excitable cells, including cardiac and skeletal muscle, where the receptors are used for specialized cellular functions such as cell contraction (Rosemblit et al, 1999;Lee et al, 2003;Leung et al, 2009;Cheung et al, 2011). Although we demonstrate that IP 3 Rs are functional from day 1 onwards, and that RyRs appear to only be functional at day 8, our current data does not tell us the specific role of either receptor with respect to early differentiation of HES2 ESCs.…”

Disruption of Intracellular Calcium Regulation Is Integral to Aminoglycoside-Induced Hair Cell Death

“…Embryos were injected at the one-cell stage with either ~1nL of 25mM NP-EGTA (dissolved in 100mM KCl, 40mM Hepes, pH 7.2) or ~1nL of 25mM diazo-2 (dissolved in 100mM KCl, 10mM EGTA, 10mM MOPS, pH 7.2) as previously described (Leung et al, 2009). At 5dpf, compounds were activated via photolysis.…”

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

“…Furthermore, the sudden appearance of the SP1 transients initially in somites 4 to 10, follow a period of quiescent Ca 2+ signaling as far as the zebrafish trunk is concerned. We have previously reported that a period of relatively localized stochastic Ca 2+ signaling occurs in the formed somites and to a much lesser extent in the pre-somitic mesoderm during the early segmentation period, i.e., during the 1 to 8 somite stages (~10 to 13 hpf; Leung et al, 2009). However, both we and others have reported that prior to the sudden appearance of the pan-somitic SP1 signals at ~17.5 hpf, there is essentially a background resting level of Ca 2+ within the zebrafish trunk (i.e., from all tissues) for at least 1 hour prior to the onset of SP1 (Figs.…”

“…However, both we and others have reported that prior to the sudden appearance of the pan-somitic SP1 signals at ~17.5 hpf, there is essentially a background resting level of Ca 2+ within the zebrafish trunk (i.e., from all tissues) for at least 1 hour prior to the onset of SP1 (Figs. 3Ai, 12Bi and 12Ci; Gilland et al, 1999;Leung et al, 2009).…”