No abstract

Using complementary luminescent- and fluorescent-based Ca(2+) imaging techniques, we have re-examined the Ca(2+) dynamics that occur during the Blastula Period (BP) of zebrafish development. We confirm that aperiodic, localized Ca(2+) transients are generated predominately in the superficial epithelial cells (SECs). At the start of the BP, these Ca(2+) transients are distributed homogeneously throughout the entire superficial epithelium. Following the mid-blastula transition (MBT), however, their distribution becomes asymmetrical, where a significantly greater number are generated in the presumptive dorsal quadrant of the superficial epithelium. This asymmetry in Ca(2+) signaling lasts for around 60 min, after which the total number of transients generated from the entire superficial epithelium falls to less than one per minute until the end of the BP. We have thus called this asymmetry the "dorsal-biased Ca(2+) signaling window". The application of pharmacological agents indicates that the post-MBT SEC Ca(2+) transients are generated via the phosphatidylinositol (PI) signaling pathway. This suggests that the previously reported ventralizing function attributed to the homogeneously distributed PI pathway-generated SEC Ca(2+) transients is most likely to occur earlier in development, prior to the MBT.

The generation of a required series of localized Ca(2+) transients during cytokinesis in zebrafish embryos suggests that Ca(2+) plays a necessary role in regulating this process. Here, we report that cortical actin remodeling, characterized by the reorganization of the contractile band and the formation during furrow deepening of pericleavage F-actin enrichments (PAEs), requires a localized increase in intracellular Ca(2+), which is released from IP(3)-sensitive stores. We demonstrate that VAMP-2 vesicle fusion at the deepening furrow also requires Ca(2+) released via IP(3) receptors, as well as the presence of PAEs and the action of calpains. Finally, by expressing a dominant-negative form of the kinesin-like protein, kif23, we demonstrate that its recruitment to the furrow region is required for VAMP-2 vesicle transport; and via FRAP analysis, that kif23 localization is also Ca(2+)-dependent. Collectively, our data demonstrate that a localized increase in intracellular Ca(2+) is involved in regulating several key events during furrow deepening and subsequent apposition.