Imaging Patterns of Ca2+ Transients During the Blastula Period in Zebrafish Embryos

It has been suggested that Ca2+ events occuring during the blastula period of zebrafish development might be involved in mediating the cell-to-cell adhesion between the enveloping layer (EVL) cells and/or might somehow play a role in dorsoventral patterning of the embryo. Using the Ca2+-sensitive luminescent reporter, aequorin, and a custom-designed photon imaging microscope (PIM), we have started to re-investigate the patterns and possible functions of the blastula period Ca2+ transients. We have demonstrated that distinct Ca2+ transients occur throughout this period in small, localized cellular domains. When these transients were characterized with respect to time, they were found to occur with the highest frequency in a 75-minute window, from the 512-cell stage to about the sphere stage. A data analysis protocol was designed such that the imaged embryos were divided into quadrants "dorsal/ventral/left/right," and the Ca2+ transients from each quadrant were then quantified with respect to time. Analysis indicates that during the period of high Ca2+ transient frequency, a greater number of Ca2+ events occur in the "dorsal" quadrant of the embryo. In addition, using the fluorescent Ca2+ reporter Oregon Green 488 dextran (10 kDa) and two-photon confocal microscopy, we showed that the Ca2+ transients appear to be confined exclusively to the EVL. This suggests that this peridermal layer may play some as yet undescribed role in the signaling cascades that determine the developmental fate of the underlying embryonic deep cells. Furthermore, we demonstrated by transmission electron microscopy that the EVL cells, but not the deep cells, possess abundant mitochondria, which are asymmetrically distributed to the inner portion of the EVL cell that adjoins the underlying deep cell population.