High Calcium Zones at the Poles of Developing Medaka Eggs.

We have injected medaka fish zygotes with recombinant aequorin and visualized the resulting patterns of luminescence to reveal patterns of free calcium during early development. We have co-injected fluorescein-labeled aequorin to correct for nonuniformities in aequorin (as opposed to calcium) distributions by visualizing the resulting patterns of fluorescence as opposed to luminescence. We have also coinjected a calcium buffer to facilitate calcium diffusion, dissipate apparent calcium gradients, and thus confirm their reality. An exploratory study shows zones of elevated free calcium at the vegetal as well as the animal pole during the first day of development and thus up to the beginning of gastrulation. A closer study during the first 6 h, and thus through ooplasmic segregation and early cleavage, shows a steady zone of high calcium at the vegetal pole and a slowly oscillating one at the animal pole. The latter is particularly strong during ooplasmic segregation and cytokinesis. This report contains the first unambiguous evidence of relatively steady zones of high cytosolic calcium during the development of an animal egg.