High Calcium Zones at the Poles of Developing Medaka Eggs

Abstract

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.

Organization of early development by calcium patterns

“…High calcium zones also appear at the animal pole but not until about half an hour after fertilization. These oscillate three to four times during the first six hours before disappearing and probably represent the slow calcium waves that accompany the first three to four cell divisions (31) (Fig. 3B).…”

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

“…In order to verify that the random nature of the SCSs was not caused by an uneven distribution of aequorin within the formed somites and notochord, zebrafish embryos were loaded at the single‐cell stage with a custom‐made FITC‐tagged AquaLite recombinant aequorin. When tagged with FITC, this aequorin loses its ability to generate light on combination with Ca 2+ , but it can be reasonably assumed that its distribution will reflect that of f ‐aequorin (Fluck et al. 1992).…”

Microinjection of cyclic ADP-ribose triggers a regenerative wave of Ca2+ release and exocytosis of cortical alveoli in medaka eggs

“…Methods for removing gonads have been previously described (Yamamoto, 1967;Kirchen & West, 1976;Fluck, 1978;Abraham et al, 1993). Gonads and gamates were placed in the same BSS used in other studies of the fertilised medaka egg (Fluck et al, 1991(Fluck et al, , 1992Abraham et al, 1993): 111 mM NaCl, 5.37 mM KCl, 1.0 mM CaCl 2 , 0.6 mM MgSO 4 , 5 mM HEPES pH 7.3. To prepare unfertilised eggs for microinjection, we transferred them through five successive washes of Ca 2+ /Mg 2+ -free BSS over a period of 1 h. The experiments were done at room temperature (19.0-25.4 °C).…”

“…Batches of three to six eggs were transferred to closely fitting holes drilled in a transparent plastic holder, and a high-pressure method (Narashige IM-200 microinjection system) was used to inject fluid into the thin, peripheral layer of ooplasm of the egg (Ridgway et al, 1977;Gilkey, 1983;Fluck et al, 1991Fluck et al, , 1992Fluck et al, , 1994. Approximately 1.4-1.9 nl of fluid was injected into the vegetal hemisphere of the egg, approximately 60°arc from the vegetal pole.…”