Through the injection of f-aequorin (a calcium-specific luminescent reporter), and the use of a photon-imaging microscope, I have visualized transient localized elevations of free cytosolic calcium in the forming blastodisc and in the animal hemisphere cortex that correlate with ooplasmic segregation. The introduction of an appropriate concentration of the weak (K_D = 1.5 μM) calcium buffer 5,5'-dibromo-BAPTA results in the dissipation of these calcium domains, and inhibits ooplasmic streaming and the subsequent formation of a blastodisc at the animal pole. These inhibitory actions are dependent on the final cytosolic concentration of buffer within the egg: ≥ 1.3 mM blocks ooplasmic streaming; 1.3 mM eggs segregate normally. Injection of 5,5'-dimethyl-BAPTA (K_D = 0.15 μM) to a final con...[ Read more ]

Through the injection of f-aequorin (a calcium-specific luminescent reporter), and the use of a photon-imaging microscope, I have visualized transient localized elevations of free cytosolic calcium in the forming blastodisc and in the animal hemisphere cortex that correlate with ooplasmic segregation. The introduction of an appropriate concentration of the weak (K_D = 1.5 μM) calcium buffer 5,5'-dibromo-BAPTA results in the dissipation of these calcium domains, and inhibits ooplasmic streaming and the subsequent formation of a blastodisc at the animal pole. These inhibitory actions are dependent on the final cytosolic concentration of buffer within the egg: ≥ 1.3 mM blocks ooplasmic streaming; < 1.3 mM eggs segregate normally. Injection of 5,5'-dimethyl-BAPTA (K_D = 0.15 μM) to a final concentration of 1.5 mM as a control has no effect on ooplasmic streaming. These results suggest that localized domains of elevated free cytosolic calcium are essential for ooplasmic segregation in zebrafish.

I also present direct evidence, via staining with rhodamine-phalloidin indicating that a sub-surface contraction band is generated by an actin-based network located in the animal hemisphere cortex, which I suggest is modulated via the localized calcium elevation. Furthermore, zygotes incubated in the microfilament-disrupting agent cytochalasin B (20 μg/ml) do not generate a contraction band nor segregate to form a blastodisc. By observing the movements of both natural and introduced ooplasmic inclusions, I calculated bulk-streaming velocities of 30 - 40 μm/min toward the animal pole in the axial streamers, and 3 - 4 μm/min toward the vegetal pole in the peripheral ooplasm. I demonstrate that the peripheral counter-flow feeds into the base of the streamers, and as a result ooplasm does not accumulate at the vegetal pole. Furthermore, treatment with cytochalasin B clearly disrupts movement in both directions.

From these results, a hypothetical model is presented that links the calcium transients observed to the contraction of a cortically located actin microfilament network in the animal hemisphere, as a possible mechanism for modulating and driving bipolar ooplasmic segregation in zebrafish zygotes.