On the mechanism of ooplasmic segregation in single-cell zebrafish embryos

It has been previously shown that localized elevations of free cytosolic calcium are associated with a morphological contraction in the forming blastodisc and animal hemisphere cortex during ooplasmic segregation in zebrafish zygotes. It was subsequently proposed, in a hypothetical model, that these calcium transients might be linked to the contraction of a cortically located actin microfilament network as a potential driving force for segregation. Here, by labeling single-cell embryos during the major phase of segregation with rhodamine-phalloidin, direct evidence is presented to indicate that the surface contraction was generated by an actin-based cortical network. Furthermore, while zygotes incubated with colchicine underwent normal ooplasmic segregation, those incubated with cytochalasin B did not generate a constriction band or segregate to form a blastodisc. During segregation at the single-cell stage, ooplasm simultaneously moved in two directions: toward the blastodisc within the so-called axial streamers, and toward the vegetal pole in the peripheral ooplasm. The velocities of both axial and peripheral streaming movements are reported. By injection of a fluorescein isothiocyanate (FITC)-labeled 2000 kDa dextran into the peripheral ooplasm it was demonstrated that a portion of it feeds into the bases of the extending streamers, which helps to explain the lack of accumulation of ooplasm at the vegetal pole. These new data were incorporated into the original model to link the bipolar ooplasmic movements with the calcium-modulated, actin-mediated contraction of the animal hemisphere cortex as a means of establishing and driving ooplasmic segregation in zebrafish.