Characterization of Ca²⁺-dependent phospholipase A₂ activity during zebrafish embryogenesis

We have developed a simple fluorescent assay for detection of phospholipase A₂ (PLA₂) activity in zebrafish embryos that utilizes a fluorescent phosphatidylcholine substrate. By using this assay in conjunction with selective PLA₂ inhibitors and Western blot analysis, we identified the principal activity in zebrafish embryogenesis as characteristic of the Ca²⁺-dependent cytosolic PLA₂ (cPLA₂) subtype. Embryonic cPLA₂ activity remained constant from the 1-cell stage until the onset of somitogenesis, at which time it increased sharply. This increase was preceded by the expression of a previously identified zebrafish cPLA₂ homologue (Nalefski, E., Sultzman, L., Martin, D., Kriz, R., Towler, P., Knopf, J., and Clark, J. (1994) J. Biol. Chem. 269, 18239-18249). By using a quenched BODIPY-labeled phosphatidylcholine that fluoresces only upon cleavage by PLA₂, lipase activity was visualized in the cells of living embryos where it localized to perinuclear membranes.