Developmental regulation of zebrafish annexins and cytosolic phospholipase A2

Potent cellular signals arc1 derived from membrane phospholipids through the action of phospholipase ,A₂ (PLA2). yet little is known about the func tion of this enzyme during development, or of proteins such as Annexins thai modulate its activity. To examine lipid mediated signaling during embryogenesis, we have studied expression of cPLA2 and 4 annexin (am) genes cloned from a zebrafish cDNA library. Zebrafish cPLA2 is expressed in presumptive neural crest cells during early somitogenesis. Zebrafish anxl is expressed in the outermost ectodermal layer at gastrulation, and later in the blood island, developing tail, and pectoral fins. Like am/, anxll is expressed in the outer ectoderm and the blood island, however it is also expressed in notochord. The arixKgene is specifically expressed in olfactory placodes, while anxXf has the most widespread expression (notochord, blood island, somites and the pectoral fins). Some anx genes are maternally expressed, and all genes become highly expressed by late somite stages. Taking advantage of the optical clarity of the zebrafish embryo, we are attempting to develop a visual assay for PLA2 activity in living embryos. In preliminary studies using single embryo extracts, PLA-2 activity was determined by TLC using a fluorescent phospholipid substrate. PLA2 activity was detected at all developmental stages and was largely inhibited (80 - 93% at 40 uM) by the arachidonic acid analog AACOOF. To elucidate their function during development, we have initiated mutagcnesis screens in the zebrafish for mutations that aller PLA₂ activity or delete PLA₂ or annexin genes.