IMMUNOCYTOCHEMICAL ANALYSIS OF AXONAL OUTGROWTH IN SYNAPTOTAGMIN MUTATIONS

Synaptotagmin is a synaptic vesicle specific protein that binds calciu m and phospholipids in vitro and is required for calcium-regulated fus ion of synaptic vesicles with the presynaptic membrane. We have examin ed the possible requirement for synaptotagmin in axonal outgrowth by f ollowing neuronal development in Drosophila embryos deficient for the synaptotagmin gene, We find that synaptotagmin is expressed abundantly in axons and growth cones before synapse formation in wild-type embry os. Using antibodies to the intravesicular domain of synaptotagmin to label live embryos, we demonstrate that vesicle populations containing synaptotagmin actively undergo exocytosis during axonogenesis. We hav e used immunocytochemical techniques to examine the distribution of th e axonal protein Fasciclin II, the presynaptic membrane protein syntax in, and the synaptic vesicle protein cysteine string protein, in synap totagmin null mutations. The distribution of these proteins is similar in wild-type and synaptotagmin mutant embryos, suggesting that synapt otagmin is not required for axonogenesis in the CNS or PNS. Based on t hese findings, we suggest that the molecular mechanisms underlying ves icular-mediated membrane expansion during axonal outgrowth are distinc t from those required for synaptic vesicle fusion during neurotransmit ter release.