This proposal examines the cellular and molecular mechanisms that govern the establishment and plasticity of synapses in a model genetic system, the Drosophila neuromuscular junction. These developmental problems will be studied using manipulations at the cellular and molecular level, applied with single cell precision during embryonic and larval development. There are 2 specific goals of the proposal. First, we will perform a series of targeted transgene expression experiments that will test hypotheses about the role of electrical activity in regulating both pre-and postsynaptic properties, as well as test the interplay of activity with retrograde signaling involving BMP growth factors. The second aim will address the role of activity in the refinement of synaptic connections, examining remodeling of neuromuscular contacts through an activity-dependent retrograde repulsion by Semaphorin IIa from muscle, and through an activity-dependent organization of IgCAMs on the muscle fiber surface. To perform these experiments, we have developed several molecular tools that allow us to test hypotheses about the role of electrical activity during neuromuscular development and functional plasticity. These include constructs used to either suppress or enhance membrane excitability, targeted as specific times in development to either side of the synapse. In addition, we have developed an inducible bipartite gene expression system to perform experiments testing both the spatial and temporal requirements for induced genes. This approach has been enhanced with our recent isolation of several hundred inducible drivers, to target specific motoneurons, interneurons, or muscles. These tools give us considerable power to test hypotheses about the role of excitability in synapse elimination during embryonic development, as well as to test the interplay of activity and retrograde signaling during synaptic modification. The proposal is structured as a series of well-defined hypotheses to be tested, that will help resolve the molecular and cellular mechanisms that govern synaptogenesis in a model genetic system.