This proposal examines the cellular and molecular mechanisms that govern synaptic development. In Drosophila a simple system of motoneurons and muscle fibers develops during embryogenesis. Connectivity is precise, with each motoneuron matched to one or more individually identified muscle fibers. Because of the small number of pre and postsynaptic cells in each hemisegment, it is possible to observe the choices made by motoneuron growth cones with single cell resolution, and to challenge the neurons with altered targets. In this proposal we will study the problem of synaptic connectivity from two standpoints. First, we will examine the nature of cellular recognition in Drosophila embryos by observing growth cone behavior in response to muscle fiber manipulation. Growth cones will be forced to choose targets that have been altered by means of either focal laser ablation and/or genetic methods. These tests include denervation, target deprivation, and techniques that either duplicate or halve the number of muscle fiber targets. Using these methods we will be able to judge how individual growth cones select their targets, and to determine how they refine their contacts into differentiated synapses. The second approach is to examine the nature of molecular recognition, by altering the expression of membrane and cell surface proteins which are expressed by developing synaptic partners in the embryo. The analysis will focus on several cell adhesion molecules, including the fasciclins, and a collection of Drosophila enhancer detector lines which label subsets of muscle fibers and/or motoneurons. The study will include both genetic approaches, and a technique for molecular ablation, chromophore assisted laser inactivation. The latter method is used to inactivate cell surface proteins at specific times and locations by means of focal nanosecond excitation of tagged molecules. These tests are performed with single cell resolution, and will be combined with vital imaging using laser confocal microscopy. By analyzing how growth cones distinguish between cellular surfaces during development, we will expand our understanding of how nervous systems establish appropriate axonal trajectories and synaptic contacts.
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