This proposal tests the "chemoaffinity" theory with single cell resolution in vivo. The mechanisms of synaptic target recognition is a major issue in developmental neurobiology. The "chemoaffinity" hypothesis postulates that each neuronal growth cone has an ability to respond to unique molecular labels of its target cells. Many diffusible and cell surface molecules have been proposed as candidate target recognition molecules. However, in most cases it is technically difficult to test their specific roles at the level of individual cells in vivo. The embryonic Drosophila neuromuscular system provides an ideal model system, being amenable to high resolution experimentation. This simple system consists of uniquely identified motoneurons and muscle cells. Connectivity is precise to the level of individual cells. Powerful genetics, allowing precise in vivo manipulations of a molecule under focus, and accessibility to high resolution cell biological assays facilitate direct tests of the candidate molecules at the level of single neurons and single genes. Already more than a dozen candidate target recognition molecules are identified in this system, based on their unique expression patterns and molecular characterization. They include diverse cell surface molecules such as Fasciclin III (Ig-CAM) and Toll (leucine-rich repeat containing cell surface molecule). Most recently, Chiba et al. (1995) have shown that Fasciclin III can function as a specific and "attractive" target recognition molecule. The proposed research will take the advantage of the Drosophila system. Rather than screening for additional candidate target recognition molecules, the study will instead dissect into the molecular nature of synaptic target selection by using both Fasciclin llI and Toll as a molecular model. The identified motoneuron growth cones will be challenged with their microenvironment being variously manipulated for either Fasciclin III or Toll expression (Specific Aims l & 2). This is achieved by combined use of misexpression through genomic transformation and loss- of-function mutations. The results will be assessed at the single cell level, through intracellular dye injection and immunocytochemistry, as well as functional and ultrastructural analyses of the identified synapses. In addition, live visualization of the identified growth cones in both undissected embryos and in vivo/vitro hybrid culture system will be performed. All together they provide comprehensive assessment of the molecular events during synaptic target recognition. Most of the methodology to be employed is already very familiar to the investigators. However, several new assays will be developed and/or fine-tuned to improve resolution of analysis further (Specific Aim 3). The results are anticipated to uncover how synaptic target recognition molecules work in vivo. They will begin to define aspects of these molecules that are crucial for their functions. Also, whether or not members of multiple gene families have evolved to serve functionally redundant roles will be tested. Ultimately, the results from the proposed research are hoped to help identifying synaptic target recognition molecules and understanding their molecular mechanisms in the human and other vertebrate brains.