We are interested in the general question of how individual neurons in a complex nervous system are differentiated from one another. A number of cell surface molecules have been described in various species which share the interesting property of having many possible forms made from a single gene, indicating that such diversity from a single gene could be a general mechanism allowing different neurons to be different from one another. We are focusing on the Drosophila DSCAM gene, which encodes a cell adhesion molecule that is essential for axon guidance. Remarkably, 38,016 possible alternative splice variants allow extraordinary diversity in the immunoglobulin-like domains of the DSCAM protein. A key question that we have begun to address and propose to address in detail in this grant application is how this remarkable potential for diversity is used by different neuron types, and indeed by different individual neurons of a given type. We have developed a sensitive single cell RT-PCR approach, which takes advantage of a custom made microarray that we created. Our preliminary studies lead us to estimate that each neuron expresses 50 or more distinct mRNAs chosen from a spectrum of thousands of splice variants distinctive of its cell type. This can allow every cell's DSCAM repertoire to be different from those of its neighbors. These preliminary results are exciting because they show that we can analyze small cell populations and individual neurons, and because they already have given us insights into the way that DSCAM diversity differentiates single neurons of a given type. Following on these results, we will: -Critically evaluate and extend our preliminary analyses of Drosophila neurons -analyze DSCAM splicing in specific populations of Drosophila neurons including single cell analyses -study the role of splicing factors in vitro using RNAi and study an interesting learning mutant -explore alternative splicing at the single cell level in mammalian neurons by studying neurexins.
