Dysregulation of the molecular processes that contribute to synaptic development, maturation, and stabilization underlie many neurological diseases like autism and schizophrenia. Formation and long-term maintenance of synapses requires the concerted effort of many proteins ? cell motility factors guide growth cones toward post- synaptic cells, signaling molecules sense when synapses are formed, and structural proteins strengthen and stabilize the developing synapse. Analyzing the genetics of these processes is difficult, though, as many of these genes are essential or redundant. Despite decades of forward genetic screening in C. elegans and other model organisms, there are still many open questions regarding the genetics of the synapse. For instance, what signal does a growth cone migrate toward? How does a newly-formed synapse signal that it needs stabilization? How do deficiencies in these processes contribute to disease etiology? Finding the genes that will help to answer these questions using current genetic screening tools is untenable. We require next- generation genetic tools that will allow us dissect and analyze these processes. I will develop MosTrap, a novel gene-trap mutagenesis method that can be used for forward genetic screening. When inserted in a gene, the MosTrap transposon inactivates the gene and expresses both a fluorescent marker to report expression patterns and a phenotypic rescue gene that can be used to select for inactivation of genes expressed in a cell type of interest. This rescue balances the gene deletion, potentially enabling the creation of a knockout allele for all genes. Using MosTrap, I will create a collection of balanced knockout alleles in neuronal genes. During the creation of this collection, I will phenotype knockout strains for defects in synaptic function, as well as characterize which genes are essential and in what cells each gene is expressed. I will then screen the mutants in this collection for knockouts in genes that are required for synaptic stability. Using longitudinal and live imaging I will characterize the synaptic stability defects in each strain. Together, these data will identify new synaptic stability genes, as well as inform the basic biology of how synapses become unstable, potentially uncovering new mechanisms that may be utilized to treat diseases like autism. The reagents and methods I develop during this work enable the creation of a genome-wide collection of balanced knockout alleles.
To fully understand a gene?s function, one must first understand its knockout phenotype and expression pattern. This proposal develops next-generation genetic screening tools to enable the creation of a genome- wide balanced knockout collection in C. elegans, and uses this collection to identify and characterize genes required for synaptic function and stability.
