A Global Analysis of Synthetic Genetic Interactions & a Genetic Analysis of Muscle Arm Development in Caenorhabditis elegans

Understanding gene function and genetic relationships is elemental in our efforts to better understand biological systems. Here, I describe a reliable high-throughput approach, Systematic Genetic Interaction analysis (SGI), capable of revealing both weak and strong genetic interactions in the nematode Caenorhabditis elegans. I also present evidence that UNC-73 functions cell-autonomously in an UNC-40 pathway to direct muscle arm extension in C. elegans. Previous efforts to systematically describe genetic interactions between redundant genes on a global scale either have focused on core biological processes in protozoans or have surveyed catastrophic interactions in metazoans. I investigated synthetic genetic interactions between eleven ‘query’ mutants in conserved signal transduction pathways and hundreds of ‘target’ genes compromised by RNAi. A network of 1246 genetic interactions was uncovered through an unbiased global analysis of the interaction matrix, establishing the largest metazoan genetic interaction network to date. To investigate how genetic interactions connect genes on a systems-wide level, the SGI network was superimposed with existing networks of physical, genetic, phenotypic and co-expression interactions. Fifty-six putative functional modules were identified within the superimposed network, one of which regulates fat accumulation and is coordinated by bar-1(ga80)/β-catenin interactions. This led to the discovery that SGI interactions link distinct functional modules on a global scale, which is a previously unappreciated level of organization within metazoan systems. In addition, I present evidence that the properties of genetic networks are conserved between C. elegans and S. cerevisiae, but that the connectivity of the interactions within the current networks is not. Although the buffering between functional modules may differ between species, studying these differences may provide insight into the evolution of divergent form and function.In C. elegans the postsynaptic membrane of the neuromuscular junction reaches its destination through an active process of guided cell extension. The worm has 95 body wall muscles (BWMs) that extend projections called 'muscle arms' to motor axons. The muscle arms harbour the postsynaptic elements of neuromuscular junctions. The stereotypical pattern of muscle arm extension was exploited in a forward genetic screen for new genes required for guided cell migration by looking for mutations that caused a reduction in the number of arms that extend to the motor axons. One of the resulting mutants was tr117, which extended half the number of arms compared to wild type animals. Genetic mapping, complementation tests, and sequencing revealed that tr117 was a mutation in unc-73/Trio, which encodes a guanine nucleotide exchange factor. Expression of UNC-73 specifically in the BWMs rescued the muscle arm development defects of unc-73(e936) mutants, indicating that UNC-73 functions cell-autonomously to regulate muscle arm extension. UNC-73::CFP was localized to muscle arm termini in a pattern similar to that of UNC-40/Dcc, which directs muscle arm extension. UNC-73 over-expression suppressed the Madd phenotype of unc-40 null worms and unc-73(e936) suppressed ectopic myopodia induced by UNC-40 over-expression. These results indicate that UNC-73 functions downstream of UNC-40 in a pathway that regulates muscle arm extension.