Creating Double Mutants to Analyze the Interactions in the Insulin-like Signaling Pathway in C. elegans

All organisms live in fluctuating environments and must make appropriate physiological changes in order to survive. To achieve this plasticity, they must be able to detect environmental stimuli and respond to them effectively. At the molecular level, how organisms are able to do this is not yet completely understood. We are interested in investigating this question using the model organism Caenorhabditis elegans. As a soil dwelling nematode, the worm can face rapid changes in food, temperature and population density. To deal with these changes, C. elegans undergoes an adaptation called the dauer diapause. According to environmental conditions, larvae can decide to either continue to develop to adulthood or enter the dauer larval state, which allows the animal to survive in very stressful environments. Major morphogenesis occurs such as constricting body size and entering a non-feeding state. Dauer arrest can last for long periods until conditions improve adequately to resume development. The decision between reproductive growth and dauer diapause requires robust genetic control. One of the major regulators of dauer arrest is the Insulin/IGF-like signaling pathway. This pathway is conserved in all multicellular animals, including humans. Among other tissues, a collection of neurons in the head called the amphids express insulin-like peptides (ILPs) and release in response to specific environmental stimuli. In C. elegans, there are 40 insulin-like (ins) genes that encode ILP’s. By utilizing the ability to control environmental factors and manipulate the genetics of the animal, prior work has characterized most of these genes on their contribution on dauer entry and dauer exit. We now question how the network of ILP’s is organized and what kinds of regulatory interactions occur between them. Creating double mutants of different ins genes, known to have interesting affects on dauer formation, will allow us further insight on their relationship of one another. Classic genetic crosses were used to make homozygous double mutants and all genotypes were tested via Polymerase Chain Reaction (PCR). As a result of the crosses, 10 double ins mutants were successfully made. In 2 cases, double mutants were produced by recombination of 2 tightly linked genes. The creation of these double ins mutants are essential for dauer assays that can help us understand how ILPs work together in the insulin-like pathway.