Analysis of the daf-2 insulin/ igf-1 receptor gene in Caenorhabditis elegans.

The daf-2 gene is a key regulator of growth, metabolism and longevity in the nematode Caenorhabditis elegans. The DAF-2 receptor functions in a pathway that is analogous to mammalian insulin/ insulin-like growth factor signalling and determines whether animals proceed with full reproductive development or arrest in a long-lived diapausal state known as the dauer larva. Temperature-sensitive hypomorphic mutants of daf-2 constitutively arrest as dauers when raised at non-permissive temperatures. At permissive temperatures the animals develop into adults that are long-lived compared to wild-type adults. These alleles of daf-2 can be separated into two distinct classes (1 and 2) based on their pleiotropic phenotypes. In addition to their phenotypic differences, the two classes also differ in their epistatic interactions with other genes involved in dauer formation, suggesting that the DAF-2 receptor has multiple signalling outputs. In this thesis I have investigated the nature of the daf-2 allele class difference using a range of methods, including sequence analysis and homology modelling of mutant receptors. This generated the prediction that signalling flux through the receptor is a determinant of the class difference, with class 2 alleles having an asymmetrical alteration in signal transduction through DAF-2. Experimental testing of these predictions suggest that some phenotypes such Eat and Unc may be associated with asymmetrical signalling, while others such as early larval arrest may be correlated with a reduction in receptor level at the plasma membrane. A comparative analysis of the DAF-2 receptor with its homologues from Caenorhabditis briggsae, Caenorhabditis remanei and the parasitic nematode Brugia malayi suggests the large C-terminal extension in the Caenorhabditis species, which shorter in Brugia and not present in vertebrates, is an adaptive trait that has evolved by exon duplication for rapid growth and development and may contribute to the shortevity of these species. In addition, I have also performed an analysis of ins-7 and ins-35, two putative ligands of DAF-2 that are differentially regulated by the transcription factor DAF-16, using RNAi. Knockdown of both these genes lead to slight lifespan extension (10-20%) at 20 °C in all genetic backgrounds and slight suppression (-10%) at 25 °C in long-lived daf-2 mutant backgrounds compared to controls. This suggests that INS peptides may function as agonists a 20 °C and antagonists at 25 °C, and that this behaviour may be independent of their transcriptional regulation.