Browsing by Author "Kinnunen, Oona"

The insulin/insulin-like growth factor signaling pathway plays an important role in the regulation of growth and development, metabolism, lifespan, reproduction and stress response of an organism. As the pathway is evolutionarily conserved, Drosophila melanogaster serves as a powerful model for characterizing the underlying molecular mechanisms and genetic components, which may further appear as potential therapeutic targets for metabolic disorders. Drosophila has eight genes encoding insulin-like peptides (DILPs), which display stage- and tissue-specific expression patterns. Furthermore, the expression of DILPs is regulated by nutritional status. To date, only few transcription factors have been shown to regulate the expression of the dilp genes: for instance Dachshund and Eyeless are known transcriptional activators of dilp5 and dFOXO is a known transcriptional activator of dilp6. Since transcription factors directly regulating the expression of dilps are not yet well known, there is a need to study them further. The aim of this master’s thesis was to screen for novel transcriptional regulators of the dilp genes and study their phenotypic effects in vivo. For this purpose Drosophila S2 cells were transfected with vectors containing the promoter regions of all the dilp genes and with a library of 822 transcription factors. Promoter activity of the dilp genes was further measured by using the Dual-Luciferase® Reporter Assay System. Some of the revealed potential regulators were further studied in vivo to confirm their regulatory functions by using RNA interference to silence these factors or overexpressing them and by performing quantitative real-time PCR to measure dilp expression levels. Subsequently, organismal growth and development were assessed and effects on larval metabolic profiles were monitored. The screen revealed a few potential transcriptional regulators, among which Spalt-related (Salr) was selected for further study. It was shown to act as an activator of dilp6 expression. dilp6 is an interesting target among the dilp network due to its central role as a communicator between two of the important organs involved in insulin signaling: the brain and the fat body. Subsequently, both Salr and DILP6 were shown to have a role in controlling growth and development of larvae. Based on the experiments in this thesis Spalt-related seems to have a broader role as a metabolic regulator, which is not limited to its function as a regulator of dilp6. Thus, it might act as an important link between the insulin signaling pathway and other pathways regulating growth and metabolism in Drosophila.