Erik Thomas Danielsen:
In contrast to humans, Drosophila melanogaster, commonly known as the fruit fly, only produces one major class of cholesterol-derived steroid hormones, the ecdysteroids. This makes Drosophila a simple but elegant model organism to study steroidogenesis. During development, pulses of ecdysone produced in the endocrine prothoracic gland dictate the timing of larvae molting and metamorphosis, the process of sexual maturation. This steroid producing tissue is a dynamic organ, sensing both internal and external environmental cues in order to produce a steroid hormone pulse with the right amplitude and duration required for juvenile-adult transition.
This PhD project demonstrates the power of Drosophila genetics by taking an in vivo genome-wide RNAi screening approach to uncover genes required for the function of steroid producing tissue and developmental maturation. In total, 1909 genes were found to be required for the prothoracic gland function and affected the developmental timing for the juvenile-adult transition. Among the screen hits, we focused on an uncharacterized gene, sit (CG5278), which is highly expressed in the gland and is required for ecdysone production. Sit is a homolog of mammalian very long chain fatty acid elongases, which are involved in lipid metabolism. Silencing of sit expression in the gland cells resulted in an increased accumulation of lipid droplets where cholesterol is typically stored. Further investigation revealed that knock down of sit expression caused a dysregulated flux of cholesterol uptake in the gland cells and affected the endosomal trafficking. Therefore this gene was suggested to be named stuck in traffic (sit). Sit’s role in cholesterol uptake was also supported by the observation that the developmental delayed phenotype from loss of sit expression in the gland could be completely rescued by supplement of dietary cholesterol. In conclusion, this study shows a role for the fatty acid elongase, Sit, in uptake and delivery of cholesterol, the crucial substrate for ecdysone synthesis. The pace of ecdysone production is essentially determined by the expression of the gland-specific biosynthetic enzymes. The network of transcription factors picked up in the RNAi screen was explored and Ventral veins lacking (Vvl) and Knirps were identified to be involved in regulation of ecdysteroidogenesis. Silencing either of these two transcription factors in the prothoracic gland resulted in reduced ecdysone titer and caused a developmental arrest in the early larval stages. Investigation revealed that both Vvl and Knirps regulated mRNA expression of the enzymes of the biosynthetic pathway and crucial cis-regulatory transcription factor binding-sites were identified.
Altogether, this thesis presents the discovery of genes required for the function of steroid producing tissue and developmental timing based on an in vivo genome-wide RNAi screen in Drosophila. The screen data were used to elucidate two vital steps for producing steroid hormones, which includes regulation of cholesterol uptake and trafficking to feed steroidogenesis, and regulation of expression of the biosynthetic enzymes of the steroidogenic pathway.