Genetic, genomic and molecular studies of signaling pathways controlling ecdysone biosynthesis in Drosophila melanogaster

Abstract

In insects, periodic pulses of the molting hormone ecdysone control all major developmental transitions such as the molts and metamorphosis. Ecdysone, a steroid hormone, is produced in the prothoracic gland during larval and pupal development of Drosophila. While we have a relatively good understanding of the enzymatic steps involved in ecdysone synthesis, the signaling pathways controlling this process remain poorly characterized. It has long been known that the synthesis and release of ecdysone is under the control of a brain neuropeptide called PTTH, which stimulates ecdysone synthesis by activating the Ras/Raf/MAPK cascade through its receptor Torso in prothoracic gland cells. However, direct downstream targets of this pathway remained elusive. My work demonstrated that Drosophila Hormone Receptor 4 (DHR4) represents a key target of the PTTH signaling cascade and plays a key role in appropriately timing ecdysone pulses by oscillating between nucleus and cytoplasm of prothoracic gland cells. I also showed that DHR4 negatively regulates the expression of Cyp6t3, a previously uncharacterized enzyme required for ecdysone synthesis. Therefore, I proposed that nuclear DHR4 inhibits ecdysone synthesis through repression of Cyp6t3 and possibly other target genes, and that this repressive function can be overturned by the removal of DHR4 from nuclei of prothoracic gland cells via activating the PTTH pathway. A parallel project was aimed at identifying novel components required for ecdysone production in the prothoracic gland. For this, I performed whole-genome microarray analysis of hand-dissected ring glands (which harbor the prothoracic gland) and compared the signal to microarrays of whole larvae samples, which allowed me to identify genes with specific expression in the ring gland. The function of the highest-scoring genes (~100) were then analyzed via prothoracic gland-specific RNA interference, which yielded 25 genes that have likely novel roles in ecdysone synthesis. Among these, I studied a gene with homology to human neurotrophins in more detail. Vertebrate neurotrophins are signaling molecules that carry out neuroprotective functions during brain development. Here, I describe a novel developmental role for a Drosophila neurotrophin in controlling ecdysone synthesis in the prothoracic gland. This finding represents a new layer of steroid hormone regulation, and broadens our current perspective of the mechanisms by which insect metamorphosis is regulated.