Signaling pathways controlling the formation and timing of steroid hormone pulses in Drosophila

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We use Drosophila melanogaster as a paradigm to study signaling pathways that control the production and timing of steroid hormone pulses. Steroid hormones are ancient signaling molecules that play fundamental roles in development and disease. During Drosophila larval development, the steroid hormone ecdysone is produced in the prothoracic gland in form of a series of pulses that ultimately control all developmental transitions such as the molts and metamorphosis. The neuropeptide PTTH has long been known to stimulate ecdysone production, but no direct targets of this signaling have been described. We have recently demonstrated that DHR4, a nuclear receptor, counteracts PTTH signaling to repress ecdysone production. PTTH inactivates DHR4 by triggering its removal from the nucleus to the cytoplasm, but when the signaling pathway is inactive, DHR4 remains in the nucleus. The periodic nature of PTTH signaling results in nucleocytoplasmic oscillations of DHR4 that correlate with the times when ecdysone pulses occur. However, our data suggest that PTTH is not the only contributing factor to the DHR4 oscillations, and our findings indicate that the circadian rhythm effector gene nocturnin has a role in this process. We find also direct roles for other circadian rhythm genes in the regulation of ecdysone synthesis, suggesting that the circadian machinery intersects with DHR4/PTTH controlled pathways to regulate the timing and production of steroid hormone pulses. In this proposal, we propose to examine the DHR4- and circadian controlled gene network by identifying target genes of these two pathways (aims 1 and 3). This will be achieved by a combination of tissue-specific microarrays, recombineering and genome-wide binding studies using ChIP-Seq. We also propose to analyze the mechanism by which the PTTH pathway and NOCTURNIN regulate the DHR4 oscillations (aim 2). In particular, we would like to know whether the NOCTURNIN protein facilitates nuclear entry of DHR4, and whether DHR4 is directly phosphorylated upon PTTH signaling.