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

我们使用果蝇作为范例来研究控制类固醇激素脉冲的产生和时间的信号通路。类固醇激素是古老的信号分子，在发育和疾病中起着重要作用。在果蝇幼虫发育期间，类固醇激素蜕皮激素以一系列脉冲的形式在前胸腺中产生，最终控制所有发育过渡，如蜕皮和变态。神经肽PTTH长期以来一直被认为可以刺激蜕皮激素的产生，但没有描述过这种信号传导的直接靶点。我们最近证明，DHR 4，核受体，抵消PTTH信号抑制蜕皮激素的生产。PTTH通过触发其从细胞核到细胞质的移除来使DHR 4失活，但是当信号传导通路失活时，DHR 4保留在细胞核中。PTTH信号的周期性性质导致与蜕皮激素脉冲发生的时间相关的DHR 4的核质振荡。然而，我们的数据表明，PTTH是不是唯一的因素，DHR 4振荡，我们的研究结果表明，昼夜节律效应基因turnin在这一过程中有一定的作用。我们还发现了其他昼夜节律基因在调节蜕皮激素合成中的直接作用，这表明昼夜节律机制与DHR 4/PTTH控制的通路交叉，以调节类固醇激素脉冲的时间和产生。在这个提议中，我们建议通过识别这两个途径的靶基因来检查DHR 4和昼夜节律控制的基因网络（目的1和3）。这将通过使用ChIP-Seq的组织特异性微阵列、重组工程和全基因组结合研究的组合来实现。我们还建议分析PTTH途径和NOCTURNIN调节DHR 4振荡的机制（目的2）。特别是，我们想知道NOCTURNIN蛋白是否促进DHR 4进入核，以及DHR 4是否在PTTH信号传导后直接磷酸化。

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.