The developmental transition from juvenile to adult is often accompanied by many systemic changes in morphology, metabolism, and reproduction. Curiously, both mammalian puberty and insect metamorphosis are triggered by a pulse of steroid hormones, which can harmonize gene expression profiles in the body and thus orchestrate drastic biological changes. However, understanding of how the timing of steroid hormone biosynthesis is regulated at the molecular level is poor. The principal insect steroid hormone, ecdysteroid, is biosynthesized from dietary cholesterol in the specialized endocrine organ called the prothoracic gland. The periodic pulses of ecdysteroid titers determine the timing of molting and metamorphosis. To date, at least nine families of ecdysteroidogenic enzyme genes have been identified. Expression levels of these genes correlate well with ecdysteroid titers, indicating that the transcriptional regulatory network plays a critical role in regulating the ecdysteroid biosynthesis pathway. In this article, we summarize the transcriptional regulation of ecdysteroid biosynthesis. We first describe the development of prothoracic gland cells during Drosophila embryogenesis, and then provide an overview of the transcription factors that act in ecdysteroid biosynthesis and signaling. We also discuss the external signaling pathways that target these transcriptional regulators. Furthermore, we describe conserved and/or diverse aspects of steroid hormone biosynthesis in insect species as well as vertebrates.

The temporal transition of development is flexibly coordinated in the context of the nutrient environment, and this coordination is essential for organisms to increase their survival fitness and reproductive success. Steroid hormone, a key player of the juvenile-to-adult transition, is biosynthesized in a nutrient-dependent manner; however, the underlying genetic mechanism remains unclear. Here we report that the biosynthesis of insect steroid hormone, ecdysteroid, is regulated by a subset of serotonergic neurons in Drosophila melanogaster. These neurons directly innervate the prothoracic gland (PG), an ecdysteroid-producing organ and share tracts with the stomatogastric nervous system. Interestingly, the projecting neurites morphologically respond to nutrient conditions. Moreover, reduced activity of the PG-innervating neurons or of serotonin signalling in the PG strongly correlates with a delayed developmental transition. Our results suggest that serotonergic neurons form a link between the external environment and the internal endocrine system by adaptively tuning the timing of steroid hormone biosynthesis.

Steroid hormones play key roles in development, growth, and reproduction in various animal phyla [1]. The insect steroid hormone, ecdysteroid, coordinates growth and maturation, represented by molting and metamorphosis [2]. In Drosophila melanogaster, the prothoracicotropic hormone (PTTH)-producing neurons stimulate peak levels of ecdysteroid biosynthesis for maturation [3]. Additionally, recent studies on PTTH signaling indicated that basal levels of ecdysteroid negatively affect systemic growth prior to maturation [4-8]. However, it remains unclear how PTTH signaling is regulated for basal ecdysteroid biosynthesis. Here, we report that Corazonin (Crz)-producing neurons regulate basal ecdysteroid biosynthesis by affecting PTTH neurons. Crz belongs to gonadotropin-releasing hormone (GnRH) superfamily, implying an analogous role in growth and maturation [9]. Inhibition of Crz neuronal activity increased pupal size, whereas it hardly affected pupariation timing. This phenotype resulted from enhanced growth rate and a delay in ecdysteroid elevation during the mid-third instar larval (L3) stage. Interestingly, Crz receptor (CrzR) expression in PTTH neurons was higher during the mid- than the late-L3 stage. Silencing of CrzR in PTTH neurons increased pupal size, phenocopying the inhibition of Crz neuronal activity. When Crz neurons were optogenetically activated, a strong calcium response was observed in PTTH neurons during the mid-L3, but not the late-L3, stage. Furthermore, we found that octopamine neurons contact Crz neurons in the subesophageal zone (SEZ), transmitting signals for systemic growth. Together, our results suggest that the Crz-PTTH neuronal axis modulates ecdysteroid biosynthesis in response to octopamine, uncovering a regulatory neuroendocrine system in the developmental transition from growth to maturation.

Stem cells fuel the development and maintenance of tissues. Many studies have addressed how local signals from neighboring niche cells regulate stem cell identity and their proliferative potential. However, the regulation of stem cells by tissue-extrinsic signals in response to environmental cues remains poorly understood. Here we report that efferent octopaminergic neurons projecting to the ovary are essential for germline stem cell (GSC) increase in response to mating in femaleDrosophila. The neuronal activity of the octopaminergic neurons is required for mating-induced GSC increase as they relay the mating signal from sex peptide receptor-positive cholinergic neurons. Octopamine and its receptor Oamb are also required for mating-induced GSC increase via intracellular Ca²⁺signaling. Moreover, we identified Matrix metalloproteinase-2 as a downstream component of the octopamine-Ca²⁺signaling to induce GSC increase. Our study provides a mechanism describing how neuronal system couples stem cell behavior to environmental cues through stem cell niche signaling.

Ecdysteroids, including the biologically active hormone 20-hydroxyecdysone (20E), play essential roles in controlling many developmental and physiological events in insects. Ecdysteroid biosynthesis is achieved by a series of specialized enzymes encoded by the Halloween genes. Recently, a new class of Halloween gene, noppera-bo (nobo), encoding a glutathione S-transferase (GST) in dipteran and lepidopteran species, has been identified and characterized. GSTs are well known to conjugate substrates with the reduced form of glutathione (GSH), a bioactive tripeptide composed of glutamate, cysteine, and glycine. We hypothesized that GSH itself is required for ecdysteroid biosynthesis. However, the role of GSH in steroid hormone biosynthesis has not been examined in any organisms. Here, we report phenotypic analysis of a complete loss-of-function mutant in the γ-glutamylcysteine synthetase catalytic subunit (Gclc) gene in the fruit fly Drosophila melanogaster. Gclc encodes the evolutionarily conserved catalytic component of the enzyme that conjugates glutamate and cysteine in the GSH biosynthesis pathway. Complete Gclc loss-of-function leads to drastic GSH deficiency in the larval body fluid. Gclc mutant animals show a larval-arrest phenotype. Ecdysteroid titer in Gclc mutant larvae decreases, and the larval-arrest phenotype is rescued by oral administration of 20E or cholesterol. Moreover, Gclc mutant animals exhibit abnormal lipid deposition in the prothoracic gland, a steroidogenic organ during larval development. All of these phenotypes are reminiscent to nobo loss-of-function animals. On the other hand, Gclc mutant larvae also exhibit a significant reduction in antioxidant capacity. Consistent with this phenotype, Gclc mutant larvae are more sensitive to oxidative stress response as compared to wild-type. Nevertheless, the ecdysteroid biosynthesis defect in Gclc mutant animals is not associated with loss of antioxidant function. Our data raise the unexpected hypothesis that a primary role of GSH in early D. melanogaster larval development is ecdysteroid biosynthesis, independent from the antioxidant role of GSH.

Here, we have developed DIAMonDS (DrosophilaIndividual Activity Monitoring and Detection System) comprising time-lapse imaging by a charge-coupled device (CCD) flatbed scanner and Sapphire, a novel algorithm and web application. DIAMonDS automatically and sequentially identified the transition time points of multiple life cycle events such as pupariation, eclosion, and death in individual flies at high temporal resolution and on a large scale. DIAMonDS performed simultaneous multiple scans to measure individual deaths (≤1152 flies per scanner) and pupariation and eclosion timings (≤288 flies per scanner) under various chemical exposures, environmental conditions, and genetic backgrounds. DIAMonDS correctly identified 74–85% of the pupariation and eclosion events and ~ 92% of the death events within ± 10 scanning frames. This system is a powerful tool for studying the influences of genetic and environmental factors on fruit flies and efficient, high-throughput genetic and chemical screening in drug discovery.