SUMMARYIn insects, the precise timing of molting and metamorphosis is strictly guided by a principal steroid hormone, ecdysone. Among the multiple conversion steps for synthesizing ecdysone from dietary cholesterol, the conversion of 7-dehydrocholesterol to 5-ketodiol, the so-called 'Black Box', is thought to be the important rate-limiting step. Although a number of genes essential for ecdysone synthesis have recently been revealed, much less is known about the genes that are crucial for functioning in the Black Box. Here we report on a novel ecdysteroidgenic gene, non-molting glossy (nm-g)/shroud (sro), which encodes a short-chain dehydrogenase/reductase. This gene was first isolated by positional cloning of the nm-g mutant of the silkworm Bombyx mori, which exhibits a low ecdysteroid titer and consequently causes a larval arrest phenotype. In the fruit fly, Drosophila melanogaster, the closest gene to nm-g is encoded by the sro locus, one of the Halloween mutant members that are characterized by embryonic ecdysone deficiency. The lethality of the sro mutant is rescued by the overexpression of either sro or nm-g genes, indicating that these two genes are orthologous. Both the nm-g and the sro genes are predominantly expressed in tissues producing ecdysone, such as the prothoracic glands and the ovaries. Furthermore, the phenotypes caused by the loss of function of these genes are restored by the application of ecdysteroids and their precursor 5-ketodiol, but not by cholesterol or 7-dehydrocholesterol. Altogether, we conclude that the Nm-g/Sro family protein is an essential enzyme for ecdysteroidogenesis working in the Black Box.

Stem cell maintenance is established by neighboring niche cells that promote stem cell self-renewal. However, it is poorly understood how stem cell activity is regulated by systemic, tissue-extrinsic signals in response to environmental cues and changes in physiological status. Here, we show that neuropeptide F (NPF) signaling plays an important role in the pathway regulating mating-induced germline stem cell (GSC) proliferation in the fruit fly Drosophila melanogaster. NPF expressed in enteroendocrine cells (EECs) of the midgut is released in response to the seminal-fluid protein sex peptide (SP) upon mating. This midgut-derived NPF controls mating-induced GSC proliferation via ovarian NPF receptor (NPFR) activity, which modulates bone morphogenetic protein (BMP) signaling levels in GSCs. Our study provides a molecular mechanism that describes how a gut-derived systemic factor couples stem cell behavior to physiological status, such as mating, through interorgan communication.

Steroid hormones play essential roles in a wide variety of biological processes in multicellular organisms. The principal steroid hormones in nematodes and arthropods are dafachronic acids and ecdysteroids, respectively, both of which are synthesized from cholesterol as an indispensable precursor. The first critical catalytic step in the biosynthesis of these ecdysozoan steroids is the conversion of cholesterol to 7-dehydrocholesterol. However, the enzymes responsible for cholesterol 7,8-dehydrogenation remain unclear at the molecular level. Here we report that the Rieske oxygenase DAF-36/Neverland (Nvd) is a cholesterol 7,8-dehydrogenase. The daf-36/nvd genes are evolutionarily conserved, not only in nematodes and insects but also in deuterostome species that do not produce dafachronic acids or ecdysteroids, including the sea urchin Hemicentrotus pulcherrimus, the sea squirt Ciona intestinalis, the fish Danio rerio, and the frog Xenopus laevis. An in vitro enzymatic assay system reveals that all DAF-36/Nvd proteins cloned so far have the ability to convert cholesterol to 7-dehydrocholesterol. Moreover, the lethality of loss of nvd function in the fruit fly Drosophila melanogaster is rescued by the expression of daf-36/ nvd genes from the nematode Caenorhabditis elegans, the insect Bombyx mori, or the vertebrates D. rerio and X. laevis. These data suggest that daf-36/nvd genes are functionally orthologous across the bilaterian phylogeny. We propose that the daf-36/nvd family of proteins is a novel conserved player in cholesterol metabolism across the animal phyla.Steroid hormones are crucial for development, growth, and homeostasis in multicellular organisms. Cholesterol and other sterol(s) serve as indispensable precursors for the biosynthesis of steroid hormones, and the conversion of cholesterol to the next specific intermediate is a crucial biochemical step across species (1-4). In the biosynthesis of vertebrate steroid hormones, cholesterol is commonly converted to pregnenolone by side-chain cleavage catalyzed by the cytochrome P450 monooxygenase P450scc/CYP11A1 (2). The step catalyzed by CYP11A1 is the key rate-limiting step in the synthesis of all vertebrate steroids, and it thus is controlled by numerous physiological responses throughout the life cycle (2, 5).Cholesterol is also required for steroid hormone biosynthesis in protostomes, including nematodes and arthropods (1, 6). However, the molecular mechanisms of cholesterol metabolism in these ecdysozoan animals have not been fully elucidated. The principal steroid hormones in nematodes and arthropods are dafachronic acids and ecdysteroids, respectively, both of which play pivotal roles in the regulation of developmental timing, reproduction, and longevity (1,7,8). In the biosynthesis of both dafachronic acids and ecdysteroids, which is different from the biosynthesis of vertebrate steroid hormones, cholesterol is converted to 7-dehydrocholesterol (7dC) 5 by dehydrogenation of the carbons at positions 7 and 8 (Fig. 1A) (9). Nematodes and arthropods lo...

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.

Highlights d Crz neurons contact both PG cells and PTTH neurons d The Crz-PTTH neuronal axis controls growth via basal ecdysteroid biosynthesis d PTTH neurons respond to Crz neurons during the mid-L3 stage with CrzR expression d Octopamine neurons contact with Crz neurons in the SEZ, regulating systemic growth