Neverland is an evolutionally conserved Rieske-domain protein that is essential for ecdysone synthesis and insect growth

Steroid hormones mediate a wide variety of developmental and physiological events in multicellular organisms. During larval and pupal stages of insects, the principal steroid hormone is ecdysone, which is synthesized in the prothoracic gland (PG) and plays a central role in the control of development. Although many studies have revealed the biochemical features of ecdysone synthesis in the PG, many aspects of this pathway have remained unclear at the molecular level. We describe the neverland (nvd) gene, which encodes an oxygenase-like protein with a Rieske electron carrier domain, from the silkworm Bombyx mori and the fruitfly Drosophila melanogaster. nvd is expressed specifically in tissues that synthesize ecdysone, such as the PG. We also show that loss of nvd function in the PG causes arrest of both molting and growth during Drosophila development. Furthermore, the phenotype is rescued by application of 20-hydroxyecdysone or the precursor 7-dehydrocholesterol. Given that the nvd family is evolutionally conserved, these results suggest that Nvd is an essential regulator of cholesterol metabolism or trafficking in steroid synthesis across animal phyla.     

Mutations of a Drosophila NPC1 gene confer sterol and ecdysone metabolic defects

The molecular mechanisms by which dietary cholesterol is trafficked within cells are poorly understood. Previous work indicates that the NPC1 family of proteins plays an important role in this process, although the precise functions performed by this protein family remain elusive. We have taken a genetic approach to further explore the NPC1 family in the fruit fly Drosophila melanogaster. The Drosophila genome encodes two NPC1 homologs, designated NPC1a and NPC1b, that exhibit 42% and 35% identity to the human NPC1 protein, respectively. Here we describe the results of mutational analysis of the NPC1a gene. The NPC1a gene is ubiquitously expressed, and a null allele of NPC1a confers early larval lethality. The recessive lethal phenotype of NPC1a mutants can be partially rescued on a diet of high cholesterol or one that includes the insect steroid hormone 20-hydroxyecdysone. We also find that expression of NPC1a in the ring gland is sufficient to rescue the lethality associated with the loss of NPC1a and that cholesterol levels in NPC1a mutant larvae are unchanged relative to controls. Our results suggest that NPC1a promotes efficient intracellular trafficking of sterols in many Drosophila tissues including the ring gland where sterols must be delivered to sites of ecdysone synthesis.     

Cytochrome P450 CYP307A1/Spook: a regulator for ecdysone synthesis in insects

The prothoracic gland (PG) has essential roles in synthesizing and secreting a steroid hormone called ecdysone that is critical for molting and metamorphosis of insects. However, little is known about the genes controlling ecdysteroidogenesis in the PG. To identify genes functioning in the PG of the silkworm, Bombyx mori, we used differential display PCR and focused on a cytochrome P450 gene designated Cyp307a1. Its expression level positively correlates with a change in the hemolymph ecdysteroid titer. In addition, Drosophila Cyp307a1 is encoded in the spook locus, one of the Halloween mutant family members showing a low ecdysone titer in vivo, suggesting that Cyp307a1 is involved in ecdysone synthesis. While Drosophila Cyp307a1 is expressed in the early embryos and adult ovaries, the expression is not observed in the PGs of embryos or third instar larvae. These results suggest a difference in the ecdysone synthesis pathways during larval development in these insects.     

Expression and tissue localization of renalase, a novel soluble FAD-dependent protein, in reproductive/steroidogenic systems

Renalase was initially identified in human kidney as a soluble monoamine oxidase. Here we show that renalase is predominantly expressed in reproductive/steroidogenic systems, with particularly substantial expression in oocytes, granulosa, interstitial and luteal cells of ovary, spermatogenic cells of testis, and cortex of adrenal gland, suggesting its function(s) in maturation of germ cells and steroid hormone regulation. Renalase expression increases in testes and ovaries as mice develop and its expression is further enhanced in the ovaries of pregnant mice, indicating an activity of renalase in reproduction. Gonadotropin-releasing hormone (GnRH) antagonist, cetrorelix, repressed renalase expression in mice ovaries and testes, suggesting that steroids regulate renalase expression. Leptin is an effector and modulator of steroid hormones and reproduction. Surprisingly, knockout of leptin causes a dramatic increase of renalase expression in mice testes. Taken together, our results suggest that reproductive/steroidogenic systems are also the sources for renalase secretion and renalase may play a critical role in reproduction and hormone regulation. This provides a novel insight into understanding the function of renalase.     

The Drosophila disembodied gene controls late embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic ecdysone levels

Ecdysteroids regulate a wide variety of cellular processes during arthropod development, yet little is known about the genes involved in the biosynthesis of these hormones. Previous studies have suggested that production of 20-hydroxyecdysone in Drosophila and other arthropods involves a series of cytochrome P450 catalyzed hydroxylations of cholesterol. In this report, we show that the disembodied (dib) locus of Drosophila codes for a P450-like sequence. In addition, we find that dib mutant embryos have very low titers of ecdysone and 20-hydroxyecdysone (20E) and fail to express IMP-E1 and L1, two 20E-inducible genes, in certain tissues of the embryo. In situ hybridization studies reveal that dib is expressed in a complex pattern in the early embryo, which eventually gives way to restricted expression in the prothoracic portion of the ring gland. In larval and adult tissues, dib expression is observed in the prothoracic gland and follicle cells of the ovaries respectively, two tissues known to synthesize ecdysteroids. Phenotypic analysis reveals that dib mutant embryos produce little or no cuticle and exhibit severe defects in many late morphogenetic processes such as head involution, dorsal closure and gut development. In addition, we examined the phenotypes of several other mutants that produce defective embryonic cuticles. Like dib, mutations in the spook (spo) locus result in low embryonic ecdysteroid titers, severe late embryonic morphological defects, and a failure to induce IMP-E1. From these data, we conclude that dib and spo likely code for essential components in the ecdysone biosynthetic pathway and that ecdysteroids regulate many late embryonic morphogenetic processes such as cell movement and cuticle deposition.     

A Drosophila model of the Niemann-Pick type C lysosome storage disease: dnpc1a is required for molting and sterol homeostasis

Niemann-Pick type C (NPC) disease is a fatal autosomal-recessive neurodegenerative disorder characterized by the inappropriate accumulation of unesterified cholesterol in aberrant organelles. The disease is due to mutations in either of two genes, NPC1, which encodes a transmembrane protein related to the Hedgehog receptor Patched, and NPC2, which encodes a secreted cholesterol-binding protein. Npc1 mutant mice can be partially rescued by treatment with specific steroids. We have created a Drosophila NPC model by mutating dnpc1a, one of two Drosophila genes related to mammalian NPC1. Cells throughout the bodies of dnpc1a mutants accumulated sterol in a punctate pattern, as in individuals with NPC1 mutations. The mutants developed only to the first larval stage and were unable to molt. Molting after the normal first instar period was restored to various degrees by feeding the mutants the steroid molting hormone 20-hydroxyecdysone, or the precursors of ecdysone biosynthesis, cholesterol and 7-dehydrocholesterol. dnpc1a is normally highly expressed in the ecdysone-producing ring gland. Ring gland-specific expression of dnpc1a in otherwise mutant flies allowed development to adulthood, suggesting that the lack of ecdysone in the mutants is the cause of death. We propose that dnpc1a mutants have sterols trapped in aberrant organelles, leading to a shortage of sterol in the endoplasmic reticulum and/or mitochondria of ring gland cells, and, consequently, inadequate ecdysone synthesis.     

Genetic analysis of the Drosophila 63F early puff. Characterization of mutations in E63-1 and maggie, a putative Tom22

The 63F early puff in the larval salivary gland polytene chromosomes contains the divergently transcribed E63-1 and E63-2 ecdysone-inducible genes. E63-1 encodes a member of the EF-hand family of Ca(2+)-binding proteins, while E63-2 has no apparent open reading frame. To understand the functions of the E63 genes, we have determined the temporal and spatial patterns of E63-1 protein expression, as well as undertaken a genetic analysis of the 63F puff. We show that E63-1 is expressed in many embryonic and larval tissues, but the third-instar larval salivary gland is the only tissue where increases in protein levels correlate with increases in ecdysone titer. Furthermore, the subcellular distribution of E63-1 protein changes dynamically in the salivary glands at the onset of metamorphosis. E63-1 and E63-2 null mutations, however, have no effect on development or fertility. We have characterized 40 kb of the 63F region, defined as the interval between Ubi-p and E63-2, and have identified three lethal complementation groups that correspond to the dSc-2, ida, and mge genes. We show that mge mutations lead to first-instar larval lethality and that Mge protein is similar to the Tom22 mitochondrial import proteins of fungi, suggesting that it has a role in mitochondrial function.     

Isolation and characterization of the ecdysone receptor and its heterodimeric partner ultraspiracle through development in Sciara coprophila

Regulation of DNA replication is critical, and loss of control can lead to DNA amplification. Naturally occurring, developmentally regulated DNA amplification occurs in the DNA puffs of the late larval salivary gland giant polytene chromosomes in the fungus fly, Sciara coprophila. The steroid hormone ecdysone induces DNA amplification in Sciara, and the amplification origin of DNA puff II/9A contains a putative binding site for the ecdysone receptor (EcR). We report here the isolation, cloning, and characterizing of two ecdysone receptor isoforms in Sciara (ScEcR-A and ScEcR-B) and the heterodimeric partner, ultraspiracle (ScUSP). ScEcR-A is the predominant isoform in larval tissues and ScEcR-B in adult tissues, contrary to the pattern in Drosophila. Moreover, ScEcR-A is produced at amplification but is absent just prior. We discuss these results in relation to the model of ecdysone regulation of DNA amplification.     

Synthesis of a labelled putative precursor of ecdysone—II: [3H4]3β-hydroxy-5β-cholest-7en-6-one: Critical re-evaluation of its role in Locusta migratoria

We have synthesized a tritiated form of 2,14,22,25-tetradeoxyecdysone (5β-ketol) of high specific activity (115 Ci/mmol). We have examined the capacity of various tissues of Locusta migratoria to use this 5β-ketol, a putative precursor of ecdysone, in ecdysteroid biosynthesis. While larval prothoracic glands convert the radiotracer to labelled 14-deoxyecdysone they fail to hydroxylate the molecule to ecdysone itself. Other larval tissues, embryonic tissues or vitellogenic female ovaries are unable to convert the radiotracer to ecdysone, 20-hydroxyecdysone or 2-deoxyecdysone, the terminal products of biosynthesis in different developmental stages. Using subcellular preparations of prothoracic glands or follicle cells we have been unable to show a biological C-14 hydroxylation of 5β-ketol. It thus appears that the step of C-14 hydroxylation in the biosynthesis of ecdysteroids requires a substrate other than 5β-ketol.     

Scavenger Receptors Mediate the Role of SUMO and Ftz-f1 in Drosophila Steroidogenesis

SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval–pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor), the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi–mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues.     

A conditional rescue system reveals essential functions for the ecdysone receptor (EcR) gene during molting and metamorphosis in Drosophila

In Drosophila, pulses of the steroid hormone ecdysone trigger larval molting and metamorphosis and coordinate aspects of embryonic development and adult reproduction. At each of these developmental stages, the ecdysone signal is thought to act through a heteromeric receptor composed of the EcR and USP nuclear receptor proteins. Mutations that inactivate all EcR protein isoforms (EcR-A, EcR-B1, and EcR-B2) are embryonic lethal, hindering analysis of EcR function during later development. Using transgenes in which a heat shock promoter drives expression of an EcR cDNA, we have employed temperature-dependent rescue of EcR null mutants to determine EcR requirements at later stages of development. Our results show that EcR is required for hatching, at each larval molt, and for the initiation of metamorphosis. In EcR mutants arrested prior to metamorphosis, expression of ecdysone-responsive genes is blocked and normal ecdysone responses of both imaginal and larval tissues are blocked at an early stage. These results show that EcR mediates ecdysone signaling at multiple developmental stages and implicate EcR in the reorganization of imaginal and larval tissues at the onset of metamorphosis.