The Halloween genes code for cytochrome P450 enzymes mediating synthesis of the insect moulting hormone.

The developmental events occurring during moulting and metamorphosis of insects are controlled by precisely timed changes in levels of ecdysteroids, the moulting hormones. The final four sequential hydroxylations of steroid precursors into the active ecdysteroid of insects, 20E (20-hydroxyecdysone), are mediated by four cytochrome P450 (P450) enzymes, encoded by genes in the Halloween family. Orthologues of the Drosophila Halloween genes phantom (phm; CYP306A1), disembodied (dib; CYP302A1), shadow (sad; CYP315A1) and shade (shd; CYP314A1) were obtained from the endocrinological model insect, the tobacco hornworm Manduca sexta. Expression of these genes was studied and compared with changes in the ecdysteroid titre that controls transition from the larval to pupal stage. phm, dib and sad, which encode P450s that mediate the final hydroxylations in the biosynthesis of ecdysone, were selectively expressed in the prothoracic gland, the primary source of ecdysone during larval and pupal development. Changes in their expression correlate with the haemolymph ecdysteroid titre during the fifth (final) larval instar. Shd, the 20-hydroxylase, which converts ecdysone into the more active 20E, is expressed in tissues peripheral to the prothoracic glands during the fifth instar. Transcript levels of shd in the fat body and midgut closely parallel the enzyme activity measured in vitro. The results indicate that these Halloween genes are transcriptionally regulated to support the high biosynthetic activity that produces the cyclic ecdysteroid pulses triggering moulting.

A role for betaFTZ-F1 in regulating ecdysteroid titers during post-embryonic development in Drosophila melanogaster.

Variations in ecdysteroid titers play crucial roles in arthropods by initiating and regulating molting and metamorphosis. The recent identification of genes coding for cytochrome P450 enzymes involved in Drosophila ecdysteroidogenesis provides new molecular tools to investigate the regulation of insect hormone production. In the present study, we used an enzyme immunoassay to show that the molting hormone titer is strictly correlated with the steroidogenic capacity of the ring gland. A temporal correlation between dynamics of ecdysone production and expression of genes encoding steroidogenic enzymes was observed during the third instar, suggesting that the timing of hormone production depends on transcriptional regulation of the biosynthetic enzymes. Using clonal analysis, levels of two steroidogenic enzymes, Phantom (PHM) and Disembodied (DIB), were shown to be very reduced in ftz transcription factor 1 (ftz-f1) mutant ring gland cells whereas there was no effect of the without children (woc) mutation, suggesting that FTZ-F1 regulates phm and dib expression. Since betaFTZ-F1 is the homolog of the vertebrate steroidogenic factor 1 (SF1), which plays a key role in the differentiation of vertebrate steroidogenic organs through transcriptional regulation of steroidogenic enzymes, this study emphasizes the strong parallels between insects and vertebrates with respect to the regulatory mechanisms of steroidogenesis.

Prothoracicotropic hormone stimulated extracellular signal-regulated kinase (ERK) activity: the changing roles of Ca(2+)- and cAMP-dependent mechanisms in the insect prothoracic glands during metamorphosis.

The synthesis of ecdysteroids by the lepidopteran prothoracic gland is regulated by a brain neuropeptide hormone, prothoracicotropic hormone (PTTH). In Manduca sexta glands, PTTH stimulates several events including Ca(2+) influx, Ca(2+)-dependent cAMP generation and the activation of several protein kinases. In the present study, the path by which PTTH stimulates extracellular signal-activated regulated kinase (ERK) phosphorylation was investigated using PTTH and second messenger analogs. The results indicate that Ca(2+)-dependent processes, other than cAMP generation, play the major role in PTTH stimulation of ERK phosphorylation in larval prothoracic glands, that cAMP-dependent events increase in importance during later development and that PTTH-stimulated ERK phosphorylation is highest in larval glands. The decline in PTTH-stimulated ERK phosphorylation associated with metamorphosis results from decreased ERK levels and an increased basal rate of ERK phosphorylation. The data suggest that the role or importance of components of the PTTH signal transduction cascade are not fixed and can change during development.

Biosynthetic maturation of the corpus allatum of the female adult medfly, Ceratitis capitata, and its putative control.

The major juvenile hormone (JH) homolog synthesized in vitro by the adult female Medfly (Ceratitis capitata) corpus allatum (CA) is JHB(3), with JH-III the minor homolog. Methyl-incorporation in vitro in post-eclosion virgin females is age-dependent. Basal activity occurs during the first four days post-eclosion and increases significantly thereafter, peaking at five days. Biosynthetic maturation of the mated female CA is delayed by one day and reduced considerably. The delayed response may be due to direct cerebral or neural inhibition. Synthetic Drosophila melanogaster sex peptide depresses JH biosynthesis by the Medfly female CA in vitro. Male-derived accessory gland peptides of the Medfly are transferred to the female during mating and a Medfly SP-analog may be responsible for down-regulation of JH synthesis by the CA in mated Medfly females. Mevinolin, an inhibitor of the mevalonate pathway, significantly reduces the biosynthesis of JHB(3), while farnesoic acid, a proximate precursor of JHIII, significantly stimulates the biosynthesis of both JHB(3) and JHIII in vitro.

A protein from the cabbage looper, Trichoplusia ni, regulated by a bacterial infection is homologous to 3-dehydroecdysone 3beta-reductase.

During the screening of immune-regulated genes from the cabbage looper, Trichoplusia ni, a 3-dehydroecdysone 3beta-reductase homologue (DERH) was cloned. In the course of development, 3-dehydroecdysone 3beta-reductase mediates the conversion of 3-dehydroecdysone (3dE) secreted from the prothoracic glands to ecdysone (E), which is subsequently converted to 20-hydroxyecdysone (20E), the major insect molting hormone. The cloned gene is upregulated in fat body during development and is strongly induced after the larva is challenged with bacteria. The gene codes for a 308 amino acid residue protein which shows 42.5% identity to Spodoptera littoralis 3-dehydroecdysone 3beta-reductase. Using the baculovirus expression system, the recombinant DERH was expressed. The purified protein mediates the reduction of 3-dehydromakisterone A to makisterone A, and requires NADPH as a cofactor. Western blots using an antiserum to T. ni DERH revealed the presence of the protein in larval hemolymph and integument. The data indicate that the protein is regulated developmentally and is induced after a challenge with bacteria. Immunohistochemical studies localized the enzyme exclusively in the epidermis and the cuticle.

Activation of an extracellular signal-regulated kinase (ERK) by the insect prothoracicotropic hormone.

Ecdysteroid hormones are crucial in controlling the growth, molting and metamorphosis of insects. The predominant source of ecdysteroids in pre-adult insects is the prothoracic gland, which is under the acute control of the neuropeptide hormone prothoracicotropic hormone (PTTH). Previous studies using the tobacco hornworm, Manduca sexta, have shown that PTTH stimulates ecdysteroid synthesis via a series of events, including the activation of protein kinase A and the 70 kDa S6 kinase (p70(S6k)). In this study, PTTH was shown to stimulate also mitogen-activated protein kinase (MAPK) phosphorylation and activity in the Manduca prothoracic gland. The MAPK involved appears to be an extracellular signal-regulated kinase (ERK) homologue. The ERK phosphorylation inhibitors PD 98059 and UO 126 blocked basal and PTTH-stimulated ERK phosphorylation and ecdysteroid synthesis. PTTH-stimulated ERK activity may be important for both rapid regulation of ecdysteroid synthesis and for longer-term changes in the size and function of prothoracic gland cells.

Woc (without children) gene control of ecdysone biosynthesis in Drosophila melanogaster.

The first step in ecdysteroidogenesis, i.e. the 7,8-dehydrogenation of dietary cholesterol (C) to 7-dehydrocholesterol (7dC), is blocked in Drosophila melanogaster homozygous woc (without children) third instar larval ring glands (source of ecdysone). Unlike ring glands from wild-type D. melanogaster larvae, glands from woc mutants cannot convert radiolabelled C or 25-hydroxycholesterol (25C) to 7dC or 7-dehydro-25-hydroxycholesterol (7d25C) in vitro, nor to ecdysone (E). Yet, when these same glands are incubated with synthetic tracer 7d25C, the rate of metabolism of this polar Delta(5,7)-sterol into E is identical to that observed with glands from comparably staged wild-type larvae. The absence of this enzymatic activity in vivo is probably the direct cause of the observed low whole-body ecdysteroid titers in late third instar homozygous mutant larvae, the low ecdysteroid secretory activity in vitro of brain-ring gland complexes from these animals, and the failure of the larvae to pupariate (undergo metamorphosis). Oral administration of 7dC, but not C, results in a dramatic increase in ecdysteroid production both in vivo and in vitro by the woc mutant brain-ring gland complexes and affects a partial rescue to the beginning of pupal-adult development, but no further, despite elevated whole-body ecdysteroid titers. Data previously reported (Wismar et al., 2000) indicate that the woc gene encodes a zinc-finger protein that apparently modulates the activity of the 7,8-dehydrogenase.

The mutation without children(rgl) causes ecdysteroid deficiency in third-instar larvae of Drosophila melanogaster.

Larvae homozygous for the recessive lethal allele without children(rgl) (woc(rgl)) fail to pupariate. Application of exogenous 20-hydroxyecdysone elicits puparium formation and pupation. Ecdysteroid titer measurements on mutant larvae show an endocrine deficiency in the brain-ring gland complex, which normally synthesizes ecdysone, resulting in a failure of the larvae to achieve a threshold whole body hormone titer necessary for molting. Ultrastructural investigation revealed extensive degeneration of the prothoracic cells of the ring gland in older larvae. The woc gene, located in polytene chromosomal region 97F, consists of 11 exons. A 6.8-kb transcript is expressed throughout development but is absent in the mutant woc(rgl) larvae. The woc gene encodes a protein of 187 kDa. Eight zinc fingers of the C2-C2 type point to a possible function as a transcription factor. The woc protein shows considerable homology to human proteins which have been implicated in both mental retardation and a leukemia/lymphoma syndrome.

Transcriptional activation of the Drosophila ecdysone receptor by insect and plant ecdysteroids.

A number of insect ecdysteroids, plant ecdysteroids and juvenoids were assayed for their ability to activate Drosophila nuclear receptors in transfected tissue culture cells. Discrete modifications to 20-hydroxyecdysone, the apparent natural ligand for the ecdysone receptor (EcR), conferred dramatic changes on the transcriptional activity of this receptor, suggesting that other biologically relevant EcR ligands may exist. Conversely, none of the compounds tested had a significant effect on the activity of three Drosophila orphan nuclear receptors: DHR38, DHR78 or DHR96. Taken together, these results demonstrate the selectivity of EcR for a series of natural and synthetic ecdysone agonists and suggest that as yet untested compounds may be responsible for activating DHR38, DHR78 and DHR96.

Dynamic regulation of prothoracic gland ecdysteroidogenesis: Manduca sexta recombinant prothoracicotropic hormone and brain extracts have identical effects.

Multiple assays were conducted in order to determine if the recently available recombinant prothoracicotropic hormone (rPTTH) from Manduca sexta is identical, or similar, to the natural hormone and if results from its use in a variety of assays confirm, or are inconsistent with, previous studies over the past 20years on PTTH action using brain extract. Brain extracts and rPTTH showed similar, if not identical, effects on the cell biology of Manduca prothoracic gland cells with the following results: increased levels of cAMP (adenosine 3':5' cyclic monophosphate) synthesis; requirement for extracellular Ca(2+) in in vitro studies; ecdysteroidogenesis stimulation in vitro; stimulation of general and specific protein synthesis; immunocytochemical identification of the two lateral cells in each brain hemisphere as the source of PTTH (the prothoracicotropes); the ability of antibodies to rPTTH to inhibit ecdysteroidogenesis stimulation in vitro; and the multiple phosphorylation of the ribosomal protein S6. The data revealed that brain extract and rPTTH show equivalent effects in all of the assays, indicating that this rPTTH is the natural PTTH of Manduca and that the data generated with brain extracts over the past two decades are indeed relevant.

cDNA cloning and expression of a hormone-regulated heat shock protein (hsc 70) from the prothoracic gland of Manduca sexta.

The brain neuropeptide prothoracicotropic hormone (PTTH) stimulates a rapid increase in ecdysteroid hormone synthesis that is accompanied by general and specific increases in protein synthesis, including that of a 70 kDa cognate heat shock protein (hsc 70). To further understand the possible roles of hsc 70, hsc 70 cDNA clones were isolated from a tobacco hornworm (Manduca sexta) prothoracic gland cDNA library. All sequenced clones were highly homologous to the Drosophila hsc 70-4 isoform. Manduca hsc 70 mRNA levels during the last larval instar exhibited a peak at the onset of wandering and a peak that coincided with the major pre-metamorphic peak of ecdysteroid synthesis. Manipulations of the glands' hormonal milieu showed that hsc 70 mRNA levels respond to 20-hydroxyecdysone, dibutyryl cAMP, PTTH and the JH analogue hydroprene. The protein and mRNA data suggest that hsc 70 could be involved in a negative feedback loop regulating assembly of the ecdysone receptor complex.

An in vitro analysis of ecdysteroid-elicited cell death in the prothoracic gland of Manduca sexta.

The prothoracic glands of the tobacco hornworm, Manduca sexta, secrete the precursor of the insect molting hormone and normally undergo programmed cell death (PCD) during pupal-adult metamorphosis, between days 5 and 6 after pupation. This phenomenon can be elicited prematurely in vitro by the addition of 20-hydroxyecdysone (20E) to the gland cultures. To induce nuclear condensation in vitro in the glands from day-1 pupae, the effective dose range of 20E is 0.7-7 micrograms/ml and the minimum exposure period is 24 h. Prothoracic glands from different stages of pupal-adult development express different responsiveness to exogenous ecdysteroids. By utilizing terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) and the apoptotic DNA laddering method together with transmission electron microscopy, it has been demonstrated that the ecdysteroid-induced cell death of the prothoracic glands occurs via not only apoptosis but also autophagy, i.e., the induced dying cells show both severe nuclear fragmentation and autophagic vacuole formation, characteristics typical of apoptotic and autophagic cell death. The composite data indicate that ecdysteroids regulate directly both apoptotic and autophagic mechanisms of PCD of the prothoracic glands.

Can the insect nervous system synthesize ecdysteroids?

The term "neurosteroid" refers to both classic and unique steroid molecules that are synthesized from cholesterol (C) by the central and peripheral nervous systems of higher vertebrates. Therein, they accumulate and modulate nervous activity by a variety of mechanisms other than the classic steroid receptor-mediated modulation of genomic activity, although such may also be involved. Since the insect nervous system expresses ecdysteroid receptors and responds both directly and developmentally to ecdysteroids, the possibility of ecdysteroidogenesis in the pupal and adult central and peripheral nervous system of Manduca sexta and the nervous system of Drosophila melanogaster larvae was investigated. The endogenous concentrations of the critical, dietary-derived delta 5,7-sterols ergosterol and 7-dehydrocholesterol (7dC) remained 10 to 20-fold higher in the Manduca pupal and adult nervous tissues than was found in the larval hemolymph at the cessation of feeding. In addition, it was determined that the Manduca pupal nervous system, but not that of the adult, could synthesize 3H/14C-7dC or 3H-7-dehydro-25-hydroxycholesterol (3H-7d25C) from 3H/14C-cholesterol (3H/14C-C) or the polar sterol substrate 3H-25-hydroxycholesterol (3H-25C), respectively. However, none of the nervous system samples from the two species and the several stages analyzed, a small window of neural development in these insects, were capable of incorporating any of the above tracer precursor sterols into a radiolabelled ecdysteroid, i.e. less than 0.0005%. Thus, the absence of neurosteroidogenesis by the insect nervous system stands in sharp contrast to previously described nervous system steroid hormone biosynthesis by the mammalian nervous system.

Alterations in ultraspiracle (USP) content and phosphorylation state accompany feedback regulation of ecdysone synthesis in the insect prothoracic gland.

Insect molting and metamorphosis are elicited by a class of ecdysteroids, mainly 20-hydroxyecdysone (20E), the precursor of which is synthesized in the prothoracic gland. 20E acts via the ecdysone receptor (EcR) and its heterodimer partner ultraspiracle (USP). Analysis of the prothoracic gland of Manduca sexta revealed that the developmental expression and phosphorylation of a specific USP form, p47, is positively correlated with ecdysteroidogenesis and that 20E, but not ecdysone, is responsible for initiating the translational expression and phosphorylation of p47. The latter forms a functional complex with EcR and the ligand-complex interaction results in the down regulation of ecdysteroidogenesis and the inhibition of prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis. The composite data suggest that USP plays a key role in modulating PTTH-stimulated ecdysteroid biosynthesis through the selective expression and phosphorylation of the p47 USP isoform.

Cloning of a beta1 tubulin cDNA from an insect endocrine gland: developmental and hormone-induced changes in mRNA expression.

A rapid increase in ecdysteroid hormone synthesis results when the insect prothoracic gland is stimulated with prothoracicotropic hormone (PTTH), a brain neuropeptide hormone. PTTH also stimulates the specific synthesis of several proteins, one of which is a beta tubulin. To further understand the possible roles of beta tubulin in the prothoracic gland, beta tubulin cDNA clones were isolated from a tobacco hornworm (Manduca sexta) gland cDNA library. Sequence analysis indicated that these clones were assignable to the beta1 tubulin isoform. Gland beta1 tubulin mRNA levels during the last larval instar and early pupal-adult development exhibited peaks that coincided with peaks in ecdysteroid synthesis. Manipulations of the glands hormonal milieu showed that beta1 tubulin mRNA levels respond to 20 hydroxyecdysone and PTTH. The data also support our earlier proposal that the prothoracic gland beta1 tubulin gene is ubiquitously expressed but exhibits tissue- and developmental-specific regulation of transcription and translation.

Juvenile hormone prevents the onset of programmed cell death in the prothoracic glands of Manduca sexta.

During normal pupal-adult development, programmed cell death of the prothoracic gland of Manduca sexta proceeds via apoptosis. By employing the DNA laddering technique, the earliest sign of DNA fragmentation in the cells comprising the gland occurred on Day 5 of pupal-adult development and DNA fragmentation peaked 1 day later. Since juvenile hormone (JH) is known to prevent adult development and since prothoracic gland degeneration occurs during the initial stages of adult development, we wished to determine the possible role of JH in prothoracic gland maintenance. JH was injected into pupae and the glands analyzed by DNA laddering and in situ TUNEL labeling. The administration of JH prevented apoptosis of the prothoracic gland even 11 days after JH injection into young pupae. The prothoracic glands remained intact and their ability to synthesize ecdysteroids was maintained at a fairly active level as ascertained by radioimmunoassay after in vitro incubation. The control glands had degenerated by this time and were almost devoid of ecdysteroidogenesis capability. Ultrastructural analysis confirmed that JH-treated prothoracic gland cells were rescued from the apoptotic sequence, i.e., nuclear condensation, cytoplasmic budding, and cell fragmentation. They exhibited a preserved smooth endoplasmic reticulum and intercellular channel system typical of active prothoracic gland cells. The composite data suggest that JH can both maintain and stimulate the prothoracic gland. Therefore, during normal pupal-adult metamorphosis the absence of JH is prerequisite for both the initiation and completion of prothoracic gland degeneration.

Ecdysteroids regulate yolk protein uptake by Drosophila melanogaster oocytes.

Juvenile hormones (JHs) are thought to drive the regulation of yolk protein uptake by ovaries in Drosophila melanogaster. However, the level of JH production in a mutant stock (ap(56f)) is depressed yet the flies are normally vitellogenic. The production of ecdysteroids by these ap(56f) ovaries in vitro is elevated above that of wild-type ovaries. The incubation of wild-type ovaries in the presence of 0.1mM JHB(3) increased ecdysteroid biosynthesis only during the first 18h following eclosion. Female Drosophila melanogaster undergo a pre-vitellogenic reproductive diapause when exposed to low temperature (11 degrees C) and a short-day photoperiod (L12:D12). The rate of ecdysteroid synthesis by the ovaries, but not JH production, increased within 12h of a temperature upshift to 25 degrees C from a basal level of 20+/-1pg/10 pair of ovaries/5h to a sustained level of 150+/-20pg/10 pair/5h. Vitellogenic oocytes were noted in all females within 12h of this temperature upshift. Diapause was also terminated by the injection of 1&mgr;g of 20-hydroxyecdysone into the abdomens of diapausing females as determined by an increase in ovary size, and the appearance of vitellogenic oocytes as compared to controls. These results are consistent with a revised model for the regulation of yolk protein uptake by ovaries in which ecdysteroids, and not JHs, play the prominent role.

Purification and characterization of the prothoracicotropic hormone of Drosophila melanogaster.

The prothoracicotropic hormone (PTTH) of Drosophila melanogaster is a modulator of ecdysteroid (molting hormone) synthesis and was isolated and characterized from extracts of whole larvae (approximately 4 x 10(5) larvae). The purification protocol included delipidation, salt-extraction, heat treatment, conventional column chromatography, and HPLC, and yielded about 50 microg of pure hormone. Biological activity was followed using a ring gland in vitro assay in which ecdysteroidogenesis by control ring glands as measured by radioimmunoassay was compared with ring gland incubations containing active fractions. The molecular weight of the purified PTTH was 45 kDa and N-terminal amino acid sequence analysis indicated that those analyzed sequences displayed no significant homology with known peptides or peptide hormones, including PTTH from the silkmoth, Bombyx mori. Western blot analysis indicated that the native form of Drosophila PTTH was a single 66-kDa polypeptide with N-linked carbohydrate chains and intrachain disulfide bonds. The purified 45-kDa peptide is the deglycosylated form, a result of glycosidase activity present during preparation of the PTTH extract. The deglycosylated form shows heterogeneity, presumably as a result of varying degrees of deglycosylation at the N terminus.