DHR3 is required for the prepupal-pupal transition and differentiation of adult structures during Drosophila metamorphosis.

Pulses of the steroid hormone ecdysone activate genetic regulatory hierarchies that coordinate the developmental changes associated with Drosophila metamorphosis. A high-titer ecdysone pulse at the end of larval development triggers puparium formation and induces expression of the DHR3 orphan nuclear receptor. Here we use both a heat-inducible DHR3 rescue construct and clonal analysis to define DHR3 functions during metamorphosis. Clonal analysis reveals requirements for DHR3 in the development of adult bristles, wings, and cuticle, and no apparent function in eye or leg development. DHR3 mutants rescued to the third larval instar also reveal essential functions during the onset of metamorphosis, leading to lethality during prepupal and early pupal stages. The phenotypes associated with these lethal phases are consistent with the effects of DHR3 mutations on ecdysone-regulated gene expression. Although DHR3 has been shown to be sufficient for early gene repression at puparium formation, it is not necessary for this response, indicating that other negative regulators may contribute to this pathway. In contrast, DHR3 is required for maximal expression of the midprepupal regulatory genes, EcR, E74B, and betaFTZ-1. Reductions in EcR and betaFTZ-F1 expression, in turn, lead to submaximal early gene induction in response to the prepupal ecdysone pulse and corresponding defects in adult head eversion and salivary gland cell death. These studies demonstrate that DHR3 is an essential regulator of the betaFTZ-F1 midprepupal competence factor, providing a functional link between the late larval and prepupal responses to ecdysone. Induction of DHR3 in early prepupae ensures that responses to the prepupal ecdysone pulse will be distinct from responses to the late larval pulse and thus that the animal progresses in an appropriate manner through the early stages of metamorphosis.     

The ecdysone-induced DHR4 orphan nuclear receptor coordinates growth and maturation in Drosophila

A critical determinant of insect body size is the time at which the larva stops feeding and initiates wandering in preparation for metamorphosis. No genes have been identified that regulate growth by contributing to this key developmental decision to terminate feeding. We show here that mutations in the DHR4 orphan nuclear receptor result in larvae that precociously leave the food to form premature prepupae, resulting in abbreviated larval development that translates directly into smaller and lighter animals. In addition, we show that DHR4 plays a central role in the genetic cascades triggered by the steroid hormone ecdysone at the onset of metamorphosis, acting as both a repressor of the early ecdysone-induced regulatory genes and an inducer of the betaFTZ-F1 midprepupal competence factor. We propose that DHR4 coordinates growth and maturation in Drosophila by mediating endocrine responses to the attainment of critical weight during larval development.     

Orphan nuclear receptor betaFTZ-F1 is required for muscle-driven morphogenetic events at the prepupal-pupal transition in Drosophila melanogaster

In Drosophila melanogaster, fluctuations in 20-hydroxyecdysone (ecdysone) titer coordinate gene expression, cell death, and morphogenesis during metamorphosis. Our previous studies have supported the hypothesis that betaFTZ-F1 (an orphan nuclear receptor) provides specific genes with the competence to be induced by ecdysone at the appropriate time, thus directing key developmental events at the prepupal-pupal transition. We are examining the role of betaFTZ-F1 in morphogenesis. We have made a detailed study of morphogenetic events during metamorphosis in control and betaFTZ-F1 mutant animals. We show that leg development in betaFTZ-F1 mutants proceeds normally until the prepupal-pupal transition, when final leg elongation is delayed by several hours and significantly reduced in the mutants. We also show that betaFTZ-F1 mutants fail to fully extend their wings and to shorten their bodies at the prepupal-pupal transition. We find that betaFTZ-F1 mutants are unable to properly perform the muscle contractions that drive these processes. Several defects can be rescued by subjecting the mutants to a drop in pressure during the normal time of the prepupal-pupal transition. Our findings indicate that betaFTZ-F1 directs the muscle contraction events that drive the major morphogenetic processes during the prepupal-pupal transition in Drosophila.     

Inducible expression of double-stranded RNA directs specific genetic interference in Drosophila

BACKGROUND: The introduction of double-stranded RNA (dsRNA) can selectively interfere with gene expression in a wide variety of organisms, providing an ideal approach for functional genomics. Although this method has been used in Drosophila, it has been limited to studies of embryonic gene function. Only inefficient effects have been seen at later stages of development. RESULTS: When expressed under the control of a heat-inducible promoter, dsRNA interfered efficiently and specifically with gene expression during larval and prepupal development in Drosophila. Expression of dsRNA corresponding to the EcR ecdysone receptor gene generated defects in larval molting and metamorphosis, resulting in animals that failed to pupariate or prepupae that died with defects in larval tissue cell death and adult leg formation. In contrast, expression of dsRNA corresponding to the coding region of the betaFTZ-F1 orphan nuclear receptor had no effect on puparium formation, but led to an arrest of prepupal development, generating more severe lethal phenotypes than those seen with a weak betaFTZ-F1 loss-of-function allele. Animals that expressed either EcR or betaFTZ-F1 dsRNA showed defects in the expression of corresponding target genes, indicating that the observed developmental defects are caused by disruption of the genetic cascades that control the onset of metamorphosis. CONCLUSIONS: These results confirm and extend our understanding of EcR and betaFTZ-F1 function. They also demonstrate that dsRNA expression can inactivate Drosophila gene function at later stages of development, providing a new tool for functional genomic studies in Drosophila.     

Temporally restricted expression of transcription factor betaFTZ-F1: significance for embryogenesis, molting and metamorphosis in Drosophila melanogaster

FTZ-F1, a member of the nuclear receptor superfamily, has been implicated in the activation of the segmentation gene fushi tarazu during early embryogenesis of Drosophila melanogaster. We found that an isoform of FTZ-F1, betaFTZ-F1, is expressed in the nuclei of almost all tissues slightly before the first and second larval ecdysis and before pupation. Severely affected ftz-f1 mutants display an embryonic lethal phenotype, but can be rescued by ectopic expression of betaFTZ-F1 during the period of endogenous betaFTZ-F1 expression in the wild type. The resulting larvae are not able to molt, but this activity is rescued again by forced expression of betaFTZ-F1, allowing progression to the next larval instar stage. On the other hand, premature expression of betaFTZ-F1 in wild-type larvae at mid-first instar or mid-second instar stages causes defects in the molting process. Sensitive periods were found to be around the time of peak ecdysteroid levels and slightly before the start of endogenous betaFTZ-F1 expression. A hypomorphic ftz-f1 mutant that arrests in the prepupal stage can also be rescued by ectopic, time-specific expression of betaFTZ-F1. Failure of salivary gland histolysis, one of the phenotypes of the ftz-f1 mutant, is rescued by forced expression of the ftz-f1 downstream gene BR-C during the late prepupal period. These results suggest that betaFTZ-F1 regulates genes associated with ecdysis and metamorphosis, and that the exact timing of its action in the ecdysone-induced gene cascade is important for proper development.     

Effects of transgenic overexpression of diapause hormone and diapause hormone receptor genes on non-diapause silkworm

Diapause is a state of developmental arrest that is most often observed in arthropods, especially insects. The domesticated silkworm, Bombyx mori, is a typical insect that enters diapause at an early embryonic stage. Previous studies have revealed that the diapause hormone (DH) signaling molecules, especially the core members DH and DH receptor 1 (DHR1), are crucial for the determination of embryonic diapause in diapause silkworm strains. However, whether they function in non-diapause silkworm strains remains largely unknown. Here, we generated two transgenic lines overexpressing DH or DHR1 genes in a non-diapause silkworm strain, Nistari. Our results showed that developmental expression patterns of DH and DHR1 are quite similar in transgenic silkworms: both genes are highly expressed in the mid to late stages of pupae and are most highly expressed in day-6 pupae but are expressed at very low levels in other developmental stages. Moreover, the overexpression of DH or DHR1 can affect the expression of diapause-related genes but is not sufficient to induce embryonic diapause in their offspring. This study provides new insights into the function of DH and DHR1 in a non-diapause silkworm strain.     

Isolation and developmental expression of two nuclear receptors, MHR4 and betaFTZ-F1, in the tobacco hornworm, Manduca sexta

The cDNAs for two members of the nuclear receptor superfamily were isolated from the tobacco hornworm, Manduca sexta. The deduced amino acid sequence of MHR4 shows 93-95% identity in the DNA-binding domain and the first portion of the hinge (D) region with the germ cell nuclear factor (GCNF)-related factors (GRFs) of the silkworm, Bombyx mori, and the mealworm, Tenebrio molitor, and with a genomic sequence from the fruit fly, Drosophila melanogaster. Northern blot hybridization showed that a 7.5 kb MHR4 mRNA appeared in Manduca abdominal epidermis just as the ecdysteroid titer began to decline during the larval molt, disappeared about 12 h later, then transiently reappeared shortly before larval ecdysis. During the pupal and adult molts, a similar pattern of expression was seen (the very end of the adult molt was not studied). At peak times of expression in the epidermis, MHR4 mRNA was also present in fat body and the central nervous system (CNS). The deduced amino acid sequence of Manduca FTZ-F1 is 100% and 96% identical to that of B. mori and Drosophila betaFTZ-F1, respectively, in the DNA-binding domain and the adjacent hinge region including the FTZ-F1 box. Northern blot analysis showed that the >9.5 kb betaFTZ-F1 mRNA appeared in Manduca epidermis during the decline of the ecdysteroid titer in the larval, pupal and adult molts as the first peak of MHR4 mRNA declined, then it disappeared in the larval and pupal molts before the second peak of MHR4 appeared. betaFTZ-F1 mRNA was also found in fat body and the CNS at the time of peak expression in the epidermis during the larval and pupal molts. Both MHR4 and betaFTZ-F1 mRNAs were found in the testis during the onset of spermatogenesis in the prepupal period.     

Identification and functional characterization of two orphan G-protein-coupled receptors for adipokinetic hormones from silkworm Bombyx mori

Adipokinetic hormones (AKHs) are the best studied insect neuropeptides with the function of mobilizing lipids and carbohydrates during energy-expensive activities and modulating fundamental physiological processes, such as sugar homeostasis, lipid metabolism, and reproduction. Three distinct cDNAs encoding the prepro-Bombyx AKH1-3 have been cloned and confirmed by mass spectrometric methods. Our previous research suggested the Bombyx AKH receptor is activated by AKH1 and AKH2 with high affinity but by AKH3 with quite low affinity. In this study, using stable functional expression of the receptors in HEK293 cells, we have now identified AKH3 as a specific ligand for two orphan G-protein-coupled receptors, and we therefore named them AKHR2a and AKHR2b, respectively. We demonstrated that both AKHR2a and AKHR2b were activated by AKH3 at high affinity and by AKH1 and AKH2 at low affinity, leading to an increase of intracellular cAMP levels and activation of ERK1/2 and receptor internalization, but they were not activated by Bombyx corazonin. Conversely, the Bombyx corazonin receptor was activated by corazonin but not by AKH1-3. Quantitative RT-PCR revealed that AKHR2a and AKHR2b were both highly expressed in the testis but were also detected at low levels in other tissues. These results will lead to a better understanding of the AKH/AKHR system in the regulation of fundamental physiological processes.     

The Drosophila orphan nuclear receptor DHR38 mediates an atypical ecdysteroid signaling pathway

Ecdysteroid pulses trigger the major developmental transitions during the Drosophila life cycle. These hormonal responses are thought to be mediated by the ecdysteroid receptor (EcR) and its heterodimeric partner Ultraspiracle (USP). We provide evidence for a second ecdysteroid signaling pathway mediated by DHR38, the Drosophila ortholog of the mammalian NGFI-B subfamily of orphan nuclear receptors. DHR38 also heterodimerizes with USP, and this complex responds to a distinct class of ecdysteroids in a manner that is independent of EcR. This response is unusual in that it does not involve direct binding of ecdysteroids to either DHR38 or USP. X-ray crystallographic analysis of DHR38 reveals the absence of both a classic ligand binding pocket and coactivator binding site, features that seem to be common to all NGFI-B subfamily members. Taken together, these data reveal the existence of a separate structural class of nuclear receptors that is conserved from fly to humans.     

Molecular characterization and functional analyses of a diapause hormone receptor-like gene in parthenogenetic Artemia

In arthropods, mature females under certain conditions produce and release encysted gastrula embryos that enter diapause, a state of obligate dormancy. The process is presumably regulated by diapause hormone (DH) and diapause hormone receptor (DHR) that were identified in the silkworm, Bombyx mori and other insects. However, the molecular structure and function of DHR in crustaceans remains unknown. Here, a DHR-like gene from parthenogenetic Artemia (Ar-DHR) was isolated and sequenced. The cDNA sequence consists of 1410 bp with a 1260-bp open reading frame encoding a protein consisting of 420 amino acid residues. The results of real-time PCR (qRT-PCR) and Western blot analysis showed that the mRNA and protein of Ar-DHR were mainly expressed at the diapause stage. Furthermore, we found that Ar-DHR was located on the cell membrane of the pre-diapause cyst but in the cytoplasm of the diapause cyst by analysis of immunofluorescence. In vivo knockdown of Ar-DHR by RNA interference (RNAi) and antiserum neutralization consistently inhibited diapause cysts formation. The results indicated that Ar-DHR plays an important role in the induction and maintenance of embryonic diapause in Artemia. Thus, our findings provide an insight into the regulation of diapause formation in Artemia and the function of Ar-DHR.     

Cloning and partial characterization of a gene in Bombyx mori homologous to a human adiponectin receptor

In this study, we report the cloning and characteristics of an adiponectin-like receptor gene from Bombyx mori (BmAdipoR) with highly conserved deduced amino-acid sequences and similar structure to the human adiponectin receptor (AdipoR). Structural analysis of the translated cDNA suggested it encoded a membrane protein with seven transmembrane domains. BmAdipoR was found to be expressed in multiple tissues and highly expressed in Malpighian tubules, fat body and testis. BmNPV (Bombyx mori nucleopolyhedrovirus) bacmid system combined with confocal microscopy revealed that BmAdipoR was targeted to the cell membrane. We also found that infection with BmNPV did not have an effect on BmAdipoR mRNA quantity in the midgut of susceptible Bombyx mori strain (306) at 48 h, but BmAdipoR mRNA quantity increased significantly at 72 h. We concluded that BmAdipoR gene was a membrane protein ubiquitously expressed in Bombyx mori tissues and that its expression was altered by treating with BmNPV.