Drosophila Ecdysone Research Articles

The Drosophila ecdysone receptor promotes or suppresses proliferation according to ligand level.

The steroid hormone 20-hydroxy-ecdysone (20E) promotes proliferation in Drosophila wing precursors at low titer but triggers proliferation arrest at high doses. Remarkably, wing precursors proliferate normally in the complete absence of the 20E receptor, suggesting that low-level 20E promotes proliferation by overriding the default anti-proliferative activity of the receptor. By contrast, 20E needs its receptor to arrest proliferation. Dose-response RNA sequencing (RNA-seq) analysis of exvivo cultured wing precursors identifies genes that are quantitatively activated by 20E across the physiological range, likely comprising positive modulators of proliferation and other genes that are only activated at high doses. We suggest that some of these "high-threshold" genes dominantly suppress the activity of the pro-proliferation genes. We then show mathematically and with synthetic reporters that combinations of basic regulatory elements can recapitulate the behavior of both types of target genes. Thus, a relatively simple genetic circuit can account for the bimodal activity of this hormone.

Essential functions of mosquito ecdysone importers in development and reproduction

The primary insect steroid hormone ecdysone requires a membrane transporter to enter its target cells. Although an organic anion-transporting polypeptide (OATP) named Ecdysone Importer (EcI) serves this role in the fruit fly Drosophila melanogaster and most likely in other arthropod species, this highly conserved transporter is apparently missing in mosquitoes. Here we report three additional OATPs that facilitate cellular incorporation of ecdysone in Drosophila and the yellow fever mosquito Aedes aegypti. These additional ecdysone importers (EcI-2, -3, and -4) are dispensable for development and reproduction in Drosophila, consistent with the predominant role of EcI. In contrast, in Aedes, EcI-2 is indispensable for ecdysone-mediated development, whereas EcI-4 is critical for vitellogenesis induced by ecdysone in adult females. Altogether, our results indicate unique and essential functions of these additional ecdysone importers in mosquito development and reproduction, making them attractive molecular targets for species- and stage-specific control of ecdysone signaling in mosquitoes.

Drosophila ecdysone receptor activity-based ex vivo assay to assess the endocrine disruption potential of environmental chemicals.

Insect pollinators, critical for both agricultural output and the ecosystem, are declining at an alarming levels partly due to human-made chemicals. Majority of environmental chemicals hamper the endocrine function and studies on the same in insects remain neglected. Here, we report a Drosophila-based ex vivo assay system that employs a reproductive tissue from transgenic males carrying a reporter gene (lacZ) downstream of ecdysone receptor response element (EcRE) and permits the evaluation of chemical-mediated activity modulation of all three isoforms of ecdysone receptor, which are critical for male fertility. We show agonistic [plasticizers, cypermethrin, atrazine, methyl parathion, imidacloprid, cadmium chloride, mercuric chloride or 3-(4-methylbenzylidene) camphor] or antagonistic (apigenin, tributyltin chloride) effects or lack of effect thereof (rutin hydrate, dichlorvos, lead acetate, parabens) for seven different classes of environmental chemicals on ecdysone receptor activity reflecting the specificity and sensitivity of the developed ex vivo assay. Exposure to a few of these chemicals in vivo hampers the fertility of Drosophila males, thus linking the observed endocrine disruption to a quantifiable reproductive phenotype. The developed ex vivo assay offers a quick Drosophila-based screening tool for throughput monitoring of environmental chemicals for their ability to hamper the endocrine function of insect pollinators and other invertebrates.

Direct and widespread role for the nuclear receptor EcR in mediating the response to ecdysone in Drosophila

The ecdysone pathway was among the first experimental systems employed to study the impact of steroid hormones on the genome. In Drosophila and other insects, ecdysone coordinates developmental transitions, including wholesale transformation of the larva into the adult during metamorphosis. Like other hormones, ecdysone controls gene expression through a nuclear receptor, which functions as a ligand-dependent transcription factor. Although it is clear that ecdysone elicits distinct transcriptional responses within its different target tissues, the role of its receptor, EcR, in regulating target gene expression is incompletely understood. In particular, EcR initiates a cascade of transcription factor expression in response to ecdysone, making it unclear which ecdysone-responsive genes are direct EcR targets. Here, we use the larval-to-prepupal transition of developing wings to examine the role of EcR in gene regulation. Genome-wide DNA binding profiles reveal that EcR exhibits widespread binding across the genome, including at many canonical ecdysone response genes. However, the majority of its binding sites reside at genes with wing-specific functions. We also find that EcR binding is temporally dynamic, with thousands of binding sites changing over time. RNA-seq reveals that EcR acts as both a temporal gate to block precocious entry to the next developmental stage as well as a temporal trigger to promote the subsequent program. Finally, transgenic reporter analysis indicates that EcR regulates not only temporal changes in target enhancer activity but also spatial patterns. Together, these studies define EcR as a multipurpose, direct regulator of gene expression, greatly expanding its role in coordinating developmental transitions.

A putative direct repeat element plays a dual role in the induction and repression of insect vitellogenin-1 gene expression.

Juvenile hormones (JH) regulate wide-ranging physiological and developmental processes in insects. However, molecular mechanisms underlying JH signaling remain to be determined. Vitellogenin (Vg) is primarily an egg-yolk protein, but recently proposed to serve many functions in insects. In the female American cockroach (Periplaneta americana), vitellogenin (Vg) genes are activated by JH III and suppressed by 20-hydroxyecdysone (20E) via cis-regulatory elements in a dose-dependent manner. In the present study, the upstream promoter region (935 bp) of Vg1 was cloned to elucidate the action of these hormones. A luciferase reporter assay identified an 81 bp region in the promoter region of Vg1 (-120 to -39 bp) that we found to be critical for JH III activation and 20E suppression. This 81 bp region contains a direct repeat separated by a 2-nucleotide spacer-designated Vg1HRE- that is similar to the Drosophila ecdysone response element direct repeat 4. Moreover, nuclear proteins isolated from nymphs, males, females, and Sf9 cells successfully bound to Vg1HRE, while binding was outcompeted by a 100-fold excess of cold probe or dephosphorylated nuclear protein extracts. In addition, binding was outcompeted by other ecdysone and JH response elements with similar half-site sequences (direct repeats) but to varying extents. Ultimately, we postulate that JH III indirectly activates Vg expression by interfering with or inhibiting the phosphorylation of nuclear proteins bound to Vg1HRE. Involvement of JH III in both induction of Vg1 and control of nuclear proteins binding to Vg1HRE suggest the latter to play an important role in JH signaling.

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness.

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene switches requires the transfer of two therapeutic expression units concurrently into a single cell, the practical application of the system for gene therapy is limited. To simplify the transgene expression system, we generated a gene switch designated as pEUI(+) encompassing a complete set of transgene expression modules in a single vector. Comprising of the GAL4 DNA-binding domain and modified EcR (GvEcR), a minimal VP16 activation domain fused with a GAL4 DNA-binding domain, as well as a modified Drosophila ecdysone receptor (EcR), the newly developed singular gene switch is highly responsive to the administration of a chemical inducer in a time- and dosage-dependent manner. The pEUI(+) vector is a potentially powerful tool for improving the control of transgene expression in both biological research and pre-clinical studies. Here, we present a detailed protocol for modulation of a transient and stable transgene expression using pEUI(+) vector by the treatment of tebufenozide (Teb). Additionally, we share important guidelines for the use of Teb as a chemical inducer.

Ecdysone Receptor-based Singular Gene Switches for Regulated Transgene Expression in Cells and Adult Rodent Tissues.

Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet)-regulated system. Exploiting a Drosophila ecdysone receptor (EcR)-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+) and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site). Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion.

Glial Cells Missing Gene Upregulated by Ecdysone in Drosophila

The Drosophilaglial cells missing (gcm) gene is not only essential for generating embryonic glial differentiation but also necessary and sufficient for generating glial cells during the postembryonic stage. However, the mechanisms of how the gcm gene is mediated are still elusive. This study reveals that gcm was expressed with fluctuating variation during the third instar larval and pupal stage, the 20-hydroxyecdysone (20E) treatment can upregulate gcm expression, the knockdown of EcR-A and USP1 led to a reduced transcript level of gcm in S2 cells. These results suggest that the 20E signaling pathway can mediate gcm expression through the 20E receptor EcR-A and its heterodimer USP1.

Ecdysone Control of Developmental Transitions: Lessons from Drosophila Research

The steroid hormone ecdysone is the central regulator of insect developmental transitions. Recent new advances in our understanding of ecdysone action have relied heavily on the application of Drosophila melanogaster molecular genetic tools to study insect metamorphosis. In this review, we focus on three major aspects of Drosophila ecdysone biology: (a) factors that regulate the timing of ecdysone release, (b) molecular basis of stage- and tissue-specific responses to ecdysone, and (c) feedback regulation and coordination of ecdysone signaling.

Ecdysone- and NO-Mediated Gene Regulation by Competing EcR/Usp and E75A Nuclear Receptors during Drosophila Development

The Drosophila ecdysone receptor (EcR/Usp) is thought to activate or repress gene transcription depending on the presence or absence, respectively, of the hormone ecdysone. Unexpectedly, we found an alternative mechanism at work in salivary glands during the ecdysone-dependent transition from larvae to pupae. In the absense of ecdysone, both ecdysone receptor subunits localize to the cytoplasm, and the heme-binding nuclear receptor E75A replaces EcR/Usp at common target sequences in several genes. During the larval-pupal transition, a switch from gene activation by EcR/Usp to gene repression by E75A is triggered by a decrease in ecdysone concentration and by direct repression of the EcR gene by E75A. Additional control is provided by developmentally timed modulation of E75A activity by NO, which inhibits recruitment of the corepressor SMRTER. These results suggest a mechanism for sequential modulation of gene expression during development by competing nuclear receptors and their effector molecules, ecdysone and NO.

Post-transcriptionally Regulated Expression System in Human Xenogeneic Transplantation Models

Cells have developed a mechanism to discriminate between premature termination codons (PTCs) and normal stop codons during translation, sparking vigorous research to develop drugs promoting readthrough at PTCs to treat genetic disorders caused by PTCs. It was posed that this concept could also be applied to regulated gene therapy protocols by incorporating a PTC into a therapeutic gene, so active protein would only be made after administration of a readthrough agent. The strengths of the system are highlighted here by results demonstrating: (i) background expression levels were reduced to 0.01% to 0.0005% of wild type in unselected mass populations of cells depending upon the specific stop codon utilized and its position within the gene; (ii) expression levels responded well to multiple "On" and "Off" regulation cycles in vivo in human xenograft systems; (iii) the level of induction approached three logs using aminoglycoside activators including NB54, a newly synthesized aminoglycoside with significantly reduced toxicity; and (iv) expression levels could be appreciably altered when employing different promoters in a variety of cell types. These results strongly support the contention that this system should have important clinical applications when tight control of gene expression is required.

The cholesterol trafficking protein NPC1 is required for Drosophila spermatogenesis

Niemann–Pick C (NPC) disease is a lethal neurodegenerative disorder affecting cellular sterol trafficking. Besides neurodegeneration, NPC patients also exhibit other pleiotropic conditions, indicating that NPC protein is required for other physiological processes. Previous studies indicated that a sterol shortage that in turn leads to a shortage of steroid hormones (for example, ecdysone in Drosophila) is likely to be the cause of NPC disease pathology. We have shown that mutations in Drosophila npc1, one of the two NPC disease-related genes, leads to larval lethal and male infertility. Here, we reported that npc1 mutants are defective in spermatogenesis and in particular in the membrane-remodeling individualization process. Interestingly, we found that ecdysone, the steroid hormone responsible for the larval lethal phenotype in npc1 mutants, is not required for individualization. However, supplying 7-dehydrocholesterol can partially rescue the male infertility of npc1 mutants, suggesting that a sterol shortage is responsible for the spermatogenesis defects. In addition, the individualization defects of npc1 mutants were enhanced at high temperature, suggesting that the sterol shortage may lead to temperature-sensitive defects in the membrane-remodeling process. Together, our study reveals a sterol-dependent, ecdysone-independent mechanism of NPC1 function in Drosophila spermatogenesis.

Revisiting the COP9 signalosome as a transcriptional regulator

The COP9 signalosome (CSN) is a highly conserved protein complex that was originally described as a repressor of light-dependent growth and transcription in Arabidopsis. The most studied CSN function is the regulation of protein degradation, which occurs primarily through the removal of the ubiquitin-like modifier Nedd8 from cullin-based E3 ubiquitin ligases. This activity can regulate transcription-factor stability and, therefore, transcriptional activity. Recent data suggest that the CSN also regulates transcription on the chromatin by mechanisms that are not yet clearly understood. Furthermore, the CSN subunits CSN5 and CSN2 seem to act as transcriptional coactivators and corepressors, respectively. Here, I re-evaluate the mechanisms by which the CSN acts as a transcriptional regulator, and suggest that they could extend beyond the regulation of protein stability.

Clearing the Way for Unpaused Polymerases

Heat shock loci in the polytene chromosomes of the fruit fly Drosophila undergo a characteristic change in appearance that coincides with the onset of gene expression. Petesch and Lis (2008) now show that nucleosomes are lost across the entire Hsp70 locus in an initial wave that precedes transcription by RNA polymerase II.

Specific transcriptional responses to juvenile hormone and ecdysone in Drosophila

Previous studies have shown that ecdysone (E), and its immediate downstream product 20-hydroxyecdysone (20E), can have different biological functions in insects, suggesting that E acts as a distinct hormone. Here, we use Drosophila larval organ culture in combination with microarray technology to identify genes that are transcriptionally regulated by E, but which show little or no response to 20E. These genes are coordinately expressed for a brief temporal interval at the onset of metamorphosis, suggesting that E acts together with 20E to direct puparium formation. We also show that E74B, pepck, and CG14949 can be induced by juvenile hormone III (JH III) in organ culture, and that CG14949 can be induced by JH independently of protein synthesis. In contrast, E74A and E75A show no response to JH in this system. These studies demonstrate that larval organ culture can be used to identify Drosophila genes that are regulated by hormones other than 20E, and provide a basis for studying crosstalk between multiple hormone signaling pathways.

Isoform specific control of gene activity in vivo by the Drosophila ecdysone receptor

The steroid hormone 20-hydroxyecdysone induces metamorphosis in insects. The receptor for the hormone is the ecdysone receptor, a heterodimer of two nuclear receptors, EcR and USP. In Drosophila the EcR gene encodes 3 isoforms (EcR-A, EcR-B1 and EcR-B2) that vary in their N-terminal region but not in their DNA binding and ligand binding domains. The stage and tissue specific distribution of the isoforms during metamorphosis suggests distinct functions for the different isoforms. By over-expressing the three isoforms in animals we present results supporting this hypothesis. We tested for the ability of the different isoforms to rescue the lack of dendritic pruning that is characteristic of mutants lacking both EcR-B1 and EcR-B2. By expressing the different isoforms specifically in the affected neurons, we found that both EcR-B isoforms were able to rescue the neuronal defect cell autonomously, but that EcR-A was less effective. We also analyzed the effect of over-expressing the isoforms in a wild-type background. We determined a sensitive period when high levels of either EcR-B isoform were lethal, indicating that the low levels of EcR-B at this time are crucial to ensure normal development. Over-expressing EcR-A in contrast had no detrimental effect. However, high levels of EcR-A expressed in the posterior compartment suppressed puparial tanning, and resulted in down-regulation of some of the tested target genes in the posterior compartment of the wing disc. EcR-B1 or EcR-B2 over-expression had little or no effect.

Coordinate regulation of small temporal RNAs at the onset of Drosophila metamorphosis

The lin-4 and let-7 small temporal RNAs play a central role in controlling the timing of Caenorhabditis elegans cell fate decisions. let-7 has been conserved through evolution, and its expression correlates with adult development in bilateral animals, including Drosophila [Nature 408 (2000), 86]. The best match for lin-4 in Drosophila, miR-125, is also expressed during pupal and adult stages of Drosophila development [Curr. Biol. 12 (2002), 735]. Here, we ask whether the steroid hormone ecdysone induces let-7 or miR-125 expression at the onset of metamorphosis, attempting to link a known temporal regulator in Drosophila with the heterochronic pathway defined in C. elegans. We find that let-7 and miR-125 are coordinately expressed in late larvae and prepupae, in synchrony with the high titer ecdysone pulses that initiate metamorphosis. Unexpectedly, however, their expression is neither dependent on the EcR ecdysone receptor nor inducible by ecdysone in cultured larval organs. Although let-7 and miR-125 can be induced by ecdysone in Kc tissue culture cells, their expression is significantly delayed relative to that seen in the animal. let-7 and miR-125 are encoded adjacent to one another in the genome, and their induction correlates with the transient appearance of an ∼500-nt RNA transcribed from this region, providing a mechanism to explain their precise coordinate regulation. We conclude that a common precursor RNA containing both let-7 and miR-125 is induced independently of ecdysone in Drosophila, raising the possibility of a temporal signal that is distinct from the well-characterized ecdysone–EcR pathway.

The Expression of the let-7 Small Regulatory RNA Is Controlled by Ecdysone during Metamorphosis in Drosophila melanogaster

In Caenorhabditis elegans, the heterochronic pathway controls the timing of developmental events during the larval stages. A component of this pathway, the let-7 small regulatory RNA, is expressed at the late stages of development and promotes the transition from larval to adult (L/A) stages. The stage-specificity of let-7 expression, which is crucial for the proper timing of the worm L/A transition, is conserved in Drosophila melanogaster and other invertebrates. In Drosophila, pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) control the timing of the transition from larval to pupal to adult stages. To test whether let-7 expression is regulated by ecdysone in Drosophila, we used Northern blot analysis to examine the effect of altered ecdysone levels on let-7 expression in mutant animals, organ cultures, and S2 cultured cells. Experiments were conducted to test the role of Broad-Complex (BR-C), an essential component in the ecdysone pathway, in let-7 expression. We show that ecdysone and BR-C are required for let-7 expression, indicating that the ecdysone pathway regulates the temporal expression of let-7 in Drosophila. These results demonstrate an interaction between steroid hormone signaling and the heterochronic pathway in insects.

Dirofilaria immitis encodes Di- nhr- 7, a putative orthologue of the Drosophila ecdysone-regulated E78 gene

Filarial parasites are responsible for several serious human diseases with symptoms such as lymphoedema, elephantiasis, and blindness. An understanding of how these parasites pass through developmental checkpoints may elucidate the general mechanisms of these illnesses and suggest potential targets for intervention. A useful model system for the study of human filariasis is the related nematode Dirofilaria immitis, the causative agent of dog heartworm disease. In D. immitis, molting from the third to the fourth larval stage can be induced in vitro by the insect hormone 20-OH ecdysone, suggesting that ecdysone, or some related hormone, may play a similar role in the development of D. immitis. Ecdysone has a well-characterized developmental role in insects, where it is involved in the control of molting and metamorphosis. We have identified a D. immitis orthologue of the Drosophila ecdysone response early gene E78, a member of the nuclear receptor (NR) superfamily. The D. immitis gene, Di- nhr- 7 (NR1E1) encodes at least three isoforms, including two potential negative regulatory isoforms, and is expressed in a sex-specific manner. An MBP/ Di-NHR-7 fusion protein is able to bind to DNA response elements that are recognized by the closely related mammalian NR Rev-erbα.

Differential control of gene activity by isoforms A, B1 and B2 of the Drosophila ecdysone receptor

Differential control of gene activity by isoforms A, B1 and B2 of the Drosophila ecdysone receptor