Search

We investigate the contribution of a candidate gene, fiz (fezzik), to complex polygenic adaptation to juvenile malnutrition in Drosophila melanogaster. Experimental populations maintained for >250 generations of experimental evolution to a nutritionally poor larval diet (Selected populations) evolved several-fold lower fiz expression compared to unselected Control populations. Here we show that this divergence in fiz expression is mediated by a cis-regulatory polymorphism. This polymorphism, originally sampled from a natural population in Switzerland, is distinct from a second cis-regulatory SNP previously identified in non-African D. melanogaster populations, implying that two independent cis-regulatory variants promoting high fiz expression segregate in non-African populations. Enzymatic analyses of Fiz protein expressed in E. coli demonstrate that it has ecdysone oxidase activity acting on both ecdysone and 20-hydroxyecdysone. Four of five fiz paralogs annotated to ecdysteroid metabolism also show reduced expression in Selected larvae, implying that malnutrition-driven selection favored general downregulation of ecdysone oxidases. Finally, as an independent test of the role of fiz in poor diet adaptation, we show that fiz knockdown by RNAi results in faster larval growth on the poor diet, but at the cost of greatly reduced survival. These results imply that downregulation of fiz in Selected populations was favored by selection on the nutritionally poor diet because of its role in suppressing growth in response to nutrient shortage. However, they suggest that fiz downregulation is only adaptive in combination with other changes evolved by Selected populations, which ensure that the organism can sustain the faster growth promoted by fiz downregulation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Cavigliasso et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Specifically timed pulses of the moulting hormone ecdysone are necessary for developmental progression in insects, guiding development through important milestones such as larval moults, pupation and metamorphosis. It also coordinates the acquisition of cell identities, known as cell patterning, and growth in a tissue-specific manner. In the absence of ecdysone, the ecdysone receptor heterodimer Ecdysone Receptor and Ultraspiracle represses expression of target primary response genes, which become de-repressed as the ecdysone titre rises. However, ecdysone signalling elicits both repressive and activating responses in a temporal and tissue-specific manner. To understand how ecdysone achieves such specificity, this review explores the layers of gene regulation involved in stage-appropriate ecdysone responses in Drosophila fruit flies., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Previous studies have shown that selection for starvation resistance in Drosophila melanogaster results in delayed eclosion and increased adult fat stores. It is assumed that these traits are caused by the starvation selection pressure, but its mechanism is unknown. We found that our starvation-selected (SS) population stores more fat during larval development and has extended larval development and pupal development time. Developmental checkpoints in the third instar associated with ecdysteroid hormone pulses are increasingly delayed. The delay in the late larval period seen in the SS population is indicative of reduced and delayed ecdysone signaling. An enzyme immunoassay for ecdysteroids (with greatest affinity to the metabolically active 20-hydroxyecdysone and the α-ecdysone precursor) confirmed that the SS population had reduced and delayed hormone production compared with that of fed control (FC) flies. Feeding third instar larvae on food supplemented with α-ecdysone partially rescued the developmental delay and reduced subsequent adult starvation resistance. This work suggests that starvation selection causes reduced and delayed production of ecdysteroids in the larval stage and affects the developmental delay phenotype that contributes to subsequent adult fat storage and starvation resistance., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

TAIMAN (TAI), the only insect ortholog of mammalian Steroid Receptor Coactivators (SRCs), is a critical modulator of ecdysone and juvenile hormone (JH) signaling pathways, which govern insect development and reproduction. The modulatory effect is mediated by JH-dependent TAI's heterodimerization with JH receptor Methoprene-tolerant and association with the Ecdysone Receptor complex. Insect hormones regulate insect physiology and development in concert with abiotic cues, such as photo- and thermoperiod. Here we tested the effects of JH and ecdysone signaling on the circadian clock by a combination of microsurgical operations, application of hormones and hormone mimics, and gene knockdowns in the linden bug Pyrrhocoris apterus males. Silencing taiman by each of three non-overlapping double-strand RNA fragments dramatically slowed the free-running period (FRP) to 27-29 hours, contrasting to 24 hours in controls. To further corroborate TAIMAN's clock modulatory function in the insect circadian clock, we performed taiman knockdown in the cockroach Blattella germanica. Although Blattella and Pyrrhocoris lineages separated ~380 mya, B. germanica taiman silencing slowed the FRP by more than 2 hours, suggesting a conserved TAI clock function in (at least) some insect groups. Interestingly, the pace of the linden bug circadian clock was neither changed by blocking JH and ecdysone synthesis, by application of the hormones or their mimics nor by the knockdown of corresponding hormone receptors. Our results promote TAI as a new circadian clock modulator, a role described for the first time in insects. We speculate that TAI participation in the clock is congruent with the mammalian SRC-2 role in orchestrating metabolism and circadian rhythms, and that TAI/SRCs might be conserved components of the circadian clock in animals., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Smykal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In prostate cancer, loss of the tumour suppressor gene, Retinoblastoma (Rb), and consequent activation of transcription factor E2F1 typically occurs at a late-stage of tumour progression. It appears to regulate a switch to an androgen-independent form of cancer, castration-resistant prostate cancer (CRPC), which frequently still requires androgen receptor (AR) signalling. We have previously shown that upon mating, binucleate secondary cells (SCs) of the Drosophila melanogaster male accessory gland (AG), which share some similarities with prostate epithelial cells, switch their growth regulation from a steroid-dependent to a steroid-independent form of Ecdysone Receptor (EcR) control. This physiological change induces genome endoreplication and allows SCs to rapidly replenish their secretory compartments, even when ecdysone levels are low because the male has not previously been exposed to females. Here, we test whether the Drosophila Rb homologue, Rbf, and E2F1 regulate this switch. Surprisingly, we find that excess Rbf activity reversibly suppresses binucleation in adult SCs. We also demonstrate that Rbf, E2F1 and the cell cycle regulators, Cyclin D (CycD) and Cyclin E (CycE), are key regulators of mating-dependent SC endoreplication, as well as SC growth in both virgin and mated males. Importantly, we show that the CycD/Rbf/E2F1 axis requires the EcR, but not ecdysone, to trigger CycE-dependent endoreplication and endoreplication-associated growth in SCs, mirroring changes seen in CRPC. Furthermore, Bone Morphogenetic Protein (BMP) signalling, mediated by the BMP ligand Decapentaplegic (Dpp), intersects with CycD/Rbf/E2F1 signalling to drive endoreplication in these fly cells. Overall, our work reveals a signalling switch, which permits rapid growth of SCs and increased secretion after mating, independently of previous exposure to females. The changes observed share mechanistic parallels with the pathological switch to hormone-independent AR signalling seen in CRPC, suggesting that the latter may reflect the dysregulation of a currently unidentified physiological process., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Sekar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Signaling networks are redeployed across different developmental times and places to generate phenotypic diversity from a limited genetic toolkit. Hormone signaling networks in particular have well-studied roles in multiple developmental processes. In insects, the ecdysone pathway controls critical events in late embryogenesis and throughout post-embryonic development. While this pathway has not been shown to function in the earliest stage of embryonic development in the model insect Drosophila melanogaster, one component of the network, the nuclear receptor E75A, is necessary for proper segment generation in the milkweed bug Oncopeltus fasciatus. Published expression data from several other species suggests possible conservation of this role across hundreds of millions of years of insect evolution. Previous work also demonstrates a second nuclear receptor in the ecdysone pathway, Ftz-F1, plays a role in segmentation in multiple insect species. Here we report tightly linked expression patterns of ftz-F1 and E75A in two hemimetabolous insect species, the German cockroach Blattella germanica and the two-spotted cricket Gryllus bimaculatus. In both species, the genes are expressed segmentally in adjacent cells, but they are never co-expressed. Using parental RNAi, we show the two genes have distinct roles in early embryogenesis. E75A appears necessary for abdominal segmentation in B. germanica, while ftz-F1 is essential for proper germband formation. Our results suggest that the ecdysone network is critical for early embryogenesis in hemimetabolous insects., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

We aimed to explain the reason and function of the successive expression of ecdysone-responsive transcription factors (ERTFs) and related cuticular protein (CP) genes during transformation from larva to pupa. The regulation of the expression of CP genes by ERTFs was examined by in vitro wing disc culture and reporter assay using a gene gun transduction system. Two CP genes that showed expression peaks at different stages-BmorCPG12 at W3L and BmorCPH2 at P0 stage-were selected and examined. Reporter constructs conveying putative BHR3, ßFTZ-F1, BHR39, and E74A binding sites of BmorCPG12 and BmorCPH2 showed promoter activity when introduced into wing discs. In the present study, we showed the functioning of the putative BHR3 and E74A binding sites, together with putative ßFTZ-F1 binding sites, on the activation of CP genes, and different ERTF binding sites functioned in one CP gene. From these, we conclude that BHR3, ßFTZ-F1, and E74A that are successively expressed bring about the successive expression of CP genes, resulting in insect metamorphosis. In addition to this, reporter constructs conveying putative BHR39 binding sites of BmorCPG12 and BmorCPH2 showed negative regulation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Insect metamorphosis is triggered by the production, secretion and degradation of 20-hydroxyecdysone (ecdysone). In addition to its role in developmental regulation, increasing evidence suggests that ecdysone is involved in innate immunity processes, such as phagocytosis and the induction of antimicrobial peptide (AMP) production. AMP regulation includes systemic responses as well as local responses at surface epithelia that contact with the external environment. At pupariation, Drosophila melanogaster increases dramatically the expression of three AMP genes, drosomycin (drs), drosomycin-like 2 (drsl2) and drosomycin-like 5 (drsl5). We show that the systemic action of drs at pupariation is dependent on ecdysone signalling in the fat body and operates via the ecdysone downstream target, Broad. In parallel, ecdysone also regulates local responses, specifically through the activation of drsl2 expression in the gut. Finally, we confirm the relevance of this ecdysone dependent AMP expression for the control of bacterial load by showing that flies lacking drs expression in the fat body have higher bacterial persistence over metamorphosis. In contrast, local responses may be redundant with the systemic effect of drs since reduction of ecdysone signalling or of drsl2 expression has no measurable negative effect on bacterial load control in the pupa. Together, our data emphasize the importance of the association between ecdysone signalling and immunity using in vivo studies and establish a new role for ecdysone at pupariation, which impacts developmental success by regulating the immune system in a stage-dependent manner. We speculate that this co-option of immune effectors by the hormonal system may constitute an anticipatory mechanism to control bacterial numbers in the pupa, at the core of metamorphosis evolution., Competing Interests: The authors have declared that no competing interests exist.

Spodoptera exigua is a worldwide pest afflicting edible vegetables and has developed varying levels of resistance to insecticides. Methoxyfenozide (MET), an ecdysteroid agonist, is effective against lepidopteran pests such as S. exigua . However, the mechanism of MET to S. exigua remains unclear. In this study, we analyzed the expression patterns of genes related to the ecdysone signaling pathway in transcriptome data treated with sublethal doses of MET and analyzed how expression levels of key genes affect the toxicity of MET on S. exigua . Our results demonstrated that 2639 genes were up-regulated and 2512 genes were down-regulated in S. exigua treated with LC 30 of MET. Of these, 15 genes were involved in the ecdysone signaling pathway. qPCR results demonstrated that ecdysone receptor A ( EcRA ) expression levels significantly increased in S. exigua when treated with different doses of MET, and that the RNAi-mediated silencing of EcRA significantly increased mortality to 55.43% at 72 h when L3 S. exigua larvae were exposed to MET at the LC 30 dose. Additionally, knocking down EcRA suppressed the most genes expressed in the ecdysone signaling pathway. The combination of MET and ds EcRA affected the expression of E74 and enhanced the expression of TREA . These results demonstrate that the adverse effects of sublethal MET disturb the ecdysone signaling pathway in S. exigua , and EcRA is closely related to MET toxic effect. This study increases our collective understanding of the mechanisms of MET in insect pests.

Phenotypic plasticity in response to environmental cues is common in butterflies, and is a major driver of butterfly wing pattern diversity. The endocrine signal ecdysone has been revealed as a major modulator of plasticity in butterflies. External cues such as day length or temperature are translated internally into variation in ecdysone titers, which in turn lead to alternate phenotypes such as seasonal wing patterns. Here we review the evidence showing that ecdysone-mediated plasticity of different wing pattern features such as wing color and eyespot size can evolve independently. Recent studies show that ecdysone regulates gene expression in Drosophila melanogaster via a chromatin remodeling mechanism. We thus propose that environmentally responsive ecdysone titers in butterflies may also function via chromatin regulation to promote different seasonal phenotypes. We present a model of ecdysone response evolution that integrates both gene regulatory architecture and organismal development, and propose a set of testable mechanistic hypotheses for how plastic response profiles of specific genes can evolve., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Organogenesis requires exquisite spatiotemporal coordination of cell morphogenesis, migration, proliferation, and differentiation of multiple cell types. For gonads, this involves complex interactions between somatic and germline tissues. During Drosophila ovary morphogenesis, primordial germ cells (PGCs) either are sequestered in stem cell niches and are maintained in an undifferentiated germline stem cell state or transition directly toward differentiation. Here, we identify a mechanism that links hormonal triggers of somatic tissue morphogenesis with PGC differentiation. An early ecdysone pulse initiates somatic swarm cell (SwC) migration, positioning these cells close to PGCs. A second hormone peak activates Torso-like signal in SwCs, which stimulates the Torso receptor tyrosine kinase (RTK) signaling pathway in PGCs promoting their differentiation by de-repression of the differentiation gene, bag of marbles. Thus, systemic temporal cues generate a transitory signaling center that coordinates ovarian morphogenesis with stem cell self-renewal and differentiation programs, highlighting a more general role for such centers in reproductive and developmental biology., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Innate behaviors consist of a succession of genetically-hardwired motor and physiological subprograms that can be coupled to drastic morphogenetic changes. How these integrative responses are orchestrated is not completely understood. Here, we provide insight into these mechanisms by studying pupariation, a multi-step innate behavior of Drosophila larvae that is critical for survival during metamorphosis. We find that the steroid-hormone ecdysone triggers parallel pupariation neuromotor and morphogenetic subprograms, which include the induction of the relaxin-peptide hormone, Dilp8, in the epidermis. Dilp8 acts on six Lgr3-positive thoracic interneurons to couple both subprograms in time and to instruct neuromotor subprogram switching during behavior. Our work reveals that interorgan feedback gates progression between subunits of an innate behavior and points to an ancestral neuromodulatory function of relaxin signaling.

Regeneration of Drosophila imaginal discs, larval precursors to adult tissues, activates a regeneration checkpoint that coordinates regenerative growth with developmental progression. This regeneration checkpoint results from the release of the relaxin-family peptide Dilp8 from regenerating imaginal tissues. Secreted Dilp8 protein is detected within the imaginal disc lumen, in which it is separated from its receptor target Lgr3, which is expressed in the brain and prothoracic gland, by the disc epithelial barrier. Here, we demonstrate that following damage the imaginal disc epithelial barrier limits Dilp8 signaling and the duration of regeneration checkpoint delay. We also find that the barrier becomes increasingly impermeable to the transepithelial diffusion of labeled dextran during the second half of the third instar. This change in barrier permeability is driven by the steroid hormone ecdysone and correlates with changes in localization of Coracle, a component of the septate junctions that is required for the late-larval impermeable epithelial barrier. Based on these observations, we propose that the imaginal disc epithelial barrier regulates the duration of the regenerative checkpoint, providing a mechanism by which tissue function can signal the completion of regeneration., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

In both mammals and insects, steroid hormones play a major role in directing the animal's progression through developmental stages. To maximize fitness outcomes, steroid hormone production is regulated by the environmental conditions experienced by the animal. In insects, the steroid hormone ecdysone mediates transitions between developmental stages and is regulated in response to environmental factors such as nutrition. These environmental signals are communicated to the ecdysone-producing gland via the action of neuropeptide and peptide hormone signalling pathways. While some of these pathways have been well characterized, there is evidence to suggest more signalling pathways than has previously been thought function to control ecdysone production, potentially in response to a greater range of environmental conditions. Here, we review the neuropeptide and peptide hormone signalling pathways known to regulate the production of ecdysone in the model genetic insect Drosophila melanogaster , as well as what is known regarding the environmental signals that trigger these pathways. Areas for future research are highlighted that can further contribute to our overall understanding of the complex orchestration of environmental, physiological and developmental cues that together produce a functioning adult organism.

Highly reproducible tissue development is achieved by robust, time-dependent coordination of cell proliferation and cell death. To study the mechanisms underlying robust tissue growth, we analyzed the developmental process of wing imaginal discs in Drosophila Minute mutants, a series of heterozygous mutants for a ribosomal protein gene. Minute animals show significant developmental delay during the larval period but develop into essentially normal flies, suggesting there exists a mechanism ensuring robust tissue growth during abnormally prolonged developmental time. Surprisingly, we found that both cell death and compensatory cell proliferation were dramatically increased in developing wing pouches of Minute animals. Blocking the cell-turnover by inhibiting cell death resulted in morphological defects, indicating the essential role of cell-turnover in Minute wing morphogenesis. Our analyses showed that Minute wing discs elevate Wg expression and JNK-mediated Dilp8 expression that causes developmental delay, both of which are necessary for the induction of cell-turnover. Furthermore, forced increase in Wg expression together with developmental delay caused by ecdysone depletion induced cell-turnover in the wing pouches of non-Minute animals. Our findings suggest a novel paradigm for robust coordination of tissue growth by cell-turnover, which is induced when developmental time axis is distorted., Competing Interests: The authors have declared that no competing interests exist.

'Developmental robustness' is the ability of biological systems to maintain a stable phenotype despite genetic, environmental or physiological perturbations. In holometabolous insects, accurate patterning and development is guaranteed by alignment of final gene expression patterns in tissues at specific developmental stage such as molting and pupariation, irrespective of individual rate of development. In the present study, we used faster developing Drosophila melanogaster populations that show reduction of ~22% in egg to adult development time. Flies from the faster developing population exhibit phenotype constancy, although significantly small in size. The reduction in development time in faster developing flies is possibly due to coordination between higher ecdysteroid release and higher expression of developmental genes. The two together might be ensuring appropriate pattern formation and early exit at each development stage in the populations selected for faster pre-adult development compared to their ancestral controls. We report that apart from plasticity in the rate of pattern progression, alteration in the level of gene expression may be responsible for pattern integrity even under reduced development time., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020. Published by Elsevier B.V.)

Juvenile hormones (JH) and ecdysone coordinately regulate metamorphosis in Aedes aegypti. We studied the function of an epigenetic regulator and multifunctional transactivator, CREB binding protein (CBP) in A. aegypti. RNAi-mediated knockdown of CBP in Ae. aegypti larvae resulted in suppression of JH primary response gene, Krüppel-homolog 1 (Kr-h1), and induction of primary ecdysone response gene, E93, resulting in multiple effects including early metamorphosis, larval-pupal intermediate formation, mortality and inhibition of compound eye development. RNA sequencing identified hundreds of genes, including JH and ecdysone response genes regulated by CBP. In the presence of JH, CBP upregulates Kr-h1 by acetylating core histones at the Kr-h1 promoter and facilitating the recruitment of JH receptor and other proteins. CBP suppresses metamorphosis regulators, EcR-A, USP-A, BR-C, and E93 through the upregulation of Kr-h1 and E75A. CBP regulates the expression of core eye specification genes including those involved in TGF-β and EGFR signaling. These studies demonstrate that CBP is an essential player in JH and 20E action and regulates metamorphosis and compound eye development in Ae. aegypti., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Insect molting hormone (ecdysteroids) and juvenile hormone regulate molting and metamorphic events in a variety of insect species. Mealybugs undergo sexually dimorphic metamorphosis: males develop into winged adults through non-feeding, pupa-like stages called prepupa and pupa, while females emerge as neotenic wingless adults. We previously demonstrated, in the Japanese mealybug Planococcus kraunhiae (Kuwana), that the juvenile hormone titer is higher in males than in females at the end of the juvenile stage, which suggests that juvenile hormone may regulate male-specific adult morphogenesis. Here, we examined the involvement of ecdysteroids in sexually dimorphic metamorphosis. To estimate ecdysteroid titers, quantitative RT-PCR analyses of four Halloween genes encoding for cytochrome P450 monooxygenases in ecdysteroid biosynthesis, i.e., spook, disembodied, shadow and shade, were performed. Overall, their expression levels peaked before each nymphal molt. Transcript levels of spook, disembodied and shadow, genes that catalyze the steps in ecdysteroid biosynthesis in the prothoracic gland, were higher in males from the middle of the second nymphal instar to adult emergence. In contrast, the expression of shade, which was reported to be involved in the conversion of ecdysone into 20-hydroxyecdysone in peripheral tissues, was similar between males and females. These results suggest that ecdysteroid biosynthesis in the prothoracic gland is more active in males than in females, although the final conversion into 20-hydroxyecdysone occurs at similar levels in both sexes. Moreover, expression profiles of ecdysone response genes, ecdysone receptor and ecdysone-induced protein 75B, were also analyzed. Based on these expression profiles, we propose that the changes in ecdysteroid titer differ between males and females, and that high ecdysteroid titer is essential for directing male adult development., Competing Interests: The authors have declared that no competing interests exist.

The insect steroid hormone ecdysone is a key regulator of oogenesis in Drosophila melanogaster and many other species. Despite the diversity of cellular functions of ecdysone in oogenesis, the molecular regulation of most ecdysone-responsive genes in ovarian cells remains largely unexplored. We performed a functional screen using the UAS/Gal4 system to identify non-coding cis-regulatory elements within well-characterized ecdysone-response genes capable of driving transcription of an indelible reporter in ovarian cells. Using two publicly available transgenic collections (the FlyLight and Vienna Tiles resources), we tested 62 Gal4 drivers corresponding to ecdysone-response genes EcR, usp, E75, br, ftz-f1 and Hr3. We observed 31 lines that were sufficient to drive a UAS-lacZ reporter in discrete cell populations in the ovary. Reporter expression was reproducibly observed in both somatic and germ cells at distinct stages of oogenesis, including those previously characterized as critical points of ecdysone regulation. Our studies identified several useful new reagents, adding to the UAS/Gal4 toolkit available for genetic analysis of oogenesis in Drosophila. Further, our study provides novel insight into the molecular regulation of ecdysone signaling in oogenesis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bisphenol A (BPA) is a representative endocrine disrupting chemical (EDC) that has estrogenic effects in aquatic animals. In recent years, due to the continuing usage of BPA, its analogues have been developed as alternative substances to replace its use. The molting process is a pivotal point in the development and reproduction of crustaceans. However, studies of the effects of EDCs on molting in crustaceans at the molecular level are scarce. In the present study, we examined the acute toxicity of BPA and its analogues bisphenol F (BPF) and S (BPS) to the brackish water flea Diaphanosoma celebensis. We further identified four ecdysteroid pathway - related genes (cyp314a1, EcRA, EcRB, and USP) in D. celebensis, and investigated the transcriptional modulation of these genes during molting and after exposure to BPA and its analogues for 48 h. Sequencing and phylogenetic analyses revealed that these four genes are highly conserved among arthropods and may be involved in development and reproduction in the adult stage. The mRNA expression patterns of cyp314a1, EcRA and USP were matched with the molting cycle, suggesting that these genes play a role in the molting process in the adult stage in cladocerans. Following relative real-time polymerase chain reaction (RT-PCR) analyses, BPA and its analogues were found to modulate the expression of each of these four genes differently, indicating that these compounds can disrupt the normal endocrine system function of D. celebensis. This study improves our understanding of the molecular mode of action of BPA and its analogues in D. celebensis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In many organisms, the regenerative capacity of tissues progressively decreases as development progresses. However, the developmental mechanisms that restrict regenerative potential remain unclear. In Drosophila, wing imaginal discs become unable to regenerate upon damage during the third larval stage (L3). Here, we show that production of ecdysone after larvae reach their critical weight (CW) terminates the window of regenerative potential by acting on a bistable loop composed of two antagonistic Broad-complex/Tramtrack/Bric-à-brac Zinc-finger (ZBTB) genes: chinmo and broad (br). Around mid L3, ecdysone signaling silences chinmo and activates br to switch wing epithelial progenitors from a default self-renewing to a differentiation-prone state. Before mid L3, Chinmo promotes a strong regenerative response upon tissue damage. After mid L3, Br installs a nonpermissive state that represses regeneration. Transient down-regulation of ecdysone signaling or Br in late L3 larvae enhances chinmo expression in damaged cells that regain the capacity to regenerate. This work unveils a mechanism that ties the self-renewing and regenerative potential of epithelial progenitors to developmental progression., Competing Interests: The authors have declared that no competing interests exist.

Crustacea, the subphylum of Arthropoda which dominates the aquatic environment, is of major importance in ecology and fisheries. Here we report the genome sequence of the Pacific white shrimp Litopenaeus vannamei, covering ~1.66 Gb (scaffold N50 605.56 Kb) with 25,596 protein-coding genes and a high proportion of simple sequence repeats (>23.93%). The expansion of genes related to vision and locomotion is probably central to its benthic adaptation. Frequent molting of the shrimp may be explained by an intensified ecdysone signal pathway through gene expansion and positive selection. As an important aquaculture organism, L. vannamei has been subjected to high selection pressure during the past 30 years of breeding, and this has had a considerable impact on its genome. Decoding the L. vannamei genome not only provides an insight into the genetic underpinnings of specific biological processes, but also provides valuable information for enhancing crustacean aquaculture.

Identification of signals for systemic adaption of hormonal regulation would help to understand the crosstalk between cells and environmental cues contributing to growth, metabolic homeostasis and development. Physiological states are controlled by precise pulsatile hormonal release, including endocrine steroids in human and ecdysteroids in insects. We show in Drosophila that regulation of genes that control biosynthesis and signaling of the steroid hormone ecdysone, a central regulator of developmental progress, depends on the extracellular matrix protein Obstructor-A (Obst-A). Ecdysone is produced by the prothoracic gland (PG), where sensory neurons projecting axons from the brain integrate stimuli for endocrine control. By defining the extracellular surface, Obst-A promotes morphogenesis and axonal growth in the PG. This process requires Obst-A-matrix reorganization by Clathrin/Wurst-mediated endocytosis. Our data identifies the extracellular matrix as essential for endocrine ring gland function, which coordinates physiology, axon morphogenesis, and developmental programs. As Obst-A and Wurst homologs are found among all arthropods, we propose that this mechanism is evolutionary conserved., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Dendrite pruning of Drosophila sensory neurons during metamorphosis is induced by the steroid hormone ecdysone through a transcriptional program. In addition, ecdysone activates the eukaryotic initiation factor 4E-binding protein (4E-BP) to inhibit cap-dependent translation initiation. To uncover how efficient translation of ecdysone targets is achieved under these conditions, we assessed the requirements for translation initiation factors during dendrite pruning. We found that the canonical cap-binding complex eIF4F is dispensable for dendrite pruning, but the eIF3 complex and the helicase eIF4A are required, indicating that differential translation initiation mechanisms are operating during dendrite pruning. eIF4A and eIF3 are stringently required for translation of the ecdysone target Mical, and this depends on the 5' UTR of Mical mRNA. Functional analyses indicate that eIF4A regulates eIF3-mRNA interactions in a helicase-dependent manner. We propose that an eIF3-eIF4A-dependent alternative initiation pathway bypasses 4E-BP to ensure adequate translation of ecdysone-induced genes., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The complex patterns of gene expression in metazoans are controlled by selective binding of transcription factors (TFs) to regulatory DNA. To improve the quantitative understanding of this process, we have developed a novel method that uses fluorescence anisotropy measurements in a controlled delivery system to determine TF-DNA binding energies in solution with high sensitivity and throughput. Owing to its large dynamic range, the method, named high performance fluorescence anisotropy (HiP-FA), allows for reliable quantification of both weak and strong binding; binding specificities are calculated on the basis of equilibrium constant measurements for mutational DNA variants. We determine the binding preference landscapes for 26 TFs and measure high absolute affinities, but mostly lower binding specificities than reported by other methods. The revised binding preferences give rise to improved predictions of in vivo TF occupancy and enhancer expression. Our approach provides a powerful new tool for the systems-biological analysis of gene regulation.

Ultraviolet (UV) filters are compounds used to prevent the damage produced by UV radiation in personal care products, plastics, etc. They have been associated with endocrine disruption, showing anti-estrogen activity in vertebrates and altering the ecdysone pathway in invertebrates. Although they have attracted the attention of multiple research teams there is a lack of data about how animals activate detoxification systems, especially in invertebrates. Here, analysis of the effects of two UV filters, benzophenone-3 (BP3) and 4-methylbenzylidene camphor (4MBC), on the transcriptional activity of nine genes covering the three steps of the detoxification process has been performed. Four cytochrome P450 genes belonging to different members of this family, five GST genes, and the multidrug resistance protein 1 (MRP1) gene were studied by RT-PCR to analyze their transcriptional activity in fourth instar larvae exposed to the UV filters for 8 and 24h. The obtained results show a differential response with downregulation of the different Cyp450s tested by 4MBC while BP3 seems not to modify their expression. On the other hand, some of the GST genes were affected by one or other of the filters, showing a less homogenous response. Finally, MRP1 was activated by both filters but at different times. These results demonstrate for first time that UV filters alter the expression of genes involved in the different steps of the detoxification process and that they can be processed by phase I enzymes other than Cyp450s. They also suggest that UV filters affect biotransformation processes, compromising the ability of the individual to respond to chemical stress, so further research is needed to know the extent of the damage that they can produce in the resistance of the cell to chemicals., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Whether common principles regulate the self-renewing potential of neural stem cells (NSCs) throughout the developing central nervous system is still unclear. In the Drosophila ventral nerve cord and central brain, asymmetrically dividing NSCs, called neuroblasts (NBs), progress through a series of sequentially expressed transcription factors that limits self-renewal by silencing a genetic module involving the transcription factor Chinmo. Here, we find that Chinmo also promotes neuroepithelium growth in the optic lobe during early larval stages by boosting symmetric self-renewing divisions while preventing differentiation. Neuroepithelium differentiation in late larvae requires the transcriptional silencing of chinmo by ecdysone, the main steroid hormone, therefore allowing coordination of neural stem cell self-renewal with organismal growth. In contrast, chinmo silencing in NBs is post-transcriptional and does not require ecdysone. Thus, during Drosophila development, humoral cues or tissue-intrinsic temporal specification programs respectively limit self-renewal in different types of neural progenitors through the transcriptional and post-transcriptional regulation of the same transcription factor., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Gene expression variation is a major contributor to phenotypic diversity within species and is thought to play an important role in adaptation. However, examples of adaptive regulatory polymorphism are rare, especially those that have been characterized at both the molecular genetic level and the organismal level. In this study, we perform a functional analysis of the Drosophila melanogaster CG9509 enhancer, a cis-regulatory element that shows evidence of adaptive evolution in populations outside the species' ancestral range in sub-Saharan Africa. Using site-directed mutagenesis and transgenic reporter gene assays, we determined that 3 single nucleotide polymorphisms are responsible for the difference in CG9509 expression that is observed between sub-Saharan African and cosmopolitan populations. Interestingly, while 2 of these variants appear to have been the targets of a selective sweep outside of sub-Saharan Africa, the variant with the largest effect on expression remains polymorphic in cosmopolitan populations, suggesting it may be subject to a different mode of selection. To elucidate the function of CG9509, we performed a series of functional and tolerance assays on flies in which CG9509 expression was disrupted. We found that CG9509 plays a role in larval growth and influences adult body and wing size, as well as wing loading. Furthermore, variation in several of these traits was associated with variation within the CG9509 enhancer. The effect on growth appears to result from a modulation of active ecdysone levels and expression of growth factors. Taken together, our findings suggest that selection acted on 3 sites within the CG9509 enhancer to increase CG9509 expression and, as a result, reduce wing loading as D. melanogaster expanded out of sub-Saharan Africa.

Cell migration is essential during animal development. In the Drosophila ovary, the steroid hormone ecdysone coordinates nutrient sensing, growth, and the timing of morphogenesis events including border cell migration. To identify downstream effectors of ecdysone signaling, we profiled gene expression in wild-type follicle cells compared to cells expressing a dominant negative Ecdysone receptor or its coactivator Taiman. Of approximately 400 genes that showed differences in expression, we validated 16 candidate genes for expression in border and centripetal cells, and demonstrated that seven responded to ectopic ecdysone activation by changing their transcriptional levels. We found a requirement for seven putative targets in effective cell migration, including two other nuclear hormone receptors, a calcyphosine-encoding gene, and a prolyl hydroxylase. Thus, we identified multiple new genetic regulators modulated at the level of transcription that allow cells to interpret information from the environment and coordinate cell migration in vivo., (Copyright © 2017. Published by Elsevier B.V.)

Ecdysteroids, including the biologically active hormone 20-hydroxyecdysone (20E), play essential roles in controlling many developmental and physiological events in insects. Ecdysteroid biosynthesis is achieved by a series of specialized enzymes encoded by the Halloween genes. Recently, a new class of Halloween gene, noppera-bo ( nobo ), encoding a glutathione S -transferase (GST) in dipteran and lepidopteran species, has been identified and characterized. GSTs are well known to conjugate substrates with the reduced form of glutathione (GSH), a bioactive tripeptide composed of glutamate, cysteine, and glycine. We hypothesized that GSH itself is required for ecdysteroid biosynthesis. However, the role of GSH in steroid hormone biosynthesis has not been examined in any organisms. Here, we report phenotypic analysis of a complete loss-of-function mutant in the γ -glutamylcysteine synthetase catalytic subunit ( Gclc ) gene in the fruit fly Drosophila melanogaster Gclc encodes the evolutionarily conserved catalytic component of the enzyme that conjugates glutamate and cysteine in the GSH biosynthesis pathway. Complete Gclc loss-of-function leads to drastic GSH deficiency in the larval body fluid. Gclc mutant animals show a larval-arrest phenotype. Ecdysteroid titer in Gclc mutant larvae decreases, and the larval-arrest phenotype is rescued by oral administration of 20E or cholesterol. Moreover, Gclc mutant animals exhibit abnormal lipid deposition in the prothoracic gland, a steroidogenic organ during larval development. All of these phenotypes are reminiscent to nobo loss-of-function animals. On the other hand, Gclc mutant larvae also exhibit a significant reduction in antioxidant capacity. Consistent with this phenotype, Gclc mutant larvae are more sensitive to oxidative stress response as compared to wild-type. Nevertheless, the ecdysteroid biosynthesis defect in Gclc mutant animals is not associated with loss of antioxidant function. Our data raise the unexpected hypothesis that a primary role of GSH in early D. melanogaster larval development is ecdysteroid biosynthesis, independent from the antioxidant role of GSH., (Copyright © 2017 by the Genetics Society of America.)

Triclosan (TCS) is an antibacterial agent widely used in personal care and consumer products and commonly detected in aquatic ecosystems. In the present study, the effects of TCS on endocrine-related genes of Chironomus riparius aquatic larvae, a reference organism in aquatic toxicology, were evaluated. Twenty-four-hour in vivo exposures at 10µg/L, 100µg/L, and 1000µg/L TCS revealed that this xenobiotic was able to alter the transcriptional activity of ecdysone receptor gene (EcR), the ultraspiracle gene (usp), the estrogen-related receptor gene (ERR), and the E74 early ecdysone-inducible gene, as measured by real-time RT-PCR. Moreover, the hsp70 gene, a heat shock protein gene, was upregulated after exposure to TCS. The results of the present work provide the first evidence of the potential disruptive effects of TCS in endocrine-related genes suggesting a mode of action that mimics ecdysteroid hormones in insects., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A new purple quail-like (q-lp) mutant found from the plain silkworm strain 932VR has pigment dots on the epidermis similar to the pigment mutant quail (q). In addition, q-lp mutant larvae are inactive, consume little and grow slowly, with a high death rate and other developmental abnormalities. Pigmentation of the silkworm epidermis consists of melanin, ommochrome and pteridine. Silkworm development is regulated by ecdysone and juvenile hormone. In this study, we performed RNA-Seq on the epidermis of the q-lp mutant in the 4th instar during molting, with 932VR serving as the control. The results showed 515 differentially expressed genes, of which 234 were upregulated and 281 downregulated in q-lp. BLASTGO analysis indicated that the downregulated genes mainly encode protein-binding proteins, membrane components, oxidation/reduction enzymes, and proteolytic enzymes, whereas the upregulated genes largely encode cuticle structural constituents, membrane components, transport related proteins, and protein-binding proteins. Quantitative reverse transcription PCR was used to verify the accuracy of the RNA-Seq data, focusing on key genes for biosynthesis of the three pigments and chitin as well as genes encoding cuticular proteins and several related nuclear receptors, which are thought to play key roles in the q-lp mutant. We drew three conclusions based on the results: 1) melanin, ommochrome and pteridine pigments are all increased in the q-lp mutant; 2) more cuticle proteins are expressed in q-lp than in 932VR, and the number of upregulated cuticular genes is significantly greater than downregulated genes; 3) the downstream pathway regulated by ecdysone is blocked in the q-lp mutant. Our research findings lay the foundation for further research on the developmental changes responsible for the q-lp mutant.

In insects, juvenile hormone (JH) and 20-hydroxyecdysone (20E) regulate larval growth and molting. However, little is known about how this cooperative control is terminating larval diapause especially in the bamboo borer, Omphisa fuscidentalis. In both in vivo and in vitro experiments, we here measured the expression levels of genes which were affected by juvenile hormone analogue (JHA: S-methoprene) and 20-hydroxyecdysone (20E) in diapausing O. fuscidentalis larvae. Corresponding mRNA expression changes in the subesophageal ganglion (SG) and prothoracic gland (PG) were evaluated using qRT-PCR. The data showed similar response patterns of JH receptor gene (OfMet), diapause hormone gene (OfDH-PBAN), ecdysone receptor genes (OfEcR-A and OfEcR-B1) and ecdysone inducible genes (OfBr-C, OfE75A, OfE75B, OfE75C and OfHR3). JHA induced the expressions of OfMet and OfDH-PBAN in both SG and PG, whereas ecdysone receptor genes and ecdysone inducible genes were induced by JHA only in PG. For 20E treatment group, expressions of ecdysone receptor genes and ecdysone inducible genes in both SG and PG were increased by 20E injection. In addition, the in vitro experiments showed that OfMet and OfDH-PBAN were up-regulated by JHA alone, but ecdysone receptor genes and ecdysone inducible genes were up-regulated by JHA and 20E. However, OfMet and OfDH-PBAN in the SG was expressed faster than OfMet and OfDH-PBAN in the PG and the expression of ecdysone receptor genes and ecdysone inducible genes induced by JHA was much later than observed for 20E. These results indicate that JHA might stimulate the PG indirectly via factors (OfMet and OfDH-PBAN) in the SG, which might be a regulatory mechanism for larval diapause termination in O. fuscidentalis.

In Drosophila melanogaster larvae, the ring gland (RG) is a control center that orchestrates major developmental transitions. It is a composite organ, consisting of the prothoracic gland, the corpus allatum, and the corpora cardiaca, each of which synthesizes and secretes a different hormone. Until now, the RG's broader developmental roles beyond endocrine secretion have not been explored. RNA sequencing and analysis of a new transcriptome resource from D. melanogaster wandering third instar larval RGs has provided a fascinating insight into the diversity of developmental signaling in this organ. We have found strong enrichment of expression of two gene pathways not previously associated with the RG: immune response and fatty acid metabolism. We have also uncovered strong expression for many uncharacterized genes. Additionally, RNA interference against RG-enriched cytochrome p450s Cyp6u1 and Cyp6g2 produced a lethal ecdysone deficiency and a juvenile hormone deficiency, respectively, flagging a critical role for these genes in hormone synthesis. This transcriptome provides a valuable new resource for investigation of roles played by the RG in governing insect development., (Copyright © 2017 Christesen et al.)

Many animals have an intrinsic growth checkpoint during juvenile development, after which an irreversible decision is made to upregulate steroidogenesis, triggering the metamorphic juvenile-to-adult transition. However, a molecular process underlying such a critical developmental decision remains obscure. Here we show that nutrient-dependent endocycling in steroidogenic cells provides the machinery necessary for irreversible activation of metamorphosis in Drosophila melanogaster. Endocycle progression in cells of the prothoracic gland (PG) is tightly coupled with the growth checkpoint, and block of endocycle in PG cells causes larval developmental arrest due to reduction in biosynthesis of the steroid hormone ecdysone. Moreover, inhibition of the nutrient sensor target of rapamycin (TOR) in the PG during the checkpoint period causes endocycle inhibition and developmental arrest, which can be rescued by inducing additional rounds of endocycles by Cyclin E. We propose that a TOR-mediated cell cycle checkpoint in steroidogenic tissue provides a systemic growth checkpoint for reproductive maturation., Competing Interests: The authors have declared that no competing interests exist.

The development of niches for tissue-specific stem cells is an important aspect of stem cell biology. Determination of niche size and niche numbers during organogenesis involves precise control of gene expression. How this is achieved in the context of a complex chromatin landscape is largely unknown. Here we show that the nuclear protein Combgap (Cg) supports correct ovarian niche formation in Drosophila by controlling ecdysone-Receptor (EcR)- mediated transcription and long-range chromatin contacts in the broad locus (BR-C). Both cg and BR-C promote ovarian growth and the development of niches for germ line stem cells. BR-C levels were lower when Combgap was either reduced or over-expressed, indicating an intricate regulation of the BR-C locus by Combgap. Polytene chromosome stains showed that Cg co-localizes with EcR, the major regulator of BR-C, at the BR-C locus and that EcR binding to chromatin was sensitive to changes in Cg levels. Proximity ligation assay indicated that the two proteins could reside in the same complex. Finally, chromatin conformation analysis revealed that EcR-bound regions within BR-C, which span ~30 KBs, contacted each other. Significantly, these contacts were stabilized in an ecdysone- and Combgap-dependent manner. Together, these results highlight Combgap as a novel regulator of chromatin structure that promotes transcription of ecdysone target genes and ovarian niche formation., Competing Interests: The authors have declared that no competing interests exist.

Damage to Drosophila melanogaster imaginal discs activates a regeneration checkpoint that (1) extends larval development and (2) coordinates the regeneration of the damaged disc with the growth of undamaged discs. These two systemic responses to damage are both mediated by Dilp8, a member of the insulin/insulin-like growth factor/relaxin family of peptide hormones, which is released by regenerating imaginal discs. Growth coordination between regenerating and undamaged imaginal discs is dependent on Dilp8 activation of nitric oxide synthase (NOS) in the prothoracic gland (PG), which slows the growth of undamaged discs by limiting ecdysone synthesis. Here we demonstrate that the Drosophila relaxin receptor homolog Lgr3, a leucine-rich repeat-containing G-protein-coupled receptor, is required for Dilp8-dependent growth coordination and developmental delay during the regeneration checkpoint. Lgr3 regulates these responses to damage via distinct mechanisms in different tissues. Using tissue-specific RNA-interference disruption of Lgr3 expression, we show that Lgr3 functions in the PG upstream of NOS, and is necessary for NOS activation and growth coordination during the regeneration checkpoint. When Lgr3 is depleted from neurons, imaginal disc damage no longer produces either developmental delay or growth inhibition. To reconcile these discrete tissue requirements for Lgr3 during regenerative growth coordination, we demonstrate that Lgr3 activity in both the CNS and PG is necessary for NOS activation in the PG following damage. Together, these results identify new roles for a relaxin receptor in mediating damage signaling to regulate growth and developmental timing., (Copyright © 2016 by the Genetics Society of America.)

Steroid hormone 20-hydroxyecdysone is known as the systemic regulators of insect cells; however, how to impact the fate and function of mature and stem cells is unclear. For the first time, we report ecdysone regulatory cascades in both mature midgut cell and stem cell fractions related to developmental events by using histological, immunohistochemical, biochemical and gene expression analysis methods. Ecdysone receptor-B1 (EcR-B1) and ultraspiracle 1 (USP-1) mRNAs were detected mainly in mature cells during programmed cell death (PCD). Lowered E75A and probably BR-C Z4 in mature cells appear to provide a signal to the initiation of PCD. E74B, E75B and BR-C Z2 seem to be early response genes which are involved in preparatory phase of cell death. It is likely that βFTZ-F1, E74A and BR-C Z1 are probably associated with execution of death. EcR-A and USP2 mRNAs were found in stem cells during remodeling processes but EcR-B1, USP1 and E74B genes imply an important role during initial phase of metamorphic events in stem cells. BHR3 mRNAs were determined abundantly in stem cells suggesting its primary role in differentiation. All of these results showed the determination the cell fate in Bombyx mori (Linnaeus) midgut depends on type of ecdysone receptor isoforms and ecdysone-related transcription factors., (© The Author 2016. Published by Oxford University Press on behalf of the Entomological Society of America.)

RNA interference (RNAi) is a robust tool to study gene functions as well as potential for insect pest control. Finding suitable target genes is the key step in the development of an efficient RNAi-mediated pest control technique. Based on the transcriptome of Chilo suppressalis, 24 unigenes which putatively associated with insect hormone biosynthesis were identified. Amongst these, four genes involved in ecdysteroidogenesis i.e., ptth, torso, spook and nm-g were evaluated as candidate targets for function study. The partial cDNA of these four genes were cloned and their bacterially expressed dsRNA were fed to the insects. Results revealed a significant reduction in mRNA abundance of target genes after 3 days. Furthermore, knocked down of these four genes resulted in abnormal phenotypes and high larval mortality. After 15 days, the survival rates of insects in dsspook, dsptth, dstorso, and dsnm-g groups were significantly reduced by 32%, 38%, 56%, and 67% respectively, compared with control. Moreover, about 80% of surviving larvae showed retarded development in dsRNA-treated groups. These results suggest that oral ingestion of bacterially expressed dsRNA in C. suppressalis could silence ptth, torso, spook and nm-g. Oral delivery of bacterially expressed dsRNA provides a simple and potential management scheme against C. suppressalis.

In eukaryotes the TFIID complex is required for preinitiation complex assembly which positions RNA polymerase II around transcription start sites. On the other hand, histone acetyltransferase complexes including SAGA and ATAC, modulate transcription at several steps through modification of specific core histone residues. In this study we investigated the function of Drosophila melanogaster proteins TAF10 and TAF10b, which are subunits of dTFIID and dSAGA, respectively. We generated a mutation which eliminated the production of both Drosophila TAF10 orthologues. The simultaneous deletion of both dTaf10 genes impaired the recruitment of the dTFIID subunit dTAF5 to polytene chromosomes, while binding of other TFIID subunits, dTAF1 and RNAPII was not affected. The lack of both dTAF10 proteins resulted in failures in the larval-pupal transition during metamorphosis and in transcriptional reprogramming at this developmental stage. Surprisingly, unlike dSAGA mutations, dATAC subunit mutations resulted in very similar changes in the steady state mRNA levels of approximately 5000 genes as did ablation of both dTaf10 genes, indicating that dTAF10- and/or dTAF10b-containing complexes and dATAC affect similar pathways. Importantly, the phenotype resulting from dTaf10+dTaf10b mutation could be rescued by ectopically added ecdysone, suggesting that dTAF10- and/or dTAF10b-containing complexes are involved in the expression of ecdysone biosynthetic genes. Indeed, in dTaf10+dTaf10b mutants, cytochrome genes, which regulate ecdysone synthesis in the ring gland, were underrepresented. Therefore our data support the idea that the presence of dTAF10 proteins in dTFIID and/or dSAGA is required only at specific developmental steps. We propose that distinct forms of dTFIID and/or dSAGA exist during Drosophila metamorphosis, wherein different TAF compositions serve to target RNAPII at different developmental stages and tissues.

Culex pipiens mosquitoes are important disease vectors inhabiting temperate zones, worldwide. The seasonal reduction in temperature and photoperiod accompanying late summer and early fall prompts female mosquitoes to enter diapause, a stage of developmental arrest and physiological conditioning that enhances survival during the winter months. To investigate the molecular mechanisms underlying diapause induction, we used custom whole transcriptome microarrays to identify differences in gene expression following exposure to nondiapause (long days, 25 °C) and diapause-inducing (short days, 18 °C) environmental conditions. Using a two-way ANOVA, we identified 1130 genes that were differentially expressed. We used the expression of these genes across three time points to construct a gene co-expression network comprising five modules. Genes in modules 1, 2, and 3 were largely up-regulated, while genes in modules 4 and 5 were down-regulated when compared to nondiapause conditions. Pathway enrichment analysis of the network modules revealed some potential regulatory mechanisms driving diapause induction. Module 1 was enriched for genes in the TGF-ß and Wnt signaling pathways; module 2 was enriched for genes involved in insect hormone biosynthesis, specifically, ecdysone synthesis; module 3 was enriched for genes involved in chromatin modification; and module 5 was enriched for genes in the circadian rhythm pathway. Our results suggest that TGF-β signaling and chromatin modification are key drivers for the integration of environmental signals into the diapause induction phase in C. pipiens mosquitoes.

During development, neural competence is conferred and maintained by integrating spatial and temporal regulations. The Drosophila sensory bristles that detect mechanical and chemical stimulations are arranged in stereotypical positions. The anterior wing margin (AWM) is arrayed with neuron-innervated sensory bristles, while posterior wing margin (PWM) bristles are non-innervated. We found that the COP9 signalosome (CSN) suppresses the neural competence of non-innervated bristles at the PWM. In CSN mutants, PWM bristles are transformed into neuron-innervated, which is attributed to sustained expression of the neural-determining factor Senseless (Sens). The CSN suppresses Sens through repression of the ecdysone signaling target gene broad (br) that encodes the BR-Z1 transcription factor to activate sens expression. Strikingly, CSN suppression of BR-Z1 is initiated at the prepupa-to-pupa transition, leading to Sens downregulation, and termination of the neural competence of PWM bristles. The role of ecdysone signaling to repress br after the prepupa-to-pupa transition is distinct from its conventional role in activation, and requires CSN deneddylating activity and multiple cullins, the major substrates of deneddylation. Several CSN subunits physically associate with ecdysone receptors to represses br at the transcriptional level. We propose a model in which nuclear hormone receptors cooperate with the deneddylation machinery to temporally shutdown downstream target gene expression, conferring a spatial restriction on neural competence at the PWM.

Ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) are members of a family of tetrameric intracellular Ca(2+)-release channels (CRCs). While it is well known in mammals that RyRs and IP3Rs modulate multiple physiological processes, the roles of these two CRCs in the development and physiology of insects remain poorly understood. In this study, we cloned and functionally characterized RyR and IP3R cDNAs (named TcRyR and TcIP3R) from the red flour beetle, Tribolium castaneum. The composite TcRyR gene contains an ORF of 15,285 bp encoding a protein of 5,094 amino acid residues. The TcIP3R contains an 8,175 bp ORF encoding a protein of 2,724 amino acids. Expression analysis of TcRyR and TcIP3R revealed significant differences in mRNA expression levels among T. castaneum during different developmental stages. When the transcript levels of TcRyR were suppressed by RNA interference (RNAi), an abnormal folding of the adult hind wings was observed, while the RNAi-mediated knockdown of TcIP3R resulted in defective larval-pupal and pupal-adult metamorphosis. These results suggested that TcRyR is required for muscle excitation-contraction (E-C) coupling in T. castaneum, and that calcium release via IP3R might play an important role in regulating ecdysone synthesis and release during molting and metamorphosis in insects.

Numerous studies have evaluated the endocrine effects of UV filters in vertebrates, but little attention has been paid to their possible hormonal activity in invertebrates. We examined the effects of benzophenone-3 (BP-3), one of the most common sunscreen agents, in Chironomus riparius (Insecta), a reference organism in aquatic toxicology. Salivary glands from larvae were treated with either the hormone ecdysone or BP-3 to compare the response of endocrine genes. It was found that BP-3 elicits the same effects as the natural hormone activating the expression of a set of ecdysone responsive genes. BP-3 also activated the stress gene hsp70. Interestingly, similar effects have been confirmed in vivo in embryos. Moreover, BP-3 also altered embryogenesis delaying hatching. This is the first demonstration of hormonal activity of UV filters in invertebrates, showing a mode of action similar to ecdysteroid hormones. This finding highlights the potential endocrine disruptive effects of these emergent pollutants., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG) that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl) and the nuclear receptor Knirps (Kni), have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH) signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld), controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development.

Phosphoinositide-3-kinase enhancer (PIKE) proteins encoded by the PIKE/CENTG1 gene are members of the gamma subgroup of the Centaurin superfamily of small GTPases. They are characterized by their chimeric protein domain architecture consisting of a pleckstrin homology (PH) domain, a GTPase-activating (GAP) domain, Ankyrin repeats as well as an intrinsic GTPase domain. In mammals, three PIKE isoforms with variations in protein structure and subcellular localization are encoded by the PIKE locus. PIKE inactivation in mice results in a broad range of defects, including neuronal cell death during brain development and misregulation of mammary gland development. PIKE -/- mutant mice are smaller, contain less white adipose tissue, and show insulin resistance due to misregulation of AMP-activated protein kinase (AMPK) and insulin receptor/Akt signaling. here, we have studied the role of PIKE proteins in metabolic regulation in the fly. We show that the Drosophila PIKE homolog, ceng1A, encodes functional GTPases whose internal GAP domains catalyze their GTPase activity. To elucidate the biological function of ceng1A in flies, we introduced a deletion in the ceng1A gene by homologous recombination that removes all predicted functional PIKE domains. We found that homozygous ceng1A mutant animals survive to adulthood. In contrast to PIKE -/- mouse mutants, genetic ablation of Drosophila ceng1A does not result in growth defects or weight reduction. Although metabolic pathways such as insulin signaling, sensitivity towards starvation and mobilization of lipids under high fed conditions are not perturbed in ceng1A mutants, homozygous ceng1A mutants show a prolonged development in second instar larval stage, leading to a late onset of pupariation. In line with these results we found that expression of ecdysone inducible genes is reduced in ceng1A mutants. Together, we propose a novel role for Drosophila Ceng1A in regulating ecdysone signaling-dependent second to third instar larval transition.

The steroid hormone ecdysone coordinates insect growth and development, directing the major postembryonic transition of forms, metamorphosis. The steroid-deficient ecdysoneless1 (ecd1) strain of Drosophila melanogaster has long served to assess the impact of ecdysone on gene regulation, morphogenesis, or reproduction. However, ecd also exerts cell-autonomous effects independently of the hormone, and mammalian Ecd homologs have been implicated in cell cycle regulation and cancer. Why the Drosophila ecd1 mutants lack ecdysone has not been resolved. Here, we show that in Drosophila cells, Ecd directly interacts with core components of the U5 snRNP spliceosomal complex, including the conserved Prp8 protein. In accord with a function in pre-mRNA splicing, Ecd and Prp8 are cell-autonomously required for survival of proliferating cells within the larval imaginal discs. In the steroidogenic prothoracic gland, loss of Ecd or Prp8 prevents splicing of a large intron from CYP307A2/spookier (spok) pre-mRNA, thus eliminating this essential ecdysone-biosynthetic enzyme and blocking the entry to metamorphosis. Human Ecd (hEcd) can substitute for its missing fly ortholog. When expressed in the Ecd-deficient prothoracic gland, hEcd re-establishes spok pre-mRNA splicing and protein expression, restoring ecdysone synthesis and normal development. Our work identifies Ecd as a novel pre-mRNA splicing factor whose function has been conserved in its human counterpart. Whether the role of mammalian Ecd in cancer involves pre-mRNA splicing remains to be discovered.

Sequential pulses of the steroid hormone ecdysone regulate the major developmental transitions in Drosophila, and the duration of each developmental stage is determined by the length of time between ecdysone pulses. Ecdysone regulates biological responses by directly initiating target gene transcription. In turn, these transcriptional responses are known to be self-limiting, with mechanisms in place to ensure regression of hormone-dependent transcription. However, the biological significance of these transcriptional repression mechanisms remains unclear. Here we show that the chromatin remodeling protein INO80 facilitates transcriptional repression of ecdysone-regulated genes during prepupal development. In ino80 mutant animals, inefficient repression of transcriptional responses to the late larval ecdysone pulse delays the onset of the subsequent prepupal ecdysone pulse, resulting in a significantly longer prepupal stage. Conversely, increased expression of ino80 is sufficient to shorten the prepupal stage by increasing the rate of transcriptional repression. Furthermore, we demonstrate that enhancing the rate of regression of the mid-prepupal competence factor βFTZ-F1 is sufficient to determine the timing of head eversion and thus the duration of prepupal development. Although ino80 is conserved from yeast to humans, this study represents the first characterization of a bona fide ino80 mutation in any metazoan, raising the possibility that the functions of ino80 in transcriptional repression and developmental timing are evolutionarily conserved., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The border cells of Drosophila are a model system for coordinated cell migration. Ecdysone signaling has been shown to act as the timing signal to initiate the migration process. Here we find that mutations in phantom (phm), encoding an enzyme in the ecdysone biosynthesis pathway, block border cell migration when the entire follicular epithelium of an egg chamber is mutant, even when the associated germline cells (nurse cells and oocyte) are wild-type. Conversely, mutant germline cells survive and do not affect border cell migration, as long as the surrounding follicle cells are wild-type. Interestingly, even small patches of wild-type follicle cells in a mosaic epithelium are sufficient to allow the production of above-threshold levels of ecdysone to promote border cell migration. The same phenotype is observed with mutations in shade (shd), encoding the last enzyme in the pathway that converts ecdysone to the active 20-hydroxyecdysone. Administration of high 20-hydroxyecdysone titers in the medium can also rescue the border cell migration phenotype in cultured egg chambers with an entirely phm mutant follicular epithelium. These results indicate that in normal oogenesis, the follicle cell epithelium of each individual egg chamber must supply sufficient ecdysone precursors, leading ultimately to high enough levels of mature 20-hydroxyecdysone to the border cells to initiate their migration. Neither the germline, nor the neighboring egg chambers, nor the surrounding hemolymph appear to provide threshold amounts of 20-hydroxyecdysone to do so. This "egg chamber autonomous" ecdysone synthesis constitutes a useful way to regulate the individual maturation of the asynchronous egg chambers present in the Drosophila ovary., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.