Phosphate starvation signaling increases mitochondrial membrane potential through respiration-independent mechanisms.

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.

A direct-drive GFP reporter for studies of tracheal development in Drosophila.

The Drosophila tracheal system consists of a widespread tubular network that provides respiratory functions for the animal. Its development, from ten pairs of placodes in the embryo to the final stereotypical branched structure in the adult, has been extensively studied by many labs as a model system for understanding tubular epithelial morphogenesis. Throughout these studies, a breathless (btl)-GAL4 driver has provided an invaluable tool to either mark tracheal cells during development or to manipulate gene expression in this tissue. A distinct shortcoming of this approach, however, is that btl-GAL4 cannot be used to specifically visualize tracheal cells in the presence of other GAL4 drivers or other UAS constructs, restricting its utility. Here we describe a direct-drive btl-nGFP reporter that can be used as a specific marker of tracheal cells throughout development in combination with any GAL4 driver and/or UAS construct. This reporter line should facilitate the use of Drosophila as a model system for studies of tracheal development and tubular morphogenesis.

Drosophila E93 promotes adult development and suppresses larval responses to ecdysone during metamorphosis.

Pulses of the steroid hormone ecdysone act through transcriptional cascades to direct the major developmental transitions during the Drosophila life cycle. These include the prepupal ecdysone pulse, which occurs 10 â€‹hours after pupariation and triggers the onset of adult morphogenesis and larval tissue destruction. E93 encodes a transcription factor that is specifically induced by the prepupal pulse of ecdysone, supporting a model proposed by earlier work that it specifies the onset of adult development. Although a number of studies have addressed these functions for E93, little is known about its roles in the salivary gland where the E93 locus was originally identified. Here we show that E93 is required for development through late pupal stages, with mutants displaying defects in adult differentiation and no detectable effect on the destruction of larval salivary glands. RNA-seq analysis demonstrates that E93 regulates genes involved in development and morphogenesis in the salivary glands, but has little effect on cell death gene expression. We also show that E93 is required to direct the proper timing of ecdysone-regulated gene expression in salivary glands, and that it suppresses earlier transcriptional programs that occur during larval and prepupal stages. These studies support the model that the stage-specific induction of E93 in late prepupae provides a critical signal that defines the end of larval development and the onset of adult differentiation.

Regulation of male fertility and accessory gland gene expression by the Drosophila HR39 nuclear receptor.

Successful reproduction is dependent on the transfer of male seminal proteins to females upon mating. These proteins arise from secretory tissues in the male reproductive tract, including the prostate and seminal vesicles in mammals and the accessory gland in insects. Although detailed functional studies have provided important insights into the mechanisms by which accessory gland proteins support reproduction, much less is known about the molecular mechanisms that regulate their expression within this tissue. Here we show that the Drosophila HR39 nuclear receptor is required for the proper expression of most genes that encode male accessory gland proteins. Consistent with this role, HR39 mutant males are infertile. In addition, tissue-specific RNAi and genetic rescue experiments indicate that HR39 acts within the accessory glands to regulate gene expression and male fertility. These results provide new directions for characterizing the mammalian orthologs of HR39, the SF-1 and LRH-1 nuclear receptors, both of which are required for glandular secretions and reproduction. In addition, our studies provide a molecular mechanism to explain how the accessory glands can maintain the abundant levels of seminal fluid production required to support fertility.

The Drosophila E78 nuclear receptor regulates dietary triglyceride uptake and systemic lipid levels.

BACKGROUND: Lipid levels are maintained by balancing lipid uptake, synthesis, and mobilization. Although many studies have focused on the control of lipid synthesis and mobilization, less is known about the regulation of lipid digestion and uptake. RESULTS: Here we show that the Drosophila E78A nuclear receptor plays a central role in intestinal lipid homeostasis through regulation of the CG17192 digestive lipase. E78A mutant adults fail to maintain proper systemic lipid levels following eclosion, with this effect largely restricted to the intestine. Transcriptional profiling by RNA-seq revealed a candidate gene for mediating this effect, encoding the predicted adult intestinal lipase CG17192. Intestine-specific disruption of CG17192 results in reduced lipid levels similar to that seen in E78A mutants. In addition, dietary supplementation with free fatty acids, or intestine-specific expression of either E78A or CG17192, is sufficient to restore lipid levels in E78A mutant adults. CONCLUSION: These studies support the model that E78A is a central regulator of adult lipid homeostasis through its effects on CG17192 expression and lipid digestion. This work also provides new insights into the control of intestinal lipid uptake and demonstrate that nuclear receptors can play an important role in these pathways.

Drosophila estrogen-related receptor directs a transcriptional switch that supports adult glycolysis and lipogenesis.

Metabolism and development must be closely coupled to meet the changing physiological needs of each stage in the life cycle. The molecular mechanisms that link these pathways, however, remain poorly understood. Here we show that the Drosophila estrogen-related receptor (dERR) directs a transcriptional switch in mid-pupae that promotes glucose oxidation and lipogenesis in young adults. dERR mutant adults are viable but display reduced locomotor activity, susceptibility to starvation, elevated glucose, and an almost complete lack of stored triglycerides. Molecular profiling by RNA-seq, ChIP-seq, and metabolomics revealed that glycolytic and pentose phosphate pathway genes are induced by dERR, and their reduced expression in mutants is accompanied by elevated glycolytic intermediates, reduced TCA cycle intermediates, and reduced levels of long chain fatty acids. Unexpectedly, we found that the central pathways of energy metabolism, including glycolysis, the tricarboxylic acid cycle, and electron transport chain, are coordinately induced at the transcriptional level in mid-pupae and maintained into adulthood, and this response is partially dependent on dERR, leading to the metabolic defects observed in mutants. Our data support the model that dERR contributes to a transcriptional switch during pupal development that establishes the metabolic state of the adult fly.

Functional analysis of Aarf domain-containing kinase 1 in Drosophila melanogaster.

BACKGROUND: The ADCK proteins are predicted mitochondrial kinases. Most studies of these proteins have focused on the Abc1/Coq8 subfamily, which contributes to Coenzyme Q biosynthesis. In contrast, little is known about ADCK1 despite its evolutionary conservation in yeast, Drosophila, Caenorhabditis elegans and mammals. RESULTS: We show that Drosophila ADCK1 mutants die as second instar larvae with double mouth hooks and tracheal breaks. Tissue-specific genetic rescue and RNAi studies show that ADCK1 is necessary and sufficient in the trachea for larval viability. In addition, tracheal-rescued ADCK1 mutant adults have reduced lifespan, are developmentally delayed, have reduced body size, and normal levels of basic metabolites. CONCLUSION: The larval lethality and double mouth hooks seen in ADCK1 mutants are often associated with reduced levels of the steroid hormone ecdysone, suggesting that this gene could contribute to controlling ecdysone levels or bioavailability. Similarly, the tracheal defects in these animals could arise from defects in intracellular lipid trafficking. These studies of ADCK1 provide a new context to define the physiological functions of this poorly understood member of the ADCK family of predicted mitochondrial proteins.

Constitutive activation of the Nrf2/Keap1 pathway in insecticide-resistant strains of Drosophila.

Pesticide resistance poses a major challenge for the control of vector-borne human diseases and agricultural crop protection. Although a number of studies have defined how mutations in specific target proteins can lead to insecticide resistance, much less is known about the mechanisms by which constitutive overexpression of detoxifying enzymes contributes to metabolic pesticide resistance. Here we show that the Nrf2/Keap1 pathway is constitutively active in two laboratory-selected DDT-resistant strains of Drosophila, 91R and RDDTR, leading to the overexpression of multiple detoxifying genes. Disruption of the Drosophila Nrf2 ortholog, CncC, or overexpression of Keap1, is sufficient to block this transcriptional response. In addition, a CncC-responsive reporter is highly active in both DDT-resistant strains and this response is dependent on the presence of an intact CncC binding site in the promoter. Microarray analysis revealed that ∼20% of the genes differentially expressed in the 91R strain are known CncC target genes. Finally, we show that CncC is partially active in these strains, consistent with the fitness cost associated with constitutive activation of the pathway. This study demonstrates that the Nrf2/Keap1 pathway contributes to the widespread overexpression of detoxification genes in insecticide-resistant strains and raises the possibility that inhibitors of this pathway could provide effective synergists for insect population control.

Transcriptional regulation of xenobiotic detoxification in Drosophila.

Living organisms, from bacteria to humans, display a coordinated transcriptional response to xenobiotic exposure, inducing enzymes and transporters that facilitate detoxification. Several transcription factors have been identified in vertebrates that contribute to this regulatory response. In contrast, little is known about this pathway in insects. Here we show that the Drosophila Nrf2 (NF-E2-related factor 2) ortholog CncC (cap 'n' collar isoform-C) is a central regulator of xenobiotic detoxification responses. A binding site for CncC and its heterodimer partner Maf (muscle aponeurosis fibromatosis) is sufficient and necessary for robust transcriptional responses to three xenobiotic compounds: phenobarbital (PB), chlorpromazine, and caffeine. Genetic manipulations that alter the levels of CncC or its negative regulator, Keap1 (Kelch-like ECH-associated protein 1), lead to predictable changes in xenobiotic-inducible gene expression. Transcriptional profiling studies reveal that more than half of the genes regulated by PB are also controlled by CncC. Consistent with these effects on detoxification gene expression, activation of the CncC/Keap1 pathway in Drosophila is sufficient to confer resistance to the lethal effects of the pesticide malathion. These studies establish a molecular mechanism for the regulation of xenobiotic detoxification in Drosophila and have implications for controlling insect populations and the spread of insect-borne human diseases.

The Drosophila estrogen-related receptor directs a metabolic switch that supports developmental growth.

Metabolism must be coordinated with development to provide the appropriate energetic needs for each stage in the life cycle. Little is known, however, about how this temporal control is achieved. Here, we show that the Drosophila ortholog of the estrogen-related receptor (ERR) family of nuclear receptors directs a critical metabolic transition during development. dERR mutants die as larvae with low ATP levels and elevated levels of circulating sugars. The expression of active dERR protein in mid-embryogenesis triggers a coordinate switch in gene expression that drives a metabolic program normally associated with proliferating cells, supporting the dramatic growth that occurs during larval development. This study shows that dERR plays a central role in carbohydrate metabolism, demonstrates that a proliferative metabolic program is used in normal developmental growth, and provides a molecular context to understand the close association between mammalian ERR family members and cancer.

The Drosophila NR4A nuclear receptor DHR38 regulates carbohydrate metabolism and glycogen storage.

Animals balance nutrient storage and mobilization to maintain metabolic homeostasis, a process that is disrupted in metabolic diseases like obesity and diabetes. Here, we show that DHR38, the single fly ortholog of the mammalian nuclear receptor 4A family of nuclear receptors, regulates glycogen storage during the larval stages of Drosophila melanogaster. DHR38 is expressed and active in the gut and body wall of larvae, and its expression levels change in response to nutritional status. DHR38 null mutants have normal levels of glucose, trehalose (the major circulating form of sugar), and triacylglycerol but display reduced levels of glycogen in the body wall muscles, which constitute the primary storage site for carbohydrates. Microarray analysis reveals that many metabolic genes are mis-regulated in DHR38 mutants. These include phosphoglucomutase, which is required for glycogen synthesis, and the two genes that encode the digestive enzyme amylase, accounting for the reduced amylase enzyme activity seen in DHR38 mutant larvae. These studies demonstrate that a critical role of nuclear receptor 4A receptors in carbohydrate metabolism has been conserved through evolution and that nutritional regulation of DHR38 expression maintains the proper uptake and storage of glycogen during the growing larval stage of development.

Med24 and Mdh2 are required for Drosophila larval salivary gland cell death.

The steroid hormone ecdysone triggers the rapid destruction of larval tissues through transcriptional cascades that culminate in rpr and hid expression and caspase activation. Here, we show that mutations in Mdh2 and Med24 block caspase cleavage and larval salivary gland cell death. Mdh2 encodes a predicted malate dehydrogenase that localizes to mitochondria. Consistent with this proposed function, Mdh2 mutants have significantly lower levels of ATP and accumulate late-stage citric acid cycle intermediates, suggesting that the cell death defects arise from a deficit in energy production. Med24 encodes a component of the Mediator transcriptional coactivator complex. Unexpectedly, however, expression of the key death regulator genes is normal in Med24 mutant salivary glands. This study identifies novel mechanisms for controlling the destruction of larval tissues during Drosophila metamorphosis and provides new directions for our understanding of steroid-triggered programmed cell death.

The Drosophila nuclear receptors DHR3 and betaFTZ-F1 control overlapping developmental responses in late embryos.

Studies of the onset of metamorphosis have identified an ecdysone-triggered transcriptional cascade that consists of the sequential expression of the transcription-factor-encoding genes DHR3, betaFTZ-F1, E74A and E75A. Although the regulatory interactions between these genes have been well characterized by genetic and molecular studies over the past 20 years, their developmental functions have remained more poorly understood. In addition, a transcriptional sequence similar to that observed in prepupae is repeated before each developmental transition in the life cycle, including mid-embryogenesis and the larval molts. Whether the regulatory interactions between DHR3, betaFTZ-F1, E74A and E75A at these earlier stages are similar to those defined at the onset of metamorphosis, however, is unknown. In this study, we turn to embryonic development to address these two issues. We show that mid-embryonic expression of DHR3 and betaFTZ-F1 is part of a 20-hydroxyecdysone (20E)-triggered transcriptional cascade similar to that seen in mid-prepupae, directing maximal expression of E74A and E75A during late embryogenesis. In addition, DHR3 and betaFTZ-F1 exert overlapping developmental functions at the end of embryogenesis. Both genes are required for tracheal air filling, whereas DHR3 is required for ventral nerve cord condensation and betaFTZ-F1 is required for proper maturation of the cuticular denticles. Rescue experiments support these observations, indicating that DHR3 has essential functions independent from those of betaFTZ-F1. DHR3 and betaFTZ-F1 also contribute to overlapping transcriptional responses during embryogenesis. Taken together, these studies define the lethal phenotypes of DHR3 and betaFTZ-F1 mutants, and provide evidence for functional bifurcation in the 20E-responsive transcriptional cascade.

Drosophila HNF4 regulates lipid mobilization and beta-oxidation.

Drosophila HNF4 (dHNF4) is the single ancestral ortholog of a highly conserved subfamily of nuclear receptors that includes two mammalian receptors, HNFalpha and HNFgamma, and 269 members in C. elegans. We show here that dHNF4 null mutant larvae are sensitive to starvation. Starved mutant larvae consume glycogen normally but retain lipids in their midgut and fat body and have increased levels of long-chain fatty acids, suggesting that they are unable to efficiently mobilize stored fat for energy. Microarray studies support this model, indicating reduced expression of genes that control lipid catabolism and beta-oxidation. A GAL4-dHNF4;UAS-lacZ ligand sensor can be activated by starvation or exogenous long-chain fatty acids, suggesting that dHNF4 is responsive to dietary signals. Taken together, our results support a feed-forward model for dHNF4, in which fatty acids released from triglycerides activate the receptor, inducing enzymes that drive fatty acid oxidation for energy production.

Drosophila DHR38 nuclear receptor is required for adult cuticle integrity at eclosion.

DHR38 is the only Drosophila member of the NR4A subclass of vertebrate nuclear receptors, which have been implicated in multiple biological pathways, including neuronal function, apoptosis, and metabolism. Although an earlier study identified three point mutations in DHR38, none of these were shown to be a null allele for the locus, leaving it unclear whether a complete loss of DHR38 function might uncover novel roles for the receptor. Here we show that a specific DHR38 null allele, DHR38(Y214), leads to fully penetrant pharate adult lethality, similar to the most severe phenotype associated with the EMS-induced mutations. DHR38(Y214) mutants display minor effects on ecdysone-regulated transcription at the onset of metamorphosis. In contrast, cuticle gene expression is significantly reduced in DHR38(Y214) mutant pupae. These studies define the essential functions of DHR38 and provide a genetic context for further characterization of its roles during development.

dTrf2 is required for transcriptional and developmental responses to ecdysone during Drosophila metamorphosis.

The TATA box-binding protein (TBP) related factor 2 (TRF2) has been well characterized at a biochemical level and in cultured cells. Relatively little, however, is known about how TRF2 functions in specific biological pathways during development. Here, we show that Drosophila TRF2 (dTRF2) plays an essential role in responses to the steroid hormone ecdysone during the onset of metamorphosis. Hypomorphic dTrf2 mutations lead to developmental arrest during prepupal and early pupal stages with defects in major ecdysone-triggered biological responses, including puparium formation, anterior spiracle eversion, gas bubble translocation, adult head eversion, and larval salivary gland cell death. The transcription of key ecdysone-regulated target genes is delayed and reduced in dTrf2 mutants. dTrf2 appears to be required for the proper timing and levels of ecdysone-regulated gene expression required for entry into metamorphosis.

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.

The DHR96 nuclear receptor regulates xenobiotic responses in Drosophila.

Exposure to xenobiotics such as plant toxins, pollutants, or prescription drugs triggers a defense response, inducing genes that encode key detoxification enzymes. Although xenobiotic responses have been studied in vertebrates, little effort has been made to exploit a simple genetic system for characterizing the molecular basis of this coordinated transcriptional response. We show here that approximately 1000 transcripts are significantly affected by phenobarbital treatment in Drosophila. We also demonstrate that the Drosophila ortholog of the human SXR and CAR xenobiotic receptors, DHR96, plays a role in this response. A DHR96 null mutant displays increased sensitivity to the sedative effects of phenobarbital and the pesticide DDT as well as defects in the expression of many phenobarbital-regulated genes. Metabolic and stress-response genes are also controlled by DHR96, implicating its role in coordinating multiple response pathways. This work establishes a new model system for defining the genetic control of xenobiotic stress responses.

The genomic response to 20-hydroxyecdysone at the onset of Drosophila metamorphosis.

BACKGROUND: The steroid hormone 20-hydroxyecdysone (20E) triggers the major developmental transitions in Drosophila, including molting and metamorphosis, and provides a model system for defining the developmental and molecular mechanisms of steroid signaling. 20E acts via a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle, to directly regulate target gene transcription. RESULTS: Here we identify the genomic transcriptional response to 20E as well as those genes that are dependent on EcR for their proper regulation. We show that genes regulated by 20E, and dependent on EcR, account for many transcripts that are significantly up- or downregulated at puparium formation. We provide evidence that 20E and EcR participate in the regulation of genes involved in metabolism, stress, and immunity at the onset of metamorphosis. We also present an initial characterization of a 20E primary-response regulatory gene identified in this study, brain tumor (brat), showing that brat mutations lead to defects during metamorphosis and changes in the expression of key 20E-regulated genes. CONCLUSION: This study provides a genome-wide basis for understanding how 20E and its receptor control metamorphosis, as well as a foundation for functional genomic analysis of key regulatory genes in the 20E signaling pathway during insect development.

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

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