Hypoxia delays steroid-induced developmental maturation in Drosophila by suppressing EGF signaling.

Animals often grow and develop in unpredictable environments where factors like food availability, temperature, and oxygen levels can fluctuate dramatically. To ensure proper sexual maturation into adulthood, juvenile animals need to adapt their growth and developmental rates to these fluctuating environmental conditions. Failure to do so can result in impaired maturation and incorrect body size. Here we describe a mechanism by which Drosophila larvae adapt their development in low oxygen (hypoxia). During normal development, larvae grow and increase in mass until they reach critical weight (CW), after which point a neuroendocrine circuit triggers the production of the steroid hormone ecdysone from the prothoracic gland (PG), which promotes maturation to the pupal stage. However, when raised in hypoxia (5% oxygen), larvae slow their growth and delay their maturation to the pupal stage. We find that, although hypoxia delays the attainment of CW, the maturation delay occurs mainly because of hypoxia acting late in development to suppress ecdysone production. This suppression operates through a distinct mechanism from nutrient deprivation, occurs independently of HIF-1 alpha and does not involve dilp8 or modulation of Ptth, the main neuropeptide that initiates ecdysone production in the PG. Instead, we find that hypoxia lowers the expression of the EGF ligand, spitz, and that the delay in maturation occurs due to reduced EGFR/ERK signaling in the PG. Our study sheds light on how animals can adjust their development rate in response to changing oxygen levels in their environment. Given that hypoxia is a feature of both normal physiology and many diseases, our findings have important implications for understanding how low oxygen levels may impact animal development in both normal and pathological situations.

Serotonergic neuron ribosomal proteins regulate the neuroendocrine control of Drosophila development.

The regulation of ribosome function is a conserved mechanism of growth control. While studies in single cell systems have defined how ribosomes contribute to cell growth, the mechanisms that link ribosome function to organismal growth are less clear. Here we explore this issue using Drosophila Minutes, a class of heterozygous mutants for ribosomal proteins. These animals exhibit a delay in larval development caused by decreased production of the steroid hormone ecdysone, the main regulator of larval maturation. We found that this developmental delay is not caused by decreases in either global ribosome numbers or translation rates. Instead, we show that they are due in part to loss of Rp function specifically in a subset of serotonin (5-HT) neurons that innervate the prothoracic gland to control ecdysone production. We find that these effects do not occur due to altered protein synthesis or proteostasis, but that Minute animals have reduced expression of synaptotagmin, a synaptic vesicle protein, and that the Minute developmental delay can be partially reversed by overexpression of synaptic vesicle proteins in 5-HTergic cells. These results identify a 5-HT cell-specific role for ribosomal function in the neuroendocrine control of animal growth and development.

The Immune Deficiency Pathway Regulates Metabolic Homeostasis in Drosophila.

Immune and metabolic pathways collectively influence host responses to microbial invaders, and mutations in one pathway frequently disrupt activity in another. We used the Drosophila melanogaster model to characterize metabolic homeostasis in flies with modified immune deficiency (IMD) pathway activity. The IMD pathway is very similar to the mammalian TNF-α pathway, a key regulator of vertebrate immunity and metabolism. We found that persistent activation of IMD resulted in hyperglycemia, depleted fat reserves, and developmental delays, implicating IMD in metabolic regulation. Consistent with this hypothesis, we found that imd mutants weigh more, are hyperlipidemic, and have impaired glucose tolerance. To test the importance of metabolic regulation for host responses to bacterial infection, we challenged insulin pathway mutants with lethal doses of several Drosophila pathogens. We found that loss-of-function mutations in the insulin pathway impacted host responses to infection in a manner that depends on the route of infection and the identity of the infectious microbe. Combined, our results support a role for coordinated regulation of immune and metabolic pathways in host containment of microbial invaders.

Ras/ERK-signalling promotes tRNA synthesis and growth via the RNA polymerase III repressor Maf1 in Drosophila.

The small G-protein Ras is a conserved regulator of cell and tissue growth. These effects of Ras are mediated largely through activation of a canonical RAF-MEK-ERK kinase cascade. An important challenge is to identify how this Ras/ERK pathway alters cellular metabolism to drive growth. Here we report on stimulation of RNA polymerase III (Pol III)-mediated tRNA synthesis as a growth effector of Ras/ERK signalling in Drosophila. We find that activation of Ras/ERK signalling promotes tRNA synthesis both in vivo and in cultured Drosophila S2 cells. We also show that Pol III function is required for Ras/ERK signalling to drive proliferation in both epithelial and stem cells in Drosophila tissues. We find that the transcription factor Myc is required but not sufficient for Ras-mediated stimulation of tRNA synthesis. Instead we show that Ras signalling promotes Pol III function and tRNA synthesis by phosphorylating, and inhibiting the nuclear localization and function of the Pol III repressor Maf1. We propose that inhibition of Maf1 and stimulation of tRNA synthesis is one way by which Ras signalling enhances protein synthesis to promote cell and tissue growth.

Early-life hypoxia alters adult physiology and reduces stress resistance and lifespan in Drosophila.

In many animals, short-term fluctuations in environmental conditions in early life often exert long-term effects on adult physiology. In Drosophila, one ecologically relevant environmental variable is hypoxia. Drosophila larvae live on rotting, fermenting food rich in microorganisms, an environment characterized by low ambient oxygen. They have therefore evolved to tolerate hypoxia. Although the acute effects of hypoxia in larvae have been well studied, whether early-life hypoxia affects adult physiology and fitness is less clear. Here, we show that Drosophila exposed to hypoxia during their larval period subsequently show reduced starvation stress resistance and shorter lifespan as adults, with these effects being stronger in males. We find that these effects are associated with reduced whole-body insulin signaling but elevated TOR kinase activity, a manipulation known to reduce lifespan. We also identify a sexually dimorphic effect of larval hypoxia on adult nutrient storage and mobilization. Thus, we find that males, but not females, show elevated levels of lipids and glycogen. Moreover, we see that both males and females exposed to hypoxia as larvae show defective lipid mobilization upon starvation stress as adults. These data demonstrate how early-life hypoxia can exert persistent, sexually dimorphic, long-term effects on Drosophila adult physiology and lifespan.

The EGF/Ras pathway controls growth in Drosophila via ribosomal RNA synthesis.

The Ras small G-protein is a conserved regulator of cell and tissue growth during animal development. Studies in Drosophila have shown how Ras can stimulate a RAF-MEK-ERK signalling pathway to control cell growth and proliferation in response to Epidermal Growth Factor (EGF) stimulation. This work has also defined several transcription factors that can function as downstream growth effectors of the EGF/Ras/ERK pathway by stimulating mRNA transcription. Here we report on stimulation of RNA polymerase I (Pol I)-mediated ribosomal RNA (rRNA) synthesis as a growth effector of Ras/ERK signalling in Drosophila. We show that Ras/ERK signalling promotes an increase in nucleolar size in larval wing discs, which is indicative of increased ribosome synthesis. We also find that activation of Ras/ERK signalling promotes rRNA synthesis both in vivo and in cultured Drosophila S2 cells. We show that Ras signalling can regulate the expression of the Pol I transcription factor TIF-IA, and that this regulation requires dMyc. Finally, we find that TIF-IA-mediated rRNA synthesis is required for Ras/ERK signalling to drive proliferation in both larval and adult Drosophila tissues. These findings indicate that Ras signalling can promote ribosome synthesis in Drosophila, and that this is one mechanism that contributes to the growth effects of the Ras signalling pathway.

The Sex Determination Gene transformer Regulates Male-Female Differences in Drosophila Body Size.

Almost all animals show sex differences in body size. For example, in Drosophila, females are larger than males. Although Drosophila is widely used as a model to study growth, the mechanisms underlying this male-female difference in size remain unclear. Here, we describe a novel role for the sex determination gene transformer (tra) in promoting female body growth. Normally, Tra is expressed only in females. We find that loss of Tra in female larvae decreases body size, while ectopic Tra expression in males increases body size. Although we find that Tra exerts autonomous effects on cell size, we also discovered that Tra expression in the fat body augments female body size in a non cell-autonomous manner. These effects of Tra do not require its only known targets doublesex and fruitless. Instead, Tra expression in the female fat body promotes growth by stimulating the secretion of insulin-like peptides from insulin producing cells in the brain. Our data suggest a model of sex-specific growth in which body size is regulated by a previously unrecognized branch of the sex determination pathway, and identify Tra as a novel link between sex and the conserved insulin signaling pathway.

Control of Drosophila endocycles by E2F and CRL4(CDT2).

Endocycles are variant cell cycles comprised of DNA synthesis (S)- and gap (G)-phases but lacking mitosis. Such cycles facilitate post-mitotic growth in many invertebrate and plant cells, and are so ubiquitous that they may account for up to half the world's biomass. DNA replication in endocycling Drosophila cells is triggered by cyclin E/cyclin dependent kinase 2 (CYCE/CDK2), but this kinase must be inactivated during each G-phase to allow the assembly of pre-Replication Complexes (preRCs) for the next S-phase. How CYCE/CDK2 is periodically silenced to allow re-replication has not been established. Here, using genetic tests in parallel with computational modelling, we show that the endocycles of Drosophila are driven by a molecular oscillator in which the E2F1 transcription factor promotes CycE expression and S-phase initiation, S-phase then activates the CRL4(CDT2) ubiquitin ligase, and this in turn mediates the destruction of E2F1 (ref. 7). We propose that it is the transient loss of E2F1 during S phases that creates the window of low Cdk activity required for preRC formation. In support of this model overexpressed E2F1 accelerated endocycling, whereas a stabilized variant of E2F1 blocked endocycling by deregulating target genes, including CycE, as well as Cdk1 and mitotic cyclins. Moreover, we find that altering cell growth by changing nutrition or target of rapamycin (TOR) signalling impacts E2F1 translation, thereby making endocycle progression growth-dependent. Many of the regulatory interactions essential to this novel cell cycle oscillator are conserved in animals and plants, indicating that elements of this mechanism act in most growth-dependent cell cycles.

TIF-IA-dependent regulation of ribosome synthesis in drosophila muscle is required to maintain systemic insulin signaling and larval growth.

The conserved TOR kinase signaling network links nutrient availability to cell, tissue and body growth in animals. One important growth-regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls rRNA synthesis-a limiting step in ribosome biogenesis-via the RNA Polymerase I transcription factor TIF-IA. However, the contribution of TOR-dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non-autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced levels of TIF-IA, and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle. We also observed that maintaining TIF-IA levels in muscle could partially reverse the starvation-mediated suppression of systemic insulin signaling. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function. These data suggest that muscle ribosome synthesis functions as a nutrient-dependent checkpoint for overall body growth: in nutrient rich conditions, TOR is required to maintain levels of TIF-IA and ribosome synthesis to promote high levels of systemic insulin, but under conditions of starvation stress, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis.

Why should cancer biologists care about tRNAs? tRNA synthesis, mRNA translation and the control of growth.

Transfer RNAs (tRNAs) are essential for mRNA translation. They are transcribed in the nucleus by RNA polymerase III and undergo many modifications before contributing to cytoplasmic protein synthesis. In this review I highlight our understanding of how tRNA biology may be linked to the regulation of mRNA translation, growth and tumorigenesis. First, I review how oncogenes and tumour suppressor signalling pathways, such as the PI3 kinase/TORC1, Ras/ERK, Myc, p53 and Rb pathways, regulate Pol III and tRNA synthesis. In several cases, this regulation contributes to cell, tissue and body growth, and has implications for our understanding of tumorigenesis. Second, I highlight some recent work, particularly in model organisms such as yeast and Drosophila, that shows how alterations in tRNA synthesis may be not only necessary, but also sufficient to drive changes in mRNA translation and growth. These effects may arise due to both absolute increases in total tRNA levels, but also changes in the relative levels of tRNAs in the overall pool. Finally, I review some recent studies that have revealed how tRNA modifications (amino acid acylation, base modifications, subcellular shuttling, and cleavage) can be regulated by growth and stress cues to selectively influence mRNA translation. Together these studies emphasize the importance of the regulation of tRNA synthesis and modification as critical control points in protein synthesis and growth. This article is part of a Special Issue entitled: Translation and Cancer.

Nutrient/TOR-dependent regulation of RNA polymerase III controls tissue and organismal growth in Drosophila.

The nutrient/target-of-rapamycin (TOR) pathway has emerged as a key regulator of tissue and organismal growth in metazoans. The signalling components of the nutrient/TOR pathway are well defined; however, the downstream effectors are less understood. Here, we show that the control of RNA polymerase (Pol) III-dependent transcription is an essential target of TOR in Drosophila. We find that TOR activity controls Pol III in growing larvae via inhibition of the repressor Maf1 and, in part, via the transcription factor Drosophila Myc (dMyc). Moreover, we show that loss of the Pol III factor, Brf, leads to reduced tissue and organismal growth and prevents TOR-induced cellular growth. TOR activity in the larval fat body, a tissue equivalent to vertebrate fat or liver, couples nutrition to insulin release from the brain. Accordingly, we find that fat-specific loss of Brf phenocopies nutrient limitation and TOR inhibition, leading to decreased systemic insulin signalling and reduced organismal growth. Thus, stimulation of Pol III is a key downstream effector of TOR in the control of cellular and systemic growth.

Activated STAT regulates growth and induces competitive interactions independently of Myc, Yorkie, Wingless and ribosome biogenesis.

Cell competition is a conserved mechanism that regulates organ size and shares properties with the early stages of cancer. In Drosophila, wing cells with increased Myc or with optimum ribosome function become supercompetitors that kill their wild-type neighbors (called losers) up to several cell diameters away. Here, we report that modulating STAT activity levels regulates competitor status. Cells lacking STAT become losers that are killed by neighboring wild-type cells. By contrast, cells with hyper-activated STAT become supercompetitors that kill losers located at a distance in a manner that is dependent on hid but independent of Myc, Yorkie, Wingless signaling, and of ribosome biogenesis. These results indicate that STAT, Wingless and Myc are major parallel regulators of cell competition, which may converge on signals that non-autonomously kill losers. As hyper-activated STATs are causal to tumorigenesis and stem cell niche occupancy, our results have therapeutic implications for cancer and regenerative medicine.

Drosophila RNA polymerase III repressor Maf1 controls body size and developmental timing by modulating tRNAiMet synthesis and systemic insulin signaling.

The target-of-rapamycin pathway couples nutrient availability with tissue and organismal growth in metazoans. The key effectors underlying this growth are, however, unclear. Here we show that Maf1, a repressor of RNA polymerase III-dependent tRNA transcription, is an important mediator of nutrient-dependent growth in Drosophila. We find nutrients promote tRNA synthesis during larval development by inhibiting Maf1. Genetic inhibition of Maf1 accelerates development and increases body size. These phenotypes are due to a non-cell-autonomous effect of Maf1 inhibition in the fat body, the main larval endocrine organ. Inhibiting Maf1 in the fat body increases growth by promoting the expression of brain-derived insulin-like peptides and consequently enhanced systemic insulin signaling. Remarkably, the effects of Maf1 inhibition are reproduced in flies carrying one extra copy of the initiator methionine tRNA, tRNA(i)(Met). These findings suggest the stimulation of tRNA(i)(Met) synthesis via inhibition of dMaf1 is limiting for nutrition-dependent growth during development.

Mitochondrial metabolism in Drosophila macrophage-like cells regulates body growth via modulation of cytokine and insulin signaling.

Macrophages play critical roles in regulating and maintaining tissue and whole-body metabolism in normal and disease states. While the cell-cell signaling pathways that underlie these functions are becoming clear, less is known about how alterations in macrophage metabolism influence their roles as regulators of systemic physiology. Here, we investigate this by examining Drosophila macrophage-like cells called hemocytes. We used knockdown of TFAM, a mitochondrial genome transcription factor, to reduce mitochondrial OxPhos activity specifically in larval hemocytes. We find that this reduction in hemocyte OxPhos leads to a decrease in larval growth and body size. These effects are associated with a suppression of systemic insulin, the main endocrine stimulator of body growth. We also find that TFAM knockdown leads to decreased hemocyte JNK signaling and decreased expression of the TNF alpha homolog, Eiger in hemocytes. Furthermore, we show that genetic knockdown of hemocyte JNK signaling or Eiger expression mimics the effects of TFAM knockdown and leads to a non-autonomous suppression of body size without altering hemocyte numbers. Our data suggest that modulation of hemocyte mitochondrial metabolism can determine their non-autonomous effects on organismal growth by altering cytokine and systemic insulin signaling. Given that nutrient availability can control mitochondrial metabolism, our findings may explain how macrophages function as nutrient-responsive regulators of tissue and whole-body physiology and homeostasis.

Myc-dependent regulation of ribosomal RNA synthesis during Drosophila development.

Regulating ribosome number is thought to control cellular growth. Synthesis of ribosomal RNA (rRNA) is a limiting step in ribosome biogenesis and rates of rRNA synthesis are generally altered depending on the growth status of a cell. Although studies in unicellular systems have addressed the mechanisms by which this occurs, few studies have applied a genetic approach to examine growth-dependent control of rRNA synthesis in metazoans. Here, we show that in Drosophila melanogaster Myc (dMyc) is a regulator of rRNA synthesis. Expression of dMyc is both necessary and sufficient to control rRNA synthesis and ribosome biogenesis during larval development. Stimulation of rRNA synthesis by dMyc is mediated through a rapid, coordinated increase in the levels of the Pol I transcriptional machinery. In addition, the growth effects of dMyc in larval wing imaginal discs require de novo rRNA synthesis. We suggest that during animal development, the control of rRNA synthesis and ribosome biogenesis is an essential Myc function.

Enteric bacterial infection in Drosophila induces whole-body alterations in metabolic gene expression independently of the immune deficiency signaling pathway.

When infected by intestinal pathogenic bacteria, animals initiate both local and systemic defence responses. These responses are required to reduce pathogen burden and also to alter host physiology and behavior to promote infection tolerance, and they are often mediated through alterations in host gene expression. Here, we have used transcriptome profiling to examine gene expression changes induced by enteric infection with the Gram-negative bacteria Pseudomonas entomophila in adult female Drosophila. We find that infection induces a strong upregulation of metabolic gene expression, including gut and fat body-enriched genes involved in lipid transport, lipolysis, and beta-oxidation, as well as glucose and amino acid metabolism genes. Furthermore, we find that the classic innate immune deficiency (Imd)/Relish/NF-KappaB pathway is not required for, and in some cases limits, these infection-mediated increases in metabolic gene expression. We also see that enteric infection with Pseudomonas entomophila downregulates the expression of many transcription factors and cell-cell signaling molecules, particularly those previously shown to be involved in gut-to-brain and neuronal signaling. Moreover, as with the metabolic genes, these changes occurred largely independent of the Imd pathway. Together, our study identifies many metabolic, signaling, and transcription factor gene expression changes that may contribute to organismal physiological and behavioral responses to enteric pathogen infection.

TOR signalling is required for host lipid metabolic remodelling and survival following enteric infection in Drosophila.

When infected by enteric pathogenic bacteria, animals need to initiate local and whole-body defence strategies. Although most attention has focused on the role of innate immune anti-bacterial responses, less is known about how changes in host metabolism contribute to host defence. Using Drosophila as a model system, we identify induction of intestinal target-of-rapamycin (TOR) kinase signalling as a key adaptive metabolic response to enteric infection. We find that enteric infection induces both local and systemic induction of TOR independently of the Immune deficiency (IMD) innate immune pathway, and we see that TOR functions together with IMD signalling to promote infection survival. These protective effects of TOR signalling are associated with remodelling of host lipid metabolism. Thus, we see that TOR is required to limit excessive infection-mediated wasting of host lipid stores by promoting an increase in the levels of gut- and fat body-expressed lipid synthesis genes. Our data support a model in which induction of TOR represents a host tolerance response to counteract infection-mediated lipid wasting in order to promote survival. This article has an associated First Person interview with the first author of the paper.

Transcriptome analysis of FOXO-dependent hypoxia gene expression identifies Hipk as a regulator of low oxygen tolerance in Drosophila.

When exposed to low oxygen or hypoxia, animals must alter their metabolism and physiology to ensure proper cell-, tissue-, and whole-body level adaptations to their hypoxic environment. These alterations often involve changes in gene expression. While extensive work has emphasized the importance of the HIF-1 alpha transcription factor on controlling hypoxia gene expression, less is known about other transcriptional mechanisms. We previously identified the transcription factor FOXO as a regulator of hypoxia tolerance in Drosophila larvae and adults. Here, we use an RNA-sequencing approach to identify FOXO-dependent changes in gene expression that are associated with these tolerance effects. We found that hypoxia altered the expression of over 2,000 genes and that ∼40% of these gene expression changes required FOXO. We discovered that hypoxia exposure led to a FOXO-dependent increase in genes involved in cell signaling, such as kinases, GTPase regulators, and regulators of the Hippo/Yorkie pathway. Among these, we identified homeodomain-interacting protein kinase as being required for hypoxia survival. We also found that hypoxia suppresses the expression of genes involved in ribosome synthesis and egg production, and we showed that hypoxia suppresses tRNA synthesis and mRNA translation and reduces female fecundity. Among the downregulated genes, we discovered that FOXO was required for the suppression of many ribosomal protein genes and genes involved in oxidative phosphorylation, pointing to a role for FOXO in limiting energetically costly processes such as protein synthesis and mitochondrial activity upon hypoxic stress. This work uncovers a widespread role for FOXO in mediating hypoxia changes in gene expression.

Adipose mitochondrial metabolism controls body growth by modulating systemic cytokine and insulin signaling.

Animals must adapt their growth to fluctuations in nutrient availability to ensure proper development. These adaptations often rely on specific nutrient-sensing tissues that control whole-body physiology through inter-organ communication. While the signaling mechanisms that underlie this communication are well studied, the contributions of metabolic alterations in nutrient-sensing tissues are less clear. Here, we show how the reprogramming of adipose mitochondria controls whole-body growth in Drosophila larvae. We find that dietary nutrients alter fat-body mitochondrial morphology to lower their bioenergetic activity, leading to rewiring of fat-body glucose metabolism. Strikingly, we find that genetic reduction of mitochondrial bioenergetics just in the fat body is sufficient to accelerate body growth and development. These growth effects are caused by inhibition of the fat-derived secreted peptides ImpL2 and tumor necrosis factor alpha (TNF-α)/Eiger, leading to enhanced systemic insulin signaling. Our work reveals how reprogramming of mitochondrial metabolism in one nutrient-sensing tissue can couple nutrient availability to whole-body growth.