Jason M. Tennessen

Drosophila HNF4 Regulates Lipid Mobilization and β-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.

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.

Methods for studying metabolism in Drosophila.

Recent research using Drosophila melanogaster has seen a resurgence in studies of metabolism and physiology. This review focuses on major methods used to conduct this work. These include protocols for dietary interventions, measurements of triglycerides, cholesterol, glucose, trehalose, and glycogen, stains for lipid detection, and the use of gas chromatography-mass spectrometry (GC-MS) to detect major polar metabolites. It is our hope that this will provide a useful framework for both new and current researchers in the field.

Coordinating Growth and Maturation — Insights from Drosophila

Adult body size in higher animals is dependent on the amount of growth that occurs during the juvenile stage. The duration of juvenile development, therefore, must be flexible and responsive to environmental conditions. When immature animals experience environmental stresses such as malnutrition or disease, maturation can be delayed until conditions improve and normal growth can resume. In contrast, when animals are raised under ideal conditions that promote rapid growth, internal checkpoints ensure that maturation does not occur until juvenile development is complete. Although the mechanisms that regulate growth and gate the onset of maturation have been investigated for decades, the emerging links between childhood obesity, early onset puberty, and adult metabolic disease have placed a new emphasis on this field. Remarkably, genetic studies in the fruit fly Drosophila melanogaster have shown that the central regulatory pathways that control growth and the timing of sexual maturation are conserved through evolution, and suggest that this aspect of animal life history is regulated by a common genetic architecture. This review focuses on these conserved mechanisms and highlights recent studies that explore how Drosophila coordinates developmental growth with environmental conditions.

The mouse Secreted frizzled-related protein 5 gene is expressed in the anterior visceral endoderm and foregut endoderm during early post-implantation development

The anterior visceral endoderm (AVE) plays an important role in anterior-posterior axis formation in the mouse. The AVE functions in part by expressing secreted factors that antagonize growth factor signaling in the proximal epiblast. Here we report that the Secreted frizzled-related protein 5 (Sfrp5) gene, which encodes a secreted factor that can antagonize Wnt signaling, is expressed in the AVE and foregut endoderm during early mouse development. At embryonic day (E) 5.5, Sfrp5 is expressed in the visceral endoderm at the distal tip region of the embryo and at E6.5 in the AVE opposite the primitive streak. In Lim1 embryos, which lack anterior neural tissue and sometimes form a secondary body axis, Sfrp5-expressing cells fail to move towards the anterior and remain at the distal tip of E6.5 embryos. When compared with Dkk1, which encodes another secreted Wnt antagonist molecule present in the visceral endoderm, Sfrp5 and Dkk1 expression overlap but Sfrp5 is expressed more broadly in the AVE. Between E7.5 and 8, Sfrp5 is expressed in the foregut endoderm underlying the cardiac mesoderm. At E8.5, Sfrp5 is expressed in the ventral foregut endoderm that gives rise to the liver. Additional domains of Sfrp5 expression occur in the dorsal neural tube and in the forebrain anterior to the optic placode. These findings identify a gene encoding a secreted Wnt antagonist that is expressed in the extraembryonic visceral endoderm and anterior definitive endoderm during axis formation and organogenesis in the mouse.

Coordinated Metabolic Transitions During Drosophila Embryogenesis and the Onset of Aerobic Glycolysis

Rapidly proliferating cells such as cancer cells and embryonic stem cells rely on a specialized metabolic program known as aerobic glycolysis, which supports biomass production from carbohydrates. The fruit fly Drosophila melanogaster also utilizes aerobic glycolysis to support the rapid growth that occurs during larval development. Here we use singular value decomposition analysis of modENCODE RNA-seq data combined with GC-MS-based metabolomic analysis to analyze the changes in gene expression and metabolism that occur during Drosophila embryogenesis, spanning the onset of aerobic glycolysis. Unexpectedly, we find that the most common pattern of co-expressed genes in embryos includes the global switch to glycolytic gene expression that occurs midway through embryogenesis. In contrast to the canonical aerobic glycolytic pathway, however, which is accompanied by reduced mitochondrial oxidative metabolism, the expression of genes involved in the tricarboxylic cycle (TCA cycle) and the electron transport chain are also upregulated at this time. Mitochondrial activity, however, appears to be attenuated, as embryos exhibit a block in the TCA cycle that results in elevated levels of citrate, isocitrate, and α-ketoglutarate. We also find that genes involved in lipid breakdown and β-oxidation are upregulated prior to the transcriptional initiation of glycolysis, but are downregulated before the onset of larval development, revealing coordinated use of lipids and carbohydrates during development. These observations demonstrate the efficient use of nutrient stores to support embryonic development, define sequential metabolic transitions during this stage, and demonstrate striking similarities between the metabolic state of late-stage fly embryos and tumor cells.

The C. elegans developmental timing protein LIN-42 regulates diapause in response to environmental cues

Environmental conditions can have a major impact on developmental progression in animals. For example, when C. elegans larvae encounter harsh conditions they can reversibly halt the passage of developmental time by forming a long-lived dauer larva at the end of the second larval stage. Here, we show that the period homolog lin-42, known to control developmental time, also acts as a component of a switch that mediates dauer entry. Loss of lin-42 function renders animals hypersensitive to dauer formation under stressful conditions, whereas misexpression of lin-42 in the pre-dauer stage inhibits dauer formation, indicating that lin-42 acts as a negative regulator of this life history decision. These phenotypes place LIN-42 in opposition to the ligand-free form of the nuclear receptor DAF-12, which indirectly senses environmental conditions and helps to integrate external cues into developmental decisions. Mutations that impair DAF-12 ligand binding are exquisitely sensitive to the absence of lin-42, whereas overexpression of LIN-42 can suppress the dauer constitutive phenotype of a ligand-insensitive daf-12 mutant, suggesting that LIN-42 and DAF-12 are intimate partners in controlling the decision to become a dauer larva. The functional outputs of Period family proteins and nuclear receptors also converge in other organisms, suggesting that the relationship between lin-42 and daf-12 represents an ancient genetic framework for responding to environmental stimuli.

Developmental Timing: let-7 Function Conserved through Evolution

Expression of the heterochronic microRNA let-7 is tightly correlated with the onset of adult development in many animals, suggesting that it functions as an evolutionarily conserved developmental timer. This hypothesis has now been confirmed by studies in Drosophila.

Drosophila larvae synthesize the putative oncometabolite L-2-hydroxyglutarate during normal developmental growth

Significance Oncometabolites are small molecules that promote tumor formation and growth. L-2-hydroxyglutarate (L-2HG) is a putative oncometabolite that is associated with gliomas and renal cell carcinomas, as well as a severe neurometabolic disorder known as L-2-hydroxyglutaric aciduria. However, despite that L-2HG is commonly considered a metabolic waste product, this compound was recently discovered to control immune cell fate, thereby demonstrating that it has endogenous functions in healthy animal cells. Here, we find that the fruit fly, Drosophila melanogaster, also synthesizes high concentrations of L-2HG during normal larval growth. Our discovery establishes the fly as a genetic model for studying this putative oncometabolite in healthy animal tissues. L-2-hydroxyglutarate (L-2HG) has emerged as a putative oncometabolite that is capable of inhibiting enzymes involved in metabolism, chromatin modification, and cell differentiation. However, despite the ability of L-2HG to interfere with a broad range of cellular processes, this molecule is often characterized as a metabolic waste product. Here, we demonstrate that Drosophila larvae use the metabolic conditions established by aerobic glycolysis to both synthesize and accumulate high concentrations of L-2HG during normal developmental growth. A majority of the larval L-2HG pool is derived from glucose and dependent on the Drosophila estrogen-related receptor (dERR), which promotes L-2HG synthesis by up-regulating expression of the Drosophila homolog of lactate dehydrogenase (dLdh). We also show that dLDH is both necessary and sufficient for directly synthesizing L-2HG and the Drosophila homolog of L-2-hydroxyglutarate dehydrogenase (dL2HGDH), which encodes the enzyme that breaks down L-2HG, is required for stage-specific degradation of the L-2HG pool. In addition, dLDH also indirectly promotes L-2HG accumulation via synthesis of lactate, which activates a metabolic feed-forward mechanism that inhibits dL2HGDH activity and stabilizes L-2HG levels. Finally, we use a genetic approach to demonstrate that dLDH and L-2HG influence position effect variegation and DNA methylation, suggesting that this compound serves to coordinate glycolytic flux with epigenetic modifications. Overall, our studies demonstrate that growing animal tissues synthesize L-2HG in a controlled manner, reveal a mechanism that coordinates glucose catabolism with L-2HG synthesis, and establish the fly as a unique model system for studying the endogenous functions of L-2HG during cell growth and proliferation.

Metabolomic Studies in Drosophila

Metabolomic analysis provides a powerful new tool for studies of Drosophila physiology. This approach allows investigators to detect thousands of chemical compounds in a single sample, representing the combined contributions of gene expression, enzyme activity, and environmental context. Metabolomics has been used for a wide range of studies in Drosophila, often providing new insights into gene function and metabolic state that could not be obtained using any other approach. In this review, we survey the uses of metabolomic analysis since its entry into the field. We also cover the major methods used for metabolomic studies in Drosophila and highlight new directions for future research.