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

Transcriptional regulation of xenobiotic detoxification in Drosophila

Thummel and colleagues provide insight into xenobiotic detoxification and pesticide resistance in insects. The Drosophila Nrf2 ortholog CnCC induces a transcriptional program of detoxification genes in response to xenobiotic pressure and is sufficient to confer resistance to the lethal effects of the pesticide malathion

Rigor mortis encodes a novel nuclear receptor interacting protein required for ecdysone signaling during Drosophila larval development

Pulses of the steroid hormone ecdysone trigger the major developmental transitions in Drosophila, including molting and puparium formation. The ecdysone signal is transduced by the EcR/USP nuclear receptor heterodimer that binds to specific response elements in the genome and directly regulates target gene transcription. We describe a novel nuclear receptor interacting protein encoded by rigor mortis (rig) that is required for ecdysone responses during larval development. rig mutants display defects in molting, delayed larval development, larval lethality, duplicated mouth parts, and defects in puparium formation--phenotypes that resemble those seen in EcR, usp, E75A and betaFTZ-F1 mutants. Although the expression of these nuclear receptor genes is essentially normal in rig mutant larvae, the ecdysone-triggered switch in E74 isoform expression is defective. rig encodes a protein with multiple WD-40 repeats and an LXXLL motif, sequences that act as specific protein-protein interaction domains. Consistent with the presence of these elements and the lethal phenotypes of rig mutants, Rig protein interacts with several Drosophila nuclear receptors in GST pull-down experiments, including EcR, USP, DHR3, SVP and betaFTZ-F1. The ligand binding domain of betaFTZ-F1 is sufficient for this interaction, which can occur in an AF-2-independent manner. Antibody stains reveal that Rig protein is present in the brain and imaginal discs of second and third instar larvae, where it is restricted to the cytoplasm. In larval salivary gland and midgut cells, however, Rig shuttles between the cytoplasm and nucleus in a spatially and temporally regulated manner, at times that correlate with the major lethal phase of rig mutants and major switches in ecdysone-regulated gene expression. Taken together, these data indicate that rig exerts essential functions during larval development through gene-specific effects on ecdysone-regulated transcription, most likely as a cofactor for one or more nuclear receptors. Furthermore, the dynamic intracellular redistribution of Rig protein suggests that it may act to refine spatial and temporal responses to ecdysone during development.J.A.O. was supported by a grant from the MCyT of Spain (`Ramón y Cajal'Program). J.G. was a Predoctoral Fellow and C.S.T. is an Investigator with the Howard Hughes Medical Institute.Peer reviewe

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

Peer reviewe

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

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