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The vast majority of species that belong to the plant or animal kingdom evolved with two main strategies to counter tissue damage - scar formation and regeneration. Whereas scar formation provides a fast and cost-effective repair to exit life-threatening conditions, complete tissue regeneration is time-consuming and requires vast resources to reinstall functionality of affected organs or structures. Local environments in wound healing are widely studied and findings have provided important biomedical applications. Less well understood are organismic physiological parameters and signaling circuits essential to maintain effective tissue repair. Here, we review accumulated evidence that positions the interplay of local and systemic changes in metabolism as essential variables modulating the injury response. We particularly emphasize the role of lipids and lipid-like molecules as significant components long overlooked.

In periods of malnutrition starving organisms initiate the reorganization and degradation of cellular lipids. Yet, the build-up of lipid stores is reported in animals entering early starvation stages. Anabolic lipid metabolism is regulated by many circuits including the insulin/insulin- like growth factor signaling (IIS) cascade. The regulative role of IIS ligands in food deprived animals is often neglected, since supporting evidence for this notion is largely missing.Here we show that in starving adult Drosophila melanogaster IIS is required to stabilize the transport of circulating lipids and sugars, and is essential for the initial increase of lipid stores in fat body cells. Further, we demonstrate that the Drosophila insulin-like peptides (dIlp) 2 and 5 regulate the cellular IIS cascade differentially. To its end we speculate that each hormone initiates complementary measures that increase the starvation resistance of adult flies.

Sense of taste is instructive for food perception and is encoded by five principle sensory inputs: sweet, bitter, salty, sour, and umami. However, there is mounting evidence that dietary fat is also palatable, and that many species including humans are able to discriminate the quality of free fatty acids in dedicated experimental environments. Multiple lipid-taste receptors are suggested to be capable of binding subsets of fatty acids and initiating cellular signals in specific sensory cells. Nevertheless, the structure and composition of dietary lipids is complex by nature and food consists of many other tasty macronutrients. To date, it remains an open question if or how dietary lipids induce taste sensation instructive for the feeding behavior of animals. We recorded the feeding behavior of adult Drosophila melanogaster utilizing different established feeding assays. Using lipid defined diets and the yeast strain BY4741, we found that flies are able to detect the fungal sterol ergosterol and specific saturated fatty acid species, the latter bound in more complex nutritional lipids. Moreover, we show that the neuronal expression of the CD36-member SNMP1 together with the neuronal insulin signaling activity are essential to define the perception of one selected tasty lipid. We provide evidence that the sugar and protein content in the food is instrumental to modulate insulin signaling, and propose that the sugar:protein ratio is one determining factor instructive for the feeding response of fruit flies to given dietary lipid profiles.

The experimental versatility of the fruit fly has helped to uncover the molecular basis of epithelial cell polarity. In this chapter, we provide protocols to dissect Drosophila larval salivary glands (SGs) for ex vivo culture and live imaging, and for fixing and immunostaining for analysis by fluorescence microscopy. We describe how to combine these approaches with genetic and pharmacological assays. These techniques can be applied to study signaling pathways regulating epithelial cell polarity, membrane trafficking, gland secretion, and their impacts on animal feeding behavior.

Neuronal function is highly energy demanding and thus requires efficient and constant metabolite delivery. Like their mammalian counterparts Drosophila glia are highly glycolytic and provide lactate to fuel neuronal metabolism. However, flies are able to survive for several weeks in the absence of glial glycolysis1. Here, we study how glial cells maintain sufficient nutrient supply to neurons under conditions of carbohydrate restriction. We show that glycolytically impaired glia switch to fatty acid breakdown via β-oxidation and provide ketone bodies as an alternate neuronal fuel. Moreover, flies also rely on glial β-oxidation under starvation conditions with glial loss of β-oxidation increasing susceptibility to starvation. Further, we show that glial cells act as a metabolic sensor in the brain and can induce mobilization of peripheral energy stores to ensure brain metabolic homeostasis. In summary, our study gives pioneering evidence on the importance of glial β-oxidation and ketogenesis for brain function, and survival, under adverse conditions, like malnutrition. The glial capacity to utilize lipids as an energy source seems to be conserved from flies to humans.

The Insulin signal cascade is one of the best studied metabolic circuits, and shows a remarkable high molecular and functional conservation across the animal kingdom. Insulin-producing cells respond directly to nutritional cues in circulation and receive modulatory input from connected neuronal networks. Neuronal control is rapid and integrates a wide range of variables including dietary change or environmental temperature. However, despite various detailed studies that demonstrated the potential of neuronal regulation the physiological relevance of this circuit remains elusive.In Drosophila, Insulin-like peptide 7 (dIlp7)-producing neurons are wired with Insulin-producing cells. We found a dual role for this neuronal subset: a.) activated dilp7-producing neurons are required to facilitate development at high temperatures, and if confronted with calorie-rich food that represses neuronal activity b.) their product, dIlp7, regulates Insulin signalling levels. Our work shows that Insulin-producing cells not simply integrate signals from circulating nutritional cues and neuronal inputs, but switch to neuronal control in response to dietary composition.

Cellular Insulin signaling shows a remarkable high molecular and functional conservation. Insulin-producing cells respond directly to nutritional cues in circulation and receive modulatory input from connected neuronal networks. Neuronal control integrates a wide range of variables including dietary change or environmental temperature. Although it is shown that neuronal input is sufficient to regulate Insulin-producing cells, the physiological relevance of this network remains elusive. In Drosophila melanogaster, Insulin-like peptide7-producing neurons are wired with Insulin-producing cells. We found that the former cells regulate the latter to facilitate larval development at high temperatures, and to regulate systemic Insulin signaling in adults feeding on calorie-rich food lacking dietary yeast. Our results demonstrate a role for neuronal innervation of Insulin-producing cells important for fruit flies to survive unfavorable environmental conditions.

It is known that the male seminal fluid contains factors that affect female post-mating behavior and physiology. InDrosophila,most of these factors are secreted by the two epithelial cell types that make up the male accessory gland: the main and secondary cells. Although secondary cells represent only 4% of the cells of the accessory gland, their contribution to the male seminal fluid is essential for sustaining the female post-mating response. To better understand the function of the secondary cells, here we investigate their molecular organization, particularly with respect to the intracellular membrane transport machinery. We determined that large vacuole-like structures found in the secondary cells are trafficking hubs labeled by Rab6, 7, 11 and 19. Furthermore, these cell-specific organelles are essential for the long-term post-mating behavior of females and that their formation is directly dependent upon Rab6. Our discovery adds to our understanding of Rab proteins function in secretory cells. We have created an online, open-access imaging resource as a valuable tool for the intracellular membrane and protein traffic community.

During cold acclimation fruit flies switch their feeding from yeast to plant food, however there are no robust molecular markers to monitor this in the wild. Drosophila melanogaster is a sterol auxotroph and relies on dietary sterols to produce lipid membranes, lipoproteins and molting hormones. We employed shotgun lipidomics to quantify eight major food sterols in total lipid extracts of heads and genital tracts of adult male and female flies. We found that their sterol composition is dynamic and reflective of fly diet in an organ-specific manner. Season-dependent changes observed in the organs of wild-living flies suggested that the molar ratio between yeast (ergosterol, zymosterol) and plant (sitosterol, stigmasterol) sterols is a quantifiable, generic and unequivocal marker of their feeding behavior suitable for ecological and environmental population-based studies. The enrichment of phytosterols over yeast sterols in wild-living flies at low temperatures is consistent with switching from yeast to plant diet and corroborates the concomitantly increased unsaturation of their membrane lipids.

An efficient vectorial intracellular transport machinery depends on a well-established apico-basal polarity and is a prerequisite for the function of secretory epithelia. Despite extensive knowledge on individual trafficking pathways, little is known about the mechanisms coordinating their temporal and spatial regulation. Here, we report that the polarity protein Crumbs is essential for apical plasma membrane phospholipid-homeostasis and efficient apical secretion. Through recruitingβHeavy-Spectrin and MyosinV to the apical membrane, Crumbs maintains the Rab6-, Rab11- and Rab30-dependent trafficking and regulates the lipid phosphatases Pten and Ocrl. Crumbs knock-down results in increased apical levels of PI(4,5)P2and formation of a novel, Moesin- and PI(4,5)P2-enriched apical membrane sac containing microvilli-like structures. Our results identify Crumbs as an essential hub required to maintain the organization of the apical membrane and the physiological activity of the larval salivary gland.

A calorie-rich diet is one reason for the continuous spread of metabolic syndromes in western societies. Smart food design is one powerful tool to prevent metabolic stress, and the search for suitable bioactive additives is a continuous task. The nutrient-sensing insulin signaling pathway is an evolutionary conserved mechanism that plays an important role in metabolism, growth and development. Recently, lipid cues capable to stimulate insulin signaling were identified. However, the mechanistic base of their activity remains obscure to date. Here, we show that specific AKT/ Protein kinase B isoforms are responsive to dietary lipid extract compositions and potentiate cellular insulin signaling levels. Our data add a new dimension to existing models and position Drosophila as a powerful model to study the relation between dietary lipid cues and the insulin induced cellular signal cascade.

The organisation of intracellular transport processes is adapted specifically to different cell types, developmental stages, and physiological requirements. Some protein traffic routes are universal to all cells and constitutively active, while other routes are cell-type specific, transient, and induced under particular conditions only. Small GTPases of the Rab (Ras related in brain) subfamily are conserved across eukaryotes and regulate most intracellular transit pathways. The complete sets of Rab proteins have been identified in model organisms, and molecular principles underlying Rab functions have been uncovered. Rabs provide intracellular landmarks that define intracellular transport sequences. Nevertheless, it remains a challenge to systematically map the subcellular distribution of all Rabs and their functional interrelations. This task requires novel tools to precisely describe and manipulate the Rab machinery in vivo. Here we discuss recent findings about Rab roles during development and we consider novel approaches to investigate Rab functions in vivo.

Metabolic research is a challenge because of the variety of data within experimental series and the difficulty of replicating results among scientific groups. The fruit fly, Drosophila melanogaster, is a cost-effective and reliable pioneer model to screen dietary variables for metabolic research. One of the main reasons for problems in this field are differences in food recipes, diet-associated microbial environments and the pharmacokinetic behavior of nutrients across the gut-blood barrier. To prevent such experimental shortcomings, a common strategy is to pool scores of subjects into one sample to create an average statement. However, this approach lacks information about the biological spread and may provoke misleading interpretations. We propose to use the developmental rate of individual Drosophila larvae as a metabolic sensor. To do so, we introduce here a 96-well plate-based assay, which allows screening for multiple variables including food quality, microbial load, and genetic differences. We demonstrate that on a diet that is rich in calories, pupation is sensitive to the variation of dietary lipid compounds and that genotypes considered as wild-types/controls produce different developmental profiles. Our platform is suited for later automation and represents a potent high-throughput screening tool for the pharmacology and food industry. If used systematically, our assay could become a powerful reference tool to compare the quality of used dietary configurations with published benchmark recipes.

A crucial yet ill-defined step during the development of tubular networks, such as the vasculature, is the formation of connections (anastomoses) between pre-existing lumenized tubes. By studying tracheal tube anastomosis in Drosophila melanogaster, we uncovered a key role of secretory lysosome-related organelle (LRO) trafficking in lumen fusion. We identified the conserved calcium-binding protein Unc-13-4/Staccato (Stac) and the GTPase Rab39 as critical regulators of this process. Stac and Rab39 accumulate on dynamic vesicles, which form exclusively in fusion tip cells, move in a dynein-dependent manner, and contain late-endosomal, lysosomal, and SNARE components characteristic of LROs. The GTPase Arl3 is necessary and sufficient for Stac LRO formation and promotes Stac-dependent intracellular fusion of juxtaposed apical plasma membranes, thereby forming a transcellular lumen. Concomitantly, calcium is released locally from ER exit sites and apical membrane-associated calcium increases. We propose that calcium-dependent focused activation of LRO exocytosis restricts lumen fusion to appropriate domains within tip cells.

Since the discovery of microRNAs (miRNAs) only two decades ago, they have emerged as an essential component of the gene regulatory machinery. miRNAs have seemingly paradoxical features: a single miRNA is able to simultaneously target hundreds of genes, while its presence is mostly dispensable for animal viability under normal conditions. It is known that miRNAs act as stress response factors; however, it remains challenging to determine their relevant targets and the conditions under which they function. To address this challenge, we propose a new workflow for miRNA function analysis, by which we found that the evolutionarily young miRNA family, the mir-310s (mir-310/mir-311/mir-312/mir-313), are important regulators of Drosophila metabolic status. mir-310s-deficient animals have an abnormal diet-dependent expression profile for numerous diet-sensitive components, accumulate fats, and show various physiological defects. We found that the mir-310s simultaneously repress the production of several regulatory factors (Rab23, DHR96, and Ttk) of the evolutionarily conserved Hedgehog (Hh) pathway to sharpen dietary response. As the mir-310s expression is highly dynamic and nutrition sensitive, this signal relay model helps to explain the molecular mechanism governing quick and robust Hh signaling responses to nutritional changes. Additionally, we discovered a new component of the Hh signaling pathway in Drosophila, Rab23, which cell autonomously regulates Hh ligand trafficking in the germline stem cell niche. How organisms adjust to dietary fluctuations to sustain healthy homeostasis is an intriguing research topic. These data are the first to report that miRNAs can act as executives that transduce nutritional signals to an essential signaling pathway. This suggests miRNAs as plausible therapeutic agents that can be used in combination with low calorie and cholesterol diets to manage quick and precise tissue-specific responses to nutritional changes.

Summary Membrane trafficking is key to the cell biological mechanisms underlying development. Rab GTPases control specific membrane compartments, from core secretory and endocytic machinery to less-well-understood compartments. We tagged all 27 Drosophila Rabs with YFPMYC at their endogenous chromosomal loci, determined their expression and subcellular localization in six tissues comprising 23 cell types, and provide this data in an annotated, searchable image database. We demonstrate the utility of these lines for controlled knockdown and show that similar subcellular localization can predict redundant functions. We exploit this comprehensive resource to ask whether a common Rab compartment architecture underlies epithelial polarity. Strikingly, no single arrangement of Rabs characterizes the five epithelia we examine. Rather, epithelia flexibly polarize Rab distribution, producing membrane trafficking architectures that are tissue- and stage-specific. Thus, the core machinery responsible for epithelial polarization is unlikely to rely on polarized positioning of specific Rab compartments., Graphical Abstract, Highlights • A collection of YFP-tagged rab alleles allows functional in vivo studies • FLYtRAB database links downloadable image data and annotated Rab localization terms • Rab compartment polarity in epithelia is flexible and dynamic • Similar intracellular Rab protein localization can indicate redundant Rab functions, Membrane trafficking is key to the cell biological mechanisms underlying development. As hypothesis-generating tools, Dunst et al. present a genetic resource for systematic visualization and manipulation of all membrane compartments controlled by Rab GTPases in Drosophila and a database summarizing Rab localization in different cell and tissue types.

During organogenesis, secreted signaling proteins direct cell migration towards their target tissue. In Drosophila embryos, developing muscles are guided by signals produced by tendons to promote the proper attachment of muscles to tendons, essential for proper locomotion. Previously, the repulsive protein Slit, secreted by tendon cells, has been proposed to be an attractant for muscle migration. However, our findings demonstrate that through tight control of its distribution, Slit repulsion is used for both directing and arresting muscle migration. We show that Slit cleavage restricts its distribution to tendon cells, allowing it to function as a short-range repellent that directs muscle migration and patterning, and promotes their halt upon reaching the target site. Mechanistically, we show that Slit processing produces a rapidly degraded C-terminal fragment and an active, stable N-terminal polypeptide that is tethered to the tendon cell membrane, which further protects it from degradation. Consistently, the requirement for Slit processing can be bypassed by providing an uncleavable, membrane-bound form of Slit that is stable and is retained on expressing tendon cells. Moreover, muscle elongation appears to be extremely sensitive to Slit levels, as replacing the entire full-length Slit with the stable Slit-N-polypeptide results in excessive repulsion, which leads to a defective muscle pattern. These findings reveal a novel cleavage-dependent regulatory mechanism controlling Slit spatial distribution, which may operate in other Slit-dependent processes.

The Insulin signaling pathway couples growth, development and lifespan to nutritional conditions. Here, we demonstrate a function for the Drosophila lipoprotein LTP in conveying information about dietary lipid composition to the brain to regulate Insulin signaling. When yeast lipids are present in the diet, free calcium levels rise in Blood Brain Barrier glial cells. This induces transport of LTP across the Blood Brain Barrier by two LDL receptor-related proteins: LRP1 and Megalin. LTP accumulates on specific neurons that connect to cells that produce Insulin-like peptides, and induces their release into the circulation. This increases systemic Insulin signaling and the rate of larval development on yeast-containing food compared with a plant-based food of similar nutritional content. DOI: http://dx.doi.org/10.7554/eLife.02862.001, eLife digest How does an animal sense if it is well nourished or not, and then regulate its metabolism appropriately? This process largely relies on the animal's body deciphering signals that that are transmitted between different organs in the form of molecules and hormones. Many animals—ranging from insects to mammals (including humans)—also use their brains to sense and decipher these nutritional signals. A signaling pathway involving the hormone insulin controls how various different animals grow and develop—and how long they will live—based on these animals' food intake. Insulin is produced in mammals by an organ called the pancreas. But in the fruit fly Drosophila, this hormone is produced by cells within different tissues, including the insect’s brain. The fruit fly is used to study many biological processes because it is easy to work with in a laboratory. Insulin-producing cells make and release insulin-like molecules into the insect's hemolymph (a blood-like fluid) in response to sugar and to other nutrients (which are detected via molecules generated in a fruit fly organ called the fat body). The fat body produces lipophorin, a protein which carries fat molecules in the hemolymph, and which is known to be able to move from the hemolymph to the brain and accumulate within the brain. The fat body also produces lipid transfer protein (or LTP), which transfers fats absorbed or made within the insect's gut onto lipophorin, and can also unload fat molecules to other insect cells. If LTP can also enter the brain, and what it might do there, was unclear. Brankatschk et al.now discover that LTP can cross the ‘blood brain barrier’ in fruit fly larvae and can accumulate over time on their insulin-producing cells and the neurons in direct contact with these cells. This accumulation depends on the flies’ diet: flies fed a diet made from yeast cells accumulated LTP on these neurons, while those fed only on sugar and proteins did not. Furthermore Brankatschk et al. found that when they switched flies from a yeast-based to a plant-based diet, the larvae grew more slowly and the flies lived longer. Both of the diets contained abundant calories and nutrients, but contained slightly different kinds of fat molecules. The fly larvae on the plant-based diet also accumulated less LTP on their insulin-pathway neurons, and insulin signaling was reduced. Branskatschk et al. also found that fat molecules from the yeast-based diet activated the cells of the blood brain barrier, and that this encouraged LTP to be transported the brain. Blocking LTP from crossing the blood brain barrier reduced insulin signaling, slowed the growth of the fly larvae, and extended the lifespan of the flies. These findings of Brankatschk et al. thus reveal that fat-containing molecules carry information about specific nutrients to the brain. The extent to which these mechanisms operate in other animals—such as mammals—remains to be explored. DOI: http://dx.doi.org/10.7554/eLife.02862.002

Background: Neurons require highly specialized intracellular membrane trafficking, especially at synapses. Rab GTPases are considered master regulators of membrane trafficking in all cells, and only very few Rabs have known neuron-specific functions. Here, we present the first systematic characterization of neuronal expression, subcellular localization, and function of Rab GTPases in an organism with a brain. Results: We report the surprising discovery that half of all Drosophila Rabs function specifically or predominantly in distinct subsets of neurons in the brain. Furthermore, functional profiling of the GTP/GDP-bound states reveals that these neuronal Rabs are almost exclusively active at synapses and the majority of these synaptic Rabs specifically mark synaptic recycling endosomal compartments. Our profiling strategy is based on Gal4 knockins in large genomic fragments that are additionally designed to generate mutants by ends-out homologous recombination. We generated 36 large genomic targeting vectors and transgenic rab-Gal4 fly strains for 25 rab genes. Proof-of-principle knockout of the synaptic rab27 reveals a sleep phenotype that matches its cell-specific expression. Conclusions: Our findings suggest that up to half of all Drosophila Rabs exert specialized synaptic functions. The tools presented here allow systematic functional studies of these Rabs and provide a method that is applicable to any large gene family in Drosophila. © 2011 Elsevier Ltd. All rights reserved.

The blood–brain barrier (BBB) regulates passage of nutrients and signaling molecules from the circulation into the brain. Whether lipoproteins cross the BBBin vivohas been controversial, and no clear requirement for circulating lipoproteins in brain development has been shown. We address these issues inDrosophila, which has an functionally conserved BBB, and lipoproteins that resemble those of vertebrates. We show that theDrosophilalipoprotein lipophorin exists in two isoforms. Both isoforms cross the BBB, but accumulate on distinct subsets of cells within the brain. In addition to acting as a lipid carrier, lipophorin carries both sterol-linked and GPI-linked proteins into the circulation and transports them across the BBB. Finally, lipophorin promotes neuroblast proliferation by a mechanism that does not depend on delivery of dietary lipids. Transport of lipophorin and its cargo across the BBB represents a novel mechanism by which peripherally synthesized proteins might enter the brain and influence its development. Furthermore, lipid-linkage may be an efficient method to transport therapeutic molecules across the BBB.

SummaryThe orthogonal array of axon pathways in the Drosophila CNS is constructed in part under the control of three Robo family axon guidance receptors: Robo1, Robo2 and Robo3. Each of these receptors is responsible for a distinct set of guidance decisions. To determine the molecular basis for these functional specializations, we used homologous recombination to create a series of 9 “robo swap” alleles: expressing each of the three Robo receptors from each of the three robo loci. We demonstrate that the lateral positioning of longitudinal axon pathways relies primarily on differences in gene regulation, not distinct combinations of Robo proteins as previously thought. In contrast, specific features of the Robo1 and Robo2 proteins contribute to their distinct functions in commissure formation. These specializations allow Robo1 to prevent crossing and Robo2 to promote crossing. These data demonstrate how diversification of expression and structure within a single family of guidance receptors can shape complex patterns of neuronal wiring.

Netrins are secreted axon guidance molecules required for commissure formation in a wide range of animal species, including Caenorhabditis elegans, Drosophila melanogaster and mice. They are generally thought to function as chemoattractants, acting at a distance to direct commissural axon growth toward the midline of the central nervous system. We show here, however, that D. melanogaster commissural axons still orient normally and reach the midline even in the complete absence of netrins, though some of them fail to cross the midline. Tethering endogenous netrin to the membrane selectively disrupts its long-range but not short-range activity, yet still allows normal commissure formation. We therefore propose that netrins act in commissural axon guidance as short-range cues that promote midline crossing, not as long-range chemoattractants.

Summary Epithelial tissues develop planar polarity that is reflected in the global alignment of hairs and cilia with respect to the tissue axes. The planar cell polarity (PCP) proteins form asymmetric and polarized domains across epithelial junctions that are aligned locally between cells and orient these external structures [1–3]. Although feedback mechanisms can polarize PCP proteins intracellularly and locally align polarity between cells, how global PCP patterns are specified is not understood. It has been proposed that the graded distribution of a biasing factor could guide long-range PCP [4, 5]. However, we recently identified epithelial morphogenesis as a mechanism that can reorganize global PCP patterns; in the Drosophila pupal wing, oriented cell divisions and rearrangements reorient PCP from a margin-oriented pattern to one that points distally [6]. Here, we use quantitative image analysis to study how PCP patterns first emerge in the wing. PCP appears during larval growth and is spatially oriented through the activities of three organizer regions that control disc growth and patterning. Flattening morphogen gradients emanating from these regions does not reduce intracellular polarity but distorts growth and alters specific features of the PCP pattern. Thus, PCP may be guided by morphogenesis rather than morphogen gradients.