Transiently restricting individual amino acids protects Drosophila melanogaster against multiple stressors.

Nutrition and resilience are linked, though it is not yet clear how diet confers stress resistance or the breadth of stressors that it can protect against. We have previously shown that transiently restricting an essential amino acid can protect Drosophila melanogaster against nicotine poisoning. Here, we sought to characterize the nature of this dietary-mediated protection and determine whether it was sex, amino acid and/or nicotine specific. When we compared between sexes, we found that isoleucine deprivation increases female, but not male, nicotine resistance. Surprisingly, we found that this protection afforded to females was not replicated by dietary protein restriction and was instead specific to individual amino acid restriction. To understand whether these beneficial effects of diet were specific to nicotine or were generalizable across stressors, we pre-treated flies with amino acid restriction diets and exposed them to other types of stress. We found that some of the diets that protected against nicotine also protected against oxidative and starvation stress, and improved survival following cold shock. Interestingly, we found that a diet lacking isoleucine was the only diet to protect against all these stressors. These data point to isoleucine as a critical determinant of robustness in the face of environmental challenges.

A multi-nutrient array protocol to study disease-diet interactions in Drosophila melanogaster.

The availability of a defined fully synthetic diet for the fruit fly Drosophila melanogaster allows for complete and precise manipulation of its nutritional environment. Here, we present a protocol for performing large-scale multivariate nutrient analysis via the traditional diet preparation approach, or by adding nutrient solutions to a baseline medium. We detail procedures from sample collection to data analysis. This protocol has applications for the study of nutrition-life trait interactions and nutrigenomics, to reveal interactions between genotype and diet composition. For complete details on the use and execution of this protocol, please refer to Martelli et al.1 and Martelli et al.2.

A defined diet for pre-adult Drosophila melanogaster.

Drosophila melanogaster is unique among animal models because it has a fully defined synthetic diet available to study nutrient-gene interactions. However, use of this diet is limited to adult studies due to impaired larval development and survival. Here, we provide an adjusted formula that reduces the developmental period, restores fat levels, enhances body mass, and fully rescues survivorship without compromise to adult lifespan. To demonstrate an application of this formula, we explored pre-adult diet compositions of therapeutic potential in a model of an inherited metabolic disorder affecting the metabolism of branched-chain amino acids. We reveal rapid, specific, and predictable nutrient effects on the disease state consistent with observations from mouse and patient studies. Together, our diet provides a powerful means with which to examine the interplay between diet and metabolism across all life stages in an animal model.

Identifying potential dietary treatments for inherited metabolic disorders using Drosophila nutrigenomics.

Inherited metabolic disorders are a group of genetic conditions that can cause severe neurological impairment and child mortality. Uniquely, these disorders respond to dietary treatment; however, this option remains largely unexplored because of low disorder prevalence and the lack of a suitable paradigm for testing diets. Here, we screened 35 Drosophila amino acid disorder models for disease-diet interactions and found 26 with diet-altered development and/or survival. Using a targeted multi-nutrient array, we examine the interaction in a model of isolated sulfite oxidase deficiency, an infant-lethal disorder. We show that dietary cysteine depletion normalizes their metabolic profile and rescues development, neurophysiology, behavior, and lifelong fly survival, thus providing a basis for further study into the pathogenic mechanisms involved in this disorder. Our work highlights the diet-sensitive nature of metabolic disorders and establishes Drosophila as a valuable tool for nutrigenomic studies for informing potential dietary therapies.

A Simple Method for Quantifying Larval Locomotion in Drosophila melanogaster.

The fruit fly Drosophila melanogaster is a powerful genetic model that has been used for many decades to study nervous system function, development, and behavior. There are a large number of developmental and behavioral traits that can be measured to provide a broad readout of neurological function. These include patterned motor behaviors, such as larval locomotion, which can be used to assess whether genetic or environmental factors affect nervous system function to provide an entry point for deeper mechanistic studies. Here, we describe a protocol for quantifying larval locomotion using a simple camera setup and a freely available image analysis software. This protocol can be readily applied to human disease models or in toxicology studies, for example, to broadly assess the impact of treatments on neurological function.

Immunohistochemical Analysis of the Drosophila Larval Neuromuscular Junction.

Synapses are specialized junctions between cells that mediate neurotransmission to modify brain activity and body function. Studies on synapse structure and function play an important role in understanding how neurons communicate and the consequences of their dysfunction in neurological disorders. The Drosophila larval neuromuscular junction is an excellent model for dissecting the cellular and molecular mechanisms of the synapse, with its large size, accessibility, and well-characterized genetics. This protocol describes the steps required for morphological and immunohistochemical analysis of the Drosophila larval neuromuscular junction including its dissection and multiplex labeling of synaptic proteins. This technique can be used to assess the impact of genetic manipulations on synaptic development, integrity, and plasticity, thus providing a valuable tool for probing complex neurological processes in a whole animal system.

GCN2 mediates access to stored amino acids for somatic maintenance during Drosophila ageing.

Many mechanistic theories of ageing argue that a progressive failure of somatic maintenance, the use of energy and resources to prevent and repair damage to the cell, underpins ageing. To sustain somatic maintenance an organism must acquire dozens of essential nutrients from the diet, including essential amino acids (EAAs), which are physiologically limiting for many animals. In Drosophila, adulthood deprivation of each individual EAA yields vastly different lifespan trajectories, and adulthood deprivation of one EAA, phenylalanine (Phe), has no associated lifespan cost; this is despite each EAA being strictly required for growth and reproduction. Moreover, survival under any EAA deprivation depends entirely on the conserved AA sensor GCN2, a component of the integrated stress response (ISR), suggesting that a novel ISR-mediated mechanism sustains lifelong somatic maintenance during EAA deprivation. Here we investigated this mechanism, finding that flies chronically deprived of dietary Phe continue to incorporate Phe into new proteins, and that challenging flies to increase the somatic requirement for Phe shortens lifespan under Phe deprivation. Further, we show that autophagy is required for full lifespan under Phe deprivation, and that activation of the ISR can partially rescue the shortened lifespan of GCN2-nulls under Phe deprivation. We therefore propose a mechanism by which GCN2, via the ISR, activates autophagy during EAA deprivation, breaking down a larvally-acquired store of EAAs to support somatic maintenance. These data refine our understanding of the strategies by which flies sustain lifelong somatic maintenance, which determines length of life in response to changes in the nutritional environment.

Neuroligin 2 governs synaptic morphology and function through RACK1-cofilin signaling in Drosophila.

Neuroligins are transmembrane cell adhesion proteins well-known for their genetic links to autism spectrum disorders. Neuroligins can function by regulating the actin cytoskeleton, however the factors and mechanisms involved are still largely unknown. Here, using the Drosophila neuromuscular junction as a model, we reveal that F-Actin assembly at the Drosophila NMJ is controlled through Cofilin signaling mediated by an interaction between DNlg2 and RACK1, factors not previously known to work together. The deletion of DNlg2 displays disrupted RACK1-Cofilin signaling pathway with diminished actin cytoskeleton proteo-stasis at the terminal of the NMJ, aberrant NMJ structure, reduced synaptic transmission, and abnormal locomotion at the third-instar larval stage. Overexpression of wildtype and activated Cofilin in muscles are sufficient to rescue the morphological and physiological defects in dnlg2 mutants, while inactivated Cofilin is not. Since the DNlg2 paralog DNlg1 is known to regulate F-actin assembly mainly via a specific interaction with WAVE complex, our present work suggests that the orchestration of F-actin by Neuroligins is a diverse and complex process critical for neural connectivity.

Spastin is an essential regulator of male meiosis, acrosome formation, manchette structure and nuclear integrity.

The development and function of male gametes is dependent on a dynamic microtubule network, yet how this is regulated remains poorly understood. We have recently shown that microtubule severing, via the action of the meiotic AAA ATPase protein clade, plays a crucial role in this process. Here, we sought to elucidate the roles of spastin, an as-yet-unexplored member of this clade in spermatogenesis. Using a SpastKO/KO mouse model, we reveal that spastin loss resulted in a complete loss of functional germ cells. Spastin plays a crucial role in the assembly and function of the male meiotic spindle. Consistent with meiotic failure, round spermatid nuclei were enlarged, indicating aneuploidy, but were still able to enter spermiogenesis. During spermiogenesis, we observed extreme abnormalities in manchette structure, acrosome biogenesis and, commonly, a catastrophic loss of nuclear integrity. This work defines an essential role for spastin in regulating microtubule dynamics during spermatogenesis, and is of potential relevance to individuals carrying spastin variants and to the medically assisted reproductive technology industry.

Drosophila as a diet discovery tool for treating amino acid disorders.

Amino acid disorders (AADs) are a large group of rare inherited conditions that collectively impact one in 6500 live births, often resulting in rapid neurological decline and death during infancy. For several AADs, including phenylketonuria, dietary modification prevents physiological deterioration and ameliorates symptoms. Despite this remarkable potential for treatment success, dietary therapy for most AADs remains largely unexplored. Although animal models have provided novel insights into AAD mechanisms, few have been used for therapeutic diet discovery. Here, we find that of all the animal models, Drosophila is particularly well suited for nutrigenomic disease modelling, having amino acid pathways conserved with humans, exceptional genetic tractability, and the unique availability of a synthetic customisable diet.

Noggin proteins are multifunctional extracellular regulators of cell signaling.

Noggin is an extracellular cysteine knot protein that plays a crucial role in vertebrate dorsoventral patterning. Noggin binds and inhibits the activity of bone morphogenetic proteins via a conserved N-terminal clip domain. Noncanonical orthologs of Noggin that lack a clip domain ("Noggin-like" proteins) are encoded in many arthropod genomes and are thought to have evolved into receptor tyrosine kinase ligands that promote Torso/receptor tyrosine kinase signaling rather than inhibiting bone morphogenic protein signaling. Here, we examined the molecular function of noggin/noggin-like genes (ApNL1 and ApNL2) from the arthropod pea aphid using the dorso-ventral patterning of Xenopus and the terminal patterning system of Drosophila to identify whether these proteins function as bone morphogenic protein or receptor tyrosine kinase signaling regulators. Our findings reveal that ApNL1 from the pea aphid can regulate both bone morphogenic protein and receptor tyrosine kinase signaling pathways, and unexpectedly, that the clip domain is not essential for its antagonism of bone morphogenic protein signaling. Our findings indicate that ancestral noggin/noggin-like genes were multifunctional regulators of signaling that have specialized to regulate multiple cell signaling pathways during the evolution of animals.

Macrophage self-renewal is regulated by transient expression of PDGF- and VEGF-related factor 2.

Macrophages are an ancient blood cell lineage critical for homeostasis and defence against pathogens. Although their numbers were long thought to be sustained solely by haematopoietic organs, it has recently become clear that their proliferation, or self-renewal, also plays a major role. In the Drosophila larva, macrophages undergo a phase of rapid self-renewal, making this an attractive model for elucidating the signals and regulatory mechanisms involved. However, a central self-renewal pathway has not been identified in this system. Here, we show that the PDGF- and VEGF-receptor related (Pvr) pathway fulfils this role. Our data show that two of the three known Pvr ligands, PDGF- and VEGF-related factor 2 (Pvf2) and Pvf3, are major determinants of overall macrophage numbers, yet they each act in a temporally independent manner and via distinct mechanisms. While Pvf3 is needed prior to the self-renewal period, we find that Pvf2 is critical specifically for expanding the larval macrophage population. We further show that Pvf2 is a potent macrophage mitogen that is kept at limiting quantities by its transient expression in a remarkably small number of blood cells. Together, these data support a novel mechanism for the regulation of macrophage self-renewal rates by the dynamic transcriptional control of Pvf2. Given the strong parallels that exist between Drosophila and vertebrate macrophage systems, it is likely that a similar self-renewal control mechanism is at play across animal phyla.

A cis-regulatory-directed pipeline for the identification of genes involved in cardiac development and disease.

BACKGROUND: Congenital heart diseases are the major cause of death in newborns, but the genetic etiology of this developmental disorder is not fully known. The conventional approach to identify the disease-causing genes focuses on screening genes that display heart-specific expression during development. However, this approach would have discounted genes that are expressed widely in other tissues but may play critical roles in heart development. RESULTS: We report an efficient pipeline of genome-wide gene discovery based on the identification of a cardiac-specific cis-regulatory element signature that points to candidate genes involved in heart development and congenital heart disease. With this pipeline, we retrieve 76% of the known cardiac developmental genes and predict 35 novel genes that previously had no known connectivity to heart development. Functional validation of these novel cardiac genes by RNAi-mediated knockdown of the conserved orthologs in Drosophila cardiac tissue reveals that disrupting the activity of 71% of these genes leads to adult mortality. Among these genes, RpL14, RpS24, and Rpn8 are associated with heart phenotypes. CONCLUSIONS: Our pipeline has enabled the discovery of novel genes with roles in heart development. This workflow, which relies on screening for non-coding cis-regulatory signatures, is amenable for identifying developmental and disease genes for an organ without constraining to genes that are expressed exclusively in the organ of interest.

Natural variation at the Drosophila melanogaster Or22 odorant receptor locus is associated with changes in olfactory behaviour.

In insects, many critical olfactory behaviours are mediated by the large odorant receptor (Or) gene family, which determines the response properties of different classes of olfactory receptor neurons (ORNs). While ORN responses are generally conserved within and between Drosophila species, variant alleles of the D. melanogaster Or22 locus have previously been shown to alter the response profile of an ORN class called ab3A. These alleles show potential clinal variation, suggesting that selection is acting at this locus. Here, we investigated if the changes seen in ab3A responses lead to changes in olfactory-related behaviours. We show that variation at the Or22 locus and in the ab3A neurons are not fully compensated for by other ORNs and lead to overall changes in antennal odorant detection. We further show that this correlates with differences in odorant preference behaviour and with differences in oviposition site preference, with flies that have the chimaeric short allele strongly preferring to oviposit on banana. These findings indicate that variation at the Or22 locus leads to changes in olfactory-driven behaviours, and add support to the idea that the ab3A neurons are of especial importance to the ecology of Drosophila flies.

Insulin-Like Signalling Influences the Coordination of Larval Hemocyte Number with Body Size in Drosophila melanogaster.

Blood cells, known as hemocytes in invertebrates, play important and conserved roles in immunity, wound healing and tissue remodelling. The control of hemocyte number is therefore critical to ensure these functions are not compromised, and studies using Drosophila melanogaster are proving useful for understanding how this occurs. Recently, the embryonic patterning gene, torso-like (tsl), was identified as being required both for normal hemocyte development and for providing immunity against certain pathogens. Here, we report that Tsl is required specifically during the larval phase of hematopoiesis, and that tsl mutant larvae likely have reduced hemocyte numbers due to a reduced larval growth rate and compromised insulin signaling. Consistent with this, we find that impairing insulin-mediated growth, either by nutrient deprivation or genetically, results in fewer hemocytes. This is likely the result of impaired insulin-like signaling in the hemocytes themselves, since modulation of Insulin-like Receptor (InR) activity specifically in hemocytes causes concomitant changes to their population size in developing larvae. Taken together, our work reveals the strong relationship that exists between body size and hemocyte number, and suggests that insulin-like signaling contributes to, but is not solely responsible for, keeping these tightly aligned during larval development.

The torso-like gene functions to maintain the structure of the vitelline membrane in Nasonia vitripennis, implying its co-option into Drosophila axis formation.

Axis specification is a fundamental developmental process. Despite this, the mechanisms by which it is controlled across insect taxa are strikingly different. An excellent example of this is terminal patterning, which in Diptera such as Drosophila melanogaster occurs via the localized activation of the receptor tyrosine kinase Torso. In Hymenoptera, however, the same process appears to be achieved via localized mRNA. How these mechanisms evolved and what they evolved from remains largely unexplored. Here, we show that torso-like, known for its role in Drosophila terminal patterning, is instead required for the integrity of the vitelline membrane in the hymenopteran wasp Nasonia vitripennis We find that other genes known to be involved in Drosophila terminal patterning, such as torso and Ptth, also do not function in Nasonia embryonic development. These findings extended to orthologues of Drosophila vitelline membrane proteins known to play a role in localizing Torso-like in Drosophila; in Nasonia these are instead required for dorso-ventral patterning, gastrulation and potentially terminal patterning. Our data underscore the importance of the vitelline membrane in insect development, and implies phenotypes caused by knockdown of torso-like must be interpreted in light of its function in the vitelline membrane. In addition, our data imply that the signalling components of the Drosophila terminal patterning systems were co-opted from roles in regulating moulting, and co-option into terminal patterning involved the evolution of a novel interaction with the vitelline membrane protein Torso-like.This article has an associated First Person interview with the first author of the paper.

PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy.

Cryo-electron tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-focused ion beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.

Control of growth factor signalling by MACPF proteins.

Members of the membrane attack complex/perforin-like (MACPF) protein superfamily have long captured interest because of their unique ability to assemble into large oligomeric pores on the surfaces of cells. The best characterised of these act in vertebrate immunity where they function to deliver pro-apoptotic factors or induce the cytolysis and death of targeted cells. Less appreciated, however, is that rather than causing cell death, MACPF proteins have also evolved to control cellular signalling pathways and influence developmental programmes such as pattern formation and neurogenesis. Torso-like (Tsl) from the fruit fly Drosophila, for example, functions to localise the activity of a growth factor for patterning its embryonic termini. It remains unclear whether these developmental proteins employ an attenuated form of the classical MACPF lytic pore, or if they have evolved to function via alternative mechanisms of action. In this minireview, we examine the evidence that links pore-forming MACPF proteins to the control of growth factor and cytokine signalling. We will then attempt to reconcile how the MACPF domain may have been repurposed during evolution for developmental events rather than cell killing.

Molecular and Functional Evolution at the Odorant Receptor Or22 Locus in Drosophila melanogaster.

Insect odorant receptor (Or) genes determine the responses of sensory neurons that mediate critical behaviors. The Drosophila melanogaster Or22 locus represents an interesting example of molecular evolution, with high levels of sequence divergence and copy number variation between D. melanogaster and other Drosophila species, and a corresponding high level of variability in the responses of the neuron it controls, ab3A. However, the link between Or22 molecular and functional diversity has not been established. Here, we show that several naturally occurring Or22 variants generate major shifts in neuronal response properties. We determine the molecular changes that underpin these response shifts, one of which represents a chimeric gene variant previously suggested to be under natural selection. In addition, we show that several alternative molecular genetic mechanisms have evolved for ensuring that where there is more than one gene copy at this locus, only one functional receptor is generated. Our data thus provide a causal link between the striking levels of phenotypic neuronal response variation found in natural populations of D. melanogaster and genetic variation at the Or22 locus. Since neuronal responses govern animal behavior, we predict that Or22 may be a key player in underlying one or more olfactory-driven behaviors of significant adaptive importance.

Receptor Tyrosine Kinases in Development: Insights from Drosophila.

Cell-to-cell communication mediates a plethora of cellular decisions and behaviors that are crucial for the correct and robust development of multicellular organisms. Many of these signals are encoded in secreted hormones or growth factors that bind to and activate cell surface receptors, to transmit the cue intracellularly. One of the major superfamilies of cell surface receptors are the receptor tyrosine kinases (RTKs). For nearly half a century RTKs have been the focus of intensive study due to their ability to alter fundamental aspects of cell biology, such as cell proliferation, growth, and shape, and because of their central importance in diseases such as cancer. Studies in model organisms such a Drosophila melanogaster have proved invaluable for identifying new conserved RTK pathway components, delineating their contributions, and for the discovery of conserved mechanisms that control RTK-signaling events. Here we provide a brief overview of the RTK superfamily and the general mechanisms used in their regulation. We further highlight the functions of several RTKs that govern distinct cell-fate decisions in Drosophila and explore how their activities are developmentally controlled.