Phagocytosis is an evolutionarily ancient, receptor-driven process, by which phagocytic cells recognize invading microbes and destroy them after internalization. The phagocytosis receptor Eater is expressed exclusively on Drosophila phagocytes and is required for the survival of bacterial infections. In a recent study, we explored how Eater can defend fruit flies against different kinds of bacteria. We discovered that Eater bound to certain types of bacteria directly, while for others bacterial binding was dependent on prior disruption of the bacterial envelope. Similar to phagocytes, antimicrobial peptides and lysozymes are ancient components of animal immune systems. Our results suggest that cationic antimicrobial peptides, as well as lysozymes, can facilitate Eater binding to live Gram-negative bacteria. Both types of molecules promote surface-exposure of bacterial ligands that otherwise would remain buried and hidden under an outer membrane. We propose that unmasking ligands for phagocytic receptors may be a conserved mechanism operating in many animals, including humans. Thus, studying a Drosophila phagocytosis receptor may advance our understanding of innate immunity in general.

Equalizing sex chromosome expression between the sexes when they have largely differing gene content appears to be necessary, and across species, is accomplished in a variety of ways. Even in birds, where the process is less than complete,1 a mechanism to reduce the difference in gene dose between the sexes exists. In early development, while the dosage difference is unregulated and still in flux, it is frequently exploited by sex determination mechanisms. The Drosophila female sex determination process is one clear example, determining the sexes based on X chromosome dose. Recent data show that in Drosophila, the female sex not only reads this gene balance difference, but at the same time usurps the moment. Taking advantage of the transient default state of male dosage compensation, the sex determination master-switch Sex-lethal which resides on the X, has its expression levels enhanced before it works to correct the gene imbalance.2 Intriguingly, key developmental genes which could create developmental havoc if their levels were unbalanced show more exquisite regulation,3 suggesting nature distinguishes them and ensures their expression is kept in the desirable range.

The fourth chromosome of Drosophila remains one of the most intractable regions of the fly genome to genetic analysis. The main difficulty posed to the genetic analyses of mutations on this chromosome arises from the fact that it does not undergo meiotic recombination, which makes recombination mapping impossible, and also prevents clonal analysis of mutations, a technique which relies on recombination to introduce the prerequisite recessive markers and FLP-recombinase recognition targets (FRT). Here we introduce a method that overcomes these limitations and allows for the generation of single Minute haplo-4 clones of any fourth chromosome mutant gene in tissues of developing and adult flies.

The voltage-gated Na+ channels (VGSC) are complex membrane proteins responsible for generation and propagation of the electrical signals through the brain, the skeletal muscle and the heart. The levels of sodium channels affect behavior and physical activity. This is illustrated by the maleless mutant allele (mlenapts) in Drosophila, where the decreased levels of voltage-gated Na+ channels cause temperature-sensitive paralysis.

 

Here, we report that mlenapts mutant flies exhibit developmental lethality, decreased fecundity and increased neurodegeneration. The negative effect of decreased levels of Na+ channels on development and ts-paralysis was more pronounced at 18 and 29°C than at 25°C, suggesting particular sensitivity of the mlenapts flies to temperatures above and below normal environmental conditions. Similarly, longevity of mlenapts flies was unexpectedly short at 18 and 29°C compared with flies heterozygous for the mlenapts mutation. Developmental lethality and neurodegeneration of mlenapts flies was partially rescued by increasing the dosage of para, confirming a vital role of Na+ channels in development, longevity and neurodegeneration of flies and their adaptation to temperatures.

Enhancer of rudimentary, e(r), encodes a small nuclear protein, ER, that has been implicated in the regulation of pyrimidine metabolism, DNA replication and cell proliferation. In Drosophila melanogaster, a new recessive Notch allele, Nnd-p, was isolated as a lethal in combination with an e(r) allele, e(r)p2. Both mutants are viable as single mutants. Nnd-p is caused by a P-element insertion in the 5′ UTR, 378-bp upstream of the start of translation. Together the molecular and genetic data argue that Nnd-p is a hypomorphic allele of N. The three viable notchoid alleles, Nnd-p, Nnd-1 and Nnd-3, are lethal in combination with e(r)− alleles. Our present hypothesis is that e(r) is a positive regulator of the Notch signaling pathway and that the lethality of the N e(r) double mutants is caused by a reduction in the expression of the pathway. This is supported by the rescue of the lethality by a mutation in Hairless, a negative regulator of N, and by the synthetic lethality of dx e(r) double mutants. Further support for the hypothesis is a reduction in E(spl) expression in an e(r)− mutant. Immunostaining localizes ER to the nucleus, suggesting a nuclear function for ER. A role in the Notch signaling pathway, suggests that e(r) may be expressed in the nervous system. This turns out to be the case, as immunostaining of ER shows that ER is localized to the developing CNS.

We describe a novel thermosensitive escape behavior in Drosophila larvae and a simple assay to accurately define the response temperature. When a larva is placed in a droplet of water that is subsequently heated, a stereotypical escape response is robustly elicited at 29°C. Larvae defective for the painless TRP receptor, or blocked in the function of class IV multi-dendritic sensory dendrites respond to this stimulus at reproducibly higher temperature (34°C). The escape response has novel behavioral components and a lower temperature threshold in comparison with the responses to touch with a hot needle. Furthermore the assay minimizes operator bias that is present in current tests of thermosensitive nociception and generates a precise determination of temperature at the point of response. This response is highly reproducible and directly applicable to genetic and neural circuit analysis of a simple escape behavior.

Mutations and most transgenes that induce ectopic cell death in Drosophila will produce an inhibitory effect on RNA interference (RNAi) in adjacent cells. When extensive cell death is sporadically induced using a heat shock promoted-head involution defective (hs-hid) transgene, molecular attributes of this inhibition can be studied. For a Green Fluorescent Protein (GFP) RNAi construct, cell death causes a greater accumulation of the mature mRNA and the double stranded RNA with an accompanying reduction in the homologous siRNAs. Endogenous transposable element expression is increased and there is an overall reduction in their corresponding siRNAs. The implications of this finding for the conduct of RNAi and potential reasons for its existence are discussed.

Our recent study found that 30% of young genes were essential for viability that determines development through stages from embryo to pupae in Drosophila melanogaster, revealing rapidly evolving genetic components involved in the evolution of development. Meanwhile, many young genes did not produce complete lethal phenotype upon constitutive knockdown, suggesting that they may not be essential for viability. These genes, nevertheless, were fixed by natural selection, and might play an important functional role in their adult stage. Here we present a detailed demonstration that a newly duplicated serine-type endopeptidase gene that originated in the common ancestor in the D. melanogaster subgroup 6∼11 million years ago, named Slfc, revealing a strong effect in post-eclosion. Although animals survived constitutive knockdown of Slfc to adult stage, however, their life span reduced significantly by two-thirds compared to wild-type. Furthermore, the Slfc-RNAi males dropped their fertility to less than 10% of the wild-type level, with over 80% of these males being sterile. The Slfc-RNAi females, on the other hand, showed a slight reduction in fertility. This case study demonstrates that a young gene can contribute to fitness on the three important traits of life history in adults, including the life expectancy, male fertility and female fertility, suggesting that new genes can quickly evolve and impact multiple phenotypes.

We recently developed integrase-mediated trap conversion (iTRAC) as a means of exploiting gene traps to create new genetic tools, such as new markers for imaging, drivers for gene expression and landing sites for gene and chromosome engineering. The principle of iTRAC is simple: primary gene traps are generated with transposon vectors carrying ϕC31 integrase docking sites, which are subsequently utilized to integrate different constructs into trapped loci. Thus, iTRAC allows us to reconfigure selected traps for new purposes. Two features make iTRAC an attractive approach for Drosophila research. First, its versatility permits the exploitation of gene traps in an open-ended way, for applications that were not envisaged during the primary trapping screen. Second, iTRAC is readily transferable to new species and provides a means for developing complex genetic tools in Drosophilids that lack the facility of Drosophila melanogaster genetics.

Inherited retinal degeneration in Drosophila has been explored for insights into similar processes in humans. Based on the mechanisms, I divide these mutations in Drosophila into three classes. The first consists of genes that control the specialization of photoreceptor cells including the morphogenesis of visual organelles (rhabdomeres) that house the visual signaling proteins. The second class contains genes that regulate the activity or level of the major rhodopsin, Rh1, which is the light sensor and also provides a structural role for the maintenance of rhabdomeres. Some mutations in Rh1 (NinaE) are dominant due to constitutive activity or folding defects, like autosomal dominant retinitis pigmentosa (ADRP) in humans. The third class consists of genes that control the Ca2+ influx directly or indirectly by promoting the turnover of the second messenger and regeneration of PIP2, or mediate the Ca2+-dependent regulation of the visual response. These gene products are critical for the increase in cytosolic Ca2+ following light stimulation to initiate negative regulatory events. Here I will focus on the signaling mechanisms underlying the degeneration in norpA, and in ADRP-type NinaE mutants that produce misfolded Rh1. Accumulation of misfolded Rh1 in the ER triggers the unfolded protein response (UPR), while endosomal accumulation of activated Rh1 may initiate autophagy in norpA. Both autophagy and the UPR are beneficial for relieving defective endosomal trafficking and the ER stress, respectively. However, when photoreceptors fail to cope with the persistence of these stresses, a cell death program is activated leading to retinal degeneration.

We generated FM7a and CyO balancer chromosomes bearing a Tubby1 (Tb1) dominant transgene. Flies heterozygous for these FM7a and CyO derivatives exhibit a phenotype undistinguishable from that elicited by the Tb1 mutation associated with the TM6B balancer. We tested two of these Tb-bearing balancers (FM7-TbA and CyO-TbA) for more than 30 generations and found that the Tb1 transgene they carry is stable. Thus, these new Tb-tagged balancers are particularly useful for balancing lethal mutations and distinguish homozygous mutant larvae from their heterozygous siblings.

Neural circuit mapping and manipulation are facilitated by independent control of gene expression in pre- and post-synaptic neurons. The GAL4/UAS and Q binary transcription systems have the potential to provide this capability. Of particular use in neural circuit mapping would be GAL4 and QF drivers specific for neurotransmitters and neurotransmitter receptors. Recently available Drosophila genomic BAC libraries make recombineering large genes including those specific for neurotransmitters and neurotransmitter receptors feasible. Here the functionality of cassettes that allow efficient recombineering of GAL4 and QF drivers based on kanamycin selection is demonstrated in Drosophila. The cassettes should, however, be generalizable for recombineering in other species.

As tissues and organs are formed, they acquire a specific shape that plays an integral role in their ability to function properly. A relatively simple system that has been used to examine how tissues and organs are shaped is the formation of an elongated Drosophila egg. While it has been known for some time that Drosophila egg elongation requires interactions between a polarized intracellular basal actin network and a polarized extracellular network of basal lamina proteins, how these interactions contribute to egg elongation remained unclear. Recent studies using live imaging have revealed two novel processes, global tissue rotation and oscillating basal actomyosin contractions, which have provided significant insight into how the two polarized protein networks cooperate to produce an elongated egg. This review summarizes the proteins involved in Drosophila egg elongation and how this recent work has contributed to our current understanding of how egg elongation is achieved.

Proteins perform essential cellular functions as part of protein complexes, often in conjunction with RNA, DNA, metabolites and other small molecules. The genome encodes thousands of proteins but not all of them are expressed in every cell type; and expressed proteins are not active at all times. Such diversity of protein expression and function accounts for the level of biological intricacy seen in nature. Defining protein-protein interactions in protein complexes, and establishing the when, what and where of potential interactions, is therefore crucial to understanding the cellular function of any protein—especially those that have not been well studied by traditional molecular genetic approaches. We generated a large-scale resource of affinity-tagged expression-ready clones and used co-affinity purification combined with tandem mass-spectrometry to identify protein partners of nearly 5,000 Drosophila melanogaster proteins. The resulting protein complex “map” provided a blueprint of metazoan protein complex organization. Here we describe how the map has provided valuable insights into protein function in addition to generating hundreds of testable hypotheses. We also discuss recent technological advancements that will be critical in addressing the next generation of questions arising from the map.

Hematopoiesis is well-conserved between Drosophila and vertebrates. Similar as in vertebrates, the sites of hematopoiesis shift during Drosophila development. Blood cells (hemocytes) originate de novo during hematopoietic waves in the embryo and in the Drosophila lymph gland. In contrast, the hematopoietic wave in the larva is based on the colonization of resident hematopoietic sites by differentiated hemocytes that arise in the embryo, much like in vertebrates the colonization of peripheral tissues by primitive macrophages of the yolk sac, or the seeding of fetal liver, spleen and bone marrow by hematopoietic stem and progenitor cells. At the transition to the larval stage, Drosophila embryonic hemocytes retreat to hematopoietic “niches,” i.e., segmentally repeated hematopoietic pockets of the larval body wall that are jointly shared with sensory neurons and other cells of the peripheral nervous system (PNS). Hemocytes rely on the PNS for their localization and survival, and are induced to proliferate in these microenvironments, expanding to form the larval hematopoietic system. In this process, differentiated hemocytes from the embryo resume proliferation and self-renew, omitting the need for an undifferentiated prohemocyte progenitor. Larval hematopoiesis is the first Drosophila model for blood cell colonization and niche support by the PNS. It suggests an interface where innocuous or noxious sensory inputs regulate blood cell homeostasis or immune responses. The system adds to the growing concept of nervous system dependence of hematopoietic microenvironments and organ stem cell niches, which is being uncovered across phyla.

The vinegar flies Drosophila subobscura and D. obscura frequently serve as study organisms for evolutionary biology. Their high morphological similarity renders traditional species determination difficult, especially when living specimens for setting up laboratory populations need to be identified. Here we test the usefulness of cuticular chemical profiles collected via the non-invasive method near-infrared spectroscopy for discriminating live individuals of the two species. We find a classification success for wild-caught specimens of 85%. The species specificity of the chemical profiles persists in laboratory offspring (87–92% success). Thus, we conclude that the cuticular chemistry is genetically determined, despite changes in the cuticular fingerprints, which we interpret as due to laboratory adaptation, genetic drift and/or diet changes. However, because of these changes, laboratory-reared specimens should not be used to predict the species-membership of wild-caught individuals, and vice versa. Finally, we demonstrate that by applying an appropriate cut-off value for interpreting the prediction values, the classification success can be immensely improved (to up to 99%), albeit at the cost of excluding a considerable portion of specimens from identification.

Drosophila melanogaster is ideal for studying lifespan modulated by dietary restriction (DR) and oxidative stress, and also for screening prolongevity compounds. It is critical to measure food intake in the aforementioned studies. Current methods, however, overlook the amount of the food excreted out of the flies as feces or deposited in eggs. Here we describe a feeding method using a radioactive tracer to measure gender-specific food intake, retention and excretion in response to DR and oxidative stress to account for all the ingested food. Flies were fed a full, restricted or paraquat-containing diet. The radioactivity values of the food in fly bodies, feces and eggs were measured separately after a 24-hr feeding. Food intake was calculated as the sum of these measurements. We found that most of the tracer in the ingested food was retained in the fly bodies and <8% of the tracer was excreted out of the flies as feces and eggs in the case of females during a 24-hr feeding. Under a DR condition, flies increased food intake in volume to compensate for the reduction of calorie content in the diet and also slightly increased excretion. Under an oxidative stress condition, flies reduced both food intake and excretion. Under all the tested dietary conditions, males ingested and excreted 3- to 5-fold less food than females. This study describes an accurate method to measure food intake and provides a basis to further investigate prandial response to DR and prolongevity interve ntions in invertebrates.

The relationship between alcohol consumption, sensitivity and tolerance is an important question that has been addressed in humans and rodent models. Studies have shown that alcohol consumption and risk of abuse may correlate with (1) increased sensitivity to the stimulant effects of alcohol, (2) decreased sensitivity to the depressant effects of alcohol and (3) increased alcohol tolerance. However, many conflicting results have been observed. To complement these studies, we utilized a different organism and approach to analyze the relationship between ethanol consumption and other ethanol responses. Using a set of 20 Drosophila melanogaster mutants that were isolated for altered ethanol sensitivity, we measured ethanol-induced hyperactivity, ethanol sedation, sedation tolerance and ethanol consumption preference. Ethanol preference showed a strong positive correlation with ethanol tolerance, consistent with some rodent and human studies, but not with ethanol hyperactivity or sedation. No pairwise correlations were observed between ethanol hyperactivity, sedation and tolerance. The evolutionary conservation of the relationship between tolerance and ethanol consumption in flies, rodents and humans indicates that there are fundamental biological mechanisms linking specific ethanol responses.

Transcription is the first step through which the cell operates, via its repertoire of transcription complexes, to direct cellular functions and cellular identity by generating the cell-specific transcriptome. The modularity of the composition of constituents of these complexes allows the cell to delicately regulate its transcriptome. In a recent study we have examined the effects of reducing the levels of specific transcription co-factors on the function of two competing transcription complexes, namely CHIP-AP and CHIP-PNR, which regulate development of cells in the thorax of Drosophila. We found that changing the availability of these co-factors can shift the balance between these complexes leading to transition from utilization of CHIP-AP to CHIP-PNR. This is reflected in change in the expression profile of target genes, altering developmental cell fates. We propose that such a mechanism may operate in normal fly development. Transcription complexes analogous to CHIP-AP and CHIP-PNR exist in mammals and we discuss how such a shift in the balance between them may operate in normal mammalian development.

The molting hormone 20-hydroxyecdysone (20E) is an active metabolite of ecdysone and plays vital roles during ontogeny of the fruit fly Drosophila, coordinating critical developmental transitions such as molting and metamorphosis. Although 20E is known to exist throughout life in both male and female flies, its functions in adult physiology and behavior remain largely elusive. Notably, findings from previous studies suggest that this hormone may be involved in adult stress responses. Consistent with this possibility, we have found that ecdysone signaling in adult flies is activated by “stressful” social interactions and plays a role in the formation of long-term courtship memory.1 In addition, we recently reported that ecdysone signaling contributes to the regulation of sleep, affecting transitions between sleep and wakefulness.2 Here we first summarize our findings on the unconventional roles of 20E in regulating memory and sleep in adult flies. We then discuss speculative ideas concerning the stress hormone-like features of 20E, as well as the possibility that ecdysone signaling contributes to remodeling of the adult nervous system, at both the functional and structural levels, through epigenetic mechanisms.

Obesity has reached pandemic proportions globally and is often associated with lipotoxic heart diseases. In the obese state, caloric surplus is accommodated in the adipocytes as triglycerides. As the storage capacity of adipocytes is exceeded or malfunctioning, lipids begin to infiltrate and accumulate in non-adipose tissues, including the myocardium of the heart, leading to organ dysfunction. While the disruption of caloric homeostasis has been widely viewed as a principal mechanism in contributing to peripheral tissue steatosis and lipotoxicity, our recent studies in Drosophila have led to the novel finding that deregulation of phospholipid homeostasis may also significantly contribute to the pathogenesis of lipotoxic cardiomyopathy. Fly mutants that bear perturbations in phosphatidylethanolamine (PE) biosynthesis, such as the easily-shocked (eas) mutants defective in ethanolamine kinase, incurred aberrant activation of the sterol regulatory element binding protein (SREBP) pathway, thereby causing chronic lipogenesis and cardiac steatosis that culminates in the development of lipotoxic cardiomyopathy.1 Here, we describe the potential relationship between SREBP and other eas-associated phenotypes, such as neuronal excitability defects. We will further discuss the additional implications presented by our work toward the effects of altered lipid metabolism on cellular growth and/or proliferation in response to defective phospholipid homeostasis.

Myogenesis in Drosophila embryos requires fusion between Founder cells (FCs) and Fusion Competent myoblasts (FCMs) to form multinucleate myotubes. Myoblast fusion is well characterized in embryos, and many factors required for this process have been identified; however, a number of questions pertaining to the mechanisms of fusion remain and are challenging to answer in the embryo. We have developed a modified primary cell culture protocol to address these questions in vitro. Using this system, we determined the optimal time for examining fusion in culture and confirmed that known fusion proteins are expressed and localized as in embryos. Importantly, we disrupted the actin and microtubule networks with the drugs latrunculin B and nocodazole, respectively, confirming that actin is required for myoblast fusion and showing for the first time that microtubules are also required for this process in Drosophila. Finally, we show that myotubes in culture adopt and maintain specific muscle identities.

Drosophila embryonic hemocytes have emerged as a potent system to analyse the roles of key regulators of the actin and microtubule cytoskeletons live and in an in vivo context (Table 1 and references therein). The relative ease with which live imaging can be used to visualize the invasive migrations of these highly motile macrophages and their responses to wound and chemoattractant signals make them a particularly appropriate and genetically tractable cell type to study in relation to pathological conditions such as cancer metastasis and inflammation.1–3 In order to understand how signaling pathways are integrated for a coordinated response, a question with direct relevance to autoimmune dysfunction, we have sought to more fully characterize the inputs these cells receive in vivo over the course of their developmental dispersal. These studies have recently revealed that hemocyte migration is intimately associated with the development of the ventral nerve cord (VNC ), a structure used by hemocytes to disperse over the embryo that itself requires this association for its correct morphogenesis. Crucially the VNC must separate from the epidermis to create a channel for hemocyte migration, revealing how constriction of extracellular space can be used to control cell migration in vivo.4

The ability to detect changes in oxygen concentration in the environment is critical to the survival of all animals. This requires cells to express a molecular oxygen sensor that can detect shifts in oxygen levels and transmit a signal that leads to the appropriate cellular response. Recent biochemical, genetic and behavioral studies have shown that the atypical soluble guanylyl cyclases function as oxygen detectors in Drosophila larvae triggering a behavioral escape response when exposed to hypoxia. These studies also identified the sensory neurons that innervate the terminal sensory cones as likely chemosensors that mediate this response. Here I summarize the data that led to these conclusions and also highlight evidence that suggests additional, as yet unidentified, proteins are also required for detecting increases and decreases in oxygen concentrations.