Drosophila Life Cycle Research Articles

Hidden features of NAD-RNA epitranscriptome in Drosophila life cycle

Nicotinamide adenine dinucleotide (NAD), a nucleotide-containing metabolite, can be incorporated into the RNA 5'-terminus to result in NAD-capped RNA (NAD-RNA). Since NAD has been heightened as one of the most essential metabolites in cells, its linkage to RNA represents a critical but poorly studied modification at the epitranscriptomic level. Here, we design a highly sensitive method, DO-seq, to capture NAD-RNAs. Using Drosophila, we identify thousands of previously unexplored NAD-RNAs and their dynamics in the fly life cycle, from embryo to adult. We show the evidence that chromosomal clustering might be the structural basis by which co-expression can couple with NAD capping on physically and functionally linked genes. Furthermore, we note that NAD capping of cuticle genes inversely correlates with their gene expression. Combined, we propose NAD-RNA epitranscriptome as a hidden layer of regulation that underlies biological processes. DO-seq empowers the identification of NAD-capped RNAs, facilitating functional investigation into this modification.

Preparation of polyclonal antibodies against the Drosophila deacetylases SIRT 6 and SIRT 7

SIRT6 and SIRT7, as members of the Sirtuins family, are indispensable for the growth and development of Drosophila. They play crucial roles in maintaining genome stability, regulating metabolic senescence, and controlling tumorigenesis. To investigate their involvement in the Drosophila life cycle, we focused on describing the expression and purification of recombinant Drosophila SIRT6 and SIRT7 proteins. Subsequently, these proteins were utilized for generating polyclonal antibodies against Drosophila SIRT6 and SIRT7. The recombinant expression plasmid was introduced into E. coli cells to enable the production of SIRT6 and SIRT7 proteins. Following immunizations of New Zealand white rabbits and guinea pigs with the recombinant proteins as antigens, specific polyclonal antisera against both proteins were obtained. After purification, the specificity of SIRT6 and SIRT7 was confirmed using ELISA and western blot analyses, demonstrating strong specificity. These antibodies hold promise for the development of detection assays required for further research.

Dynamic changes in genomic 5-hydroxymethyluracil and N6-methyladenine levels in the Drosophila melanogaster life cycle and in response to different temperature conditions

In this study, the level of DNA modifications was investigated in three developmental stages of Drosophila melanogaster (larvae, pupae, imago) and in an in vitro model (Schneider 2 cells). Analysis was carried out using two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. Our method made it possible, for the first time, to analyze a broad spectrum of DNA modifications in the three stages of Drosophila. Each stage was characterized by a specific modification pattern, and the levels of these compounds fluctuated throughout the D. melanogaster life cycle. The level of DNA modification was also compared between insects bred at 25 °C (optimal temperature) and at 18 °C, and the groups differed significantly. The profound changes in N6-methyladenine and 5-hydroxymethyluracil levels during the Drosophila life cycle and as a result of breeding temperature changes indicate that these DNA modifications can play important regulatory roles in response to environmental changes and/or biological conditions. Moreover, the supplementation of Schneider 2 cells with 1 mM L-ascorbic acid caused a time-dependent increase in the level of 5-(hydroxymethyl)-2′-deoxyuridine. These data suggest that a certain pool of this compound may arise from the enzymatic activity of the dTET protein.

PvdM of fluorescent pseudomonads is required for the oxidation of ferribactin by PvdP in periplasmic pyoverdine maturation

Fluorescent pseudomonads such as Pseudomonas aeruginosa or Pseudomonas fluorescens produce pyoverdine siderophores that ensure iron-supply in iron-limited environments. After its synthesis in the cytoplasm, the nonfluorescent pyoverdine precursor ferribactin is exported into the periplasm, where the enzymes PvdQ, PvdP, PvdO, PvdN, and PtaA are responsible for fluorophore maturation and tailoring steps. While the roles of all these enzymes are clear, little is known about the role of PvdM, a human renal dipeptidase–related protein that is predicted to be periplasmic and that is essential for pyoverdine biogenesis. Here, we reveal the subcellular localization and functional role of PvdM. Using the model organism P. fluorescens, we show that PvdM is anchored to the periplasmic side of the cytoplasmic membrane, where it is indispensable for the activity of the tyrosinase PvdP. While PvdM does not share the metallopeptidase function of renal dipeptidase, it still has the corresponding peptide-binding site. The substrate of PvdP, deacylated ferribactin, is secreted by a ΔpvdM mutant strain, indicating that PvdM prevents loss of this periplasmic biosynthesis intermediate into the medium by ensuring the efficient transfer of ferribactin to PvdP in vivo. We propose that PvdM belongs to a new dipeptidase-related protein subfamily with inactivated Zn2+ coordination sites, members of which are usually genetically linked to TonB-dependent uptake systems and often associated with periplasmic FAD-dependent oxidoreductases related to d-amino acid oxidases. We suggest that these proteins are necessary for selective binding, exposure, or transfer of specific d- and l-amino acid–containing peptides and other periplasmic biomolecules in manifold pathways.

Systematic Identification of Microproteins during the Development of Drosophila melanogaster.

Short open reading frame-encoded peptides (SEPs) are microproteins with less than 100 amino acids that play an essential role in the growth and development of organisms. There are plenty of short open reading frames in Drosophila melanogaster that potentially code polypeptides. We chose 11 time points during the life cycle of Drosophila to investigate microproteins, particularly those related to development. Finally, we identified a total of 410 microproteins, of which 27 were noncoding RNA-encoded proteins. Of the 410 microproteins, 74 were expressed in all stages from embryo to adults, whereas 300 microproteins were only found in one or two time points. Approximately, one-third of the microproteins were not reported previously and 44 were obtained from de novo sequencing, validated by synthetic peptides. These microproteins are related to the main bioprocesses of growth and development, such as multicellular organism reproduction, postmating behavior, and oviposition. Over half of the microproteins have predicted functional domains and are conserved across species, suggesting that these microproteins have critical functions in fly development. This work enriches the D. melanogaster proteome and provides a significant data resource for growth and development research.

Drosophila E93 promotes adult development and suppresses larval responses to ecdysone during metamorphosis

Pulses of the steroid hormone ecdysone act through transcriptional cascades to direct the major developmental transitions during the Drosophila life cycle. These include the prepupal ecdysone pulse, which occurs 10 ​hours after pupariation and triggers the onset of adult morphogenesis and larval tissue destruction. E93 encodes a transcription factor that is specifically induced by the prepupal pulse of ecdysone, supporting a model proposed by earlier work that it specifies the onset of adult development. Although a number of studies have addressed these functions for E93, little is known about its roles in the salivary gland where the E93 locus was originally identified. Here we show that E93 is required for development through late pupal stages, with mutants displaying defects in adult differentiation and no detectable effect on the destruction of larval salivary glands. RNA-seq analysis demonstrates that E93 regulates genes involved in development and morphogenesis in the salivary glands, but has little effect on cell death gene expression. We also show that E93 is required to direct the proper timing of ecdysone-regulated gene expression in salivary glands, and that it suppresses earlier transcriptional programs that occur during larval and prepupal stages. These studies support the model that the stage-specific induction of E93 in late prepupae provides a critical signal that defines the end of larval development and the onset of adult differentiation.

Disruption of Drosophila larval muscle structure and function by UNC45 knockdown

BackgroundProper muscle function is heavily dependent on highly ordered protein complexes. UNC45 is a USC (named since this region is shared by three proteins UNC45/CRO1/She4P) chaperone that is necessary for myosin incorporation into the thick filaments. UNC45 is expressed throughout the entire Drosophila life cycle and it has been shown to be important during late embryogenesis when initial muscle development occurs. However, the effects of UNC45 manipulation at later developmental times, after muscle development, have not yet been studied.Main resultsUNC45 was knocked down with RNAi in a manner that permitted survival to the pupal stage, allowing for characterization of sarcomere organization in the well-studied third instar larvae. Second harmonic generation (SHG) microscopy revealed changes in the striated pattern of body wall muscles as well as a reduction of signal intensity. This observation was confirmed with immunofluorescence and electron microscopy imaging, showing diminished UNC45 signal and disorganization of myosin and z-disk proteins. Concomitant alterations in both synaptic physiology and locomotor function were also found. Both nerve-stimulated response and spontaneous vesicle release were negatively affected, while larval movement was impaired.ConclusionsThis study highlights the dependency of normal sarcomere structure on UNC45 expression. We confirm the known role of UNC45 for myosin localization and further show the I-Z-I complex is also disrupted. This suggests a broad need for UNC45 to maintain sarcomere integrity. Newly discovered changes in synaptic physiology reveal the likely presence of a homeostatic response to partially maintain synaptic strength and muscle function.

Drosophila model for studying the link between lipid metabolism and development.

Developmental processes are cascades of biological changes linked with information transfer, growth, and differentiation during the life cycle of an organism. Lipid metabolism plays a vital role in the life cycle of organisms. Drosophila models grant numerous advantages in investigating the underlying mechanisms of each process as well as their connections. In each section of this review, we will discuss multiple studies revealing the function of lipid-related genes in different stages of early development: spermatogenesis, oogenesis, embryogenesis along with late development in life cycle of Drosophila.

Functional analysis of RIP toxins from the Drosophila endosymbiont Spiroplasma poulsonii

BackgroundInsects frequently live in close relationship with symbiotic bacteria that carry out beneficial functions for their host, like protection against parasites and viruses. However, in some cases, the mutualistic nature of such associations is put into question because of detrimental phenotypes caused by the symbiont. One example is the association between the vertically transmitted facultative endosymbiont Spiroplasma poulsonii and its natural host Drosophila melanogaster. Whereas S. poulsonii protects its host against parasitoid wasps and nematodes by the action of toxins from the family of Ribosome Inactivating Proteins (RIPs), the presence of S. poulsonii has been reported to reduce host’s life span and to kill male embryos by a toxin called Spaid. In this work, we investigate the harmful effects of Spiroplasma RIPs on Drosophila in the absence of parasite infection.ResultsWe show that only two Spiroplasma RIPs (SpRIP1 and SpRIP2) among the five RIP genes encoded in the S. poulsonii genome are significantly expressed during the whole Drosophila life cycle. Heterologous expression of SpRIP1 and 2 in uninfected flies confirms their toxicity, as indicated by a reduction of Drosophila lifespan and hemocyte number. We also show that RIPs can cause the death of some embryos, including females.ConclusionOur results indicate that RIPs released by S. poulsonii contribute to the reduction of host lifespan and embryo mortality. This suggests that SpRIPs may impact the insect-symbiont homeostasis beyond their protective function against parasites.

The somatic mobilization of transposable element mariner-Mos1 during the Drosophila lifespan and its biological consequences

Transposable elements (TEs) are mobile DNA sequences on genomes. Some elements are able to transpose in somatic cells, a process known as somatic transposition (ST), which has been associated with detrimental biological effects. The mariner-Mos1 element of Drosophila promotes transposition in somatic and germline cells and is an excellent model for studies related to the biological consequence of somatic excision (SE). In this work, we used temperature stress to induce increasing transposition of mariner-Mos1 during different stages of the development of D. simulans, aiming to quantify SE during lifespan. Furthermore, strains of D. melanogaster exhibiting differential expression of mariner-Mos1 were employed for estimating some biological consequences of mariner mobilization. It is shown that SE of mariner-Mos1 was not constant during development; the larval phase had the highest rates while the pupal stage exhibited lower rates, and in the embryonic stage, no difference was detected. SE can be detrimental, as suggested by correlation in SE level and reduction in behavioral activities and embryonic viability. This study showed that mariner-Mos1 SE accumulates during the Drosophila life cycle, and can be involved in detrimental effects.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae.

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle – the Drosophila pupa – offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term <em>In Vivo</em> Tracking of Inflammatory Cell Dynamics Within <em>Drosophila</em> Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle – the Drosophila pupa – offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism.

Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which may function as a mitochondrial iron sensor since it is inactivated by iron; (iii) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (iv) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle.

Histone Demethylase Activity of Utx Is Essential for Viability and Regulation of HOX Gene Expression in Drosophila.

The trimethylation of histone H3 at lysine 27 (H3K27me3) by Polycomb Repressive Complex 2 (PRC2) is essential for the repression of Polycomb target genes. However, the role of enzymatic demethylation of H3K27me3 by the KDM6-family demethylases Utx, Uty, and JmjD3 is less clear. Studies in both mice and worms led to the proposal that KDM6 proteins, but not their H3K27me3 demethylase activity, is critical for normal development. Here, we investigated the requirement of the demethylase activity of the single KDM6 family member Utx in Drosophila. We generated Drosophila expressing a full-length but catalytically inactive Utx protein and found that these mutants show the same phenotypes as animals lacking the Utx protein. Specifically, animals lacking maternally deposited active Utx demethylase in the early embryo show stochastic loss of HOX gene expression that appears to be propagated in a clonal fashion. This suggests that Utx demethylase activity is critical for the removal of ectopic H3K27 trimethylation from active HOX genes during the onset of zygotic gene transcription, and thereby prevents the inappropriate installment of long-term repression by Polycomb. Conversely, maternally deposited catalytically active Utx protein suffices to permit animals that lack zygotic expression of enzymatically active Utx to develop into morphologically normal adults, which eclose from the pupal case but die shortly thereafter. Utx demethylase activity is therefore also essential to sustain viability in adult flies. Together, these analyses identify the earliest embryonic stages and the adult stage as two phases during the Drosophila life cycle that critically require H3K27me3 demethylase activity.

Population Effects of Calcium Phosphate Nanoparticles in Drosophila melanogaster: The Effects of Phase Composition, Crystallinity, and the Pathway of Formation.

Unpredictable biological response due to the finest nanostructural variations is one of the hallmarks of nanoparticles. Because of this erratic behavior of nanoparticles in living systems, thorough analyses of biosafety must precede the analyses of the pharmacotherapeutic efficacy and simple animal models are ideal for such purposes. Drosophila melanogaster, the common fruit fly, is an animal model capable of giving a fast, high-throughput response as to the safety and efficacy of drug delivery carriers and other pharmacological agents, while minimizing the suffering imposed onto animals in more complex in vivo models. Here we studied the effects on the viability and fertility of D. melanogaster due to variations in phase composition, crystallinity, and the pathway of formation of four different calcium phosphate (CP) nanopowders consumed orally. To minimize the effect of other nanostructural variables, CP nanopowders were made to possess highly similar particle sizes and morphologies. The composition of CP affected the fecundity of flies, but so did crystallinity and the pathway of formation. Both the total number of eclosed viable flies and pupae in populations challenged with hydroxyapatite (HAP) greatly exceeded those in control populations. Viability was adversely affected by the only pyrophosphate tested (CPP) and by the metastable and the most active of all CP nanopowders analyzed: the amorphous CP (ACP). The pupation peak was delayed and the viable fly to-pupa ratio increased in all the CP-challenged populations. F1 CPP population, whose viability was most adversely affected by the CP consumption, when crossed, produced the largest number of F2 progeny under regular conditions, possibly pointing to stress as a positive evolutionary drive. The positive effect of HAP on fertility of fruit flies may be due to its slow absorption and the activation of calmodulin during the transit of oocytes through the reproductive tract of fertilized females. Exerted in the prepupation stage, the effect of CP is thus traceable beyond the instar larval stage and to the oogenesis stage of the Drosophila lifecycle.

Cellular aspects of gonadal atrophy in Drosophila P-M hybrid dysgenesis

Gonadal atrophy is the most typical and dramatic manifestation of intraspecific hybrid dysgenesis syndrome leading to sterility in Drosophila melanogaster dysgenic progeny. The P-M system of hybrid dysgenesis is primarily associated with germ cell degeneration during the early stages of Drosophila embryonic development at elevated temperatures. In the present study, we have defined the phase of germ cell death as beginning at the end of embryogenesis immediately following gonad formation. However, the temperature-dependent screening of germ cell developmental patterns in the dysgenic background showed that early germ cells are susceptible to the hybrid dysgenesis at any Drosophila life-cycle stage, including in the imago. Electron microscopy of germ cells after dysgenesis induction revealed significant changes in subcellular structure, especially mitochondria, prior to cellular breakdown. The mitochondrial pathology can promote the activation of cell death pathways in dysgenic germ cells, which leads to gonadal atrophy.

Maintenance of a Drosophila melanogaster Population Cage.

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a <em>Drosophila melanogaster</em> Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

From Embryo to Adult: Hematopoiesis along the Drosophila Life Cycle

Studies on Drosophila hematopoiesis have thus far focused on the embryonic and larval origin of hemocytes, the fly blood cells. In this issue of Developmental Cell, Ghosh etal. (2015) identify adult hematopoietic hubs containing progenitors that can differentiate into different blood cell types.

Fat Body Development and its Function in Energy Storage and Nutrient Sensing in Drosophila melanogaster

The fat body of Drosophila has been considered as the equivalent to the vertebrate adipose tissue and liver in its storage and major metabolic functions. It is a dynamic and multifunctional tissue which functions in energy storage, immune response and as a nutritional sensor. As a major endocrine organ in Drosophila, the fat body can produce various proteins, lipids and carbohydrates, synthesize triglyceride, diacylglycerol, trehalose and glycogen in response to energetic demands. It also secretes significant proteins governing oocyte maturation or targeting nutritional signals in the regulation of the metabolism. At different developmental stages and under different environmental conditions the fat body can interplay with other tissues in monitoring and responding to the physiological needs of the body’s growth and to coordinate the metabolism of development. The Drosophila fat body exists as a model relating to human lipometabolic disease, puberty and maturation and age-related diseases such as cancer, obesity and diabetes. In this review, we summarize the fat body formation and maturation in the Drosophila life cycle and provide an overview of fat body function as an energy reservoir and nutrient sensor. We also discuss the signaling pathways and key regulatory factors involved.