Myoblast fusion in Drosophila

The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process [1], [2], [3] and [4]. With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.     

Interaction between Sleep and Metabolism in Drosophila with Altered Octopamine Signaling

Sleep length and metabolic dysfunction are correlated, but the causal relationship between these processes is unclear. Octopamine promotes wakefulness in the fly by acting through the insulin-producing cells (IPCs) in the fly brain. To determine if insulin signaling mediates the effects of octopamine on sleep:wake behavior, we assayed flies in which insulin signaling activity was genetically altered. We found that increasing insulin signaling does not promote wake, nor does insulin appear to mediate the wake-promoting effects of octopamine. Octopamine also affects metabolism in invertebrate species, including, as we show here, Drosophila melanogaster. Triglycerides are decreased in mutants with compromised octopamine signaling and elevated in flies with increased activity of octopaminergic neurons. Interestingly, this effect is mediated at least partially by insulin, suggesting that effects of octopamine on metabolism are independent of its effects on sleep. We further investigated the relative contribution of metabolic and sleep phenotypes to the starvation response of flies with altered octopamine signaling. Hyperactivity (indicative of foraging) induced by starvation was elevated in octopamine receptor mutants, despite their high propensity for sleep, indicating that their metabolic state dictates their behavioral response under these conditions. Moreover, flies with increased octopamine signaling do not suppress sleep in response to starvation, even though they are normally hyper-aroused, most likely because of their high triglyceride levels. Together, these data suggest that observed correlations between sleep and metabolic phenotypes can result from shared molecular pathways rather than causality, and environmental conditions can lead to the dominance of one phenotype over the other.     

Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics,screening, and stability

The previously uncharacterized Drosophila melanogaster Epsilon-class glutathione transferases E6 and E7 were immobilized on nanoporous alumina. The nanoporous anodized alumina membranes were derivatized with 3-aminopropyl-triethoxysilane, and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes via ε-amino groups. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzyme in solution. A limited kinetic study was also carried out on immobilized human GST S1-1 (also known as hematopoietic prostaglandin D synthase). The stability of the immobilized enzymes was virtually identical to that of enzymes in solution, and no leakage of enzyme from the matrix could be observed.     

Are we there yet? Initial targeting of the Male-Specific Lethal and Polycomb group chromatin complexes in Drosophila

Chromatin-binding proteins must navigate the complex nuclear milieu to find their sites of action, and a constellation of protein factors and other properties are likely to influence targeting specificity. Despite considerable progress, the precise rules by which binding specificity is achieved have remained elusive. Here, we consider early targeting events for two groups of chromatin-binding complexes in Drosophila: the Male-Specific Lethal (MSL) and the Polycomb group (PcG) complexes. These two serve as models for understanding targeting, because they have been extensively studied and play vital roles in Drosophila, and their targets have been documented at high resolution. Furthermore, the proteins and biochemical properties of both complexes are largely conserved in multicellular organisms, including humans. While the MSL complex increases gene expression and PcG members repress genes, the two groups share many similarities such as the ability to modify their chromatin environment to create active or repressive domains, respectively. With legacies of in-depth genetic, biochemical and now genomic approaches, the MSL and PcG complexes will continue to provide tractable systems for understanding the recruitment of multiprotein chromatin complexes to their target loci.     

Insect haemocytes: what type of cell is that?

Classification of insect larvae circulating haemocytes is the subject of controversy, and the terminology used to designate each cellular type is often different from one species to another. However, a survey of the literature on insect haemocytes suggests that there are resemblances for most of the cell types and functions, in different insect species. In this review paper, we compare the structure and functions of circulating haemocytes in those insect species that are, by far, the most often used species for insect physiology studies, i.e. lepidopteran species and Drosophila. We show that there is high degree of homology of haemocyte types and suggest possible synonymies in terminology among species from these taxa.     

The endosymbiont Wolbachia increases insulin/IGF-like signalling in Drosophila

Insulin/IGF-like signalling (IIS) is an evolutionarily conserved pathway that has diverse functions in multi-cellular organisms. Mutations that reduce IIS can have pleiotropic effects on growth, development, metabolic homeostasis, fecundity, stress resistance and lifespan. IIS is also modified by extrinsic factors. For instance, in the fruitfly Drosophila melanogaster, both nutrition and stress can alter the activity of the pathway. Here, we test experimentally the hypothesis that a widespread endosymbiont of arthropods, Wolbachia pipientis, can alter the degree to which mutations in genes encoding IIS components affect IIS and its resultant phenotypes. Wolbachia infection, which is widespread in D. melanogaster in nature and has been estimated to infect 30 per cent of strains in the Bloomington stock centre, can affect broad aspects of insect physiology, particularly traits associated with reproduction. We measured a range of IIS-related phenotypes in flies ubiquitously mutant for IIS in the presence and absence of Wolbachia. We show that removal of Wolbachia further reduces IIS and hence enhances the mutant phenotypes, suggesting that Wolbachia normally acts to increase insulin signalling. This effect of Wolbachia infection on IIS could have an evolutionary explanation, and has some implications for studies of IIS in Drosophila and other organisms that harbour endosymbionts.     

A new genetic model for calcium induced autophagy and ER-stress in Drosophila photoreceptor cells

Cytoplasmic Ca²⁺ overload is known to trigger autophagy and ER-stress. Furthermore, ER-stress and autophagy are commonly associated with degenerative pathologies, but their role in disease progression is still a matter of debate, in part, owing to limitations of existing animal model systems. The Drosophila eye is a widely used model system for studying neurodegenerative pathologies. Recently, we characterized the Drosophila protein, Calphotin, as a cytosolic immobile Ca²⁺ buffer, which participates in Ca²⁺ homeostasis in Drosophila photoreceptor cells. Exposure of calphotin hypomorph flies to continuous illumination, which induces Ca²⁺ influx into photoreceptor cells, resulted in severe Ca²⁺-dependent degeneration. Here we show that this degeneration is autophagy and ER-stress related. Our studies thus provide a new model in which genetic manipulations trigger changes in cellular Ca²⁺ distribution. This model constitutes a framework for further investigations into the link between cytosolic Ca²⁺, ER-stress and autophagy in human disorders and diseases.     

Calpains expression during Xenopus laevis development

Calpains are cytoplasmic proteases activated by calcium, implicated in cell differentiation and apoptosis. The best characterized enzymes are calpains 1-3. The aim of this work was to localize calpains 1-3 during the development of Xenopus laevis in order to clarify the function of these three proteases. For the first time, we detected the localization of the three proteases at the protein level between one-cell stage and adult age. Their expression was weak at early stages, then increased at tadpole stage and decreased through metamorphosis and adult life. The calpain's expression was maximal during the period characterized by the appearance of organs and modelling process. These observations suggest that calpains play a crucial role during development.     

Drosophila melanogaster genes encoding three troponin-C isoforms and a calmodulin-related protein

Using low-stringency hybridization and polymerase chain reaction (PCR)-based DNA amplification, we have isolated threeDrosophila melanogaster genes that encode troponin-C isoforms and one specifying a protein that is closely related to calmodulin. Two of the troponin-C genes, located within the 47D and 73F subdivisions of chromosomes 2 and 3, respectively, encode very closely related isoforms. That specified by the 47D gene accumulates almost exclusively in larval muscles, while that encoded by the 73F gene is present in both larvae and adults. The third gene, located within the 41C subdivision of chromosome 2, encodes a more distantly related troponin-C isoform that accumulates only within adults. The gene that encodes the calmodulin-related protein is located within the 97A subdivision of chromosome three. The protein encoded by this gene has a different primary sequence from that of conventional calmodulin, which is specified by a gene located within the 49A subdivision of chromosome 2. Our report is the first to describe insect troponin-C isoforms and further avails genetic methods for investigating thein vivo functions of the troponin-C/myosin light-chain/calmodulin protein superfamily.This work was supported by grants from the NIH and Muscular Dystrophy Association to E. F.Sequences described herein have been filed in the EMBL and GenBank databases under Accession Numbers X76042, X76043, X76044, and X76045.     

Ectopic Repo suppresses expression of castor gene in Drosophila central nervous system

The ventral nerve cord (VNC) of the Drosophila embryo is derived from neuroblasts (NBs). NBs divide in a stem cell lineage to generate a series of ganglion mother cells (GMCs), each of which divides once to produce a pair of neurons or glial cells. One of the NB genes, castor (cas), is expressed in a subset of NBs and has never been identified in neurons and the peripheral nervous system; cas plays a role in axonogenesis. But its limited expression along the dorsal-ventral axis within the central nervous system has not been investigated yet. In the present study, we examined the expression patterns of both genes using confocal microscopy to determine the effects of repo mutation on cas expression. Cas was mainly expressed in layers different from repo-expressed layers during early embryogenesis: repo was expressed mostly from deep to mid layers, while cas, from mid to superficial layers. Loss-of-function of repo did not result in an ectopic expression of cas, but rather, a scattering of cas-expressing cells. However, repo gain-of-function mutation caused repression of cas. In addition, repo-expressing cells seemed to block the migration of cas-expressing cells.     

Density-mediated carry-over effects explain variation in breeding output across time in a seasonal population

In seasonal environments, where density dependence can operate throughout the annual cycle, vital rates are typically considered to be a function of the number of individuals at the beginning of each season. However, variation in density in the previous season could also cause surviving individuals to be in poor physiological condition, which could carry over to influence individual success in the following season. We examine this hypothesis using replicated populations of Drosophila melanogaster, the common fruitfly, over 23 non-overlapping generations with distinct breeding and non-breeding seasons. We found that the density at the beginning of the non-breeding season negatively affected the fresh weight of individuals that survived the non-breeding season and resulted in a 25% decrease in per capita breeding output among those that survived to the next season to breed. At the population level, per capita breeding output was best explained by a model that incorporated density at the beginning of the previous non-breeding season (carry-over effect, COE) and density at the beginning of the breeding season. Our results support the idea that density-mediated COEs are critical for understanding population dynamics in seasonal environments.     

Effectors of alcohol-induced cell killing in Drosophila

Heavy alcohol consumption provokes an array of degenerative pathologies but the signals that couple alcohol exposure to regulated forms of cell death are poorly understood. Using Drosophila as a model, we genetically establish that the severity of ethanol challenge dictates the type of death that occurs. In contrast to responses seen under acute exposure, cytotoxic responses to milder challenges required gene encoding components of the apoptosome, Dronc and Dark. We conducted a genome-wide RNAi screen to capture targets that specifically mediate ethanol-induced cell death. One effector, Drat, encodes a novel protein that contains an ADH domain but lacks essential residues in the catalytic site. In cultured cells and neurons in vivo, depletion of Drat conferred protection from alcohol-induced apoptosis. Adults mutated for Drat showed both improved survival and enhanced propensities toward sedation after alcohol challenge. Together, these findings highlight novel effectors that support regulated cell death incited by alcohol stress in vitro and in vivo.     

The deubiquitinase emperor's thumb is a regulator of apoptosis in Drosophila

We have characterized the gene emperor's thumb (et) and showed that it is required for the regulation of apoptosis in Drosophila. Loss-of-function mutations in et result in apoptosis associated with a decrease in the concentration of DIAP1. Overexpression of one form of et inhibits apoptosis, consistent with et having an anti-apoptotic function; however, overexpression of a second form of et induces apoptosis, indicating that the two forms of et may have competing functions. et encodes a protein deubiquitinase, suggesting it regulates apoptosis by controlling the stability of apoptotic regulatory proteins.     

Increased dopaminergic signaling impairs aversive olfactory memory retention in Drosophila

Dopamine is necessary for the aversive olfactory associative memory formation in Drosophila, but its effect on other stages of memory is not known. Herein, we studied the effect of enhanced dopaminergic signaling on aversive olfactory memory retention in flies. We used l-3,4-dihydroxyphenylalanine (l-DOPA) to elevate dopamine levels: l-DOPA-treated flies exhibited a normal learning performance, but a decrease in 1-h memory. Dopamine transporter (DAT) mutant flies or flies treated with the DAT inhibitor desipramine exhibited poor memory retention. Flies subjected to heat stress after training exhibited a decrease in memory. Memory was restored by blocking dopaminergic neuronal output during heat stress, suggesting that dopamine is involved in heat stress-induced memory impairment in flies. Taken together, our findings suggest that increased dopaminergic signaling impairs aversive olfactory memory retention in flies.