Reversible commitment of neural and epidermal progenitor cells during embryogenesis of Drosophila melanogaster

Summary Cell-cell interactions are involved in mediating developmental fate. An example is the decision of the neuroectodermal cells of Drosophila to develop as neural or epidermal progenitors, where cellular interactions participate in the process of acquisition of either cell fate. The results of heterochronic cell transplantations we describe here suggest that both neuroblasts and epidermoblasts are not irreversibly committed to a particular developmental fate. Rather, they retain the ability to interact with neighbouring cells and, under our experimental conditions, are capable of switching their fate during a relatively long period of time, i.e. until the end of embryonic stage 11.     

A novel Drosophila Girdin-like protein is involved in Akt pathway control of cell size

The Akt signaling pathway is well known to regulate cell proliferation and growth. Girdin, a novel substrate of Akt, plays a crucial role in organization of the actin cytoskeleton and cell motility under the control of Akt. We here identified a novel Girdin-like protein in Drosophila (dGirdin), which has two isoforms, dGirdin PA and dGirdin PB. dGirdin shows high homology with human Girdin in the N-terminal and coiled-coil domains, while diverging at the C-terminal domain. On establishment of transgenic fly lines, featuring knockdown or overexpression of dGirdin in vivo, overexpression in the wing disc cells induced ectopic apoptosis, implying a role in directing apoptosis. Knockdown of dGirdin in the Drosophila wing imaginal disc cells resulted in reduction of cell size. Furthermore, this was enhanced by half reduction of the Akt gene dose, suggesting that Akt positively regulates dGirdin. In the wing disc, cells in which dGirdin was knocked down exhibited disruption of actin filaments. From these in vivo analyses, we conclude that dGirdin is required for actin organization and regulation of appropriate cell size under control of the Akt signaling pathway.     

Model for the regulation of size in the wing imaginal disc of Drosophila

For animal development it is necessary that organs stop growing after they reach a certain size. However, it is still largely unknown how this termination of growth is regulated. The wing imaginal disc of Drosophila serves as a commonly used model system to study the regulation of growth. Paradoxically, it has been observed that growth occurs uniformly throughout the disc, even though Decapentaplegic (Dpp), a key inducer of growth, forms a gradient. Here, we present a model for the control of growth in the wing imaginal disc, which can account for the uniform occurrence and termination of growth. A central feature of the model is that net growth is not only regulated by growth factors, but by mechanical forces as well. According to the model, growth factors like Dpp induce growth in the center of the disc, which subsequently causes a tangential stretching of surrounding peripheral regions. Above a certain threshold, this stretching stimulates growth in these peripheral regions. Since the stretching is not completely compensated for by the induced growth, the peripheral regions will compress the center of the disc, leading to an inhibition of growth in the center. The larger the disc, the stronger this compression becomes and hence the stronger the inhibiting effect. Growth ceases when the growth factors can no longer overcome this inhibition. With numerical simulations we show that the model indeed yields uniform growth. Furthermore, the model can also account for other experimental data on growth in the wing disc.     

amnesiac regulates sleep onset and maintenance in Drosophila melanogaster

The adenylate cyclase/cAMP signaling pathway and adult mushroom bodies (MBs) have been shown to play an important role in sleep regulation in Drosophila. The amnesiac (amn) gene, encodes a neuropeptide that is homologous with vertebrate pituitary adenylate cyclase-activating peptide (PACAP), is expressed in dorsal paired medial (DPM) neurons and is required for the middle-term memory (MTM) in flies. However, the role of amn on regulation of sleep is as yet unknown. Here we provide evidence that amn plays a major role on sleep maintenance and onset in Drosophila. Flies with the amnesiac allele, loss-of-function amn^X8 mutation, showed a fragmented sleep pattern and short sleep latency. Moreover, homeostatic regulation was disrupted in amn^X8 mutants after sleep deprivation. Sleep maintenance was also influenced by disruption of neurotransmission in DPM neurons with increased sleep bout number and decreased sleep bout length. Furthermore, age-related sleep fragmentation and initiation were inhibited in amn^X8 mutant flies. These data suggest that amn is required in initiation and maintenance of sleep.     

Automated identification of social interaction criteria in Drosophila melanogaster

The study of social behaviour within groups has relied on fixed definitions of an ‘interaction’. Criteria used in these definitions often involve a subjectively defined cut-off value for proximity, orientation and time (e.g. courtship, aggression and social interaction networks) and the same numerical values for these criteria are applied to all of the treatment groups within an experiment. One universal definition of an interaction could misidentify interactions within groups that differ in life histories, study treatments and/or genetic mutations. Here, we present an automated method for determining the values of interaction criteria using a pre-defined rule set rather than pre-defined values. We use this approach and show changing social behaviours in different manipulations of Drosophila melanogaster. We also show that chemosensory cues are an important modality of social spacing and interaction. This method will allow a more robust analysis of the properties of interacting groups, while helping us understand how specific groups regulate their social interaction space.     

Genetic analysis of mitochondrial protein misfolding in Drosophila melanogaster

Protein misfolding has a key role in several neurological disorders including Parkinson's disease. Although a clear mechanism for such proteinopathic diseases is well established when aggregated proteins accumulate in the cytosol, cell nucleus, endoplasmic reticulum and extracellular space, little is known about the role of protein aggregation in the mitochondria. Here we show that mutations in both human and fly PINK1 result in higher levels of misfolded components of respiratory complexes and increase in markers of the mitochondrial unfolded protein response. Through the development of a genetic model of mitochondrial protein misfolding employing Drosophila melanogaster, we show that the in vivo accumulation of an unfolded protein in mitochondria results in the activation of AMP-activated protein kinase-dependent autophagy and phenocopies of pink1 and parkin mutants. Parkin expression acts to clear mitochondria with enhanced levels of misfolded proteins by promoting their autophagic degradation in vivo, and refractory to Sigma P (ref(2)P), the Drosophila orthologue of mammalian p62, is a critical downstream effector of this quality control pathway. We show that in flies, a pathway involving pink1, parkin and ref(2)P has a role in the maintenance of a viable pool of cellular mitochondria by promoting organellar quality control.     

The Hox gene Ultrabithorax modulates the shape and size of the third leg of Drosophila by influencing diverse mechanisms

The developmental mechanisms that regulate the relative size and shape of organs have remained obscure despite almost a century of interest in the problem and the fact that changes in relative size represent the dominant mode of evolutionary change. Here, I investigate how the Hox gene Ultrabithorax (Ubx) instructs the legs on the third thoracic segment of Drosophila melanogaster to develop with a different size and shape from the legs on the second thoracic segment. Through loss-of-function and gain-of-function experiments, I demonstrate that different segments of the leg, the femur and the first tarsal segment, and even different regions of the femur, regulate their size in response to Ubx expression through qualitatively different mechanisms. In some regions, Ubx acts autonomously to specify shape and size, whereas in other regions, Ubx influences size through nonautonomous mechanisms. Loss of Ubx autonomously reduces cell size in the T3 femur, but this reduction seems to be partially compensated by an increase in cell numbers, so that it is unclear what effect cell size and number directly have on femur size. Loss of Ubx has both autonomous and nonautonomous effects on cell number in different regions of the basitarsus, but again there is not a strong correlation between cell size or number and organ size. Total organ size appears to be regulated through mechanisms that operate at the level of the entire leg segment (femur or basitarsus) relatively independently of the behavior of individual subpopulations of cells within the segment.     

Drosophila perlecan modulates FGF and hedgehog signals to activate neural stem cell division

Mutations in the Drosophila trol gene cause cell cycle arrest of neuroblasts in the larval brain. Here, we show that trol encodes the Drosophila homolog of Perlecan and regulates neuroblast division by modulating both FGF and Hh signaling. Addition of human FGF-2 to trol mutant brains in culture rescues the trol proliferation phenotype, while addition of a MAPK inhibitor causes cell cycle arrest of the regulated neuroblasts in wildtype brains. Like FGF, Hh activates stem cell division in the larval brain in a Trol-dependent fashion. Coimmunoprecipitation studies are consistent with interactions between Trol and Hh and between mammalian Perlecan and Shh that are not competed with heparin sulfate. Finally, analyses of mutations in trol, hh, and ttv suggest that Trol affects Hh movement. These results indicate that Trol can mediate signaling through both of the FGF and Hedgehog pathways to control the onset of stem cell proliferation in the developing nervous system.     

The neuropeptide SIFamide modulates sexual behavior in Drosophila

The expression of Drosophila neuropeptide AYRKPPFNGSIFamide (SIFamide) was shown by both immunohistology and in situ hybridization to be restricted to only four neurons of the pars intercerebralis. The role of SIFamide in adult courtship behavior in both sexes was studied using two different approaches to perturb the function of SIFamide; targeted cell ablation and RNA interference (RNAi). Elimination of SIFamide by either of these methods results in promiscuous flies; males perform vigorous and indiscriminant courtship directed at either sex, while females appear sexually hyper-receptive. These results demonstrate that SIFamide is responsible for these behavioral effects and that the four SIFamidergic neurons and arborizations play an important function in the neuronal circuitry controlling Drosophila sexual behavior.     

Discrete functions of rictor and raptor in cell growth regulation in Drosophila

The TOR signaling pathway regulates cell growth and metabolism in response to various nutrient signals by forming complexes with either rictor or raptor. To distinguish the physiological roles of the complexes formed by the two different TOR partners, we compared the in vivo functions of rictor and raptor in Drosophila. In rictor-null mutants, Akt-induced tissue hyperplasia was reduced and Akt-Ser-505 phosphorylation was decreased. Furthermore, FOXO-dependent apoptosis, which is inhibited by Akt, was augmented in the rictor-null background, indicating that rictor is essential for the Akt-FOXO signaling module. We found that neither S6K-dependent cell growth nor S6K-Thr-398 phosphorylation was affected in rictor-null mutants. However, the knockdown of another TOR binding partner, raptor, decreased S6K-Thr-398 phosphorylation and inhibited S6K-induced cell overgrowth. Collectively, our findings strongly support that the association of TOR with rictor or raptor plays pivotal roles in TOR-mediated cell apoptosis and growth control by differentially regulating Akt- and S6K-dependent signaling pathways, respectively.     

Proteomics of immune-challenged Drosophila melanogaster larvae hemolymph

In the last decade, the fruit fly Drosophila melanogaster has emerged as a promising invertebrate model for the investigation of innate immunity, in part because of its well characterised genetics. The information provided by the innumerous reports on Drosophila's immune response indicates that a large number of genes, in addition to the well-known antimicrobial peptide genes, are both up- and down-regulated upon immune challenge. Nevertheless, their contribution to fighting off infection has not been seriously addressed. With the application of recent advances in proteomics, the effects of an immune challenge in the overall modification of Drosophila 2-DE protein patterns were investigated. The aim of this study was to investigate hemolymph proteins differentially expressed between control and immunised larvae sets, which could be related solely to the Drosophila immune response. The list of immune-related protein spots included heat shock proteins and other proteins with chaperone properties, serine proteases, phenol oxidase, and Drosophila antioxidant system components, which accounted for 21% of the total of 70 identified proteins, metabolic enzymes implicated in pathways such as cellular respiration, fatty-acid oxidation, protein biosynthesis, and structural proteins.     

Lineage analysis of transplanted individual cells in embryos of Drosophila melanogaster

Summary A method is presented which allows the study of the progeny of single cells during Drosophila embryogenesis. Cells from various larval anlagen of donor embryos labelled with a lineage tracer are individually transplanted from defined positions into similar, or different, positions in unlabelled hosts. The clones produced by these cells can be seen in whole mounts or in sections of fixed material, when using a histochemical marker (i.e. HRP), and/or in living embryos, when using fluorescent lineage tracers. The characteristics of the clones disclose lineage parameters, such as division patterns, morphogenetic movements and differentiation. The method is especially useful for testing the respective roles of positional information and cell lineage on the commitment of progenitor cells by transplanting these cells into heterotopic positions or into hosts of different genotypes.     

A natural genetic polymorphism affects retroactive interference in Drosophila melanogaster

As environments change, animals update their internal representations of the external world. New information about the environment is learned and retained whereas outdated information is disregarded or forgotten. Retroactive interference (RI) occurs when the retrieval of previously learned information is less available owing to the acquisition of recently acquired information. Even though RI is thought to be a major cause of forgetting, its functional significance is still under debate. We find that natural allelic variants of the Drosophila melanogaster foraging gene known to affect rover and sitter behaviour differ in RI. More specifically, rovers who were previously shown to experience greater environmental heterogeneity while foraging display RI whereas sitters do not. Rover responses are biased towards more recent learning events. These results provide an ecological context to investigate the function of forgetting via RI and a suitable genetic model organism to address the evolutionary relevance of cognitive tasks.     

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.