Gene Expression Profiles of Drosophila third instar larvae hemocytes to explain hemocyte development.
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE10853 | GEO | 2008/03/18
SECONDARY ACCESSION(S): PRJNA107349
REPOSITORIES: GEO
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Project description:We measured gene expression profiles of third instar larvae hemocytes from selected strains related to hemocyte development. Every strain was purchased from commercial companies. And we used microarray data to find what biological processes show abnormalities first, and which gene expressions show similarities and differences between selected strains. Experiment Overall Design: 200~300 Drosophila third instar larvae hemocytes from each of five lines were dissected to get at least 1 microgram of total RNA. Because all samples were hybridized to single-dye Affymetrix chip, control data were compared with data from each mutant.
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Project description:Toll pathway is a key mediator of antiplasmodial immunity and its mechanism of action is dependent on hemocytes (mosquito white blood cells). Anopheles gambiae were injected with dsRNA for Cactus, an inhibitor of the Toll pathway. Cactus silencing over activates the Toll pathway. The goal of this experiment is to evaluate hemocyte transcriptional changes between control mosquitoes injected with a non-related dsRNA (dsLacZ and dsCactus injected mosquitoes. We also divide hemocytes from control and experimental conditions into 2 groups: bound (hemocytes that attach to glass - granulocyte populations) and unbound (hemocytes that remain in suspension - prohemocytes and oenocytoids). Files from Unbound fractions are labelled: UB and from bound fractions are labelled B. Some of the samples were run twice and have 2 gz files.
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Project description:The squid, Euprymna scolopes, and the luminescent bacterium, Vibrio fischeri, form a highly specific beneficial light organ symbiosis. Not only does the host have to select V. fischeri from the environment, but it must also prevent subsequent colonization by non-symbiotic microorganisms. Host macrophage-like hemocytes are believed to play a role in mediating the symbiosis with V. fischeri. Previous studies have shown that the colonization state of the light organ influences the host’s hemocyte response to the symbiont. To further understand the molecular mechanisms behind this process, two quantitative mass spectrometry-based proteomic techniques, isobaric tags for relative and absolute quantification (iTRAQ) and label-free spectral counting, were used to compare and quantify the adult hemocyte proteomes from colonized (sym) and uncolonized (antibiotic-treated/cured) squid. Overall, iTRAQ allowed for the quantification of 1,024 proteins with 2 or more peptides. Thirty-seven unique proteins were determined to be significantly different between sym and cured hemocytes (p-value < 0.05), with 20 more abundant proteins and 17 less abundant in sym hemocytes. The label-free approach resulted in 1,241 proteins that were identified in all replicates. Of 185 unique proteins present at significantly different amounts in sym hemocytes (as determined by spectral counting), 92 were more abundant and 93 were less abundant. Comparisons between iTRAQ and spectral counting revealed that 30 of the 37 proteins quantified by iTRAQ exhibited similar trends identified by the label-free method. Both proteomic techniques mutually identified 16 proteins that were significantly different between the two groups of hemocytes (p-value <0.05). The presence of V. fischeri in the host light organ influenced the abundance of proteins associated with the cytoskeleton, adhesion, lysosomes, proteolysis, and the innate immune response. These data provide evidence that colonization by V. fischeri alters the hemocyte proteome and reveals proteins that may be important for maintaining host-symbiont specificity.
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Project description:The aim of the experiment is to establish the impact of the interaction between the hemocyte differentiation factor gcm and the Toll pathway in the immune cells of the Drosophila larva. The hemocytes were collected from 3rd instar larvae of the following genotypes: Toll10B/+, gcm26/+ and gcm26/+;Toll10B/+.
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Project description:Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. The metamorphosis of the fruit fly represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, the mechanisms that coordinate development and immune cell activity in the transition from larva to adult in Drosophila remain to elucidate. The steroid hormone ecdysone is known to act as a key coordinator of metamorphosis. This hormone activates a nuclear receptor, the Ecdysone Receptor (EcR), which acts as a heterodimer with its partner Ultraspiracle (USP). Together, they activate the transcription of primary response genes, which in turn activate the transcription of a battery of late response genes. We have revealed that regulation of macrophage-like cells (hemocytes) by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. We have shown that in response to ecdysone signalling, hemocytes rapidly up regulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential to hemocyte immune functions and survival after infection. To better understand the ecdysone regulation of hemocyte activities, we have performed gene expression analysis. In order to identify the genes which expression change at the onset of metamorphosis, we have sorted hemocytes from 3rd instar larvae and from young prepupae and compared their gene expression. Moreover, and in order to identify which genes are regulated by the ecdysone signalling, we have used individuals expressing a dominant negative form of the Ecdysone Receptor specifically in their hemocytes. We have sorted hemocytes from 3rd instar and young prepupae of this genotype to compare their gene expression to the gene expression in larvae and prepupae from the control individuals. Hemocytes were isolated by FACS from selected 3rd instar larvae (at the late feeding stage) and prepupae (from 1h to 2h after puparium formation - APF) corresponding to two different genotypes: individuals w;HmlDeltaGal4, UAS-GFP/+ that express GFP specifically in hemocytes (genotype control), and individuals w;HmlDeltaGal4; UAS-GFP/UAS-EcRB1DN W650A which hemocytes express an Ecdysone Receptor Dominant Negative construct in addition to the GFP (EcRDN). For each of the four conditions we performed three biological replicates.
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Project description:Drosophila embryos that were lacking the hemocyte lineage were compared to wild-type embryos. Keywords: genetic modification
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