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A single-cell survey of Drosophila blood

ABSTRACT: Drosophila blood cells, called the hemocytes, are composed of three distinct cell populations: plasmatocytes (PM), crystal cells (CC), and lamellocytes (LM). These cell types have been extensively characterized based on the expression of a few marker genes and cell morphologies, which are inadequate to classify the complete hemocyte repertoire. Further, intermediate states along the course of maturation of these cell types have been poorly characterized. Here, we used single-cell RNA sequencing (scRNA-seq) to map the Drosophila larval hemocytes across different inflammatory conditions. We identified all known cell types, resolved PMs into different states based on the expression of genes involved in cell cycle, antimicrobial response, and metabolism together with the detection of CC and LM intermediates. Further, we discovered rare cells within CC and LM populations that express the fibroblast growth factor (FGF) ligand branchless (bnl) and receptor breathless (btl), respectively. We demonstrate that both bnl and btl are required for mediating effective immune responses against parasitoid wasp eggs in vivo, highlighting a novel role for FGF signaling in inter-hemocyte crosstalk. Our scRNA-seq analysis reveals the diversity of hemocyte populations and provides a rich resource of gene expression profiles for a systems-level understanding of their functions.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE146596 | GEO | 2020/05/16

REPOSITORIES: GEO

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Project description:Goal: Natural populations of Drosophila melanogaster vary widely in their capacity to resist infection by the parasitic wasp Leptopilina boulardi. To date, little is known about the genetic, cellular and molecular basis underpinning the variation in susceptibility to parasitic wasps. In D.melanogaster populations artificially selected for parasitoid resistance, an increase in the number of circulating hemocytes (blood cells) is observed. One possibility is that this is accompanied by changes in hemocyte activation state, resulting in a successful defence response when exposed to parasitic wasps. We tested this possibility by generating populations that are highly resistant and highly susceptible to the parasitic wasps. We generated these populations after 26 generations of experimental evolution, starting from a single interbred wild caught population. Here, we study how selection for resistance to the parasitic wasp L. boulardi changes the number and type of circulating hemocytes in D. melanogaster larvae using scRNA-seq. Methods: Wild caught D. melanogaster females were collected from Cambridge, UK, to establish an outbreed population. From this, three replicated populations were selected for resistance to L. boulardi strain NSRef for 26 generations (Selection). Another three populations were maintained in the laboratory for 26 generations without being exposed to parasitoid-associated selection pressures (No Selection). At generation 26, second instar D. melanogaster larvae (48-63 hours after fertilization) from each population were infected with L. boulardi for three hours (Infection) or maintained without infection (No infection). 48 hours after, circulating hemocytes from third instar D. melanogaster larvae (96-111 hours after fertilization) from each population were collected in PBS and cleaned in OptiPrep solution (1.09g/ml). 10X Single Cell GEX v2 libraries were prepared and sequenced. CellRanger v2 was used to generate sample cell count matrices. Seurat v3 was used to integrate, normalise and cluster the cell types. Results: We identified novel plasmatocyte cell types, immature and mature lamellocytes and crystal cells. We identify a single lineage of cellular differentiation starting from a self-cycling plasmatocyte population, enriched for genes involved in extracellular matrix organisation, and ending in a mature lamellocyte population that has enriched expression of genes involved in signalling, hemostasis and gluconeogenesis. Immature lamellocytes were overrepresented in populations selected for resistance to parasitoids. Infection with parasitoid resulted in an increase in both immature and mature lamellocytes in circulation. Conclusions: Selection for resistance to the parasitic wasps resulted in the constitutive activation of the D. melanogaster immune system, where plasmatocytes differentiate into immature lamellocytes even where they are not exposed to parasitic wasps. We speculate these constitutive changes assist in mounting a successful defence response when exposed to parasitic wasps.

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A single-cell survey of Drosophila blood

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