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Whole-transcriptome RNA-seq analysis of zebrafish microglia subpopulations

ABSTRACT: We generate whole-transcriptome RNA-seq profiles of microglia subpopulations in the adult zebrafish brain (4-month-old) to study their heterogeneity. Cells were isolated as ccl34b.1+mpeg1+ populations or ccl34b.1-mpeg1+ populations from the whole brain of double transgenic TgBAC(ccl34b.1:eGFP);Tg(mpeg1:DsRedx) fish. ccl34b.1+mpeg1+ and ccl34b.1-mpeg1+ microglia were grouped into two distinct clusters in the Principal component analysis (PCA), and we identified substantial differentially-expressed genes between these two microglial populations using the DESeq2 package. To further unveil their differential roles during inflammation, we injected E. coli into the brain ventricles of adult zebrafish and isolated both populations for whole-transcriptome RNA-seq. This RNA-seq profile provides valuable information for dissecting their respective functions in vivo.

ORGANISM(S): Danio rerio

PROVIDER: GSE156158 | GEO | 2020/11/24

REPOSITORIES: GEO

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Project description:Macrophage expressed gene 1 (MPEG1) encodes an evolutionary conserved protein with a predicted Membrane Attack Complex/Perforin domain associated with host defence against invading pathogens. In vertebrates, MPEG1 is an integral membrane protein of macrophages, but how it contributes to the macrophage defence mechanisms remains unknown. Zebrafish have three copies of MPEG1, two of which (mpeg1 and mpeg1.2) are expressed in macrophages whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens, Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is down-regulated during infection with both pathogens, mpeg1.2 is infection inducible. Up-regulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1, and requires the Toll-like receptor adaptor molecule MyD88 and transcription factor NFM-NM-:B. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in increased bacterial burden. In S. typhimurium infection, both mpeg1 and mpeg1.2 knockdown increase bacterial burdens, but mpeg1 morphants show an increased survival rate. The combined results of these two in vivo infection models support the anti-bacterial function of the Mpeg1 family and indicate that the intricate cross-regulation of the two mpeg1 copies aids the zebrafish host in combatting infection Embryos were injected at the one cell stage with a morpholino targeting mpeg1, or with the standard control morpholino from GeneTools, or with a morpholino targeting ptpn6 (Kanwal et al., 2013, J. Immunol 190:1631-45) for comparison. Subsequently, at 24 hours post fertilisation (hpf) the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein, or mock-injected with PBS/2%PVP. After injections embryos were transferred into fresh egg water containing 0.003% 1-phenyl-2-thiourea (Sigma-Aldrich) to prevent melanisation and incubated for 4 days at 28M-BM-0C. After the incubation period, infected and uninfected morphants, mutants and their controls were imaged and groups of 30 embryos were snap-frozen in liquid nitrogen and RNA was isolated for Illumina RNAseq analysis.

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Whole-transcriptome RNA-seq analysis of zebrafish microglia subpopulations

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