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.2020 Feb;68(2):298-315.
doi: 10.1002/glia.23717. Epub 2019 Sep 11.

Gene expression profiling reveals a conserved microglia signature in larval zebrafish

Affiliations

Gene expression profiling reveals a conserved microglia signature in larval zebrafish

Julie Mazzolini et al. Glia.2020 Feb.

Abstract

Microglia are the resident macrophages of the brain. Over the past decade, our understanding of the function of these cells has significantly improved. Microglia do not only play important roles in the healthy brain but are involved in almost every brain pathology. Gene expression profiling allowed to distinguish microglia from other macrophages and revealed that the full microglia signature can only be observed in vivo. Thus, animal models are irreplaceable to understand the function of these cells. One of the popular models to study microglia is the zebrafish larva. Due to their optical transparency and genetic accessibility, zebrafish larvae have been employed to understand a variety of microglia functions in the living brain. Here, we performed RNA sequencing of larval zebrafish microglia at different developmental time points: 3, 5, and 7 days post fertilization (dpf). Our analysis reveals that larval zebrafish microglia rapidly acquire the core microglia signature and many typical microglia genes are expressed from 3 dpf onwards. The majority of changes in gene expression happened between 3 and 5 dpf, suggesting that differentiation mainly takes place during these days. Furthermore, we compared the larval microglia transcriptome to published data sets of adult zebrafish microglia, mouse microglia, and human microglia. Larval microglia shared a significant number of expressed genes with their adult counterparts in zebrafish as well as with mouse and human microglia. In conclusion, our results show that larval zebrafish microglia mature rapidly and express the core microglia gene signature that seems to be conserved across species.

Keywords: RNA sequencing; brain; evolution; microglia; transcriptome; zebrafish.

© 2019 The Authors. Glia published by Wiley Periodicals, Inc.

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Figures

Figure 1
Figure 1
Development of the microglia population in larval zebrafish. (a) Representative confocal images are shown to illustrate zebrafish larval head development from 1 to 7 days postfertilization (dpf) and microglial cell distribution throughout the developing brain. Upper panels correspond to a brightfield transmission image and lower panels represent the maximum intensity projection of 4C4+ microglia (magenta) at each developmental stage. Microglia start colonizing the brain (dotted line) at 3 dpf whereas signal can be detected in the retina from 1 dpf onwards. Scale bar represents 50 μm. (b) Upper panels show 4C4 antibody immunohistochemistry, lower panels show segmented images of microglia morphology at 3, 5, and 7 dpf using the Imaris surface tool. Microglia morphology changes from amoeboid (1 dpf) to ramified (7 dpf) with an intermediate feature at 5 dpf. Scale bar represents 10 μm. (c) Schematic representation of the protocol used to isolate 4C4+ microglia from larval zebrafish brains at 3, 5, and 7 dpf. All images represent maximum intensity projections of confocal stacks. Images were captured using a Zeiss LSM710 confocal microscope with a 20×/NA 0.8 objective [Color figure can be viewed athttp://wileyonlinelibrary.com]
Figure 2
Figure 2
Zebrafish microglia transcriptome at 3, 5, and 7 dpf. (a) Principal component analysis (PCA) score plot obtained from normalized transformed read counts of isolated microglia RNA from 600 zebrafish embryos at 3 (green), 5 (yellow), and 7 (magenta) dpf,n = 3. The PCA score plot shows that replicates from 3, 5, and 7 dpf are clustered and separated according to their developmental stages. (b) Venn diagram shows unique and intersecting genes (3,097) differentially expressed (DE) from microglia transcriptome at 3, 5, and 7 dpf (FDR < 0.05, Fold Change > |2|). (c) Heatmap of DE genes from microglia transcriptome comparisons between 3, 5, and 7 dpf. This heatmap reveals six groups of different expression profiles. These groups correspond to the groups shown in the Venn diagram in (b), which provides the number of genes within these groups. See also Table S1. FDR, false discovery rate [Color figure can be viewed athttp://wileyonlinelibrary.com]
Figure 3
Figure 3
Larval zebrafish microglia show expression of microglia core signature genes. (a) Dot plots of normalized transformed read counts of a representative set of microglia genes (black; orange) and erythro‐myeloid progenitor (EMP) genes (blue) that show significant differences during development and dot plots of normalized transformed read counts of a representative set of microglia (green) and macrophage (purple) genes that show no significant differences between 3, 5, and 7 dpf. The means ± SD of three independent experiments are plotted. (b) mRNA expression levels forapoeb,csf1ra,mpeg1.1,hexb,p2ry12,plnxb2a,spi1b, andirf8 from isolated macrophages at 28 hpf and microglia at 3, 5, and 7 dpf determined by qPCR (n = 3 for each gene). Fold change was measured in relation to 3 dpf microglia using the comparative (ΔΔCT) method. The means ± SD of three independent experiments are plotted [Color figure can be viewed athttp://wileyonlinelibrary.com]
Figure 4
Figure 4
Expression profile of genes involved in microglial processes during development. Heatmap representing normalized transformed read counts of 87 genes involved in different microglia processes at 3, 5, and 7 dpf [Color figure can be viewed athttp://wileyonlinelibrary.com]
Figure 5
Figure 5
Gene Ontology (GO) categories analysis of DE larval zebrafish microglia genes. (a‐i) Pie chart representation for significant enrichment of GO for DE genes from microglia transcriptome comparison between 5 and 3 dpf (FDR < 0.05, fold change > |2|). Only categories containing at least 10 genes are represented. (a‐ii) Heatmap of DE genes belonging to the GO category “immune system process” containing the highest number of genes (155) from microglia transcriptome comparison between 5 and 3 dpf. (b‐i) Pie chart representation for significant enrichments GO for DE genes from microglia transcriptome comparison between 7 and 3 dpf (FDR < 0.05, fold change > |2|). Only categories containing at least 10 genes are represented. (b‐ii) Heatmap of genes belonging to the GO category “Response to stimulus” containing the highest number of genes (317) from microglia transcriptome comparisons between 7 and 3 dpf [Color figure can be viewed athttp://wileyonlinelibrary.com]
Figure 6
Figure 6
Differential gene expression during microglia development in the larval zebrafish shows differences to microglia development in the mouse. Venn diagrams showing unique and intersecting DE genes from zebrafish microglia transcriptome at 3 (green), 5 (yellow), and 7 (magenta) dpf and DE genes from mouse microglia (blue) in the (Y) yolk sac (a‐i), in the (P2) postnatal stage 2 (a‐ii), in the (A1) adult stage 1 (a‐iii) and in the (A2) adult stage 2 (a‐iv, FDR < 0.05, Fold Change > |2|). Mouse data obtained from Matcovitch‐Natan et al. (2016). Significant gene enrichments are shown in bold (FDR < 0.05) [Color figure can be viewed athttp://wileyonlinelibrary.com]
Figure 7
Figure 7
Larval zebrafish microglia share a significant number of DE genes with adult zebrafish microglia, adult mouse microglia, and human microglia. (a) Venn diagram showing unique and intersecting DE genes from zebrafish microglia transcriptome at 3 (green), 5 (yellow), and 7 (magenta) dpf versus other brain cells in comparison to DE genes from adult zebrafish (AZ) microglia transcriptome versus other brain cells (Oosterhof et al., 2016; FDR < 0.05, Fold change >2). Significant gene enrichments are shown in bold (FDR < 0.05). (b) Pie chart representation of GO categories for higher expressed genes in 7 dpf versus adult zebrafish microglia. (c) Venn diagram showing unique and intersecting DE genes from zebrafish microglia transcriptome at 3 (green), 5 (yellow), and 7 (magenta) dpf versus other brain cells in comparison to DE genes from adult mouse (M) microglia transcriptome versus other brain cells (Zhang et al., 2014). Significant gene enrichments are shown in bold (FDR < 0.05). (d) Pie chart representation of GO categories in 7 dpf zebrafish versus adult mouse microglia. (e) Venn diagram showing unique and intersecting DE genes from zebrafish microglia transcriptome at 3 (green), 5 (yellow), and 7 (magenta) dpf versus other adult brain cells in comparison to DE genes from human (H) microglia transcriptome versus other adult brain cells (Galatro et al., 2017). Significant gene enrichments are shown in bold (FDR < 0.05). (f) Pie chart representation of GO categories for higher expressed genes in 7 dpf zebrafish versus adult human microglia [Color figure can be viewed athttp://wileyonlinelibrary.com]
Figure 8
Figure 8
Conserved cellular, immune system, development, and metabolic process core genes across species. Venn diagrams showing unique and intersecting genes from 7 dpf zebrafish microglia transcriptome versus adult zebrafish microglia transcriptome (green), 7 dpf zebrafish microglia transcriptome versus mouse microglia transcriptome (yellow) and 7 dpf zebrafish microglia transcriptome versus human microglia transcriptome (magenta) belonging to the GO category “Cellular process” (a), “Immune system process” (b), “Metabolic process” (c), and “Development” (d). The core genes shared between zebrafish, mouse, and human for the different categories are listed. (e) mRNA expression levels forparvg andlpcat2 from isolated macrophages at 28 hpf and microglia at 3, 5, and 7 dpf determined by qPCR (n = 3 for each gene). Fold change was measured in relation to 3 dpf microglia using the comparative (ΔΔCT) method. The means ± SD of three independent experiments are plotted [Color figure can be viewed athttp://wileyonlinelibrary.com]
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