Leukocyte heterogeneity in the adult zebrafish brain using blood lineage-specific transgenic lines.

A. Schematic overview of the experiment. First, cd45:DsRed+ cells were sorted, cytospined and stained with May-Grunwald Giemsa (MGG). In parallel, lines carrying the cd45:DsRed transgene in combination with blood lineage-specific GFP reporters were analyzed by flow cytometry. B. Morphology of brain-sorted cd45:DsRed+ cells stained with MGG. Microglia and/or macrophages, monocytes, dendritic cells, neutrophils and lymphocytes were identified. The scale bar represents 5 µm. C. Flow cytometry analysis on brain cell suspensions from adult Tg(mpeg1:GFP; cd45:DsRed) identifying mpeg1:GFP+; cd45:DsRed+mononuclear phagocytes (green gate). D. Proportion of brain immune cell types, as determined by flow cytometry analysis on cell suspensions from fish carrying cd45:DsRed+ and a lineage-specific GFP reporter (n=4 fish). The percentage relative to total cd45:DsRed+ leukocytes is shown, with the exception of Tg(ighm:GFP; cd45:DsRed) which are not normalized as the cd45:DsRed transgene is not expressed in the B cell lineage. E. Flow cytometry analysis of brain cell suspensions from an adult Tg(p2ry12:p2ry12-GFP; cd45:DsRed) fish, identifying p2ry12:p2ry12-GFP+; cd45:DsRed+ microglial cells (light green gate). n refers to the number of biological replicates. Data in (D) are mean ± SEM.

Diversity of brain leukocytes as shown by single-cell transcriptomics.

A. Schematic overview of the experimental approach. Single-cell profiling of total brain cd45:DsRed+ leukocytes (pool from 3 individual fish) was performed using the 10X Genomics platform. B. Split Uniform Manifold Approximation Projection (UMAP) of brain cd45:DsRed+ cells with annotated cell populations. Clusters in grey shade are not indicative of a specific cell type and were not annotated. C. UMAP plots depicting the expression pattern of ptprc, also known as cd45 (leukocytes), mpeg1.1 (mononuclear phagocytes), mpx (neutrophils) and lck (T and NK lymphocytes). Gene expression levels from low to high are indicated by a color gradient from yellow to purple (normalized counts in log1p). D. Heat map of the top differentially up-regulated genes in each cluster (row=gene, column=cell type). Color scale (gradual from purple to yellow) indicates the expression level (average log2 fold change).

Single-cell RNA sequencing identifies several lymphocyte subpopulations in the adult brain.

A-C. UMAP visualization of the expression of selected genes in the annotated T cell clusters Tcells1 and Tcells2 (zap70, cd4-1 and cd8a), NK cluster (ccl38.6, il2rb, gzm3.3) and ILC-like cluster (il4, il13 and gata3). Color scale (gradual from yellow to purple) indicates the expression level for each gene (normalized counts in log1p). D. Violin plots representing the expression levels of known lymphocyte markers (normalized counts in log1p) within the different clusters. E. Comparison of the relative expression of lck, zap70, gata3, il13 and il4 transcripts between brain cd45:DsRed+ cells isolated by FACS from T cell-deficient rag2-/- mutants (red bars) and their wild-type siblings (grey bars). Each data point represents an individual fish (n=6) and error bars indicate SEM. *** P<0.001, **** P<0.0001 (Two-tailed unpaired t-test).

Heterogenous subsets of mononuclear phagocytes exist in the zebrafish brain.

A-C. UMAP visualization of the expression of selected genes in the microglia (apoeb and lgals3bpb) (A), non-microglia macrophage (marco and f13a1b) (B) and DC-like (xcr1a.1 and siglec15l) (C) cell clusters. Color scale (gradual from yellow to purple) indicates the expression level for each gene (normalized counts in log1p). D. Violin plot analysis comparing the expression levels of selected genes (y-axis, normalized counts in log1p) between the different mononuclear phagocyte cell clusters. E. Volcano plot showing the differentially expressed (DE) genes between microglia (MG) and non-microglia macrophages (MF). Lines indicate significantly DE genes (log2 fold-change >|0.5|, -log10 Padj <0.001). Red dots represent up-regulated genes and blue dots down-regulated genes. Labels show representative DE genes identified in the analysis. F. Volcano plot showing the DE genes between microglia (MG) and DC-like cells (DC-like). Lines indicate significantly DE genes (log2 fold-change >|0.5|, -log10 Padj <0.001).

DC-like cells localize together with microglia within the brain parenchyma.

A-F. Immunofluorescence on transversal brain sections (14 µm) from Tg(mpeg1:GFP) (A-C) or Tg(p2ry12:p2ry12-GFP) (D-F) transgenic adult fish co-immunostained with anti-GFP (green) and anti-Lcp1 (magenta) antibodies. A-C. All mpeg1:GFP+ mononuclear phagocytes in the brain parenchyma display Lcp1 immunostaining, as expected. D-F. Similarly, all microglial cells, identified by GFP expression in the brain parenchyma of Tg(pr2y12:p2ry12-GFP) fish, are Lcp1+, as expected. G-J. In sections of adult Tg(p2ry12:p2ry12-GFP; mpeg1:mCherry) double transgenic animals, GFP labeling is not observed in all mCherry+ cells. GFP (green), mCherry (grey), Lcp1 (magenta) and merge of the three channels. All images were taken using a 20X objective and correspond to orthogonal projections. White arrowheads point to microglial cells (GFP+; Lcp1+or GFP+; mCherry+; Lcp1+) and yellow arrowheads to DC-like cells (GFP-; Lcp1+ or GFP-; mCherry+; Lcp1+). Scale bars: 50 µm. L-O. Confocal imaging of a midbrain vibratome section (100 µm) from an adult Tg(mhc2dab:GFP; cd45:DsRed) brain. GFP (green), DsRed (magenta) and merge of the two channels are shown. Images correspond to orthogonal projections, white arrowheads point to GFP+; DsRed+ cells and yellow arrowheads to GFP-; DsRedhigh. Scale bar in (K): 100 µm, scale bar in (L-N): 50 µm.

Transcriptomic analysis of microglia (p2ry12+; cd45+ or mhc2dab+; cd45low) and DC-like cells (mhc2dab+; cd45high).

A. Schematic overview of the experiments. Microglia were isolated using Tg(p2ry12:p2ry12-GFP; cd45:DsRed) or Tg(mhc2dab:GFP; cd45:DsRed) transgenic fish, and DC-like cells using the Tg(mhc2dab:GFP; cd45:DsRed) reporter line. B. Representative flow cytometry plot identifying microglial cells in brain cell suspensions from Tg(p2ry12:p2ry12-GFP; cd45:DsRed) fish. C. Representative flow cytometry plot identifying mhc2dab:GFP+; cd45:DsRedlow microglia from mhc2dab:GFP+; cd45:DsRedhigh DC-like cells in brain cell suspensions from Tg(mhc2dab:GFP; cd45:DsRed) fish. D. Volcano plot showing the differentially expressed (DE) genes between mhc2dab+; cd45high DC-like cells and p2ry12+; cd45+ microglia. Red dots represent up-regulated genes and blue dots down-regulated genes. Lines indicate significantly DE genes (log2 fold-change >|2|, -log10 Padj <0.01). Labels show marker genes for DC-like cells and microglia identified in the scRNA-sequencing analysis. E. Volcano plot showing the DE genes between mhc2dab+; cd45high DC-like cells (blue) and mhc2dab+cd45lowmicroglia (red). Lines indicate significantly DE genes (log2 fold-change >|2|, -log10 Padj <0.01).

Brain DC-like cells are lost in batf3-/-mutant fish.

A-J. Immunofluorescence on transverse brain sections (14 µm) from adult wild-type (A-E) and batf3-/- mutant (F-J) fish carrying the Tg(p2ry12:p2ry12-GFP; mpeg1:mCherry) double transgene and immunostained for GFP (green), mCherry (grey) and Lcp1 (magenta). Illustrative case of the merge of the three channels (A, F) allowing to identify GFP+; mCherry+; Lcp1+ microglia (white arrowheads) versus GFP-; mCherry+; Lcp1+ DC-like cells (yellow arrowheads). While DC-like cells are found in high numbers within the ventral part of control parenchyma (A), these are dramatically decreased following genetic loss of batf3 (F). Scale bars: 100 µm. (B-E, G-J). Single channels high magnification of the insets in A (B-E) and F (G-J). Scale bars: 50 µm. Images were taken using a 20X objective and correspond to orthogonal projections. K-M. Quantification of cell density for GFP+; mCherry+; Lcp1+ microglia and GFP-; mCherry+; Lcp1+ DC-like cells in the dorsal midbrain area or optic tectum (K), ventral midbrain area (L) and the entire section (M) of control (grey bars) and batf3-/- (green bars) fish. Each dot represents a single fish and data are mean ± SEM. * P<0.05 (Mann-Whitney test), *** P<0.0001 (Two-tailed unpaired t-test). N. Flow cytometry analysis of brain cell suspensions from wild-type and batf3-/-adult fish carrying the Tg(p2ry12:p2ry12-GFP; mpeg1:mCherry) reporter. The GFP+; mCherry+ fraction identifies microglia (green circle), whereas the GFP-; mCherry+ fraction contains mainly DC-like cells (blue frame). O. Percentage of microglia and DC-like cells in brain cell suspensions for each genotype, relative to the whole living brain population, as shown in (N) (wild-type, n=6; batf3-/-, n=10). *** P<0.001 (Two-tailed unpaired t-test). n refers to number of biological replicates.

Examination of microglia and DC-like cells in myeloid–deficient mutant lines.

A-D. Immunofluorescence on transverse brain sections from Tg(p2ry12:p2ry12-GFP) transgenic adult wild-type (A-D), irf8-/- (E-H), csf1ra-/- (I-L), csf1rb-/- (M-P) and csf1ra-/-; csf1rb-/- (csf1rDM) (Q-T) fish, co-stained with anti-GFP (green) and Lcp1 (magenta) antibodies. A, E, I, M, Q. For each genotype, illustrative case of the merge of the two channels, allowing to discriminate in the parenchyma GFP+; Lcp1+ microglia (white arrowheads) from GFP-; Lcp1+ DC-like cells (yellow arrowheads). Single channels high magnification of the insets (dashed frame) in A (B-D), E (F-H), I (J-L), M (N-P) and Q (R-T). Outline yellow arrowheads indicate the absence of GFP signal in corresponding yellow arrowhead pointed cells. Scale bar in (A), (E), (I), (M) and (Q) represents 100 µm and scale bar in other images 50 µm. U-V. Quantification of the cell density for GFP+ Lcp1+ microglia and GFP-Lcp1+ DC-like cells in the dorsal (U), ventral (V) and whole area (W) of the brain for each genotype (n=3). Data in U-W are mean ± SEM. *P<0.05, **P<0.01 (Klustal-Wallis test with Dunn’s post-hoc).