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Fig. 1 | Acta Neuropathologica Communications

Fig. 1

From: Unique microglia recovery population revealed by single-cell RNAseq following neurodegeneration

Fig. 1

Single-cell analysis identified disease stage-specific microglial populations in a transient model of neurodegeneration. a Scheme of single microglial cell gene expression analysis after facial nerve axotomy (FNX) in 8 weeks old female CX3CR1GFP/+ mice. Microglia from contralateral facial nuclei (FN) of non-operated healthy mice (0 d) were used as baseline control for steady state transcriptome. Microglia from both FN of mice at peak of disease (7 d after FNX) and onset of recovery (30 d after FNX) were analyzed. A coronal brain section from 7 d after FNX at peak of disease is shown to indicate the locations of the FN (orange dotted circles) from which GFP+ CD45lo CD11b+ microglia were index-sorted by FACS for RNA sequencing. b Quantification of GFP+ FN microglia after FNX. Each symbol represents mean count per animal. N = 4 mice per group. Two-way ANOVA and one-tailed paired t-tests showed significant difference between time and between FN at peak of disease (7 d) and onset of recovery (30 d). c Representative images of GFP+ FN microglia (green) at peak of disease (7 d) after FNX. 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstain is in blue. Scale bar: 30 μm. d t-distributed stochastic neighbor embedding (t-SNE) representations of 944 microglial cells from contralateral (left) and ipsilateral (right) FN based on transcriptomic analysis. The proximity of cells reflects transcriptome similarity as measured by Pearson’s correlation. Cells from contralateral FN are represented by open circles in black, red and green for disease-free (0 d), peak of disease (7 d) and onset of recovery (30 d), respectively. Cells from the injured FN are shown as open squares in red and green for 7 and 30 d and contributed significantly to the distinct “tail” population. Cells from all groups were distributed uniformly in the cloud. See Table 1 for contribution of cells per mouse. N = 3 mice per stage. e Cluster analysis based on transcriptome similarities of all 944 microglial cells revealed 10 clusters (C1-C10) of which C4 (52 cells), C8 (27 cells) and C9 (15 cells) belong to the disease-associated tail of the t-SNE map (top). Tail clusters exhibit 101 differentially expressed genes (see Additional file 1: Figure S1) in comparison to the cloud clusters (P < 0.05). Microglial cells from injured FN at 7 d and 30 d after FNX contribute to the tail as in (d). Cloud clusters (850 cells) reveal a homogeneous population of microglia in contralateral and lesion groups with minor heterogeneity in gene expression levels. The heat map shows the enrichment of microglial cells belonging to each group in clusters C1-C10 (bottom). The color legend depicts an enrichment score [−log10(p-value+ 10− 3)], where the P-value is calculated by a hypergeometric test. t-SNE representations of total FACS sorted microglia (944 cells) show the relative fluorescence intensities (color legend) of surface markers CD45, CD11b and MHC class II, and endogenous GFP mapped to single CX3CR1+ microglia. g MA plot of the 101 differentially expressed genes (see Additional file 1: Figure S1) that distinguish the cloud and tail (comprising C4, C8 and C9 contributed by microglial cells from injured FN at 7 d and 30 d after FNX) transcriptomes in (e). Ten selected upregulated (red) and down-regulated (blue) genes with a minimum of two-fold change (Benjamini-Hochberg-corrected P < 0.05) are indicated. Non-regulated expressed genes are shown in gray. h Representative Gene Ontology (GO) terms for the neurodegeneration-associated microglial gene signature represented by the 101 differentially expressed genes in (g)

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