Extended Data Fig. 4: Generation and comparative analysis of Mrc1^CreERT2 mice.

(a) Targeting schemes. The T2A-CreERT2 cassette was knocked in into the Mrc1 locus just before the stop codon. From the resulting polycistronic RNA, a fusion protein is translated, which gives rise to MRC1 (CD206) and the CreERT protein through the activity of the self-cleaving T2A peptide. (b) Representative gating strategy for FACS-sorting of CD206⁺ CAMs from adult Mrc1^+/+, Mrc1^CreERT2/+ or Mrc1^{CreRT2/CreERT2} mice for RNA-expression analysis and for measuring CD206 surface expression levels. (c) Heat map of the top 1,000 differentially regulated genes across the respective genotypes. (d) Volcano plots depicting differentially regulated genes between the groups (14,639 variables) (n = 3 for Mrc1^+/+ and Mrc1^CreERT2/+, n = 4 for Mrc1^{CreERT2/ CreERT2}). NS = not significant. (e) Left: Representative histograms showing CD206 surface expression levels on CAMs isolated using the gating strategy depicted in (b). Right: Quantification geometric mean fluorescence intensities (gMFI). Symbols represent individual mice (means ± s.e.m. are shown). N = 4 mice per genotype. One-way ANOVA with Tukey’s multiple comparisons test. (f) Immunofluorescence images showing IBA1⁺ (white) microglia, CD206⁺ (red) pvMΦ and CD206⁺ mMΦ in the cortex of Mrc1^+/+, Mrc1^CreERT2/+ or Mrc1^{CreERT2/CreERT2} mice. Images representative of 4 mice per genotype. Scale bar: 20 μm (g) Bar graphs depicting the expression levels of CAM signature genes. Dots represent individual mice (mean ± s.d.). (n = 3 for Mrc1^+/+ and Mrc1^CreERT2/+, n = 4 for Mrc1^{CreERT2/ CreERT2}). (h) Quantification of microglia, pvMΦ and mMΦ in the cortex of adult Mrc1^+/+ (black circles), Mrc1^CreERT2/+ (grey circles) or Mrc1^{CreERT2/CreERT2} (white circles) mice. Each symbol represents one mouse (n = 4 for all genotypes). Three sections per mouse were quantified (means ± s.e.m.). One-way ANOVA with Tukey’s multiple comparisons test. (i) Scheme of the experimental set-up. (j) Direct fluorescence microscopic visualization reveals tdT signals (red) in CD206⁺ pvMΦ and mMΦ (green), but not in microglia (IBA1, green), astrocytes (SOX9, green), neurons (NeuN, green), or oligodendrocyte (APC, green) in the cortex of adult Mrc1^{CreERT2/CreERT2}R26^tdT/tdT mice. Col IV is shown in blue. Filled arrowheads indicate double positive pvMΦ and mMΦ, empty triangles show single positive cells. Images representative of three mice. Scale bars: 20 µm. (k) Quantification of recombination efficacy in several CNS cells of Mrc1^{CreERT2/CreERT2}R26^tdT/tdT and Mrc1^CreERT2/+R26^tdT/tdT mice (with or without TAM), respectively. Each symbol represents one mouse (Mrc1^CreERT2/+R26^tdT/tdT with TAM, n = 7; Mrc1^CreERT2/+R26^tdT/tdT without TAM, n = 2; Mrc1^{CreERT2/ CreERT2}R26^tdT/tdT with TAM, n = 3; Mrc1^{CreERT2/ CreERT2}R26^tdT/tdT without TAM, n = 3). Three sections per mouse were quantified (means ± s.e.m.). (l) Flow cytometry-based quantification of tdT-labelled blood immune cells. Each symbol represents one mouse (Mrc1^CreERT2/+R26^tdT/tdT, n = 6; Mrc1^{CreERT2/CreERT2}R26^tdT/tdT, n = 3. Data are shown as mean ± s.e.m.

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