Fig. 2: Individual non-PAM give rise to clones of expanding PAM at the plaque sites.

a, Representative images of Confetti⁺ microglia in Cx3cr1^CreERT2R26^Confetti5×FAD⁺ mice. Immunofluorescence for anti-GFP (green) and thioflavine-S/thiazine red/Methoxy-X04 (blue) are shown with the following markers (cyan). Top: APOE and CLEC7A. Bottom: P2RY12. Blue arrowheads indicate non-PAM; red arrowheads highlight PAM. Scale bars, 50 µm. b, Top: representative images for anti-GFP (green) and thiazine red (blue) together with CD11c (cyan). Bottom: images of Iba-1⁺CD11c⁺ PAM restricted to amyloid plaques in Cx3cr1^CreERT2R26^Confetti5×FAD⁺ animals. Thiazine red (blue), Iba-1 (red) and CD11c (cyan) are shown. Blue arrowheads indicate non-PAM and red arrowheads indicate CD11c⁺ PAM. Scale bars, 100 µm. c, Left: illustrative image of Confetti⁺ microglia in Cx3cr1^CreERT2R26^Confetti5×FAD⁺ animals. Iba-1 (red), anti-GFP (green), Methoxy-X04 (blue) and Tmem119 (cyan) are shown. Scale bars, 100 µm. Right: individual channels are shown. d, Voronoi grid visualizing plaque sizes and the positioning of Confetti⁻ and Confetti⁺ Tmem119⁺Iba-1⁺ microglia (non-PAM) and Confetti⁻ and Confetti⁺ Tmem119⁻Iba-1⁺ microglia (PAM) in 20-week-old Cx3cr1^CreERT2R26^Confetti5×FAD⁺ mice. Tmem119⁺Iba-1⁺ (white), CFP⁺Tmem119⁺Iba-1⁺ (cyan), YFP⁺Tmem119⁺Iba-1⁺ (yellow), Tmem119⁻Iba-1⁺ (gray), CFP⁺Tmem119⁻Iba-1⁺ (green) and YFP⁺Tmem119⁻Iba-1⁺ (orange) microglia are shown. Volume of Methoxy-X04⁺ plaques is color coded as indicated in the legend. e, Quantification of Tmem119⁺ PAM (red) and non-PAM (blue) in Cx3cr1^CreERT2R26^Confetti5×FAD⁺ mice (n = 12). Dots represent individual animals. Mean ± s.e.m. is shown. ***P < 0.0001. f, Experimental scheme of Tmem119^CreERT2R26^Confetti5×FAD⁺ mice. Scheme was created with BioRender.com. g, Characteristic images of Tmem119^CreERT2R26^Confetti5×FAD⁺ animals 8 weeks after TAM application. Images are shown for Methoxy-X04 (blue), anti-GFP (green), Tmem119 (cyan) and Iba-1 (red). Scale bars, 100 µm. Data are shown from one independent experiment. h, Characteristic images of Confetti⁺Iba-1⁺ microglia (arrowheads) clones at amyloid plaques in Tmem119^CreERT2R26^Confetti5×FAD⁺ animals 8 weeks after TAM application. Clones are found in all Confetti colors: RFP (red), mCFP (cyan), YFP (yellow) and nGFP (green). Immunofluorescence for Iba-1 (cyan, top) and magenta (middle and bottom) and Methoxy-X04 (blue) are shown. Scale bars, 50 µm. Data are shown from one independent experiment. i, Typical confocal pictures of single labeled Confetti⁺Iba-1⁺ non-PAM (blue arrowheads) in the cortex (top) and cerebellum (bottom) of 44-week-old Tmem119^CreET2RR26^Confetti5×FAD⁺ animals 8 weeks after TAM application. Immunofluorescence for Iba-1 (magenta), Methoxy-X04 (blue) and YFP (yellow). Scale bars, 50 µm. Data are shown from one independent experiment. j, Representative images from Tmem119^CreERT2R26^Confetti5×FAD⁺ animals 2 weeks (left) or 8 weeks (right) after TAM application. Confetti labeling was observed in both Iba-1⁺ (magenta) non-PAM (blue arrowheads) and Iba-1⁺ PAM (red arrowheads). Immunofluorescence for Methoxy-X04 (blue), YFP (yellow) and mCFP (cyan) is shown. Scale bars, 50 µm. k, Quantification of same-colored Confetti⁺ PAM per clone in Tmem119^CreERR26R^Confetti5×FAD⁺ animals 2 weeks (N = 62) and 8 weeks (N = 48) after TAM application. Mean ± s.e.m. is shown. Symbols represent individual clones from biological replicates (n = 2). P = 0.0021. l, Quantification of the percentage of same-colored Confetti⁺ PAM clones that were either associated (red) or not associated (dark red) with an individual same-colored Confetti⁺ non-PAM. Pie charts represent distribution at 2 weeks (left) and 8 weeks (right) after TAM injection.

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