Fig. 3. The gene expression signature associated with XO4⁺ microglia is reversible and is acquired through phagocytosis of amyloid plaques.

a Schematic representing the experimental design involving addition of 2 × 10⁴ microglia to NIAD4-stained organotypic hippocampal slice cultures (OHSCs), followed by FACS isolation of carboxyfluorescein succinimidyl ester (CFSE)-labelled replenished and CFSE^- endogenous microglia that differentially phagocytose endogenous NIAD4-labelled plaques after 5 days co-culture with wild-type (WT) or 5xFAD OHSCs, created with BioRender.com. b, c k-nearest-neighbour (kNN) graph rendered using a force-directed layout (SPRING)¹⁴², coloured by single cell consensus (SC3) cluster (b), and log₂-transformed ΔCt values of selected DEGs (c). Each dot represents 10 sorted cells, and data are from (d) n = 120 cells, (e) n = 240 cells, (f) n = 110 cells, (g) n = 280 cells sorted during 3 independent experiments. Replicates from independent experiments are closed circles, technical replicates are open circles. The XO4⁺ score is defined as the x-axis position of each sorted population on the kNN graph. The colour scales are log₂(ΔCt), Mafb min ΔCt = 0.0003, max ΔCt = 3.37; Cx3cr1 min ΔCt = 0.0001, max ΔCt = 9.69; Cst7 min ΔCt = 0.0002, max ΔCt = 4.56; Igf1 min ΔCt = 0.0001, max ΔCt = 1.07. d–g Experimental schematic, XO4⁺ score and proportion Cluster 1 and Cluster 2 membership of groups of exogenous and endogenous (d) WT microglia added into a WT OHSC, (e) WT microglia added into 5xFAD slices, recapitulating the gene expression signature associated with XO4⁺ microglia upon plaque phagocytosis. f XO4⁺ phenotype is stable in exogenous CFSE⁺NIAD4⁺ 5xFAD microglia recovered from 5xFAD slices, but (g) is lost in CFSE⁺ 5xFAD microglia recovered from WT slices. N.D., not detected. Data are presented as mean ± SEM.