Expression of MSX3 is dynamically regulated in microglia during EAE. A, Screening of microglia genes that are differentially expressed after IL-4 (10 ng/ml) or IFNγ (100 ng/ml) treatment. The volcano plots showing genes that are regulated (more than twofold and p < 0.05) after IL-4 or IFNγ treatment for 24 h. Probes for some M1 or M2 marker genes are shown (arrows). B, Heat map of the nuclear genes that are upregulated after IL-4 treatment (p < 0.05) and that are downregulated (p < 0.05) or unchanged after IFNγ treatment for 24 h. Untreated microglia (Ctrl) were used as the control. C, Western blot analysis of MSX3 expression in microglia after no stimulation, IFNγ stimulation, or IL-4 stimulation for 24 h. n = 3 blots. MSX3 is 22 kDa. D, MSX3 mRNA expression from microglia stimulated with TGF-β1 (5 ng/ml), IL-4 (10 ng/ml), IL-17A (10 ng/ml), and IFNγ (100 ng/ml) for 24 h. E, The time course of MSX3 mRNA expression on microglia stimulated with IL-4 (10 ng/ml). C–E, *p < 0.05; **p < 0.01; ***p < 0.005 versus MSX3 in naive microglia. F, Flow cytometry analysis of microglia isolated from the spinal cord of EAE mice. Mononuclear cells were isolated, stained for CD11b and CD45, and analyzed using FACS. CD11b⁺CD45^low resting microglia (a), CD11b⁺CD45^hi-activated microglia and peripheral macrophages (b), and CD11b⁻CD45^hi lymphocytes (c) are shown. G, Relative MSX3 and FIZZ-1 mRNA expression in microglia from spinal cord of control mice (CFA without MOG) and EAE mice on day 11 (preclinic), day 16 (EAE peak), or day 40 (chronic EAE) after induction. *p < 0.05; **p < 0.01; ***p < 0.005 versus microglia from control mice. n = 8 mice per group. AU, Arbitrary units. The data are represented as the mean ± SEM.