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. 2019 Mar 7;216(4):900–915. doi: 10.1084/jem.20181290

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© 2019 Arnold et al.

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Figure 6. — Microglial dysmaturation due to loss of TGFβ signaling is critically time dependent. (A) Diagram of Tgfbr2 gene inactivation and mutant analysis. Tamoxifen was injected at P1 for early induction in B or at P30 for adult induction in C. Mice were sacrificed and analyzed at indicated time points after tamoxifen injection. (B and C) Staining of cortical brain sections from Tgfbr2iΔMG and Cre⁺ control littermates 120 d after tamoxifen administration following early (B) or adult (C) induction. Left panels: tdTomato recombination reporter (tdTom, red) marks all microglia and immature (APOE, green) and mature (P2RY12, blue) microglia markers to identify type A (dysmature) or type B microglia, respectively. Right panels (insets from Fig. S5): pSMAD3 (green) coimmunostaining reveals reduction in pSMAD3 staining intensity in type A (dysmature) microglia compared with type B microglia and controls. Percent (%) recombination (upper right graphs in B and C) based on % F4/80⁺, CD45⁺, CD11b⁺ cells that are also tdTomato positive (cells isolated and analyzed by flow cytometry as in Fig. S3). Mean pSMAD3 per nucleus (lower right graphs in B and C) based on fluorescent intensity of individual recombined (tdTom⁺) microglia coexpressing P2RY12 (blue, control and type B cells), or lacking P2RY12 expression (type A cells). Bars, 100 µm or 25 µm (pSMAD3, right panels). Error bars indicate SE. *P < 0.05; ***P < 0.005; ****P < 0.0005. ANOVA with Tukey’s post hoc test (B); Student’s t test (C); n = 4 animals for each group.