Foxn1 Regulates Lineage Progression in Cortical and Medullary Thymic Epithelial Cells But Is Dispensable for Medullary Sublineage Divergence
Figure 3
Reduced Foxn1 expression results in impaired development of both medullary and cortical TEC in postnatal Foxn1R/R thymi.
(A-G, Ai-Hi) Immunohistochemical (A-E,G,H) and/or flow cytometric (Ai-Hi, F) analysis of postnatal Foxn1R/R thymi showing anti-CD205, Ly51, CDR1, anti-Aire, anti-K14, anti-MHC Class II (MHCII), anti-Dll4, anti-ß5t, anti-PanK and anti-CD40 staining. (A–E,G,H) Right panels show co-stains of left panels, right Aire panel shows detail from left panel. Scale bars 150 µm except for anti-CD205 and Ly51, which are 75 µm and Aire right panel which is 11.9 µm. (Ai-Hi, F) Plots show data after gating against CD45+ and Ter119+ and for EpCam+ cells. Lower panel in Ei shows data after gating on CD45-EpCamlo cells (i.e. cTEC). Red lines, Foxn1R/R; blue lines, WT (Foxn1R/+ rather than WT is shown for Ly51, and is indistinguishable from WT); grey lines, isotype control. All analyses are on 6 week old thymi except E and Ei which are on postnatal day 10-11 thymi. Data shown are representative of at least three independent experiments, except (F), n = 1. (I) 4 week old Foxn1R/R;ROSA26CreErt2 mice were injected intraperitoneally with 1.5 mg 4-hydroxy tamoxifen (4OHT) and sacrificed 2 days later; bottom panels show un-injected littermate controls. Images show immunostaining with CDR1, anti-pancytokeratin, Ly51, anti-MHC Class II and anti-Aire. Data are representative of at least 3 mice for each condition. (J) 6 week old Foxn1R/R;ROSA26CreErt2 (R/R) thymi were injected with 1.5mg 4OHT or carrier only and sacrificed 2 days later. Controls were provided by uninjected Foxn1R/+;ROSA26CreErt2 (R/+) littermates. Frozen sections were stained with anti-Aire and anti-PanK, as in (I), and the numbers of Aire+ TEC per mm2 of medullary area were determined by counting for three medullary areas from two sections from each of three mice for each condition. Untreated R/R versus R/+, p = 0.022; 4OHT-treated R/R versus R/+, p = 0.28; 4OHT-treated R/R versus untreated R/R, p = 0.025. (K) Mature and immature postnatal cTEC (EpCAM+Ly51+MHC Class IIhi and EpCAM+Ly51+MHC Class IIlo cells respectively) and mTEC (EpCAM+Ly51-MHC Class IIhi and EpCAM+Ly51-MHC Class IIlo cells respectively) were purified from 4-8 week old WT C57BL/6 mice by flow cytometry and the relative Foxn1 mRNA expression levels in each cell population determined by QRT-PCR. Foxn1 is expressed at higher levels in WT cTEC than WT mTEC, and at higher levels in mature than in immature TEC in both compartments. Foxn1 expression levels are shown relative to alpha-tubulin and are normalized to MHC Class IIhi cTEC. Data shown are the mean of two technical replicates and are representative of three independent experiments. Error bars show SD.