Foxn1 Regulates Lineage Progression in Cortical and Medullary Thymic Epithelial Cells But Is Dispensable for Medullary Sublineage Divergence (original) (raw)
Figure 6
Foxn1 is required for exit from the earliest TEC progenitor states and regulates the size of the differentiated TEC compartment during ontogeny.
(A) Absolute numbers of Plet-1+ EpCam+MHC Class II- (MHCII)(open bars) and Plet-1-EpCam+ (black bars) cells per thymus at E13.5 and E15.5 for each genotype. Data shown are representative of at least three independent experiments. Error bars show SD. (B) Cell cycle analysis of Foxn1 allelic variants. Images show flow cytometric analysis of E15.5 WT, Foxn1R/+, Foxn1R/R, Foxn1R/- and Foxn1-/- thymi using anti-BrdU-APC and 7-AAD 1 hour after injection with BrdU. Plots are representative plots for each genotype after gating on EpCam+ cells; pairs of plots show data collected on the same day. (C,D,F) QRT-PCR analysis of E13.5 TEC (EpCam+ cells) or E13.5 thymic mesenchymal cells (PDGFRalpha+ cells) for the genes shown. Data are normalized to alpha-tubulin and shown relative to E13.5 WT EpCam+ cells (C,D) or E13.5 WT PDGFRalpha+ cells (F) and show the means from two (Pax1) or three (Dll4, CCL25, Fgf2) biological replicates for each gene. Data for all genes are representative of at least three biological replicates performed using either the Lightcycler or Fluidigm platforms. Error bars show SD. (C’,D’,E) QRT-PCR analysis of E12.5 Plet1+ TEC for the genes shown. Data are normalized to EVA [64] and shown relative to E12.5 WT Plet1+ cells. Data show mean of three technical replicates from one experiment on RNA from pooled thymi for each genotype. CCL25 and Dll4, data are representative of two independent analyses using different analysis platforms. (G) QRT-PCR analysis of mouse embryonic stem cells after transient transfection with either Foxn1 or GFP. Data show Foxn1, Dll4 and CCL25 expression levels relative to ß actin and show means of two (Foxn1) or three (Dll4, CCL25) biological repeats. Error bars show SD. Dll4, p = 0.009 (Foxn1 over-expression versus GFP over-expression); CCL25 p = 0.038 (Foxn1 over-expression versus GFP over-expression). (H) Cartoon shows regulatory network indicated by data shown.