Foxn1 Is Dynamically Regulated in Thymic Epithelial Cells during Embryogenesis and at the Onset of Thymic Involution

Abstract

Thymus function requires extensive cross-talk between developing T-cells and the thymic epithelium, which consists of cortical and medullary TEC. The transcription factor FOXN1 is the master regulator of TEC differentiation and function, and declining Foxn1 expression with age results in stereotypical thymic involution. Understanding of the dynamics of Foxn1 expression is, however, limited by a lack of single cell resolution data. We have generated a novel reporter of Foxn1 expression, Foxn1G, to monitor changes in Foxn1 expression during embryogenesis and involution. Our data reveal that early differentiation and maturation of cortical and medullary TEC coincides with precise sub-lineage-specific regulation of Foxn1 expression levels. We further show that initiation of thymic involution is associated with reduced cTEC functionality, and proportional expansion of FOXN1-negative TEC in both cortical and medullary sub-lineages. Cortex-specific down-regulation of Foxn1 between 1 and 3 months of age may therefore be a key driver of the early stages of age-related thymic involution.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Generation and validation of Foxn1G reporter mice.

(A) Schematic representation of the Foxn1G allele. A LoxP flanked cassette containing the 5’ engrailed 2 splice acceptor site (SA), eGFP, an internal ribosome entry site coupled to the puromycin resistance fusion protein (IRES-PuroR), and the CMAZ transcriptional pause (Stop) [19] was inserted into intron 1b of the Foxn1 locus of mouse ES cells. E, exon. (B) Flow cytometric analysis of E13.5 Foxn1G/+ thymic primordia. Plots show data after gating against Lineage+ cells (Lin) and on total EpCAM+ cells. Left plot shows analysis with UEA1 and anti-CD205. WT, wild type. Red line shows FMO. (C) Flow cytometric analysis of thymi from 1 month old Foxn1G/+ or WT mice. Plots display data from total EpCAM+Lin- or EpCAM-Lin+, as shown. Absolute number of EpCAM+ cells for 1 month old Foxn1G/+ mice, 6.06x104±2.10 x 104. (D) Flow cytometric analysis of 3 month old Foxn1G/+ thymi after staining with the markers shown. Plots show subpopulations of EpCAM+ cells, as shown. Absolute number of EpCAM+ cells for 3 month old Foxn1G/+ mice, 5.41x104±6.97x103. (E) Median fluorescence intensities (MFI) for data shown in (D). (F) RT-qPCR analysis showing relative Foxn1 mRNA expression level in the populations shown, after purification by flow cytometry. (B) n = 4, (C) n = 5, (D,E) n = 6, (F) n = 5 independent biological experiments.

Fig 2. Dynamic regulation of Foxn1 is evident in the early stages of TEC differentiation.

(A-D) Flow cytometric analysis of E13.5, E15.5 and E17.5 fetal Foxn1G/+ thymic primordia for the markers shown. Plots show data after gating against Lineage+ and on total EpCAM+ cells. WT, wild type. (A-C) n = 3, (D) n = 4 independent biological experiments.

Fig 3. Foxn1neg TEC subpopulations emerge postnatally in both cTEC and mTEC compartments.

(A-C) Flow cytometric analysis of thymi from 12 week-old adult Foxn1G/+ (A,B) or Foxn1Cre;mTmG (C) mice, for the markers shown. Data are shown after gating against Lineage+ and on total EpCAM+ cells (A-C) and further gating on GFPneg cells (B). Absolute number of GFPneg cells in 3 month old mice, 1.31x104±3.01x103. WT, wild type. Red line in (A) shows FMO. (A,B) n = 6, (C) n = 3 independent biological experiments.

Fig 4. Proportional expansion of Foxn1neg TEC occurs at the onset of age-related thymic involution.

(A) Thymus involution in Foxn1G/+ mice occurs with normal kinetics. (C, D, E) Flow cytometric analysis of Foxn1G/+ thymi at the ages shown; the proportion of GFP- TEC increases with age. Red (C, D) and grey (E) lines show FMO. (B) Absolute number of GFP+ and GFP- TEC isolated from Foxn1G/+ thymi at the timepoints shown. Absolute numbers are as follows: 1 month; GFP+ 4.94x104±1.81x104, GFP- 1.06x104±3.07x103. 3 months; GFP+ 4.03x104±8.86x103, GFP- 1.31x104±3.01 x 103. 12 months; GFP+ 1.55x104±2.36x103, GFP- 6.55x103±1.88x103. 24 months; GFP+ 4.52x103±2.75x103, GFP- 1.81x103±1.10x103. (A,B,C) n = 3, (D,E) n = 2 independent biological experiments.

Fig 5. Proportional expansion of Foxn1neg cTEC and mTEC is driven by different mechanisms.

(A,B) Plots show proportion of GFP-(A) and numbers of GFP+ and GFP- (B) mTEC and cTEC at the ages shown, as determined by flow cytometric analysis. (C) Flow cytometric analysis showing proportion of active caspase-3+ cells in the populations shown. (C’) shows data in (C) presented to indicate the relative levels of apoptosis in GFP+ and GFP- cTEC and mTEC. (D) Flow cytometric analysis of thymi from 6 week old mice showing proportion of Ki67+ cells in the populations shown. (E) GFP expression profile in cTEC and mTEC at the ages shown. (F) Quantification of data displayed in (E) showing median fluorescence intensity (MFI) of GFP+ cTEC and mTEC at 1 month and 3 months. (A) n = 5, (B) n = 7, (C) n = 3 (D) n = 4, (E, F) n = 5 independent biological experiments.

Fig 6. Changes in gene expression with age.

(A-H) Graphs show RT-qPCR analysis of the markers shown in WT mice at 1 month and 3 months. Error bars show SD. (A-G) n = 5, (H) n = 3 independent biological experiments.