Proportional expansion of <i>Foxn1</i>^<i>neg</i> cTEC and mTEC is driven by different mechanisms.

<p><b>(A,B)</b> Plots show proportion of GFP^- <b>(A)</b> and numbers of GFP⁺ and GFP^- (B) mTEC and cTEC at the ages shown, as determined by flow cytometric analysis. (<b>C</b>) Flow cytometric analysis showing proportion of active caspase-3⁺ cells in the populations shown. (<b>C’</b>) shows data in (<b>C</b>) presented to indicate the relative levels of apoptosis in GFP⁺ and GFP^- cTEC and mTEC. <b>(D)</b> Flow cytometric analysis of thymi from 6 week old mice showing proportion of Ki67⁺ cells in the populations shown. <b>(E)</b> 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. <b>(A)</b> n = 5, (B) n = 7, (<b>C</b>) n = 3 (<b>D</b>) n = 4, (<b>E, F</b>) n = 5 independent biological experiments.</p

Changes in gene expression with age.

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

Proportional expansion of <i>Foxn1</i>^<i>neg</i> TEC occurs at the onset of age-related thymic involution.

<p><b>(A)</b> Thymus involution in <i>Foxn1</i>^<i>G/+</i> mice occurs with normal kinetics. <b>(C, D, E)</b> Flow cytometric analysis of <i>Foxn1</i>^<i>G/+</i> thymi at the ages shown; the proportion of GFP- TEC increases with age. Red (C, D) and grey (E) lines show FMO. (<b>B</b>) Absolute number of GFP⁺ and GFP^- TEC isolated from <i>Foxn1</i>^<i>G/+</i> 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. <b>(A,B,C)</b> n = 3, (<b>D,E</b>) n = 2 independent biological experiments.</p

Dynamic regulation of <i>Foxn1</i> is evident in the early stages of TEC differentiation.

<p><b>(A-D)</b> Flow cytometric analysis of E13.5, E15.5 and E17.5 fetal <i>Foxn1</i>^<i>G/+</i> thymic primordia for the markers shown. Plots show data after gating against Lineage⁺ and on total EpCAM⁺ cells. WT, wild type. <b>(A-C)</b> n = 3, (<b>D</b>) n = 4 independent biological experiments.</p

Generation and validation of <i>Foxn1</i>^<i>G</i> reporter mice.

<p><b>(A)</b> Schematic representation of the <i>Foxn1</i>^<i>G</i> 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-Puro^R), and the CMAZ transcriptional pause (Stop) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151666#pone.0151666.ref019" target="_blank">19</a>] was inserted into intron 1b of the <i>Foxn1</i> locus of mouse ES cells. E, exon. <b>(B)</b> Flow cytometric analysis of E13.5 <i>Foxn1</i>^<i>G/+</i> 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. <b>(C)</b> Flow cytometric analysis of thymi from 1 month old <i>Foxn1</i>^<i>G/+</i> or WT mice. Plots display data from total EpCAM⁺Lin^- or EpCAM^-Lin⁺, as shown. Absolute number of EpCAM⁺ cells for 1 month old <i>Foxn1</i>^<i>G/+</i> mice, 6.06x10⁴±2.10 x 10⁴. (<b>D)</b> Flow cytometric analysis of 3 month old <i>Foxn1</i>^<i>G/+</i> thymi after staining with the markers shown. Plots show subpopulations of EpCAM⁺ cells, as shown. Absolute number of EpCAM⁺ cells for 3 month old <i>Foxn1</i>^<i>G/+</i> mice, 5.41x10⁴±6.97x10³. <b>(E)</b> Median fluorescence intensities (MFI) for data shown in <b>(D). (F)</b> RT-qPCR analysis showing relative <i>Foxn1</i> mRNA expression level in the populations shown, after purification by flow cytometry. <b>(B)</b> n = 4, (<b>C</b>) n = 5, (<b>D,E</b>) n = 6, (<b>F</b>) n = 5 independent biological experiments.</p

<i>Foxn1</i>^<i>neg</i> TEC subpopulations emerge postnatally in both cTEC and mTEC compartments.

<p><b>(A-C)</b> Flow cytometric analysis of thymi from 12 week-old adult <i>Foxn1</i>^<i>G/+</i> (<b>A,B)</b> or <i>Foxn1</i>^<i>Cre</i><i>;mTmG</i> (<b>C</b>) mice, for the markers shown. Data are shown after gating against Lineage⁺ and on total EpCAM⁺ cells <b>(A-C)</b> and further gating on GFP^neg cells <b>(B)</b>. Absolute number of GFP^neg cells in 3 month old mice, 1.31x10⁴±3.01x10³. WT, wild type. Red line in (<b>A</b>) shows FMO. <b>(A,B)</b> n = 6, (<b>C</b>) n = 3 independent biological experiments.</p