Foxn1 Regulates Lineage Progression in Cortical and Medullary Thymic Epithelial Cells But Is Dispensable for Medullary Sublineage Divergence

The forkhead transcription factor Foxn1 is indispensable for thymus development, but the mechanisms by which it mediates thymic epithelial cell (TEC) development are poorly understood. To examine the cellular and molecular basis of Foxn1 function, we generated a novel and revertible hypomorphic allele of Foxn1. By varying levels of its expression, we identified a number of features of the Foxn1 system. Here we show that Foxn1 is a powerful regulator of TEC differentiation that is required at multiple intermediate stages of TE lineage development in the fetal and adult thymus. We find no evidence for a role for Foxn1 in TEC fate-choice. Rather, we show it is required for stable entry into both the cortical and medullary TEC differentiation programmes and subsequently is needed at increasing dosage for progression through successive differentiation states in both cortical and medullary TEC. We further demonstrate regulation by Foxn1 of a suite of genes with diverse roles in thymus development and/or function, suggesting it acts as a master regulator of the core thymic epithelial programme rather than regulating a particular aspect of TEC biology. Overall, our data establish a genetics-based model of cellular hierarchies in the TE lineage and provide mechanistic insight relating titration of a single transcription factor to control of lineage progression. Our novel revertible hypomorph system may be similarly applied to analyzing other regulators of development

Long-Term Persistence of Functional Thymic Epithelial Progenitor Cells In Vivo under Conditions of Low FOXN1 Expression

Normal thymus function reflects interactions between developing T-cells and several thymic stroma cell types. Within the stroma, key functions reside in the distinct cortical and medullary thymic epithelial cell (TEC) types. It has been demonstrated that, during organogenesis, all TECs can be derived from a common thymic epithelial progenitor cell (TEPC). The properties of this common progenitor are thus of interest. Differentiation of both cTEC and mTEC depends on the epithelial-specific transcription factor FOXN1, although formation of the common TEPC from which the TEC lineage originates does not require FOXN1. Here, we have used a revertible severely hypomorphic allele of Foxn1, Foxn1R, to test the stability of the common TEPC in vivo. By reactivating Foxn1 expression postnatally in Foxn1R/- mice we demonstrate that functional TEPCs can persist in the thymic rudiment until at least 6 months of age, and retain the potential to give rise to both cortical and medullary thymic epithelial cells (cTECs and mTECs). These data demonstrate that the TEPC-state is remarkably stable in vivo under conditions of low Foxn1 expression, suggesting that manipulation of FOXN1 activity may prove a valuable method for long term maintenance of TEPC in vitro

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

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

Thymus formation upon Cre-mediated reversion of the <i>Foxn1^R</i> allele in <i>R/−; CreERt2</i> mice.

<p>3–4 months old <i>R/−; CreERt2</i> mice were injected with 4OHT at the doses shown. Images show immunohistochemical analysis of thymi or thymic rudiments from 4OHT mice 7 weeks post-injection or from a 6 week old C57BL/6 wild type (WT) control. Staining for (<b>A</b>) pan-cytokeratin (PANK; green) and PLET1 (red). Scale bars, 150 µm. (<b>B</b>) K5 (green) and K8 (red). Scale bars, 300 µm. (<b>C</b>) PANK (green) and UEA-1 (red). Scale bars, 150 µm. (<b>D</b>) PANK (green) and MHC Class II (MHCII, red). Scale bars, 150 µm. Arrowheads in (<b>A</b>) and (<b>B</b>) indicate areas of undifferentiated thymic rudiment. DAPI reveals nuclei (blue) in panels (<b>A–D</b>). (<b>E</b>) PANK (green) and CD45 (red). Scale bars 100 µm. Note that CD45⁺ cells are found associated with but not within the epithelium in thymic rudiments from carrier only and 0.25 µg 4OHT injected mice. 0.25 mg and 0.5 mg 4OHT injected mice and WT controls, n>3. 1.5 mg and 2 mg 4OHT injected mice, n = 1 for each condition; equivalent data were obtained from mice injected with 1.0 mg 4OHT (n = 3).</p

Spontaneous reversion of the R allele in aged <i>R/-; CreERt2</i> mice.

<p>Images show representative immunohistochemical analysis of carrier-only injected 6 month-old <i>R/−; CreERt2</i> mice analyzed one month after injection. (<b>A</b>) Carrier-only injected mice exhibited a thymus structure containing both cortical and medullary regions (cytokeratin 14 (K14) and CDR1 indicate medullary and cortical TEC respectively, while pan cytokeratin identifies all TEC). Scale bar: 150 um. <b>B</b>, the thymic region of a carrier-only injected mouse, showing a series of small thymic lobes. Scale bar: 300 µm. (<b>C,D</b>) Plots show analysis of thymocytes for CD4 and CD8 expression after gating on live CD45⁺ cells for (<b>C</b>) two randomly selected, untreated 11 month old <i>R/−; CreERt2</i> mice and (<b>D</b>) two untreated <i>R/−</i> mice (7 m and 11 m old, respectively). Each of the untreated <i>R/−; CreERt2</i> mice contained DP cells within the CD45⁺ population (33.9% and 85.6%, respectively) while the <i>R/−</i> mice contained no DP cells (mean±SD: Untreated <i>R/−; CreERt2</i> mice, 6 months old, 48.20%±40.88%, n = 5; 11 m old, 61.78%±39.34%, n = 2. <i>R/−</i> controls 1.09%±1.55%, n = 2). Of note is that the CD4⁺ and CD8⁺ single positive populations present in the aged <i>R/−</i> mice are also commonly observed in aged <i>nu/nu</i> mice, and are thought to arise by homeostatic expansion of extrathymically-generated T cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114842#pone.0114842-GuyGrand1" target="_blank">[30]</a>. (<b>E</b>) Representative images of thymic rudiments from 5 month-old <i>R/−</i> mice after immunohistochemical analysis with the markers shown.</p

Quantification of DP cell number over time.

<p>Thymi from wild type, or uninjected and carrier-injected <i>R/−; CreERt2</i> mice of the ages shown were dissected, and thymocyte subset profile was determined by flow cytometric analysis after staining for CD4 and CD8. The number of CD4⁺CD8⁺ DP thymocytes present in each thymus was determined. Graphs show data from (<b>A</b>) male wild type C57BL/6 mice; p<0.05 for 6 weeks versus 43 weeks old, (<b>B</b>) male uninjected and carrier-injected <i>R/−; CreERt2</i> mice and (<b>C</b>) female uninjected and carrier-injected <i>R/−; CreERt2</i> mice. Each dot represents an individual mouse and the lines show the means. The dotted lines in (<b>B</b>) and (<b>C</b>) show the number of cells present in the DP gate of <i>R/−</i> controls in which Cre recombinase could not be expressed. Closed triangle on all graphs, <i>R/−</i> control. Data from uninjected and carrier-injected <i>R/−; CreERt2</i> mice with equivalent or fewer cells in the DP gate to <i>R/−</i> controls (i.e. on or below the dotted lines) were not included in the analysis presented in Fig. 5, as these mice were considered not to have undergone tamoxifen-independent Cre-mediated recombination (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114842#pone-0114842-t001" target="_blank">Table 1</a>).</p

Thymi generated on reversion of the Foxn1 R allele in <i>R/−; CreERt2</i> mice support normal T cell development.

<p>Thymi from 6 week old C57BL/6 wild type, and <i>R/− reERt2</i> mice injected with 4OHT at the doses shown were dissected. Thymocytes were collected and processed for flow cytometric analysis. Plots show staining with the markers shown after gating out dead cells and on CD45⁺ cells. (<b>A</b>) Thymi from mice injected with carrier-only contained no CD4⁺ CD8⁺ DP cells (percentage of DP cells: Carrier-only injected, 0.0125±0.015 n = 4; Wild-type, 85.96±1.74 n = 7), while thymi from 1.5 mg 4OHT injected mice contained DP and SP cells. (<b>B</b>) Plots show staining with CD44 and CD25 after gating against a lineage cocktail (lin). (lin  = CD3, CD4, CD8, NK1.1, Ter119, CD19, Mac1). Thymic rudiments from carrier-injected mice contained no DN2, DN3 or DN4 thymocytes. The CD45⁺ cells in the DN1 gate most likely represent circulating CD45⁺ cells present in the tissue dissected along with the thymic rudiment, as no CD45⁺ cells were observed within the epithelial component of the rudiment itself; it is possible that these cells might be hematopoietic progenitors attracted by the undifferentiated TEC but not licenced to colonise the epithelial rudiment itself. Thymi from 1.5 mg 4OHT injected <i>R/−; CreERt2</i> mice contained all normal DN populations. n>3 for WT and carrier-injected; n = 1 for 1.5 mg (equivalent data were obtained from mice injected with 0.5 mg [n = 3] and 1.0 mg 4OHT [n = 3]).</p

AIRE expression in the unreverted <i>R/−; CreERt2</i> thymic rudiment.

<p>3–4 months old <i>R/−; CreERt2</i> mice were injected with 4OHT at the doses shown (<b>A</b>, carrier-only, <b>B</b>, 0.25 mg 4OHT, <b>C</b>, 2 mg 4OHT). Images show immunohistochemical analysis of thymi or thymic rudiments from 4OHT mice 7 weeks post-injection. Staining is shown for AIRE, counterstained for cytokeratin 8 (K8) or CD205 as shown. DAPI reveals nuclei (blue). (<b>A</b>) Top and bottom panels show representative images of AIRE⁻ regions (which comprised the majority of sections), and a rare AIRE⁺ cell, respectively. (<b>B</b>) Top and middle panels show representative images of AIRE⁻ regions and AIRE⁺ cells, respectively. Bottom panel shows higher power image of middle panel. Carrier-only and 0.25 mg 4OHT injected mice, n = 2; reverted mice (i.e. injected with ≥0.5 mg 4OHT), n>3 (shown for 2 mg injected). Scale bars 100 µm except where shown.</p

Evidence for continued tamoxifen-independent recombination with age in <i>R/−; CreERt2</i> mice.

<p>The table shows the total number of uninjected and carrier-only injected <i>R/−; CreERt2</i> mice analyzed within each age group, for male and female mice. Uninjected and carrier-injected <i>R/−; CreERt2</i> with equivalent or fewer cells in the DP gate to <i>R/−</i> controls were considered not to have undergone tamoxifen-independent Cre-mediated recombination. When the results for males and females are combined, the proportion of mice showing evidence of tamoxifen-independent Cre-mediated recombination varies significantly with age and there is a trend for this to increase with age. *p-value was calculated to compare the recombination frequencies among the four ages by 2×4 Fisher's Exact test.</p><p>Evidence for continued tamoxifen-independent recombination with age in <i>R/−; CreERt2</i> mice.</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