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

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

Initial seeding of the embryonic thymus by immune-restricted lympho-myeloid progenitors

The final stages of restriction to the T cell lineage occur in the thymus after the entry of thymus-seeding progenitors (TSPs). The identity and lineage potential of TSPs remains unclear. Because the first embryonic TSPs enter a non-vascularized thymic rudiment, we were able to directly image and establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before their entry into the thymus and activation of Notch signaling. T-IPs did not include multipotent stem cells or molecular evidence of T cell-restricted progenitors. Instead, single-cell molecular and functional analysis demonstrated that most fetal T-IPs expressed genes of and had the potential to develop into lymphoid as well as myeloid components of the immune system. Moreover, studies of embryos deficient in the transcriptional regulator RBPJ demonstrated that canonical Notch signaling was not involved in pre-thymic restriction to the T cell lineage or the migration of T-IPs

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

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

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

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

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

Initial seeding of the embryonic thymus by immune-restricted lympho-myeloid progenitors

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