Thymopoiesis in mice depends on a Foxn1-positive thymic epithelial cell lineage

Abstract

The thymus is essential for T-cell development. Here, we focus on the role of the transcription factor Foxn1 in the development and function of thymic epithelial cells (TECs) of the mouse. TECs are of endodermal origin; they initially express Foxn1 and give rise to orthotopic (thoracic) and additional (cervical) thymi. Using Foxn1-directed cytoablation, we show that during embryogenesis, cervical thymi develop a few days after the thoracic lobes, and that bipotent epithelial progenitors of cortical and medullary compartments express Foxn1. We also show that following acute selective near-total ablation during embryogenesis, complete regeneration of TECs does not occur, providing an animal model for human thymic aplasia syndromes. Finally, we address the functional role of Foxn1-negative TECs that arise postnatally in the mouse. Lineage tracing shows that such Foxn1-negative TECs are descendants of Foxn1-positive progenitors; furthermore, Foxn1-directed subacute intoxication of TECs by polyglutamine-containing EGFP proteins indicates that a presumptive Foxn1-independent lineage does not contribute to thymopoietic function of the adult thymus. Our findings therefore support the notion that Foxn1 is the essential transcription factor regulating the differentiation of TECs and that its expression marks the major functional lineage of TECs in embryonic and adult thymic tissue.

Fig. 1.

Varying times of onset of Foxn1 expression in the epithelial cells of thoracic and cervical thymi. (A) Hypothetical scheme of the development of organ anlagen from the epithelium of the third pharyngeal pouch in mouse embryos. A common anlage (yellow) differentiates into separate parathyroid (green) and thymic (red) domains, but a small undifferentiated primordium remains; at a later stage, this gives rise to the anlage of the cervical thymus (Upper). Foxn1-directed cytoablation during early stages of embryogenesis destroys the primordium of the thoracic thymus but does not affect the subsequent development of the cervical thymus (Lower). (B) RNA in situ hybridization analysis of adjacent transverse sections of an E15.5 mouse embryo. The cervical thymus with Foxn1-positive epithelium is located directly adjacent to the parathyroid (see enlargements underneath). Hybridization with Ttf1, which is expressed in the primordium of the thyroid and the epithelium of the larynx, is shown for orientation (it is expressed at low levels in parathyroid and cervical thymus). Note that at this stage of development, the thoracic thymus has already descended to the mediastinum. This section is representative of a group of four embryos. (Scale bars: Upper, 100 μm; Lower, 50 μm.) (C) Macroscopic view of the mediastinal region of Foxn1:eGFP single- and Foxn1:eGFP, Foxn1:DTR double-transgenic embryos treated with the indicated doses of diphtheria toxin (DT). Pregnant female mice were injected with DT three times at E10.5, E11.5, and E12.5; photographs were taken under UV illumination at E14.5. The pictures shown are representative of groups of two, three, and five embryos dissected for the three treatment/genotype combinations, respectively. (Scale bar: 100 μm.) (D) Macroscopic view of thoracic and neck regions of Foxn1:eGFP single- and Foxn1:eGFP, Foxn1:DTR double-transgenic embryos treated with DT as in C. Photographs were taken under UV (Left and Right) or bright-light (Center) illumination. Anatomical landmarks are indicated, and the two cervical thymi are marked with arrows. The embryos shown are representative of groups of >100 (Left) and 20 (Center and Right). (Scale bar: 500 μm.)

Fig. 2.

Thymic epithelial progenitors express Foxn1. (A) Thymic lobes from E15.5 Foxn1:CFP-NTR transgenic mice were transplanted under the kidney capsule of nude mice. After 1 wk, animals were treated with vehicle or vehicle plus CB1954 at 500 μg four times per week for 3 wk. After a recovery period, kidneys were removed and photographed under bright-light and UV illumination. (Scale bar: 2.5 mm.) (B) Number of CD4/CD8 double-positive T cells isolated from thymus transplants (such as those shown in A) 4 (Left) or 9–10 (Right) weeks after the last treatment. The number of thymic lobes (n) analyzed, the presence (+) or absence (−) of the Foxn1:CFP-NTR transgene (tg), and treatment with vehicle (−) or CB1954 (+) for the respective groups are noted. (C) Macroscopic views of thoracic regions of sibling wild-type or Foxn1:CFP-NTR transgenic mice at 2 mo of age photographed under bright-light (Upper) and UV illumination (Lower). During the embryonic period, pregnant wild-type mothers received i.p. injections of CB1954 at 500 μg per mouse at E12.5, E13.5, and E14.5. Thymic tissue is outlined with solid lines; the heart is indicated with broken lines. Note that no thymic tissue could be detected for the animal shown in the center panel. (Scale bar: 2.5 mm.) (D) Macroscopic appearance of a small thoracic thymus (about 1/8 the size of that in wild-type sibling) observed at the age of 2 wk in a Foxn1:CFP-NTR animal treated as in C. In the photograph taken under UV illumination, note several clusters of strongly fluorescent epithelial cells, corresponding to individual medullary islets. (Scale bar: 500 μm.) (E) Number of CD4/CD8 double-positive thymocytes recovered from adolescent and adult mice exposed in utero to CB1954 as in C. The number of animals in each group is shown underneath the graph. Animals from group 1 were exposed to CB1954 in utero at E12.5, E13.5, and E14.5; animals from group 2 at E13.5, E.14.5, and E15.5.

Fig. 3.

Foxn1-negative cells in the adult thymus are descendants of Foxn1-positive cells. Flow cytometric determination of the percentage of fluorescent TECs (Epcam⁺CD45⁻) for the indicated genotypes at two different time points. For representative flow cytometric profiles, see Fig. S1.

Fig. 4.

Expression of polyglutamine-containing proteins causes thymic malfunction. (A) Thymopoietic activity in age- and sex-matched mice transgenic for the indicated constructs, expressed relative to control. Values for CD4/CD8 double-positive thymocytes (Upper) and thymopoietic index [number of CD4/CD8 double-positive thymocytes divided by number of thymic epithelial cells (Lower)] are shown. The number of mice per data point (mean ± SD were applicable) is indicated and applies to both panels. (B) Abnormal architecture of thymic microenvironment in Foxn1:Q82-eGFP transgenic mice at 2 mo of age. Note the impaired separation of cortex (marked by K8 staining) and medulla (distinguished by strong GFP staining) (Upper). The altered shapes of medullary and cortical TECs are particularly evident in higher-power views of corticomedullary junctions (Lower). The sections are derived from the 2-mo-old animals also shown in C. The tissue architecture of Foxn1:Q19-eGFP transgenic mice is normal and comparable to Foxn1:eGFP mice. (Scale bars: Upper, 200 μm; Lower, 100 μm.) m, medulla; c, cortex. (C) Distribution of thymocyte subsets in sex-matched mice transgenic for the indicated constructs, analyzed at 2 and 5 mo of age. The numbers of total thymocytes are given above the panels; the relative proportions of thymocyte subsets are indicated in percentages (red numbers). For this experiment, flow cytometry profiles are representative of four animals in each group. Results from wild-type, Foxn1:eGFP, and Foxn1:Q19-eGFP transgenic mice are similar. (D) Macroscopic appearance of thymic tissue in aged mice. Note the size of the thymus and the widespread fluorescence of thymic epithelial cells in Foxn1:eGFP mice (50 wk of age) and the involuted thymus (outlined by dashed line) of a Foxn1:Q82-eGFP mouse that contains only few fluorescent cell clusters (42 wk of age). (Scale bar: 100 μm.)