Thymic epithelial cell differentiation, growth and function depend on the expression of the transcription factor Foxn1, however its target genes have never been physically identified. Using novel static and inducible genetic model systems and chromatin studies, we provide now a genome wide map of direct Foxn1 target genes for postnatal thymic epithelia and define the Foxn1 binding motif. We detail the function of Foxn1 in these cells and demonstrate that in addition to the transcriptional control of genes involved in the attraction and lineage commitment of T cell precursors, Foxn1 regulates the expression of genes involved in antigen processing and thymocyte selection. Thus, critical events in thymic lympho-stromal cross-talk and T cell selection are indispensably choreographed by Foxn1.

The thymus provides multiple microenvironments that are essential for the development and repertoire selection of T lymphocytes. The thymic cortex induces the generation and positive selection of T lymphocytes, whereas the thymic medulla establishes self-tolerance among the positively selected T lymphocytes. Cortical thymic epithelial cells (cTECs) and medullary TECs (mTECs) constitute the major stromal cells that structurally form and functionally characterize the cortex and the medulla, respectively. cTECs and mTECs are both derived from the endodermal epithelium of the third pharyngeal pouch. However, the molecular and cellular characteristics of the progenitor cells for the distinct TEC lineages are unclear. Here we report the preparation and characterization of mice that express the recombinase Cre instead of β5t, a proteasome subunit that is abundant in cTECs and not detected in other cell types, including mTECs. By crossing β5t-Cre knock-in mice with loxP-dependent GFP reporter mice, we found that β5t-Cre-mediated recombination occurs specifically in TECs but not in any other cell types in the mouse. Surprisingly, in addition to cTECs, β5t-Cre-loxP-mediated GFP expression was detected in almost all mTECs. These results indicate that the majority of mTECs, including autoimmune regulator-expressing mTECs, are derived from β5t-expressing progenitor cells.T hymic epithelial cells (TECs) are derived from the endodermal epithelium of the third pharyngeal pouch (1-3). Cortical TECs (cTECs) provide a microenvironment that induces the generation of T cells and the positive selection of functionally competent T cells, whereas medullary TECs (mTECs) essentially contribute to the establishment of self-tolerance by the deletion of self-reactive T cells and the generation of regulatory T cells (4, 5). The nuclear protein Autoimmune regulator (Aire) expressed by a subpopulation of mTECs is essential, especially during the perinatal period, for the establishment of selftolerance in T cells (6, 7). Although the importance of the forkhead transcription factor Foxn1 for the development of both cTECs and mTECs has been established (2, 8), it remains unknown how the endodermal epithelium of the third pharyngeal pouch gives rise to cTECs and mTECs. In particular, the molecular and cellular mechanisms underlying the separate development of the cTEC and mTEC lineages remain unclear.We previously reported β5t, a proteasome subunit expressed in cTECs (9, 10). β5t is pivotal for the positive selection of immunocompetent CD8 + T cells (11,12). β5t mRNA and protein are prominently expressed in cTECs and not detected in other cell types, including mTECs (10). To examine whether β5t-expressing cells could contribute to the development of cells other than cTECs, we engineered mice in which the β5t-encoding genomic sequence was replaced with the sequence that encodes the loxP-specific recombinase Cre. Analyzing mice that are crossed to carry the β5t-Cre knock-in allele and the loxPdependent GFP reporter allele, we demonstrate that β5t-Cr...

Medullary thymic epithelial cells (mTECs) play an essential role in establishing self-tolerance in T cells. mTECs originate from bipotent TEC progenitors that generate both mTECs and cortical TECs (cTECs), although mTEC-restricted progenitors also have been reported. Here, we report in vivo fate-mapping analysis of cells that transcribe β5t, a cTEC trait expressed in bipotent progenitors, during a given period in mice. We show that, in adult mice, most mTECs are derived from progenitors that transcribe β5t during embryogenesis and the neonatal period up to 1 week of age. The contribution of adult β5t(+) progenitors was minor even during injury-triggered regeneration. Our results further demonstrate that adult mTEC-restricted progenitors are derived from perinatal β5t(+) progenitors. These results indicate that the adult thymic medullary epithelium is maintained and regenerated by mTEC-lineage cells that pass beyond the bipotent stage during early ontogeny.