FoxN1 mediates thymic cortex-medulla differentiation through modifying a developmental pattern based on epithelial tubulogenesis.

The mechanisms that determine the commitment of thymic epithelial precursors to the two major thymic epithelial cell lineages, cTECs and mTECs, remain unknown. Here we show that FoxN1 nu mutation, which abolishes thymic epithelium differentiation, results in the formation of a tubular branched structure according to a typical branching morphogenesis and tubulogenesis developmental pattern. In the presence of FoxN1, in alymphoid NSG and fetal Ikaros-/- thymi, there is no lumen formation and only partial apical differentiation. This initiates cortex-medulla differentiation inducing expression of medullary genes in the apically differentiating cells and of cortical genes in the non-apically differentiating cells, which will definitely differentiate in wt and postnatal Ikaros-/- mice. Therefore, the thymus development is based on a branching morphogenesis and tubulogenesis developmental pattern: FoxN1 expression in the thymic primordium inhibits tubulogenesis and induces the expression of genes involved in TEC differentiation, which culminates with the expression of functional cell markers, i.e., MHCII, CD80, Aire in both postnatal Ikaros-/- and WT thymi after arrival of lymphoid progenitor cells.

Eph/Ephrins-Mediated Thymocyte-Thymic Epithelial Cell Interactions Control Numerous Processes of Thymus Biology.

Numerous studies emphasize the relevance of thymocyte-thymic epithelial cell (TECs) interactions for the functional maturation of intrathymic T lymphocytes. The tyrosine kinase receptors, Ephs (erythropoietin-producing hepatocyte kinases) and their ligands, ephrins (Eph receptor interaction proteins), are molecules known to be involved in the regulation of numerous biological systems in which cell-to-cell interactions are particularly relevant. In the last years, we and other authors have demonstrated the importance of these molecules in the thymic functions and the T-cell development. In the present report, we review data on the effects of Ephs and ephrins in the functional maturation of both thymic epithelial microenvironment and thymocyte maturation as well as on their role in the lymphoid progenitor recruitment into the thymus.

Conditioned deletion of ephrinB1 and/or ephrinB2 in either thymocytes or thymic epithelial cells alters the organization of thymic medulla and favors the appearance of thymic epithelial cysts.

Our understanding about medullary compartment, its niches composition and formation is still limited. Previous studies using EphB2 and/or EphB3 knockout mice showed an abnormal thymic development that affects mainly to the epithelial component, including the cortex/medulla distribution, thymic epithelial cell (TEC) morphology and different epithelial-specific marker expression. We have already demonstrated that the lack of ephrinB1 and/or ephrinB2, either on thymocytes or on TECs, alters the cell intermingling processes necessary for thymus organization and affect cortical TEC subpopulations. In the present work, we have used the Cre-LoxP model to selectively delete ephrinB1 and/or ephrinB2 in thymocytes (EfnB1(thy/thy), EfnB2(thy/thy), EfnB1(thy/thy)EfnB2(thy/thy) mice) or TECs (EfnB1(tec/tec), EfnB2(tec/tec), EfnB1(tec/tec)EfnB2(tec/tec) mice) and have analyzed their role on the medullary compartment. In all the studied mutants, medullary areas are smaller and more compact than in the wt thymuses. In most of them, we observe abundant big cysts and a higher proportion of UEA(hi)MTS10(-) cells than in wt mice, which are often forming small cysts. On EfnB1(tec/tec)EfnB2(tec/tec), changes affecting organ size and medullary compartment start at perinatal stage. Our data shed some light on knowledge about wt medulla histological structure and cysts meaning and formation process and on the role played by ephrinB in them.

3D immunofluorescence analysis of early thymic morphogenesis and medulla development.

The thymus represents an epithelial microenvironment specialized in the generation of T-cells. The mechanisms or signals that determine the initial differentiation of the two well distinguished histological compartments of the thymus, cortex and medulla, remain unknown. Here, we report a three-dimensional analysis of the distribution of some established thymic epithelial markers in relation to thymic anatomical development during the first steps of thymus organogenesis. In the thymic primordium, initial lumen is lined by claudin (Cld)3/4+K5+ cells, after thymus growth and lobulation they form a continuous branched structure that increases its length and branching degree. Within it, the presence of luminal structures can be distinguished, even at E13.5. The medullary marker mouse thymic stroma 10 (MTS10) is upregulated in these Cld3/4+ lumen forming cells in a proximal-distal sequence. This structural organisation is histologically similar to that described in other epithelial organs undergoing a branching morphogenesis process. These results indicate that the thymic medulla can be evidenced as a continuous branched structure from early stages and suggest a thymic developmental program based on or containing elements of a branching morphogenesis program modified by the presence of lymphoid cells, in which medullary epithelial cell commitment is initially determined by lumen formation.

Ephrin-B-dependent thymic epithelial cell-thymocyte interactions are necessary for correct T cell differentiation and thymus histology organization: relevance for thymic cortex development.

Previous analysis on the thymus of erythropoietin-producing hepatocyte kinases (Eph) B knockout mice and chimeras revealed that Eph-Eph receptor-interacting proteins (ephrins) are expressed both on T cells and thymic epithelial cells (TECs) and play a role in defining the thymus microenvironments. In the current study, we have used the Cre-LoxP system to selectively delete ephrin-B1 and/or ephrin-B2 in either thymocytes (EfnB1(thy/thy), EfnB2(thy/thy), and EfnB1(thy/thy)EfnB2(thy/thy) mice) or TECs (EfnB1(tec/tec), EfnB2(tec/tec), and EfnB1(tec/tec)EfnB2(tec/tec) mice) and determine the relevance of these Eph ligands in T cell differentiation and thymus histology. Our results indicate that ephrin-B1 and ephrin-B2 expressed on thymocytes play an autonomous role in T cell development and, expressed on TECs, their nonautonomous roles are partially overlapping. The effects of the lack of ephrin-B1 and/or ephrin-B2 on either thymocytes or TECs are more severe and specific on thymic epithelium, contribute to the cell intermingling necessary for thymus organization, and affect cortical TEC subpopulation phenotype and location. Moreover, ephrin-B1 and ephrin-B2 seem to be involved in the temporal appearance of distinct cortical TECs subsets defined by different Ly51 levels of expression on the ontogeny.