Development Of Thymic Epithelial Cells Research Articles

MiR-205 Supports Thymopoiesis Following Stress by Positively Regulating Foxn1 Expression

Abstract T cells are a critical component of the adaptive immune system, developing within the thymus. Immature thymocytes interact with an interconnected meshwork of thymic epithelial cells (TECs) to establish the T cell repertoire. The developmental process is extremely stress sensitive, as the thymus can undergo a rapid involution in response to diverse inflammatory conditions, and a more prolonged hypoplasia during aging. The type of stress predicates whether TECs, thymocytes, and/or myeloid cell populations are affected. Several microRNAs (miRs) have been identified in the thymus based on their ability to mitigate the stress damage. We identified miR-205 as a stress responsive miR specifically expressed in TECs. Mice generated with a conditional ablation of miR-205 in TECs exhibit an age and sex-dependent thymic hypoplasia beginning at 8 weeks. Under stress conditions involving a type I interferon response (dsRNA mimic; polyI:C), the TEC-miR-205 deficient mice had a severe thymic atrophy compared to littermate controls. The TEC-miR-205 deficient mice displayed a delayed recovery of single positive CD4 and CD8 thymocytes, likely resulting from a block in the cortical TEC expansion. qPCR and gene expression comparisons revealed that the miR-205 deficient TECs had significant changes in chemokine/chemokine receptor and antigen processing pathways. MiR-205 positively regulated Foxn1 transcription factor expression, the master regulator of TEC development and function. Interestingly, miR-205 is encoded within a long non-coding RNA (lncRNA), 4631405K08Rik. Current experiments will reveal the mechanisms by which the miR and lncRNA are transcriptionally regulated, and how these affect Foxn1 expression to support thymopoiesis.

Development and developmental potential of cortical thymic epithelial cells.

The thymic cortex provides a microenvironment for the development and positive selection of immature T cells. Cortical thymic epithelial cells (cTECs), which structurally and functionally support the thymic cortical microenvironment, originate from endodermal epithelial progenitors that arise in the third pharyngeal pouch. Recent studies have revealed that thymic epithelial progenitors pass through a stage where the cells express cTEC-associated molecules prior to lineage separation into cTECs and medullary TECs (mTECs). Here, we review the molecular signatures of cTECs and highlight the development and developmental potential of cTECs.

Eph/ephrin-B-mediated cell-to-cell interactions govern MTS20(+) thymic epithelial cell development.

Thymus development is a complex process in which cell-to-cell interactions between thymocytes and thymic epithelial cells (TECs) are essential to allow a proper maturation of both thymic cell components. Although signals that control thymocyte development are well known, mechanisms governing TEC maturation are poorly understood, especially those that regulate the maturation of immature TEC populations during early fetal thymus development. In this study, we show that EphB2-deficient, EphB2LacZ and EphB3-deficient fetal thymuses present a lower number of cells and delayed maturation of DN cell subsets compared to WT values. Moreover, deficits in the production of chemokines, known to be involved in the lymphoid seeding into the thymus, contribute in decreased proportions of intrathymic T cell progenitors (PIRA/B(+)) in the mutant thymuses from early stages of development. These features correlate with increased proportions of MTS20(+) cells but fewer MTS20(-) cells from E13.5 onward in the deficient thymuses, suggesting a delayed development of the first epithelial cells. In addition, in vitro the lack of thymocytes or the blockade of Eph/ephrin-B-mediated cell-to-cell interactions between either thymocytes-TECs or TECs-TECs in E13.5 fetal thymic lobes coursed with increased proportions of MTS20(+) TECs. This confirms, for the first time, that the presence of CD45(+) cells, corresponding at these stages to DN1 and DN2 cells, and Eph/ephrin-B-mediated heterotypic or homotypic cell interactions between thymocytes and TECs, or between TECs and themselves, contribute to the early maturation of MTS20(+) TECs.

MTORC1 in Thymic Epithelial Cells Is Critical for Thymopoiesis, T-Cell Generation, and Temporal Control of γδT17 Development and TCRγ/δ Recombination.

Thymus is crucial for generation of a diverse repertoire of T cells essential for adaptive immunity. Although thymic epithelial cells (TECs) are crucial for thymopoiesis and T cell generation, how TEC development and function are controlled is poorly understood. We report here that mTOR complex 1 (mTORC1) in TECs plays critical roles in thymopoiesis and thymus function. Acute deletion of mTORC1 in adult mice caused severe thymic involution. TEC-specific deficiency of mTORC1 (mTORC1KO) impaired TEC maturation and function such as decreased expression of thymotropic chemokines, decreased medullary TEC to cortical TEC ratios, and altered thymic architecture, leading to severe thymic atrophy, reduced recruitment of early thymic progenitors, and impaired development of virtually all T-cell lineages. Strikingly, temporal control of IL-17-producing γδT (γδT17) cell differentiation and TCRVγ/δ recombination in fetal thymus is lost in mTORC1KO thymus, leading to elevated γδT17 differentiation and rearranging of fetal specific TCRVγ/δ in adulthood. Thus, mTORC1 is central for TEC development/function and establishment of thymic environment for proper T cell development, and modulating mTORC1 activity can be a strategy for preventing thymic involution/atrophy.

CCR7 Controls Thymus Recirculation, but Not Production and Emigration, of Foxp3(+) T Cells.

SummaryCurrent models of Foxp3+ regulatory T cell (Treg) development involve CCR7-mediated migration of thymocytes into the thymus medulla to enable essential interactions with medullary epithelium. However, increased Foxp3+ thymic Treg numbers in Ccr7−/− mice challenge this view, and the role of CCR7 in Treg development, emigration, and/or recirculation is unknown. Here, we have examined CCR7 and Rag2pGFP levels during Treg development and generated Rag2pGFPCcr7−/− mice to study its impact on the intrathymic Treg pool. We reveal surprising developmental heterogeneity in thymocytes described as Treg precursors, showing that they contain recirculating CCR6+CCR7−Rag2pGFP− T cells. Although CCR7 defines bona fide Rag2GFP+ Treg precursors, it is not required for Treg production and emigration. Rather, we show that lack of CCR7 renders the thymus more receptive to Treg thymus homing. Our study reveals a role for CCR7 in limiting Treg recirculation back to the thymus and enables separation of the mechanisms controlling Treg production and thymic recirculation.

Requirement of Stat3 Signaling in the Postnatal Development of Thymic Medullary Epithelial Cells.

Thymic medullary regions are formed in neonatal mice as islet-like structures, which increase in size over time and eventually fuse a few weeks after birth into a continuous structure. The development of medullary thymic epithelial cells (TEC) is dependent on NF-κB associated signaling though other signaling pathways may contribute. Here, we demonstrate that Stat3-mediated signals determine medullary TEC cellularity, architectural organization and hence the size of the medulla. Deleting Stat3 expression selectively in thymic epithelia precludes the postnatal enlargement of the medulla retaining a neonatal architecture of small separate medullary islets. In contrast, loss of Stat3 expression in cortical TEC neither affects the cellularity or organization of the epithelia. Activation of Stat3 is mainly positioned downstream of EGF-R as its ablation in TEC phenocopies the loss of Stat3 expression in these cells. These results indicate that Stat3 meditated signal via EGF-R is required for the postnatal development of thymic medullary regions.

Sonic Hedgehog regulates thymic epithelial cell differentiation

Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus.

Alterations of Thymic Epithelial Cells in Lipopolysaccharide-induced Neonatal Thymus Involution.

Background:Vascular endothelial growth factor (VEGF) in the thymus was mainly produced by the thymic epithelial cells (TECs), the predominant component of the thymic microenvironment. The progression of TECs and the roles of VEGF in the neonatal thymus during sepsis have not been reported. This study aimed to explore the alterations of TECs and VEGF level in the neonatal thymus involution and to explore the possible mechanisms at the cellular level.Methods:By establishing a model of clinical sepsis, the changes of TECs were measured by hematoxylin-eosin staining, confocal microscopy, and flow cytometry. Moreover, the levels of VEGF in serum and thymus were assessed based on enzyme-linked immunosorbent assay and Western blotting.Results:The number of thymocytes and TECs was significantly decreased 24 h after lipopolysaccharide (LPS) challenge, (2.40 ± 0.46)×107 vs. (3.93 ± 0.66)×107 and (1.16 ± 0.14)×105 vs. (2.20 ± 0.19)×105, P < 0.05, respectively. Cortical TECs and medullary TECs in the LPS-treated mice were decreased 1.5-fold and 3.9-fold, P < 0.05, respectively, lower than those in the controls. The number of thymic epithelial progenitors was also decreased. VEGF expression in TECs was down-regulated in a time-dependent manner.Conclusion:VEGF in thymic cells subsets might contribute to the development of TECs in neonatal sepsis.

Foxn1 Protein Expression in the Developing, Aging, and Regenerating Thymus.

The forkhead box N1 (Foxn1) protein is the key regulator of thymic epithelial cell (TEC) development, yet how Foxn1 functions remains largely unknown. All mature TECs arise from Foxn1-expressing progenitors/immature TECs and it is widely assumed that TECs as a whole are defined by Foxn1 expression. However, data on the Foxn1 protein are virtually lacking. In this study, we developed novel tools to visualize Foxn1 protein expression at single-cell resolution. We generated Foxn1 knock-in mice expressing a C-terminal hemagglutinin-tagged Foxn1 protein, and a cytometry-grade monoclonal anti-Foxn1 Ab. We evaluated Foxn1 expression patterns in TEC subsets and its dynamics during normal thymus development, aging, injury, and regeneration. Upon challenges, upregulation of Foxn1 was a common feature of thymus regeneration, but the timing of Foxn1 expression changed and the responding TEC subsets depended on the type of treatment. Whereas dexamethasone and recombinant human fibroblast growth factor 7 promoted expansion of Foxn1(+)Ly51(+)CD80(-) TECs, castration led to expansion of Foxn1(+)Ly51(-)CD80(+) TECs. Collectively, Foxn1 expression is highly heterogeneous in the normal thymus, with large fractions of Foxn1(low) or Foxn1(-) TECs accumulating with age. Furthermore, Foxn1 expression is responsive to perturbations.

Thymic Epithelial Cells Are a Nonredundant Source of Wnt Ligands for Thymus Development.

Wnt signaling has been implicated in T cell development. However, it remained unclear which cell type is the major source of Wnt ligands and to what extent thymic epithelial cell (TEC) development is dependent on Wnt signaling. In this study, we analyzed the role of Wnt ligands provided by TECs for the development of T cells and TECs without manipulating the intracellular Wnt signaling machinery in either cell type. To this end, we used conditional knockout mice (FoxN1-Gpr177) in which TECs are unable to secrete Wnt ligands. Gpr177 (Evi/Wls) is a Wnt-specific cargo receptor that is required for the secretion of Wnt ligands. We found that TECs are the main source of Wnt ligands in the thymus, which serves a nonredundant role, and lack of TEC-provided Wnt ligands led to thymic hypotrophy, as well as a reduced peripheral T cell pool. Despite being reduced in numbers, T cells that developed in the absence of TEC-secreted Wnt ligands were functionally competent, and the subset composition of the peripheral T cell pool was not affected. Thus, our data suggest that T cell development is not directly dependent on TEC-provided Wnt ligands. Rather, TEC-secreted Wnt ligands are essential for normal thymus development and normal peripheral T cell frequencies but are dispensable for T cell function in the periphery.

Adult thymic epithelial cell (TEC) progenitors and TEC stem cells: Models and mechanisms for TEC development and maintenance.

The thymus is the primary lymphoid organ for generating self-restricted and self-tolerant functional T cells. Its two distinct anatomical regions, the cortex and the medulla, are involved in positive and negative selection, respectively. Thymic epithelial cells (TECs) constitute the framework of this tissue and function as major stromal components. Extensive studies for more than a decade have revealed how TECs are generated during organogenesis; progenitors specific for medullary TECs (mTECs) and cortical TECs (cTECs) as well as bipotent progenitors for both lineages have been identified, and the signaling pathways required for the development and maturation of mTECs have been elucidated. However, little is known about the initial commitment of mTECs and cTECs during ontogeny, and how regeneration of both lineages is sustained in the postnatal/adult thymus. Recently, stem cell activities in TECs have been demonstrated, and TEC progenitors have been identified in the postnatal thymus. In this review, recent advances in studying the development and maintenance of TECs are summarized, and the possible mechanisms of thymic regeneration and involution are discussed.

Loss of ΔNp63 impairs thymic epithelial progenitor survival and differentiation (HEM7P.234)

Abstract The thymic stroma consists primarily of thymic epithelial cells (TECs) that originate from endodermal progenitors in the 3rd pharyngeal pouch (pp). A common epithelial progenitor in fetal thymus differentiates into networks of cortical (cTEC) and medullary (mTEC) cells, which provide distinct maturation signals that support T cell generation. The transcription factor p63 is expressed in cTECs and mTECs and global deletion results in a severely hypoplastic thymus. Alternative promoters generate two p63 isoforms, TAp63 containing a transactivation (TA) domain and ΔNp63, lacking a TA domain. The roles of TAp63 and ΔNp63 in thymus organogenesis have not been determined. We find that TAp63 is undetectable in fetal or steady state adult thymus and deletion of TAp63 in fetal TECs does not affect thymus size or TEC development. In contrast, ΔNp63, is highly expressed by E9.5 and persists in cTECs and mTECs in the postnatal thymus. Using Foxn1Cre to delete ΔNp63fl/fl in TEC progenitors of the 3rd pp results in a severely hypoplastic thymic phenotype by E14.5. Patterning of 3rd pp endoderm into thymus- and parathyroid-fated domains is not disrupted. Plet1+ progenitor TECs are found at a normal frequency at E12.5; however, Plet1- TECs lacking ΔNp63 undergo apoptosis at a higher frequency than wildtype. Furthermore, surviving ΔNp63 null TECs fail to differentiate beyond an early progenitor state. We conclude that ΔNp63 is necessary for survival and differentiation of developing TECs.

Endothelial cells promote endogenous thymic regeneration via BMP4 signaling and activation of Foxn1 (HEM7P.224)

Abstract Although the thymus has a remarkable capacity for repair following damage, the mechanisms underlying this endogenous regeneration remain poorly understood. Here we reveal a critical role for endothelial cells (ECs) in aiding thymic recovery following acute injury. We found that ECs represent a highly damage-resistant niche in the thymus and, moreover, rather than just being passive conduits that deliver oxygen and nutrients, ECs are active participants in organ function producing distinct paracrine factors that orchestrate tissue repair. In particular, we demonstrated that after damage ECs induce upregulation by cortical thymic epithelial cells (TECs) of Foxn1, a key transcription factor implicated in TEC development and thymic regeneration. Further investigation revealed that the mechanism by which ECs induced upregulation of Foxn1 was via a soluble factor that could be inhibited by Noggin, strongly suggesting the involvement of BMP signaling. Consistent with this, ECs were found to be potent producers of BMP4 after thymic damage and inhibition of BMP4 signals by ECs, achieved either pharmacologically or genetically, led to significantly worse thymic recovery after acute injury caused by TBI. These findings not only provide the first evidence of the role of ECs in endogenous thymic regeneration but, given the significant clinical need for strategies to promote immunity, also offer an innovative therapy to boost thymic function.

Thymocyte requirements for normal thymic medullary epithelial cell development (HEM7P.227)

Abstract The thymic medullary compartment has a central role in tolerizing developing TCRαβ+ CD4 and CD8 single positive (SP) thymocytes. In turn, SP thymocytes are required for normal maturation of medullary thymic epithelial cells (mTEC). In the absence of TCRαβ+ SP thymocytes, the thymic medullary compartment is much smaller and numbers of mTEC are dramatically reduced. We recently demonstrated that the requirement for TCRαβ+ SP thymocytes in mTEC development can be overcome when Traf3, an inhibitor of NIK-dependent NF-κB signaling, is absent in TEC. In contrast to TCRα KO mice, TCRα KO/TEC-specific Traf3 KO mice have mTEC numbers comparable to that of wildtype mice. Traf3 inactivation also overcomes the requirements for LTβR and CD40 in mTEC development. Interestingly, however, we find that mTEC cell numbers are not rescued in TCRβ KO/TEC-specific Traf3 KO mice. TCRβ KO mice, like TCRα KO mice, lack SP thymocytes but also lack post-β-checkpoint TCRβ+ DN and DP thymocytes. This result suggests that a cell type(s) other than a TCRαβ+ SP thymocyte is required for normal mTEC development even in the absence of Traf3 and that this requirement is mediated by molecular interactions other than those involving LTβR and CD40. Studies to determine the identity of this thymic cell population and the corresponding molecular pathways involved in mTEC development are underway.

Cell types and cell signals important for normal thymic medullary epithelial cell development (HEM7P.231)

Abstract Thymic crosstalk between medullary thymic epithelial cells (mTEC) and mature SP thymocytes is critical for tolerance in developing T cells as well as for mTEC development. Several TNF family receptor-ligand pairs, including RANK-RANKL, LTαβ-LTβR and CD40L-CD40, have been shown to be important in thymic crosstalk, in part via RelB mediated non-canonical NFκB pathway. Recently, we identified CD28-CD80/86 as an additional pathway important for mTEC development. CD80/86, LTβR, CD40, RANK, as well as MHCII (also shown to be important for mTEC development) are expressed by both mTEC and by bone marrow (BM) derived cells in the thymus. Thus, it is important to ask which cell types need to express these molecules in order to support mTEC development. To address the question, we have generated and analyzed BM chimeras or conditional knockout mice in which either non-BM-derived cells (including thymic epithelium) or BM-derived cells express the molecule in question. Interestingly, for CD40 and CD80/86, we find that expression on either non-BM or BM-derived cells is sufficient to promote mTEC development. For LTβR, and RANK, we find that expression on radiation-resistant non-BM derived cells is required. The ability of MHCII expression on specific cell populations to drive mTEC development is currently being examined. Our findings suggest that models of thymic crosstalk must consider not only communication between TEC and thymocytes but with other BM derived cell types as well.

NF-κB-inducing kinase in thymic stroma establishes central tolerance by orchestrating cross-talk with not only thymocytes but also dendritic cells.

Essential roles of NF-κB-inducing kinase (NIK) for the development of medullary thymic epithelial cells (mTECs) and regulatory T cells have been highlighted by studies using a strain of mouse bearing a natural mutation of the NIK gene (aly mice). However, the exact mechanisms underlying the defect in thymic cross-talk leading to the breakdown of self-tolerance in aly mice remain elusive. In this study, we demonstrated that production of regulatory T cells and the final maturation process of positively selected conventional αβ T cells are impaired in aly mice, partly because of a lack of mature mTECs. Of note, numbers of thymic dendritic cells and their expression of costimulatory molecules were also affected in aly mice in a thymic stroma-dependent manner. The results suggest a pivotal role of NIK in the thymic stroma in establishing self-tolerance by orchestrating cross-talk between mTECs and dendritic cells as well as thymocytes. In addition, we showed that negative selection was impaired in aly mice as a result of the stromal defect, which accounts for the development of organ-specific autoimmunity through a lack of normal NIK.

Biological significance of FoxN1 gain-of-function mutations during T and B lymphopoiesis in juvenile mice.

FoxN1 is cell-autonomously expressed in skin and thymic epithelial cells (TECs), essential for their development. Inborn mutation of FoxN1 results in hair follicle and TEC development failure, whereas insufficient postnatal FoxN1 expression induces thymic atrophy, resulting in declined T lymphopoiesis. Although upregulating FoxN1 expression in the aged FoxN1-declined thymus rejuvenates T lymphopoiesis, whether its over- and ectopic-expression in early life is beneficial for T lymphopoiesis is unknown. Using our newly generated Rosa26-STOPflox–FoxN1 mice, in which over- and ectopic-expression of FoxN1 can be induced by various promoter-driven Cre-mediated deletions of the roadblock STOPflox in early life, we found that K14Cre-mediated inborn FoxN1 overexpression induced neonatal lethality, exhibited abnormal permeability in the skin and abnormal nursing. Ubiquitous deletion of the STOPflox mediated by progressive uCreERT leakage in juvenile mice affected thymus and bone marrow normality, resulting in an increased ratio of medullary/cortical TECs, along with declined T and B lymphopoiesis. Although the K5CreERT-mediated FoxN1 overexpression mice had a normal lifespan, induction of K5CreERT activation in juveniles adversely influenced total thymoycte development and produced ichthyosis-like skin. Therefore, FoxN1 has temporal and tissue-specific activity. Over- and ectopic-expression of FoxN1 in early life adversely influence immature TEC, T and B cell, and skin epithelial development.

An organized and functional thymus generated from FOXN1-reprogrammed fibroblasts.

SummaryA central goal of regenerative medicine is to generate transplantable organs from cells derived or expanded in vitro. Although numerous studies have demonstrated production of defined cell-types in vitro1, creation of a fully intact organ has not been reported. The transcription factor Forkhead box N1 (FOXN1) is critically required for development of thymic epithelial cells (TECs)2,3 a key cell-type of the thymic stroma4. Here, we show that enforced Foxn1 expression is sufficient to reprogramme fibroblasts into functional TECs, an unrelated cell-type across a germ-layer boundary. These Foxn1-induced TECs (iTECs) supported efficient development of both CD4+ and CD8+ T cells in vitro. Upon transplantation, iTEC established a complete, fully organized and functional thymus, that contained all of the TEC sub-types required to support T cell differentiation and populated the recipient immune system with T cells. iTEC thus demonstrate that cellular reprogramming approaches can be used to generate an entire organ, and open the possibility of widespread use of thymus transplantation to boost immune function in patients.

Cell types important for fostering normal thymic medullary epithelial cell development (HEM4P.242)

Abstract Tolerance in developing T cells is dependent on medullary thymic epithelial cells (mTEC) and mTEC development in turns requires signals from mature SP thymocytes. Several TNF family receptor-ligand pairs have been shown to be important players in the thymic crosstalk required to foster mTEC development including RANK-RANKL, LTαβ-LTβR and CD40L-CD40. In addition to confirming the important roles of TNF family members in thymic crosstalk, we have recently identified CD28-CD80/86 interactions as an additional pathway important for normal thymic medullary compartment development. CD80 and CD86, similar to LTβR, CD40 and RANK, are expressed by mTEC as well as by bone marrow (BM) derived cells in the thymus. To address the question of which cell type(s) need to express CD80/86, LTβR, CD40 and RANK in order to support mTEC development, we have generated and analyzed BM chimeras or conditional knockout mice in which either non-BM-derived cells (including thymic epithelium) or BM-derived cells express the molecule in question. For LTβR and RANK, we find that expression on radiation-resistant non-BM-derived cells, is required. Interestingly, however, we find that for CD80/86 and CD40, expression on either non-BM-derived or BM-derived cells is sufficient to promote mTEC development. Our findings suggest that models of thymic crosstalk must consider not only communication between TEC and thymocytes but with other bone marrow derived cell types as well.

Over-, ectopic-expression of FoxN1 at early life adversely influences lymphopoiesis (HEM3P.280)

Abstract FoxN1 expresses in thymic epithelial cell (TEC) and skin epithelium to regulate their development. Prenatal mutation in FoxN1 results in thymic development failure and nude phenotype, while postnatal insufficient FoxN1 expression induces thymic atrophy, resulting in declined T-lymphopoiesis. Although enhancing FoxN1 expression in aged thymus promotes functional rejuvenation, whether FoxN1 over-expression at early age or ectopic expression in the bone marrow is beneficial for lymphopoiesis is unknown. Using our newly generated R26-STOPflox-FoxN1 (gain-of-function) mutant mice, which have an over-expressed FoxN1 after various promoter-driven Cre-mediated deletion of the roadblock STOPflox, we found that K14Cre-mediated prenatal FoxN1 over-expression resulted in a newborn lethal phenotype, displaying abnormal permeability in the skin. Ubiquitous deletion of the STOPflox mediated by uCreERT progressive leak in infant mice influenced TEC development, and T- and B-lymphopoiesis. Although the K5CreERT mediated FoxN1 over-expression in adult mice may be beneficial for postponing thymic aging, early induction of K5CreERT activation in infants induced adverse influences to thymoycte and hair follicle development. Therefore, FoxN1 has a stage and tissue sensitivity. Over- and ectopic-expression of FoxN1 at early life adversely influences immature thymic, T-, and B-cell, and skin epithelial development.