Harsh Vaidya

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.

GeneProf: analysis of high-throughput sequencing experiments

Existing tools are hence not sufficient to make highthroughput sequencing data fully accessible to the entire research community. To address these challenges, we developed GeneProf, which combines (i) an easy-to-use data analysis suite that automates the analysis process with (ii) a comprehensive resource of integrated, readily interpretable and reusable analysis results. GeneProf ’s user interface is web-based, obviating the need to install specialized software (Supplementary Figs. 1 and 2). The system uses a dedicated, remote compute cluster to carry out large-scale genomic analyses, simultaneously dealing with many computationally demanding tasks. GeneProf is available online (http://www.geneprof.org/) and is free for academic researchers. Technical details are available in Supplementary Discussion and Supplementary Figure 3. GeneProf simplifies the construction of complex workflows by providing assistive web forms (‘wizards’) that conceal the underlying complexities of workflow programming. These wizards reconceive common, best-practice analysis steps as a series of logical stages, which researchers can customize easily by answering only a few basic questions. Users may change wizard-generated workflows to suit specialized requirements, maintaining full methodological flexibility. GeneProf: analysis of high-throughput sequencing experiments

An organized and functional thymus generated from FOXN1-reprogrammed fibroblasts

A central goal of regenerative medicine is to generate transplantable organs from cells derived or expanded in vitro. Although numerous studies have demonstrated the production of defined cell types in vitro, the 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), a key cell type of the thymic stroma. 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. On transplantation, iTECs established a complete, fully organized and functional thymus, that contained all of the TEC subtypes required to support T-cell differentiation and populated the recipient immune system with T cells. iTECs 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.

Dynamics of thymus organogenesis and colonization in early human development

The thymus is the central site of T-cell development and thus is of fundamental importance to the immune system, but little information exists regarding molecular regulation of thymus development in humans. Here we demonstrate, via spatial and temporal expression analyses, that the genetic mechanisms known to regulate mouse thymus organogenesis are conserved in humans. In addition, we provide molecular evidence that the human thymic epithelium derives solely from the third pharyngeal pouch, as in the mouse, in contrast to previous suggestions. Finally, we define the timing of onset of hematopoietic cell colonization and epithelial cell differentiation in the human thymic primordium, showing, unexpectedly, that the first colonizing hematopoietic cells are CD45+CD34int/-. Collectively, our data provide essential information for translation of principles established in the mouse to the human, and are of particular relevance to development of improved strategies for enhancing immune reconstitution in patients.

Identification of a Bipotent Epithelial Progenitor Population in the Adult Thymus

Summary Thymic epithelial cells (TECs) are critically required for T cell development, but the cellular mechanisms that maintain adult TECs are poorly understood. Here, we show that a previously unidentified subpopulation, EpCam+UEA1−Ly-51+PLET1+MHC class IIhi, which comprises <0.5% of adult TECs, contains bipotent TEC progenitors that can efficiently generate both cortical (c) TECs and medullary (m) TECs. No other adult TEC population tested in this study contains this activity. We demonstrate persistence of PLET1+Ly-51+ TEC-derived cells for 9 months in vivo, suggesting the presence of thymic epithelial stem cells. Additionally, we identify cTEC-restricted short-term progenitor activity but fail to detect high efficiency mTEC-restricted progenitors in the adult thymus. Our data provide a phenotypically defined adult thymic epithelial progenitor/stem cell that is able to generate both cTECs and mTECs, opening avenues for improving thymus function in patients.

FOXN1 in thymus organogenesis and development

Development of the primary T‐cell repertoire takes place in the thymus. The linked processes of T‐cell differentiation and T‐cell repertoire selection each depend on interactions between thymocytes and thymic stromal cells; in particular, with the epithelial cells of the cortical and medullary thymic compartments (cortical and medullary thymic epithelial cells; cTECs and mTECs, respectively). The importance of the thymic epithelial cell lineage in these processes was revealed in part through analysis of nude (nu/nu) mice, which are congenitally hairless and athymic. The nude phenotype results from null mutation of the forkhead transcription factor FOXN1, which has emerged as a pivotal regulator both of thymus development and homeostasis. FOXN1 has been shown to play critical roles in thymus development, function, maintenance, and even regeneration, which positions it as a master regulator of thymic epithelial cell (TEC) differentiation. In this review, we discuss current understanding of the regulation and functions of FOXN1 throughout thymus ontogeny, from the earliest stages of organogenesis through homeostasis to age‐related involution, contextualising its significance through reference to other members of the wider Forkhead family.

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.

Development of Thymic Epithelial Cells

The thymus is the primary lymphoid organ in which the T cell repertoire is generated. The complex cellularity of this organ is uniquely designed to facilitate T cell development: defects in thymus development or function can cause immunodeficiencies ranging from the absence of T cell–mediated immunity to broad-spectrum autoimmune disease. Peak thymus size and output occurs early in life, after which the thymus undergoes a natural process of involution. This results in the progressive loss of functional thymus tissue and correspondingly in decreased production of new naive T cells with age – contributing to the diminished capacity of the aged immune system to adequately respond to new antigenic challenge. Age-related thymic involutions, together with the thymic involutions associated with cytotoxic therapies (e.g., radio- or chemotherapy), have raised interest in development of clinically useful protocols for boosting thymus function in vivo , by reactivating or protecting the endogenous thymus or via thymus transplantation. Improvements in this area are likely to depend on an intimate understanding of the mechanisms regulating development and maintenance of the thymus, and in particular of its stromal components. We here review current understanding of these mechanisms, focusing specifically on the epithelial component of the thymic stroma since this highly specialized lineage directs thymus development and is required critically to mediate many of the organs specialist functions.