C. Clare Blackburn

Identification and Characterization of Thymic Epithelial Progenitor Cells

T cell differentiation and repertoire selection depend critically on several distinct thymic epithelial cell types, whose lineage relationships are unclear. We have investigated these relationships via functional analysis of the epithelial populations within the thymic primordium. Here, we show that mAbs MTS20 and MTS24 identify a population of cells that, when purified and grafted ectopically, can differentiate into all known thymic epithelial cell types, attract lymphoid progenitors, and support CD4(+) and CD8(+) T cell development in nude mice. In contrast, other epithelial populations in the thymic primordium can fulfill none of these functions. These data establish that the MTS20(+)24(+) population is sufficient to generate a functional thymus in vivo and thus argue strongly that all thymic epithelial cell types derive from a common progenitor cell.

Developing a new paradigm for thymus organogenesis

The mature thymic epithelium is complex, with two major compartments — the cortex and the medulla — each containing several functionally distinct epithelial-cell types. There is considerable debate as to the embryonic origins of these different thymic epithelial-cell subpopulations. The textbook view is a dual origin, with cortical thymic epithelium arising from the ectoderm and medullary thymic epithelium originating in the endoderm. However, the literature has been divided on this issue since it was first considered. In this review, we discuss recent embryological, functional, genetic and molecular data that collectively support a new model of thymus organogenesis and patterning.

Gcm2 and Foxn1 mark early parathyroid- and thymus-specific domains in the developing third pharyngeal pouch

The thymus and parathyroids originate from a common primordium that develops from the third pharyngeal pouch in mice and humans. The molecular mechanism that specifies this primordium into distinct organ domains is not known. The Gcm2 and Foxn1 transcription factors are required for development of the parathyroid and thymus respectively, and are attractive candidates for this role. However, their embryonic expression patterns during pharyngeal pouch development and early thymus and parathyroid organogenesis have not been described. Here we report that Gcm2 is expressed specifically in the developing second and third pharyngeal pouches at E9.5, and is further confined to a small domain of the third pouch endoderm by E10.5. In contrast, Foxn1 is not expressed until after the common primordium is formed, beginning at E11.25. Our results show that Gcm2 and Foxn1 expression mark two complementary domains that prefigure parathyroid and thymus regions within the common primordium before morphological distinctions are present.

Extensive Hematopoietic Stem Cell Generation in the AGM Region via Maturation of VE-Cadherin+CD45+ Pre-Definitive HSCs

Elucidating the mechanisms underlying hematopoietic stem cell (HSC) specification and expansion in the embryo has been hampered by the lack of analytical cell culture systems that recapitulate in vivo development. Here, we describe an ex vivo model that facilitates a rapid and robust emergence of multipotent long-term repopulating HSCs in the embryonic AGM region. Because this method includes a cell dissociation step prior to reconstruction of a three-dimensional functional tissue and preserves both stromal and hematopoietic elements, it allowed us to identify the direct ancestry of the rapidly expanding HSC pool. We demonstrate that extensive generation of definitive HSCs in the AGM occurs predominantly through the acquisition of stem characteristics by the VE-cadherin+CD45+ population.

Contribution of Neural Crest-Derived Cells in the Embryonic and Adult Thymus

Neural crest (NC)-derived mesenchyme has previously been shown to play an important role in the development of fetal thymus. Using Wnt1-Cre and Sox10-Cre mice crossed to Rosa26eYfp reporter mice, we have revealed NC-derived mesenchymal cells in the adult murine thymus. We report that NC-derived cells infiltrate the thymus before day 13.5 of embryonic development (E13.5) and differentiate into cells with characteristics of smooth muscle cells associated with large vessels, and pericytes associated with capillaries. In the adult organ at 3 mo of age, these NC-derived perivascular cells continue to be associated with the vasculature, providing structural support to the blood vessels and possibly regulating endothelial cell function.

Microenvironmental reprogramming of thymic epithelial cells to skin multipotent stem cells

The thymus develops from the third pharyngeal pouch of the anterior gut and provides the necessary environment for thymopoiesis (the process by which thymocytes differentiate into mature T lymphocytes) and the establishment and maintenance of self-tolerance. It contains thymic epithelial cells (TECs) that form a complex three-dimensional network organized in cortical and medullary compartments, the organization of which is notably different from simple or stratified epithelia. TECs have an essential role in the generation of self-tolerant thymocytes through expression of the autoimmune regulator Aire, but the mechanisms involved in the specification and maintenance of TECs remain unclear. Despite the different embryological origins of thymus and skin (endodermal and ectodermal, respectively), some cells of the thymic medulla express stratified-epithelium markers, interpreted as promiscuous gene expression. Here we show that the thymus of the rat contains a population of clonogenic TECs that can be extensively cultured while conserving the capacity to integrate in a thymic epithelial network and to express major histocompatibility complex class II (MHC II) molecules and Aire. These cells can irreversibly adopt the fate of hair follicle multipotent stem cells when exposed to an inductive skin microenvironment; this change in fate is correlated with robust changes in gene expression. Hence, microenvironmental cues are sufficient here to re-direct epithelial cell fate, allowing crossing of primitive germ layer boundaries and an increase in potency.

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.

The earliest thymic T cell progenitors sustain B cell and myeloid lineage potential

The stepwise commitment from hematopoietic stem cells in the bone marrow to T lymphocyte–restricted progenitors in the thymus represents a paradigm for understanding the requirement for distinct extrinsic cues during different stages of lineage restriction from multipotent to lineage-restricted progenitors. However, the commitment stage at which progenitors migrate from the bone marrow to the thymus remains unclear. Here we provide functional and molecular evidence at the single-cell level that the earliest progenitors in the neonatal thymus had combined granulocyte-monocyte, T lymphocyte and B lymphocyte lineage potential but not megakaryocyte-erythroid lineage potential. These potentials were identical to those of candidate thymus-seeding progenitors in the bone marrow, which were closely related at the molecular level. Our findings establish the distinct lineage-restriction stage at which the T cell lineage–commitment process transits from the bone marrow to the remote thymus.

A developmental look at thymus organogenesis: where do the non-hematopoietic cells in the thymus come from?

The origins of the non-hematopoietic cell types that comprise the thymic stroma remain a topic of considerable controversy. Three recent studies, using lineage analysis and other methods to determine the developmental potential of specific cell types within the thymus, have provided strong evidence of a single endodermal origin for all thymic epithelial cells. Together with other investigations that merge immunological and developmental biology approaches, these studies have suggested a new model of thymus organogenesis, and have begun to uncover the molecular pathways that control this process.

Regeneration of the aged thymus by a single transcription factor

Thymic involution is central to the decline in immune system function that occurs with age. By regenerating the thymus, it may therefore be possible to improve the ability of the aged immune system to respond to novel antigens. Recently, diminished expression of the thymic epithelial cell (TEC)-specific transcription factor Forkhead box N1 (FOXN1) has been implicated as a component of the mechanism regulating age-related involution. The effects of upregulating FOXN1 function in the aged thymus are, however, unknown. Here, we show that forced, TEC-specific upregulation of FOXN1 in the fully involuted thymus of aged mice results in robust thymus regeneration characterized by increased thymopoiesis and increased naive T cell output. We demonstrate that the regenerated organ closely resembles the juvenile thymus in terms of architecture and gene expression profile, and further show that this FOXN1-mediated regeneration stems from an enlarged TEC compartment, rebuilt from progenitor TECs. Collectively, our data establish that upregulation of a single transcription factor can substantially reverse age-related thymic involution, identifying FOXN1 as a specific target for improving thymus function and, thus, immune competence in patients. More widely, they demonstrate that organ regeneration in an aged mammal can be directed by manipulation of a single transcription factor, providing a provocative paradigm that may be of broad impact for regenerative biology.