Seminars in Immunology 17 (2005) 95–102 Thymopoiesis in 4 dimensions Colleen M. Witt ∗ , Ellen A. Robey Division of Immunology, Department of Molecular and Cell Biology, 479 Life Sciences Addition, University of California, Berkeley, CA 94720, USA Abstract Developing T cells undergo long-range migrations in the thymus that are tightly linked to their developmental program. In this review, we discuss the anatomical positioning of developing T cells in the thymus, review what we know about how cells move in the thymus, and point out unresolved questions. We also discuss new imaging technologies and interdisciplinary approaches and discuss how the promise they offer to addressing these questions. © 2004 Elsevier Ltd. All rights reserved. Keywords: Thymocyte; Cell migration; Thymic architecture; Development 1. Introduction Thymopoiesis involves the tightly regulated movement of cells over considerable distances to achieve the final pro- duction of mature T cells. In this review we focus on what is known, and not known, of the spatial organization within the thymus, how thymic compartmentalization is maintained, and how cell movement proceeds across the thymic landscape and in concert with development. We also discuss new imag- ing technologies and interdisciplinary methodologies and the promise they offer in addressing many of the spatial–temporal aspects of thymocyte development. 2. Thymus ontogeny and compartmentalization Thymic organogenesis begins on E10.5 as the re- sult of epithelial–mesenchymal interactions between the third pharyngeal pouch endoderm and neural crest-derived mesynchyme [1]. Seeding of the thymus anlage by hematopoietic precursors begins on E11.0 [2,3] followed by further phenotypic maturation of thymic epithelium mediated by Wnt-induced expression of the FoxN1 gene [4–6]. Com- Abbreviations: DN, double-negative (CD4 - CD8 - ); DP, double-positive (CD4 + CD8 + ); SP, single-positive (CD4 + CD8 - or CD4 - CD8 + ); CMJ, (cor- tical/medullary junction); SCZ, subcapsulary zone ∗ Corresponding author. Tel.: +1 510 643 6957; fax: +1 510 643 9500. E-mail address: cwitt@uclink.berkeley.edu (C.M. Witt). mitment of hematopoietic precursors to the T lineage is es- tablished by E14.5 and marks the transition from thymocyte- independent to thymocyte-dependent epithelial development. Beyond this point of development, patterning and differenti- ation of thymic epithelium occurs in parallel with and is fully dependent upon further thymocyte development [7,8]. By 3 weeks of age the adult thymus is compartmental- ized with a DC-rich boundary separating an inner region of medullary tissue from the outer cortex (Fig. 1). The two major regions contain distinct types of stromal elements and there is a considerable degree of epithelial heterogeneity within the compartments, particularly within the medulla [9,10]. Little is known as to how cortico-medullary boundaries are maintained, but recently it has been shown that Eph-A re- ceptors and their ephrin ligands are expressed in thymus. Given their differential expression in thymic stromal subsets [11,12], and their known role in maintaining compartmental- ization in other developmental contexts [13,14] it is tempting to speculate that they play a role in maintaining tissue bound- aries in the thymus. 3. Thymocyte trafficking 3.1. From bone marrow to thymus A developing thymocte travels thousands of microns through the thymus before becoming a mature functional T cell. Before entering the thymus, progenitors are released 1044-5323/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.smim.2004.09.008