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Thymus development and function
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Thymus development and function Thomas Boehm Thymopoiesis is a complex and highly dynamic process. It involves reciprocal tissue interactions between epithelial cells derived from the endoderm of the anterior foregut and neural crest-derived mesenchyme to form the thymic rudiment. This organ anlage attracts thymocyte progenitors and supports their differentiation and functional maturation into a self-tolerant diverse repertoire of T cells. In recent years, a more detailed picture of the molecular mechanisms determining the formation of the thymic rudiment and those controlling the maturation of the epithelial compartment has emerged and these are briefly summarized here. This review also addresses new experimental approaches toward a better understanding of thymopoiesis and discusses the impact of new animal models. Address Department of Developmental Immunology, Max-Planck-Institute of Immunobiology, Stuebeweg 51, D-79108 Freiburg, Germany Corresponding author: Boehm, Thomas ([email protected])
Current Opinion in Immunology 2008, 20:178–184 This review comes from a themed issue on Lymphocyte Development Edited by Klaus Rajewsky and Harald von Boehmer Available online 9th April 2008 0952-7915/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2008.03.001
Introduction The thymus is a primary lymphoid organ that is found in all vertebrates (with the exception of jawless fish, such as the lamprey); its emergence in evolution parallels the appearance of VDJ recombination as a novel means of somatically diversifying antigen receptors [1]. The thymus has a unique capacity to support the development of self-tolerant T cells expressing a diverse repertoire of antigen receptors. Research on thymopoiesis deals with several distinct areas. The first aspect concerns the elaboration of the thymic anlage as a site for incoming T cell progenitors and the subsequent development of a multicomponent stromal compartment. The second aspect concerns the nature of T cell progenitors during embryonic and adult stages and the nature of the entry site(s). The third aspect deals with the interaction of T cell progenitors and the stromal microenvironment to arrive at the many different types of mature thymocytes that are ready to leave the thymus. The fourth aspect of thymopoiesis concerns the mechanisms controlling the exit of Current Opinion in Immunology 2008, 20:178–184
mature thymocytes. Although all of these aspects are briefly touched upon here, the main focus lies on the formation and differentiation of the thymic microenvironment.
In and out of the thymic anlage/thymus There are apparently various mechanisms governing the colonization of the thymus before and after vascularization (which occurs at E15–16 in the mouse). Before vascularization, T cell progenitors migrate through mesenchymal tissue before they reach the epithelial thymic anlage. The chemokine Ccl25 is expressed in the thymic anlage [2] and is the ligand for the chemokine receptor Ccr9, which is expressed on T cell progenitors. Lack of Ccr9 reduces but does not abolish thymus homing [3], indicating that other cues are important, perhaps involving guidance (Ccl21 [4]) in addition to attraction. Another such factor that has been considered in this process is Cxcl12, the ligand for Cxcr4; however, thymus homing is not affected in mice lacking either gene [5]. Interestingly, a small population of hematopoietic cells in the perithymic mesenchyme and in the thymic anlage lacks Ccr7, Ccr9, and Cxcr4; how these cells home to the thymus is unclear [6]. It appears probable therefore that multiple factors cooperate in regulating the process of homing to the thymus rudiment. After vascularization, the relative importance of Ccr9 and Ccr7 for thymus homing appears to decrease, as mice deficient for these genes do not exhibit a reduced number of T cells in the thymus from late gestation onwards [4]. Indeed, the structure of the niche via which T cell progenitors enter into the adult thymus still remains largely undefined [7], but possibly involves perivascular spaces [8]; a multistep homing process has been proposed for at least one presumptive thymus-settling cell type [9]. Exactly which cell type represents the thymus-settling cell remains an issue of much debate [10]; likewise, whether T cell commitment occurs before or after entry into the thymus also remains unclear [11,12], although this may differ for embryonic and adult phases [13]. The molecular mechanisms of thymocyte egress from the thymus have not been fully defined; however, the sphingosine-1-phosphate (S1P) receptor, whose expression is controlled by the Klf2 transcription factor [14], plays an important role [15,16]. Where exactly emigration occurs in the thymus is unclear [8], as it can occur also from the cortex in mice deficient in Ccr7 [17]. Recent experiments have also indicated that, on average, a naı¨ve ab T cell precursor emigrates only four to five days after becoming a single-positive thymocyte [18]. www.sciencedirect.com
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Thymus development during early embryogenesis The epithelial compartment of the thymus originates from the endodermal layer of the anterior foregut [19]. During pharyngeal pouch development, these cells delaminate from the inner layer of the foregut and grow into the underlying mesenchymal tissue compartment that is of neural crest origin. In the mouse, the prospective thymus anlage is located next to that of the parathyroid in the third pharyngeal pouch. It begins to express Foxn1 at about E11.5. Foxn1 encodes a transcription factor whose function is essential for subsequent epithelial differentiation; without it, colonization of the anlage by thymocyte progenitors fails [2] and thymopoiesis is aborted, resulting in severe immunodeficiency [20]. It is still unknown which signal(s) determine(s) the site and also the size of the thymic anlage and whether these or other signals initiate Foxn1 expression. Clearly, genetic abnormalities involving the formation of pharyngeal pouches (such as deficiencies in Tbx1, Pbx1, Eya1, Six1) also have an impact on thymus formation [21–23]. Wnt and BMP signals have also been implicated in initiating [24] and/or maintaining [25,26] Foxn1 expression in the thymic epithelium, respectively, but their role in specifying the thymic field has not been unequivocally established. Shh signaling during early embryonic development is involved in restricting the size of the thymic
field, as measured by Foxn1 expression [27]. Although the interplay of various signaling pathways thus requires further scrutiny, the surprisingly consistent presence of supernumerary thymic lobes in mice [28] hints at the complexities of this process. The analysis of the signal inducing the development of cervical thymi, which appear to be delayed in development as compared to the mediastinal thymic tissues, might shed light on the mechanism of the induction of thymic anlagen [29]. Likewise, comparative analyses in species with multiple sites of thymic lobes might prove informative [28]. The overall size of the thymus is probably determined by the action of p63 and Fgf signaling through the Fgfr2IIIb receptor, with p63 acting upstream of Fgfr2 [30,31]. Another factor impacting on thymus size is the presence of fetal mesenchyme ensheathing the thymic epithelial anlage. It provides Fgf signals and it has been demonstrated that the presence of this cell type, which expresses Pdgfra correlates with increased proliferation of the epithelial cells [32]. Heterozygosity of wild-type Foxn1 leads to a substantial reduction in the number of thymocytes without affecting their differentiation [33]. It is unclear whether this is caused by reduced numbers of epithelial cells in Foxn1+/ mice; if so, it would be important to investigate a possible genetic interaction of Foxn1 with the p63/Fgfr2 pathway (Figure 1).
Figure 1
Developmental control points during thymic epithelial development. (a) Early in embryogenesis, the thymic field is specified in the endoderm of the anterior foregut, possibly via several signaling pathways. The induction of Foxn1 expression is indicative of the differentiation of thymic epithelial cells, which develop next to the parathyroid anlage. Additional specification events from an undifferentiated common progenitor source might lead to the occurrence of supernumerary parathyroid and thymic lobes. (b) Differentiation pathways of a bipotent thymic epithelial progenitor. The TEC progenitor (red) probably expresses Foxn1, whose expression is maintained by BMP signaling. TEC progenitor expansion might be controlled by p63 that acts upstream of Fgf signaling. Although the molecular mechanism controlling the bifurcation into cortical and medullary epithelial lineages is not known, Wnt signaling might be involved. The distinct cortical (pink) and medullary (blue) epithelial progenitors are indicated. The medullary lineage differentiates further under the control of lymphotoxin and TNF signals and arrives at a postmitotic stage characterized by Aire expression. These terminally differentiated cells are postmitotic and have a short half-life, which may facilitate crosspresentation of peripheral tissue antigens. The functional consequences of altered differentiation, particularly of the medullary compartment have been reviewed recently [65]. www.sciencedirect.com
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Bipotent thymic epithelial progenitors Broadly speaking, thymic epithelium in the adult thymus can be divided into cortical and medullary epithelium. Recent experiments employing quite different experimental systems showed that single epithelial cells are capable of differentiating into the full range of epithelial cells characteristic of a functional thymic microenvironment. Clonal analysis using single E12 thymic epithelial cells showed that they can differentiate into cortical and medullary cell types when injected into a normal fetal thymic lobe [34]. Postnatal reactivation of Foxn1 in single cells of Foxn1-deficient thymic cysts led to the development of small functional thymic lobes [35]. Furthermore, lineage-tracing experiments [35] confirmed the existence of medullary epithelial progenitors [36] and provided evidence for a cortical counterpart. Do bipotent thymic epithelial cells express the Foxn1 transcription factor? The answer to this important question is still unknown, but the circumstantial evidence referred to above suggests they do here first, early thymic epithelial cells all express Foxn1 [2,20] and they have precursor activity [34]; second, cells in the Foxn1 / anlage express Foxn1 and can be induced to differentiate normally upon re-supply of this gene [35]. It should be noted, however, that, at present, thymic epithelial progenitor cells cannot be prospectively defined [37].
Differentiation and maturation of the thymic epithelial compartment The early thymus anlage consists of an epithelial core and a mesenchymal sheath. The microenvironment of the thymus becomes more complex with age and then additionally includes cell types of the vasculature and other hematopoietic derivatives such as dendritic cells and macrophages. The major developmental checkpoint with respect to the functional epithelial anlage is the bifurcation that gives rise to cortical and medullary epithelium. It occurs in the absence of lymphocytes [38]. However, at present there is no information as to which signal(s) regulate(s) the bifurcation into cortical and medullary epithelial lineages. Differentiation still occurs, at least to some extent, in the absence of p63 [30,31] and Fgfr2IIIb [39], but cannot occur normally when Wnt signaling is affected by the loss of the negative regulator Kremen-1, particularly in the cortex [40]. Clearly, a systematic analysis of the role of different signaling pathways (singly and in combination) will be required to settle this important issue. BMP signaling seems to be required early on in the differentiation pathway for the maintenance of Foxn1 expression with direct consequences to thymic epithelial differentiation [25]. It is at present unclear whether this involves Fgf signaling also in vivo [41]. Whether Foxn1 expression is required in all epithelial cells in later life is currently unknown; in this regard, it will be interesting to investigate whether the Current Opinion in Immunology 2008, 20:178–184
epithelial cells lining cysts occurring during thymic involution still express Foxn1. The degree to which the maturation, as opposed to the initial differentiation, of cortical and medullary epithelial cells is cell-autonomous, appears to differ substantially. In mice with blocks in early thymocyte development, the cortex is largely normal, while the medulla is hypoplastic [42]. The molecular mechanisms of this process, often described as epithelial-lymphocytic crosstalk, have recently been investigated. While no genetic defect affecting the functional maturation of cortical epithelial cells has yet been described (note that positive selection also occurs in Kremen-1 deficient mice [40]), a number of molecules influence differentiation of the medulla to the extent that its altered function leads to immunopathology. The LTbR signaling pathway is required for full maturation of the medulla and seems to be required for a proper architecture of the medulla. It does not affect the expression of Aire and the associated downstream peripheral tissue antigens (PTAs) [43,44]. Rather, signaling via the Rank/Rankl pathway [45] including its downstream effector Traf6 [46] appears to be involved in regulating Aire expression via the action of cells with the phenotype of lymphoid tissue inducer cells. These data and related results [47] all support the so-called terminal differentiation model for PTA expression [48] and argue against an alternative view [49] that PTA expression occurs in immature mTECs. The two models have recently been directly tested and it was shown that thymic epithelial cells expressing Aire are postmitotic cells with high turnover [50]. These results essentially support the former model (Figure 1).
New experimental approaches to study thymus development In recent years, several new experimental strategies have been developed that substantially enhance the armamentarium of thymus research. Artificial thymic organoids
Recent experiments have explored the possibility of creating an artificial thymic organoid. Previous studies of ectopic thymopoiesis made use of reaggregated thymic epithelial cells that were transplanted under the kidney capsule. These aggregates are capable of attracting T cell progenitors from the host and of supporting normal thymopoiesis. Clark et al. extended this approach by creating, in a solid foam-like support, an artificial three-dimensional network composed of skin keratinocytes and fibroblasts into which hematopoietic progenitors were seeded [51]. After in vitro culture for several weeks, functional T cells developed. The artificial microenvironment was shown to express Foxn1 as well as Aire and PTAs as markers of differentiated thymic epithelium. The impetus for further development of this or related www.sciencedirect.com
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systems lies in the fact that immunosuppressed patients might eventually be supplied with an alternative and readily modifiable source of T cells. Furthermore, it might facilitate the analysis of the effects of genetic alterations in any of the components added to this in vitro system. Nude complementation
The effect of embryonic lethal mutations in the lymphocyte lineage can be studied by blastocyst complementation [52]. In this scheme, embryonic stem cells carrying the desired mutation(s) are injected into blastocysts defective for Rag1 or Rag2 genes. The latter mutation prevents maturation of T and B cells; therefore, all stages of lymphocyte development, except the very early ones, are supplied by the mutant ES cells. Thus, lymphocyte lineage specific effects of otherwise lethal mutations can be investigated. Rodewald’s group has devised a similar system to examine gene function in thymic epithelial cells [53]. By injecting suitably modified ES cells into blastocysts defective for Foxn1, differentiating thymic epithelium can only be supplied by the ES cell progeny. Thus it was shown that VEGF-A produced by thymic epithelial cells is required for a normal thymic vasculature. Transgenic modification of thymic epithelium
Several promoters have until now been used to drive the expression of transgenes into thymic epithelial cells. A
recent addition to this list is the Foxn1 promoter [25]. Foxn1 cDNA expressed under the control of this promoter rescued the thymus defect in Foxn1-deficient mice (Figure 2); subsequently, it was used to interfere with signaling pathways in the developing thymic anlage [25]. Employing the Cre/lox strategy, expression of Cre recombinase enables genomic loci to be tissue-specifically modified in thymic epithelial cells (unpublished results). New animal models
In recent years, zebrafish and medaka have emerged as important additions to the list of model animals for immunological research. While fish, for instance, trout and salmon have been studied by immunologists for quite some time, zebrafish and medaka have the unique advantage that they are genetically tractable and this has been used for large-scale mutagenesis screens [54– 56]. On the basis of pioneering studies demonstrating the overall conservation of antigen receptor and MHC genes, it was anticipated that the genetic basis of other processes might be similar if not identical between fish and mammals. Indeed, while some specific features of the fish immune system have been uncovered, the level of evolutionary conservation in the hematopoietic system appears to be quite high [57]. Two important features make these models attractive for thymus research. First, large-scale forward genetic screens can be conducted in these animals at reasonable
Figure 2
Functional validation of a Foxn1 promoter fragment. The expression of a Foxn1 cDNA fragment under the control of the Foxn1 promoter [25] rescues the hair phenotype of Foxn1-deficient mice (top panels) and restores normal thymopoiesis (bottom panels). The size of the thymus in Foxn1 / ; Foxn1::Foxn1 transgenic mice varies but always leads to normal T cell development (CC Bleul, T Boehm, unpublished data). www.sciencedirect.com
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expense [54–56] and the first results are encouraging [58,59]; the availability of large collections of mutagenized males (or sperm preparations thereof) allows reverse genetics to be performed [60], albeit not at the level of mouse with its facility of precise targeted genetic modifications. A second advantage is the fact that the developing larvae are transparent and can thus be used for live imaging using fish expressing transgenes (fluorescent marker proteins under the control of inducible or tissue-specific promoters) in a stable or transient fashion [61]. Although two-photon live imaging in vivo is increasingly being used to study the details of cellular behavior during the initiation of immune reactions in the mouse [62], similar analyses of the thymus have so far proved impossible, because of its inconvenient location in the rib cage. In the future, such analyses might be possible using thymic tissues transplanted under the skin of the scalp to allow better visual access. In the meantime, the fish systems have been used to investigate the immigration of the first thymocyte progenitors into the thymic rudiment and to map out the routes of this immigration in the developing fish larvae [63,64]. Ultimately, these models might be used to also investigate the process of thymic epithelial differentiation and the elaboration of the thymic microenvironment over time.
Acknowledgements Work in the author’s laboratory is supported by the Max-Planck-Society, the Deutsche Forschungsgemeinschaft, and the European Union.
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58. Schorpp M, Bialecki M, Diekhoff D, Walderich B, Odenthal J, Maischein HM, Zapata AG, Boehm T: Conserved functions of Ikaros in vertebrate lymphocyte development: genetic evidence for distinct larval and adult phases of T cell development and two lineages of B cells in zebrafish. J Immunol 2006, 177:2463-2476. This paper describes the characterization of the first zebrafish mutant isolated from a large-scale forward ENU-mutagenesis screen. It demonstrates the feasibility of such screens to uncover lymphocyte-specific mutants and also indicates that the molecular mechanisms governing lymphocyte development are highly conserved between fish and mammals. 59. Trede NS, Medenbach J, Damianov A, Hung LH, Weber GJ, Paw BH, Zhou Y, Hersey C, Zapata A, Keefe M et al.: Network of coregulated spliceosome components revealed by zebrafish mutant in recycling factor p110. Proc Natl Acad Sci U S A 2007, 104:6608-6613. 60. Wienholds E, Schulte-Merker S, Walderich B, Plasterk RH: Targetselected inactivation of the zebrafish rag1 gene. Science 2002, 297:99-102. 61. Langenau DM, Ferrando AA, Traver D, Kutok JL, Hezel J-PD, Kanki JP, Zon LI, Look AT, Trede NS: In vivo tracking
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of T cell development, ablation, and engraftment in transgenic zebrafish. Proc Natl Acad Sci U S A 2004, 101:7369-7374. 62. Gross S, Moss BL, Piwnica-Worms D: Veni, vidi, vici: in vivo molecular imaging of immune response. Immunity 2007, 27:533-538. 63. Kissa K, Murayama E, Zapata A, Corte´s A, Perret E, Machu C, Herbomel P: Live imaging of emerging hematopoietic stem cells and early thymus colonization. Blood 2007, 111: 1147-1156. See annotation to Ref. [64]. 64. Li J, Iwanami N, Hoa VQ, Furutani-Seiki M, Takahama Y: Noninvasive intravital imaging of thymocyte dynamics in medaka. J Immunol 2007, 179:1605-1615. This paper and Ref. [63] demonstrate the power of live imaging in developing zebrafish larva to study homing processes to the thymus. 65. Anderson G, Lane PJL, Jenkinson EJ: Generating intrathymic microenvironments to establish T-cell tolerance. Nat Rev Immunol 2007, 7:954-963. This paper discusses the immunopathological consequences of altered mTEC differentiation.
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Thymus development and function Thomas Boehm Thymopoiesis is a complex and highly dynamic process. It involves reciprocal tissue interactions between epithelial cells derived from the endoderm of the anterior foregut and neural crest-derived mesenchyme to form the thymic rudiment. This organ anlage attracts thymocyte progenitors and supports their differentiation and functional maturation into a self-tolerant diverse repertoire of T cells. In recent years, a more detailed picture of the molecular mechanisms determining the formation of the thymic rudiment and those controlling the maturation of the epithelial compartment has emerged and these are briefly summarized here. This review also addresses new experimental approaches toward a better understanding of thymopoiesis and discusses the impact of new animal models. Address Department of Developmental Immunology, Max-Planck-Institute of Immunobiology, Stuebeweg 51, D-79108 Freiburg, Germany Corresponding author: Boehm, Thomas ([email protected])
Current Opinion in Immunology 2008, 20:178–184 This review comes from a themed issue on Lymphocyte Development Edited by Klaus Rajewsky and Harald von Boehmer Available online 9th April 2008 0952-7915/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2008.03.001
Introduction The thymus is a primary lymphoid organ that is found in all vertebrates (with the exception of jawless fish, such as the lamprey); its emergence in evolution parallels the appearance of VDJ recombination as a novel means of somatically diversifying antigen receptors [1]. The thymus has a unique capacity to support the development of self-tolerant T cells expressing a diverse repertoire of antigen receptors. Research on thymopoiesis deals with several distinct areas. The first aspect concerns the elaboration of the thymic anlage as a site for incoming T cell progenitors and the subsequent development of a multicomponent stromal compartment. The second aspect concerns the nature of T cell progenitors during embryonic and adult stages and the nature of the entry site(s). The third aspect deals with the interaction of T cell progenitors and the stromal microenvironment to arrive at the many different types of mature thymocytes that are ready to leave the thymus. The fourth aspect of thymopoiesis concerns the mechanisms controlling the exit of Current Opinion in Immunology 2008, 20:178–184
mature thymocytes. Although all of these aspects are briefly touched upon here, the main focus lies on the formation and differentiation of the thymic microenvironment.
In and out of the thymic anlage/thymus There are apparently various mechanisms governing the colonization of the thymus before and after vascularization (which occurs at E15–16 in the mouse). Before vascularization, T cell progenitors migrate through mesenchymal tissue before they reach the epithelial thymic anlage. The chemokine Ccl25 is expressed in the thymic anlage [2] and is the ligand for the chemokine receptor Ccr9, which is expressed on T cell progenitors. Lack of Ccr9 reduces but does not abolish thymus homing [3], indicating that other cues are important, perhaps involving guidance (Ccl21 [4]) in addition to attraction. Another such factor that has been considered in this process is Cxcl12, the ligand for Cxcr4; however, thymus homing is not affected in mice lacking either gene [5]. Interestingly, a small population of hematopoietic cells in the perithymic mesenchyme and in the thymic anlage lacks Ccr7, Ccr9, and Cxcr4; how these cells home to the thymus is unclear [6]. It appears probable therefore that multiple factors cooperate in regulating the process of homing to the thymus rudiment. After vascularization, the relative importance of Ccr9 and Ccr7 for thymus homing appears to decrease, as mice deficient for these genes do not exhibit a reduced number of T cells in the thymus from late gestation onwards [4]. Indeed, the structure of the niche via which T cell progenitors enter into the adult thymus still remains largely undefined [7], but possibly involves perivascular spaces [8]; a multistep homing process has been proposed for at least one presumptive thymus-settling cell type [9]. Exactly which cell type represents the thymus-settling cell remains an issue of much debate [10]; likewise, whether T cell commitment occurs before or after entry into the thymus also remains unclear [11,12], although this may differ for embryonic and adult phases [13]. The molecular mechanisms of thymocyte egress from the thymus have not been fully defined; however, the sphingosine-1-phosphate (S1P) receptor, whose expression is controlled by the Klf2 transcription factor [14], plays an important role [15,16]. Where exactly emigration occurs in the thymus is unclear [8], as it can occur also from the cortex in mice deficient in Ccr7 [17]. Recent experiments have also indicated that, on average, a naı¨ve ab T cell precursor emigrates only four to five days after becoming a single-positive thymocyte [18]. www.sciencedirect.com
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Thymus development during early embryogenesis The epithelial compartment of the thymus originates from the endodermal layer of the anterior foregut [19]. During pharyngeal pouch development, these cells delaminate from the inner layer of the foregut and grow into the underlying mesenchymal tissue compartment that is of neural crest origin. In the mouse, the prospective thymus anlage is located next to that of the parathyroid in the third pharyngeal pouch. It begins to express Foxn1 at about E11.5. Foxn1 encodes a transcription factor whose function is essential for subsequent epithelial differentiation; without it, colonization of the anlage by thymocyte progenitors fails [2] and thymopoiesis is aborted, resulting in severe immunodeficiency [20]. It is still unknown which signal(s) determine(s) the site and also the size of the thymic anlage and whether these or other signals initiate Foxn1 expression. Clearly, genetic abnormalities involving the formation of pharyngeal pouches (such as deficiencies in Tbx1, Pbx1, Eya1, Six1) also have an impact on thymus formation [21–23]. Wnt and BMP signals have also been implicated in initiating [24] and/or maintaining [25,26] Foxn1 expression in the thymic epithelium, respectively, but their role in specifying the thymic field has not been unequivocally established. Shh signaling during early embryonic development is involved in restricting the size of the thymic
field, as measured by Foxn1 expression [27]. Although the interplay of various signaling pathways thus requires further scrutiny, the surprisingly consistent presence of supernumerary thymic lobes in mice [28] hints at the complexities of this process. The analysis of the signal inducing the development of cervical thymi, which appear to be delayed in development as compared to the mediastinal thymic tissues, might shed light on the mechanism of the induction of thymic anlagen [29]. Likewise, comparative analyses in species with multiple sites of thymic lobes might prove informative [28]. The overall size of the thymus is probably determined by the action of p63 and Fgf signaling through the Fgfr2IIIb receptor, with p63 acting upstream of Fgfr2 [30,31]. Another factor impacting on thymus size is the presence of fetal mesenchyme ensheathing the thymic epithelial anlage. It provides Fgf signals and it has been demonstrated that the presence of this cell type, which expresses Pdgfra correlates with increased proliferation of the epithelial cells [32]. Heterozygosity of wild-type Foxn1 leads to a substantial reduction in the number of thymocytes without affecting their differentiation [33]. It is unclear whether this is caused by reduced numbers of epithelial cells in Foxn1+/ mice; if so, it would be important to investigate a possible genetic interaction of Foxn1 with the p63/Fgfr2 pathway (Figure 1).
Figure 1
Developmental control points during thymic epithelial development. (a) Early in embryogenesis, the thymic field is specified in the endoderm of the anterior foregut, possibly via several signaling pathways. The induction of Foxn1 expression is indicative of the differentiation of thymic epithelial cells, which develop next to the parathyroid anlage. Additional specification events from an undifferentiated common progenitor source might lead to the occurrence of supernumerary parathyroid and thymic lobes. (b) Differentiation pathways of a bipotent thymic epithelial progenitor. The TEC progenitor (red) probably expresses Foxn1, whose expression is maintained by BMP signaling. TEC progenitor expansion might be controlled by p63 that acts upstream of Fgf signaling. Although the molecular mechanism controlling the bifurcation into cortical and medullary epithelial lineages is not known, Wnt signaling might be involved. The distinct cortical (pink) and medullary (blue) epithelial progenitors are indicated. The medullary lineage differentiates further under the control of lymphotoxin and TNF signals and arrives at a postmitotic stage characterized by Aire expression. These terminally differentiated cells are postmitotic and have a short half-life, which may facilitate crosspresentation of peripheral tissue antigens. The functional consequences of altered differentiation, particularly of the medullary compartment have been reviewed recently [65]. www.sciencedirect.com
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Bipotent thymic epithelial progenitors Broadly speaking, thymic epithelium in the adult thymus can be divided into cortical and medullary epithelium. Recent experiments employing quite different experimental systems showed that single epithelial cells are capable of differentiating into the full range of epithelial cells characteristic of a functional thymic microenvironment. Clonal analysis using single E12 thymic epithelial cells showed that they can differentiate into cortical and medullary cell types when injected into a normal fetal thymic lobe [34]. Postnatal reactivation of Foxn1 in single cells of Foxn1-deficient thymic cysts led to the development of small functional thymic lobes [35]. Furthermore, lineage-tracing experiments [35] confirmed the existence of medullary epithelial progenitors [36] and provided evidence for a cortical counterpart. Do bipotent thymic epithelial cells express the Foxn1 transcription factor? The answer to this important question is still unknown, but the circumstantial evidence referred to above suggests they do here first, early thymic epithelial cells all express Foxn1 [2,20] and they have precursor activity [34]; second, cells in the Foxn1 / anlage express Foxn1 and can be induced to differentiate normally upon re-supply of this gene [35]. It should be noted, however, that, at present, thymic epithelial progenitor cells cannot be prospectively defined [37].
Differentiation and maturation of the thymic epithelial compartment The early thymus anlage consists of an epithelial core and a mesenchymal sheath. The microenvironment of the thymus becomes more complex with age and then additionally includes cell types of the vasculature and other hematopoietic derivatives such as dendritic cells and macrophages. The major developmental checkpoint with respect to the functional epithelial anlage is the bifurcation that gives rise to cortical and medullary epithelium. It occurs in the absence of lymphocytes [38]. However, at present there is no information as to which signal(s) regulate(s) the bifurcation into cortical and medullary epithelial lineages. Differentiation still occurs, at least to some extent, in the absence of p63 [30,31] and Fgfr2IIIb [39], but cannot occur normally when Wnt signaling is affected by the loss of the negative regulator Kremen-1, particularly in the cortex [40]. Clearly, a systematic analysis of the role of different signaling pathways (singly and in combination) will be required to settle this important issue. BMP signaling seems to be required early on in the differentiation pathway for the maintenance of Foxn1 expression with direct consequences to thymic epithelial differentiation [25]. It is at present unclear whether this involves Fgf signaling also in vivo [41]. Whether Foxn1 expression is required in all epithelial cells in later life is currently unknown; in this regard, it will be interesting to investigate whether the Current Opinion in Immunology 2008, 20:178–184
epithelial cells lining cysts occurring during thymic involution still express Foxn1. The degree to which the maturation, as opposed to the initial differentiation, of cortical and medullary epithelial cells is cell-autonomous, appears to differ substantially. In mice with blocks in early thymocyte development, the cortex is largely normal, while the medulla is hypoplastic [42]. The molecular mechanisms of this process, often described as epithelial-lymphocytic crosstalk, have recently been investigated. While no genetic defect affecting the functional maturation of cortical epithelial cells has yet been described (note that positive selection also occurs in Kremen-1 deficient mice [40]), a number of molecules influence differentiation of the medulla to the extent that its altered function leads to immunopathology. The LTbR signaling pathway is required for full maturation of the medulla and seems to be required for a proper architecture of the medulla. It does not affect the expression of Aire and the associated downstream peripheral tissue antigens (PTAs) [43,44]. Rather, signaling via the Rank/Rankl pathway [45] including its downstream effector Traf6 [46] appears to be involved in regulating Aire expression via the action of cells with the phenotype of lymphoid tissue inducer cells. These data and related results [47] all support the so-called terminal differentiation model for PTA expression [48] and argue against an alternative view [49] that PTA expression occurs in immature mTECs. The two models have recently been directly tested and it was shown that thymic epithelial cells expressing Aire are postmitotic cells with high turnover [50]. These results essentially support the former model (Figure 1).
New experimental approaches to study thymus development In recent years, several new experimental strategies have been developed that substantially enhance the armamentarium of thymus research. Artificial thymic organoids
Recent experiments have explored the possibility of creating an artificial thymic organoid. Previous studies of ectopic thymopoiesis made use of reaggregated thymic epithelial cells that were transplanted under the kidney capsule. These aggregates are capable of attracting T cell progenitors from the host and of supporting normal thymopoiesis. Clark et al. extended this approach by creating, in a solid foam-like support, an artificial three-dimensional network composed of skin keratinocytes and fibroblasts into which hematopoietic progenitors were seeded [51]. After in vitro culture for several weeks, functional T cells developed. The artificial microenvironment was shown to express Foxn1 as well as Aire and PTAs as markers of differentiated thymic epithelium. The impetus for further development of this or related www.sciencedirect.com
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systems lies in the fact that immunosuppressed patients might eventually be supplied with an alternative and readily modifiable source of T cells. Furthermore, it might facilitate the analysis of the effects of genetic alterations in any of the components added to this in vitro system. Nude complementation
The effect of embryonic lethal mutations in the lymphocyte lineage can be studied by blastocyst complementation [52]. In this scheme, embryonic stem cells carrying the desired mutation(s) are injected into blastocysts defective for Rag1 or Rag2 genes. The latter mutation prevents maturation of T and B cells; therefore, all stages of lymphocyte development, except the very early ones, are supplied by the mutant ES cells. Thus, lymphocyte lineage specific effects of otherwise lethal mutations can be investigated. Rodewald’s group has devised a similar system to examine gene function in thymic epithelial cells [53]. By injecting suitably modified ES cells into blastocysts defective for Foxn1, differentiating thymic epithelium can only be supplied by the ES cell progeny. Thus it was shown that VEGF-A produced by thymic epithelial cells is required for a normal thymic vasculature. Transgenic modification of thymic epithelium
Several promoters have until now been used to drive the expression of transgenes into thymic epithelial cells. A
recent addition to this list is the Foxn1 promoter [25]. Foxn1 cDNA expressed under the control of this promoter rescued the thymus defect in Foxn1-deficient mice (Figure 2); subsequently, it was used to interfere with signaling pathways in the developing thymic anlage [25]. Employing the Cre/lox strategy, expression of Cre recombinase enables genomic loci to be tissue-specifically modified in thymic epithelial cells (unpublished results). New animal models
In recent years, zebrafish and medaka have emerged as important additions to the list of model animals for immunological research. While fish, for instance, trout and salmon have been studied by immunologists for quite some time, zebrafish and medaka have the unique advantage that they are genetically tractable and this has been used for large-scale mutagenesis screens [54– 56]. On the basis of pioneering studies demonstrating the overall conservation of antigen receptor and MHC genes, it was anticipated that the genetic basis of other processes might be similar if not identical between fish and mammals. Indeed, while some specific features of the fish immune system have been uncovered, the level of evolutionary conservation in the hematopoietic system appears to be quite high [57]. Two important features make these models attractive for thymus research. First, large-scale forward genetic screens can be conducted in these animals at reasonable
Figure 2
Functional validation of a Foxn1 promoter fragment. The expression of a Foxn1 cDNA fragment under the control of the Foxn1 promoter [25] rescues the hair phenotype of Foxn1-deficient mice (top panels) and restores normal thymopoiesis (bottom panels). The size of the thymus in Foxn1 / ; Foxn1::Foxn1 transgenic mice varies but always leads to normal T cell development (CC Bleul, T Boehm, unpublished data). www.sciencedirect.com
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expense [54–56] and the first results are encouraging [58,59]; the availability of large collections of mutagenized males (or sperm preparations thereof) allows reverse genetics to be performed [60], albeit not at the level of mouse with its facility of precise targeted genetic modifications. A second advantage is the fact that the developing larvae are transparent and can thus be used for live imaging using fish expressing transgenes (fluorescent marker proteins under the control of inducible or tissue-specific promoters) in a stable or transient fashion [61]. Although two-photon live imaging in vivo is increasingly being used to study the details of cellular behavior during the initiation of immune reactions in the mouse [62], similar analyses of the thymus have so far proved impossible, because of its inconvenient location in the rib cage. In the future, such analyses might be possible using thymic tissues transplanted under the skin of the scalp to allow better visual access. In the meantime, the fish systems have been used to investigate the immigration of the first thymocyte progenitors into the thymic rudiment and to map out the routes of this immigration in the developing fish larvae [63,64]. Ultimately, these models might be used to also investigate the process of thymic epithelial differentiation and the elaboration of the thymic microenvironment over time.
Acknowledgements Work in the author’s laboratory is supported by the Max-Planck-Society, the Deutsche Forschungsgemeinschaft, and the European Union.
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