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A developmental look at thymus organogenesis: where do the non-hematopoietic cells in the thymus come from?
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A developmental look at thymus organogenesis: where do the non-hematopoietic cells in the thymus come from? Opinion Nancy R Manley and C Clare Blackburny 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. Addresses Correspondence: Nancy R Manley: Department of Genetics, B420A Life Sciences Building, University of Georgia, Athens, GA 30602, USA e-mail: [email protected] y Institute for Stem Cell Research, The University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh EH9 3JQ, UK
will then integrate this model with current knowledge of the transcription factor pathways that mediate thymus development.
The basics of thymus organogenesis The morphological events of early thymus organogenesis occur between days 10 and 13.5 of embryonic development (E10 and E13.5, respectively), and are outlined in Figure 1. In mice, the cell types known, or thought, to be required for initiation of organogenesis are present by E10 (Figure 1a), with overt organ formation beginning at around E11. This earliest phase of organogenesis culminates in the formation of two primordia, each surrounded by a condensing mesenchymal capsule (Figure 1b), that still contact both the surface ectoderm and the pharyngeal endoderm. By E13.5 the parathyroid and thymus are separated into physically distinct organs (Figure 1c) and soon afterwards they reach their approximate adult positions within the embryo.
Current Opinion in Immunology 2003, 15:225–232 This review comes from a themed issue on Lymphocyte development Edited by Ellen Robey and Mark Schlissel 0952-7915/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0952-7915(03)00006-2
Abbreviations E day of embryonic development FGF fibroblast growth factor NCC neural crest cell RT-PCR reverse transcriptase polymerase chain reaction TEC thymic epithelial cell
Introduction The role of the thymus in T-cell development is well established. However, although fetal and adult thymocyte development has been studied in great detail, investigation of the early embryonic development of the thymus has been relatively neglected. Until recently, only a few studies of thymus organogenesis existed, and these often came to conflicting conclusions. In this article, we discuss recent studies that address lineage relationships in both the mesenchymal and epithelial cells of the thymus. In particular, we review the initial evidence for and against the two proposed models of early thymus organogenesis, and present recent evidence that overwhelmingly supports one model over the other. We www.current-opinion.com
Applying developmental principles to the study of thymus organogenesis The origins and relationships of the constituent cell types of any mature organ can only be unambiguously identified via lineage analysis; that is, the use of experimental methods to trace the embryonic origins of the cells that contribute to organ formation. Histological analysis of cell and tissue morphology or gene expression/marker studies alone are not sufficient to determine lineage, as these methods rely on properties of cells that can, and often do, change during embryonic development. In this regard, the fundamental difference between ectoderm, endoderm and mesoderm, which are designations of cell lineage or germ layer of origin, and epithelial and mesenchymal, which are descriptions of cell morphology, is of crucial importance. Just as cells can change their gene expression profile, they can change their morphology. For example, epithelial to mesenchymal transformations occur during kidney organogenesis, and neural crest cells (NCCs) transform from an epithelial cell type in the neural tube to a migratory mesenchymal cell type. However, cells cannot change their lineage — NCCs are always ectodermal, whether in an epithelial or mesenchymal morphology. Lineage analysis must therefore be based on cell labeling methods that allow the identification of specific cells and their descendents regardless of their morphology or normal patterns of gene expression (reviewed in [1]). Two of the most common methods for analyzing lineage in mice, Current Opinion in Immunology 2003, 15:225–232
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Figure 1
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The early stages of thymus/parathyroid organogenesis in mice. (a) Diagram of a parasaggital section of an E11.5 left third pouch and associated thymus/parathyroid primordium (blue), growing into the surrounding mesenchyme (green). Each primodium contains the precursors for both one thymus lobe and one parathyroid gland [5]. (b) By E12.5, the shared primordia (blue) have separated from the pharynx. Thymus and parathyroidspecific domains are still not morphologically distinguishable at this stage. (c) E13.5 embryo with parathyroids (red) separating from thymic lobes (blue); the thyroid gland is shown in pink. The pharyngeal endoderm (a), oesophagus (b) and trachea (c) are shown in beige. (b) and (c) are frontal views of pseudo-colored 3-dimensional reconstructions generated from actual mouse embryos. The thymus develops bilaterally as a result of interactions between the third pharyngeal pouch endoderm and surrounding neural crest mesenchyme. A possible role for the pharyngeal ectoderm (not shown) remains controversial.
dye labeling and Cre-recombinase-mediated lineage analysis are outlined in Box 1. The data from such lineage tracing experiments can be combined with data from other approaches, including explant or transplantation-based assays of developmental potential, analysis of mutant phenotypes, gene expression and morphological analyses, to construct a comprehensive model of organogenesis. Lineage analysis of thymus organogenesis: the neural crest
Although controversy surrounds the exact functions of NCCs in the thymus, NCCs are widely accepted as being the embryonic source for most, if not all, of the mesenchyme in the thymus. This contribution was originally identified by classical lineage analysis of NCCs in birds [2]. These chick–quail chimera studies demonstrated that NCCs form the embryonic mesenchymal capsule and are associated with vasculature in the fetal thymus, and they further demonstrated that NCCs did not contribute to the thymic reticular epithelium. More recently, a Crebased NCC lineage analysis was performed in transgenic mice [3], in which transient and specific expression of Crerecombinase in premigratory NCCs was used to activate a lacZ reporter gene. This analysis indicated that in mice, as in birds, NCCs form the capsule of the fetal thymus. Interestingly, the number of NC-derived cells in the thymic capsule was very low in newborn and postnatal thymus in comparison to the number present in the early stages of thymus organogenesis. These results suggest that NCCs are important during fetal development, but they may be reduced or even replaced by non-NCC-derived mesenchymal cells, such as mesoderm. This intriguing possibility needs to be thoroughly tested by looking specifically at the fetal, neonatal and adult thymus at a higher resolution Current Opinion in Immunology 2003, 15:225–232
than the previous studies. Therefore, new experimental approaches have re-opened the debate on the origins of thymic mesenchyme. Furthermore, evidence of a change in the origin of mesenchymal cells with the developmental age of the thymus could have implications for the function of mesenchyme within the thymus over time. Dual-origin versus single-origin models of thymic epithelial development
Considerable controversy has surrounded the origin of thymic epithelial cells, with conflicting interpretations reaching back over decades. The thymic epithelial rudiment originates from the third pharyngeal pouch, one of a series of a transient embryonic structures formed around E9 by ‘outpocketing’ of the pharyngeal endoderm. These pouches are matched by a series of opposing ectodermal invaginations called the pharyngeal clefts (see Figure 2 and Table 1). Although the origin of the thymic rudiment in the third pharyngeal pouch endoderm is well established, two opposing views dispute the contribution of the third pharyngeal cleft ectoderm. The first and most widely accepted model suggests that the thymus has a dual origin, with the cortical epithelium deriving from ectoderm and the medullary epithelium deriving from endoderm. The second model states that the thymic epithelium has a solely endodermal origin. Both models, together with a summary of the principal evidence supporting each one, are outlined in Table 1. Although morphological and functional evidence has been used to support both models, the most convincing arguments supporting each one are drawn from a single study. The most commonly cited evidence in favor of the dualorigin model is a comparative histological analysis of www.current-opinion.com
Developmental principles in thymus organogenesis Manley and Blackburn 227
Box 1 Dye labelling.
(a)
(b)
Dye labeling uses lipid-soluble fluorescent dyes to label cells in a specific region and tissue of the embryo. The embryo is then incubated in culture for a specified time (usually 1–2 days). Labeled cells and their progeny are then detected by fluorescence microscopy, either in whole-mount or in sections. In the example shown, dye is injected in the open neural tube at the hindbrain level at about E8.5 (a). After embryo culture, the dye is present in the neural tube and in neural crest cells that have migrated out of the neural tube and into the pharyngeal arches (b). This method is limited by the specificity of injection and by the ability to culture embryos for the desired developmental time window. Recombinase-mediated lineage analysis.
X uP
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lacZ
During recombinase-mediated lineage analysis, a transgenic strain carrying a tissue-specific promoter (tP) driving Cre recombinase is crossed to an indicator strain, in which a ubiquitous promoter (uP) drives expression of a marker gene only after a floxed transcriptional ‘stop’ is removed by Cre-mediated recombination. In the example shown, the Cre gene is activated only in cells at the midbrain-hindbrain boundary. The marker will be expressed in these cells as well as in all daughter cells at all subsequent stages of development. This method is limited by the availability of a very well characterized, tissue-specific promoter to drive Cre, preferably one whose expression is restricted both spatially and temporally.
thymus development in wild-type and nude mice [4,5]. This study drew two main conclusions. First, it stated that, although the medullary epithelium is derived from the endoderm of the third pharyngeal pouch, the cortical thymic epithelial compartment is derived from the pharyngeal ectoderm. Second, it concluded that the thymus defect in nude mice was due to a failure of ectodermal cells to contribute normally to the thymic rudiment; the endodermal portion of the nude thymic rudiment then fails to develop due to the absence of an ectodermderived inductive signal. This study forms the basis of the dual-origin model of thymus organogenesis currently included in most immunology textbooks [6,7]. Inherent in this model is the idea that the cortical and medullary compartments have separate embryological origins. An elegant functional study using chick–quail chimeras provides the most convincing support for a single endowww.current-opinion.com
dermal origin of the thymic epithelium [8]. In these experiments, pharyngeal endoderm was first isolated from quail embryos before development of the third pharyngeal pouch and then transplanted into the somatopleure of chick embryos. This seminal study demonstrated the extra-thymic origin of intrathymic T cells. Importantly, it also provided a clear demonstration that purified pharyngeal endoderm is sufficient for the generation of a thymus with both cortical and medullary compartments. Although the study did not directly test cell lineage, it provided a stringent assessment of the developmental potential of the pharyngeal endoderm, and therefore constitutes strong functional evidence in favor of a single origin. In our view, and that of others [9], the weight of evidence presented so far has thus favored the singleorigin over the dual-origin model, primarily because of the elegant experimental design and functional aspects Current Opinion in Immunology 2003, 15:225–232
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Figure 2
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pa1 pa2
Pax1/9 Eya1 Six1
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A model for the initial specification and patterning of the third pharyngeal pouch and the early development of the epithelial primordium. (a) Diagram of a coronal section through one half of the pharyngeal arch region of an E10.5 embryo (ectoderm is on the left, endoderm is on the right), showing the overlap in expression patterns for Hoxa3 (purple), Pax1/9, Eya 1 and Six1 (all turquoise) in the pharyngeal pouch endoderm. The third pharyngeal pouch (marked by) is the most anterior location where all of these genes are co-expressed. (b) At this stage, the third pharyngeal pouch is already at least partially patterned, as shown by the restricted expression of the parathyroid-specific marker Gcm2 (red; [14]), which is under the control of the Hox-Pax-Eya-Six pathway. Complementary expression of a thymus-specific marker has not yet been shown (blue domain), although Foxn1 can be detected in the third pouch by RT-PCR at this stage [15]. (c) At E11.25, the common primordium is beginning to develop from the pharyngeal endoderm and is detached from the ectoderm except for a very small domain (not shown). Gcm2 expression (red) remains stable, whereas Foxn1 (blue) is beginning to be expressed at high levels at the most distal aspect of the developing primordium [14]. (d) By E12, the remaining connections to the pharynx and the surface ectoderm undergo apoptosis (black), freeing the primordia to migrate ventro-medially and caudally. Condensation of the mesenchymal capsule (green) is now also apparent. The parathyroid and thymus-specific domains are clearly established by Gcm2 and Foxn1 expression, and will begin to physically separate by E13.
of the chick–quail chimera study. However, controversy has persisted because of a lack of rigorous lineage studies designed specifically to address cell lineage in the mammalian thymus.
Recent studies in mice that support the single-origin model of thymic epithelium development
Several recent papers have used new experimental approaches to investigate cell lineage relationships in
Table 1 Evidence for the two major models of thymic epithelial cell origins. Dual origin model
Type of study
References Results/conclusion
Morphological studies in pig
[41]
Morphological studies in human Morphological studies in mouse
[42]
Functional study in chick
[46]
Marker studies in mouse and human
–
[4,5]
Single origin model
Reference
– Two distinct embryological sources for epithelial elements, with inner cells of endodermal origin, outer ectodermal; this division represents primitive medulla and cortex. [43,44] Compared wild-type and nude mice; ectoderm forms cortex, endoderm forms medulla. Surgical ablation of ectoderm results in hypoplastic thymus; ectoderm is required for thymus development. –
[45]
[8]
[47–52]
Results/conclusion –
Histological analysis of human fetal development; no evidence for ectodermal contribution to thymus. Histochemical analysis of wild-type thymus development; ectoderm does not contribute to thymus. Transplanted quail endoderm ectopically into chick embryo; endoderm is sufficient to generate thymus.
Immunohistochemical analysis of human fetal thymi [47], cultured mouse thymic epithelial cells [49] and human thymic tumors [48], and ultrastructural studies [50–52]; single origin for cortical and medullary TECs.
In the figure above, red ¼ ectoderm; blue ¼ endoderm. It should be noted that this procedure can also cause damage to the underlying endoderm. ‘—’ indicates that the studies that were carried out were in favor of either the dual- or single-origin model.
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the thymic epithelium of mice. A well-designed chimera analysis demonstrated that the thymic medulla initially arises as a series of clonal islets [10], apparently arguing against the derivation of the medullary compartment from a single epithelial sheet. Strong support of a single origin for all thymic epithelium is also provided by two papers that describe a discrete population of embryonic thymic epithelial cells (TECs), identified by monoclonal antibodies MTS20 and MTS24, that can differentiate into all known TEC types and, on transplantation, is sufficient to generate a thymus capable of fully supporting T-cell development [11,12]. These cells are present as early as E12.5, at which time they are homogeneous with respect to currently available markers [11], suggesting that they represent a common progenitor cell for all mature thymic epithelia. However, clonal analysis of the developmental potential of these cells has yet to be performed. Furthermore, similar to the chick–quail chimera study described above [8], these studies only address the potency of this cell population, but do not formally address lineage. To definitively address the embryonic origins of the cortical and medullary thymic epithelial compartments, we have performed a lineage analysis in mice, using dye labeling to trace the fate of ectodermal cells during early thymus organogenesis. In this experiment, pharyngeal region surface ectoderm was specifically labeled at E10.5, and the embryos cultured for a further 30 hours. Subsequent analysis found no evidence of labeled cells in the thymus at E11.5 and, importantly, demonstrated normal thymus development during the culture period, as judged by morphology and by normal initiation of Foxn1 expression (J Gordon, V Wilson, NR Manley and CC Blackburn, unpublished data). This experiment is a direct test of cell lineage, and provides clear evidence that the third pharyngeal cleft ectoderm does not contribute cells to the thymus. To complement this study we also performed a transplant-based functional analysis of pharyngeal endoderm, modeled on the chick–quail chimera studies described above [8]. At E9.0 prospective third pharyngeal pouch endoderm was isolated by enzymatic and manual dissection and transplanted under the kidney capsule of nude mice. These transplants generated a thymus positive for both cortical and medullary markers, and capable of generating both CD4þ and CD8þ T cells. These results show that the pharyngeal pouch endoderm is able to produce a functional thymic epithelium in mice. Taken together, these complimentary approaches provide compelling evidence in support of the single-origin model of thymic epithelium development. Molecular analysis of thymus organogenesis
Analysis of Foxn1, a forkhead transcription factor identified as the gene mutated in the athymic nude mouse [13], is also consistent with a single endodermal origin. Foxn1 is of special interest, as the dual-origin model is partly based on the conclusion that the morphological defect in nude www.current-opinion.com
mice is in ectodermal development [5]. Since Foxn1 was cloned, it has been shown to be highly expressed in the endodermal rudiment from E11.25, but is not detected in the surface ectoderm by in situ hybridization (NR Manley, CC Blackburn, unpublished data; [14]), and is also expressed at low levels in the third pharyngeal pouch from E10.5 [15]. These results alone do not prove an endodermal lineage for the entire rudiment, as Foxn1 expression could be turned on de novo in all cells as they become a part of the thymic primordium, regardless of their lineage. However, a chimera analysis demonstrated a cell autonomous requirement for Foxn1 for the development of all TECs, rather than for cortical epithelium alone (as predicted by Cordier and colleagues [4,5]), and further indicated a single phenotype for all presumptive TECs unable to express Foxn1 [16]. The combination of the Foxn1 expression pattern with functional analysis is therefore inconsistent with the conclusions of the earlier histological study that supported a dual origin, thereby undermining an important supporting premise for that model. The emerging model
Thus, recent evidence from our laboratories, and from other investigators, favors a model of thymus organogenesis in which the common thymus/parathyroid primordium arises solely from the endoderm of the third pharyngeal pouch (Figure 2). This model is consistent with a common progenitor giving rise to all thymic epithelial cell types, as suggested by recent studies described above [11,12]. There is significant molecular evidence pertaining to the regulation of thymus development that is consistent with the single-origin model. Analysis of mutant phenotypes suggests that the initial development of the endodermal primordium requires the action of a Hoxa3-Pax1/9-Eya1-Six1 transcription factor cascade acting in the pouch endoderm [17–20,21]. These transcription factors are required to initiate thymus organogenesis and to pattern both the pouch and the primordium into parathyroid and thymus domains by regulating the expression of the parathyroid-specific marker Gcm2, and possibly also Foxn1 [14,21,22]. The primordium is subsequently encapsulated by NCCs, which promote early growth of the rudiment, at least in part, through the expression of Fgf7/10, and may influence subsequent patterning/differentiation ([23]; see also Update). Foxn1 is then required for the differentiation [16] and proliferation [24] of all TECs.
Conclusions As we have discussed in this review, the investigation of the earliest stages of thymus organogenesis has yielded exciting new evidence regarding the origins of both mesenchymal and epithelial cells in the thymus. Because of the progress made by these studies, very specific questions concerning lineage contributions and cellular interactions in early thymus organogenesis can now be Current Opinion in Immunology 2003, 15:225–232
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addressed. Some of the key remaining questions are outlined briefly below. First, what induces the initial formation of the primordium from the third pharyngeal pouch? It is not yet clear whether the initial information required to induce formation of the thymic rudiment is entirely intrinsic to the endoderm via the Hox-Pax-Eya-Six pathway, or if it requires an additional extrinsic signal from surrounding cell types. By analogy with other organs, and consistent with results from endoderm transplantation experiments described above, we expect that the endoderm carries an intrinsic pattern or identity [25]. However, an additional external signal from the mesenchyme, or even the ectoderm, may be required to induce organ formation. Candidate signals include fibroblast growth factors (FGFs; [23,26,27]), bone morphogenic proteins (BMPs; [28,29]), wingless transcription factors (Wnts; [15]) and sonic hedgehog (Shh; [30]). Although all of these pathways have been implicated in thymus development and/or thymocyte differentiation, none have yet been linked to the initiation of organogenesis. Investigations into the source and nature of the initiating signal(s) will thus be of great interest. Second, do NCCs provide any patterning signals to the developing primordium or do they simply promote survival and/or proliferation? What, if anything, is the physical and functional contribution of NCCs to the adult thymus? Although there is general agreement that NCCs play an important role in early thymus organogenesis, the function of thymic mesenchyme remains controversial. Classic experiments in both chick [31] and mice [32], and a more recent analysis of FGF receptor2IIIb mutant mice [23] all support a crucial role for NCCs in supporting the growth of the early thymic epithelial primordium. However, controversy surrounds a potential function for mesenchyme in directly promoting thymocyte development [33–36]. In addition, the role of mesenchyme in regulating subsequent patterning of the cortical and medullary compartments, and in the differentiation and maintenance of particular TEC types remains to be addressed. Third, what is the role of other cell types in the thymus and in thymus organogenesis? Although recent studies have provided convincing evidence that hematopoietic cells are not required for thymus organogenesis or initial TEC differentiation [37], other non-bone-marrowderived stromal cell types in the thymus have been implicated in early thymus organogenesis and patterning; the contribution of endothelial cells in particular is one of the least understood. Recent data from studies of the pancreas and liver have provided the precedent, by suggesting that endothelial cells and vasculogenesis could be important regulators of patterning and differentiation [38,39]. This theory is supported by a recent study that Current Opinion in Immunology 2003, 15:225–232
provides evidence that the development of blood vessels in the thymus provides organizing signals for the medullary compartment [40]. Further work is required to investigate the initial entry of endothelial cells into the thymic primordium and their possible functions, including potential interactions with mesenchyme. Further investigation of these and other questions should permit real progress towards the ultimate goal of linking the morphological events of early thymus organogenesis with the transcription factor and signaling pathways that control these processes.
Update Recent work used a Noggin transgene to inhibit the production of BMP-dependent neural crest cells in the hindbrain, resulting in absent or ectopic hypoplastic thymus formation, among other defects [53]. Marker gene analysis also suggested that neural crest cells may have a role in inducing or maintaining Pax1 expression in the endoderm, suggesting that the expression of at least some thymus-related genes in the pharyngeal pouch endoderm may not be regulated by a genetic pathway autonomous to the endoderm.
Acknowledgements We wish to thank Julie Gordon for helpful comments and for sharing her unpublished data. CCB is supported by the Leukaemia Research Fund; NRM is supported by National Institutes of Health/National Institute of Child Health and Human Development (HD035920). The authors are also supported by a Biomedical collaboration grant from the Wellcome Trust.
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10. Rodewald HR, Paul S, Haller C, Bluethmann H, Blum C: Thymus medulla consisting of epithelial islets each derived from a single progenitor. Nature 2001, 414:763-768. This elegant study used allophenic chimeric mice to demonstrate that the thymic medulla arises as a series of independent clonal islets. Further analysis of medullary development using reaggregated fetal thymic organ cultures grafted under the kidney capsule revealed a putative thymus medulla epithelial stem cell activity that persisted until at least E16.5. 11. Bennett AR, Farley A, Blair NF, Gordon J, Sharp L, Blackburn CC: Identification and characterization of thymic epithelial progenitor cells. Immunity 2002, 16:803-814. See annotation to [12]. 12. Gill J, Malin M, Hollander GA, Boyd R: Generation of a complete thymic microenvironment by MTS24(þ) thymic epithelial cells. Nat Immunol 2002, 3:635-642. These important studies [11,12] provide the first functional evidence that a common progenitor cell may give rise to all TEC types. The authors identify a discrete population of immature embryonic TECs (using the monoclonal antibodies MTS20 [11] and MTS24 [11,12]) which, upon transplantation, is sufficient to establish an organized, fully functional thymus containing all known TEC populations. 13. Nehls M, Pfeifer D, Schorpp M, Hedrich H, Boehm T: New member of the winged-helix protein family disrupted in mouse and rat nude mutations. Nature 1994, 372:103-107. 14. Gordon J, Bennett AR, Blackburn CC, Manley NR: Gcm2 and Foxn1 mark early parathyroid- and thymus-specific domains in the developing third pharyngeal pouch. Mech Dev 2001, 103:141-143. This gene expression study provides the first evidence that the shared parathyroid-thymus primordium is patterned into organ-specific domains from its earliest formation, and that the third pharyngeal pouch is patterned to some extent by Gcm2 expression, even before primordium formation. 15. Balciunaite G, Keller MP, Balciunaite E, Piali L, Zuklys S, Mathieu YD, Gill J, Boyd R, Sussman DJ, Hollander GA: Wnt glycoproteins regulate the expression of FoxN1, the gene defective in nude mice. Nat Immunol 2002, 3:1102-1108. 16. Blackburn CC, Augustine CL, Li R, Harvey RP, Malin MA, Boyd RL, Miller JF, Morahan G: The nu gene acts cell-autonomously and is required for differentiation of thymic epithelial progenitors. Proc Natl Acad Sci USA 1996, 93:5742-5746. 17. Chisaka O, Capecchi MR: Regionally restricted developmental defects resulting from targeted disruption of the mouse homeobox gene hox-1.5. Nature 1991, 350:473-479. 18. Manley NR, Capecchi MR: The role of Hoxa-3 in mouse thymus and thyroid development. Development 1995, 121:1989-2003. 19. Hetzer-Egger C, Schorpp M, Haas-Assenbaum A, Balling R, Peters H, Boehm T: Thymopoiesis requires Pax9 function in thymic epithelial cells. Eur J Immunol 2002, 32:1175-1181. 20. Wallin J, Eibel H, Neubu¨ ser A, Wilting J, Koseki H, Balling R: Pax1 is expressed during development of the thymus epithelium and is required for normal T cell maturation. Development 1996, 122:23-30. 21. Xu PX, Zheng W, Laclef C, Maire P, Maas RL, Peters H, Xu X: Eya1 is required for the morphogenesis of mammalian thymus, parathyroid and thyroid. Development 2002, 129:3033-3044. See annotation to [22]. 22. Su D, Ellis S, Napier A, Lee K, Manley NR: Hoxa3 and pax1 regulate epithelial cell death and proliferation during thymus and parathyroid organogenesis. Dev Biol 2001, 236:316-329. These two studies [21,22] identify the Hox-Pax-Eya-Six molecular pathway that controls both initial formation of the thymus/parathyroid primordium and patterning into organ-specific domains via control of Gcm2 expression. 23. Revest JM, Suniara RK, Kerr K, Owen JJ, Dickson C: Development of the thymus requires signaling through the fibroblast growth factor receptor r2-IIIb. J Immunol 2001, 167:1954-1961. This genetic analysis of the role of FGF signaling in early thymus organogenesis shows that an FGF signal from the mesenchyme is required for growth (but not initial formation) of the thymic rudiment. However, differentiation of TECs remains largely normal, showing that differentiation and proliferation of TECs are controlled at least in part by distinct signals. www.current-opinion.com
24. Itoi M, Kawamoto H, Katsura Y, Amagai T: Two distinct steps of immigration of hematopoietic progenitors into the early thymus anlage. Int Immunol 2001, 13:1203-1211. 25. Grapin-Botton A, Melton DA: Endoderm development: from patterning to organogenesis. Trends Genet 2000, 16:124-130. 26. Galy A, Jolivet M, Jolivet-Reynaud C, Hadden J: Fibroblast growth factor (FGF) and an FGF-like molecule in pituitary extracts stimulate thymic epithelial cell proliferation. Thymus 1990, 15:199-211. 27. Erickson M, Morkowski S, Lehar S, Gillard G, Beers C, Dooley J, Rubin JS, Rudensky A, Farr AG: Regulation of thymic epithelium by keratinocyte growth factor. Blood 2002, 100:3269-3278. 28. Graf D, Nethisinghe S, Palmer DB, Fisher AG, Merkenschlager M: The developmentally regulated expression of Twisted gastrulation reveals a role for bone morphogenetic proteins in the control of T cell development. J Exp Med 2002, 196:163-171. 29. Hager-Theodorides AL, Outram SV, Shah DK, Sacedon R, Shrimpton RE, Vicente A, Varas A, Crompton T: Bone morphogenetic protein 2/4 signaling regulates early thymocyte differentiation. J Immunol 2002, 169:5496-5504. 30. Outram SV, Varas A, Pepicelli CV, Crompton T: Hedgehog signaling regulates differentiation from double-negative to double-positive thymocyte. Immunity 2000, 13:187-197. 31. Bockman DE, Kirby ML: Dependence of thymus development on derivatives of the neural crest. Science 1984, 223:498-500. 32. Auerbach R: Morphogenetic interactions in the development of the mouse thymus gland. Dev Biol 1960, 2:271-284. 33. Suniara RK, Jenkinson EJ, Owen JJ: An essential role for thymic mesenchyme in early T cell development. J Exp Med 2000, 191:1051-1056. 34. Anderson G, Jenkinson EJ, Moore NC, Owen JJ: MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus. Nature 1993, 362:70-73. 35. Owen JJ, McLoughlin DE, Suniara RK, Jenkinson EJ: The role of mesenchyme in thymus development. Curr Top Microbiol Immunol 2000, 251:133-137. 36. Lilic M, Santori FR, Neilson EG, Frey AB, Vukmanovic S: The role of fibroblasts in thymocyte-positive selection. J Immunol 2002, 169:4945-4950. 37. Klug DB, Carter C, Gimenez-Conti IB, Richie ER: Cutting edge: thymocyte-independent and thymocyte-dependent phases of epithelial patterning in the fetal thymus. J Immunol 2002, 169:2842-2845. This study compares keratin gene expression in Ikaros and Rag2,gc null mutants to their expression in wild-type embryos, showing that initial TEC differentiation occurs normally even in the absence of lymphocyte progenitors. 38. Lammert E, Cleaver O, Melton D: Induction of pancreatic differentiation by signals from blood vessels. Science 2001, 294:564-567. 39. Matsumoto K, Yoshitomi H, Rossant J, Zaret KS: Liver organogenesis promoted by endothelial cells prior to vascular function. Science 2001, 294:559-563. 40. Anderson M, Anderson SK, Farr AG: Thymic vasculature: organizer of the medullary epithelial compartment? Int Immunol 2000, 12:1105-1110. 41. Badertscher J: The development of the thymus in the pig II: morphogenesis. Am J Anat 1915, 17:317. 42. Norris E: The morphogenesis and histogenesis of the thymus gland in man: in which the origin of the Hassall’s corpuscles of the human thymus is discovered. Contrib Embryol 1938, 27:193. 43. Kingsbury B: The development of the human pharynx I: the pharyngeal derivatives. Am J Anat 1915, 18:329-386. 44. Weller G: Development of the thyroid, parathyroid and thymus glands in man. Contrib Embryol 1933, 24:93-142. 45. Smith C: Studies on the thymus of the mammal XIV: Histology and histochemistry of embryonic and early postnatal Current Opinion in Immunology 2003, 15:225–232
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thymuses of C57BL/6 and AKR strain mice. Am J Anat 1965, 116:611-630. 46. Hammond W: Origin of thymus in the chick embryo. J Morphol 1954, 95:501-521. 47. Haynes BF, Heinly CS: Early human T cell development: analysis of the human thymus at the time of initial entry of hematopoietic stem cells into the fetal thymic microenvironment. J Exp Med 1995, 181:1445-1458. 48. Schluep M, Willcox N, Ritter MA, Newsom-Davis J, Larche M, Brown AN: Myasthenia gravis thymus: clinical, histological and culture correlations. J Autoimmun 1988, 1:445-467. 49. Ropke C, Van Soest P, Platenburg PP, Van Ewijk W. A common stem cell for murine cortical and medullary thymic epithelial cells? Dev Immunol 1995, 4:149-156.
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50. von Gaudecker B, Muller-Hermelink H: Ontogeny and organization of the stationary non-lymphoid cells in the human thymus. Cell Tissue Res 1980, 207:287-306. 51. von Gaudecker B, Steinmann GG, Hansmann ML, Harpprecht J, Milicevic NM, Muller-Hermelink HK: Immunohistochemical characterization of the thymic microenvironment. A lightmicroscopic and ultrastructural immunocytochemical study. Cell Tissue Res 1986, 244:403-412. 52. von Gaudecker B: The development of the human thymus microenvironment. Curr Top Pathol 1986, 75:1-41. 53. Ohnemus S, Kanzler B, Jerome-Majewska LA, Papaioannou VE, Boehm T, Mallo M: Aortic arch and pharyngeal phenotype in the absence of BMP-dependent neural crest in the mouse. Mech Dev 2002, 119:127-135.
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A developmental look at thymus organogenesis: where do the non-hematopoietic cells in the thymus come from? Opinion Nancy R Manley and C Clare Blackburny 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. Addresses Correspondence: Nancy R Manley: Department of Genetics, B420A Life Sciences Building, University of Georgia, Athens, GA 30602, USA e-mail: [email protected] y Institute for Stem Cell Research, The University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh EH9 3JQ, UK
will then integrate this model with current knowledge of the transcription factor pathways that mediate thymus development.
The basics of thymus organogenesis The morphological events of early thymus organogenesis occur between days 10 and 13.5 of embryonic development (E10 and E13.5, respectively), and are outlined in Figure 1. In mice, the cell types known, or thought, to be required for initiation of organogenesis are present by E10 (Figure 1a), with overt organ formation beginning at around E11. This earliest phase of organogenesis culminates in the formation of two primordia, each surrounded by a condensing mesenchymal capsule (Figure 1b), that still contact both the surface ectoderm and the pharyngeal endoderm. By E13.5 the parathyroid and thymus are separated into physically distinct organs (Figure 1c) and soon afterwards they reach their approximate adult positions within the embryo.
Current Opinion in Immunology 2003, 15:225–232 This review comes from a themed issue on Lymphocyte development Edited by Ellen Robey and Mark Schlissel 0952-7915/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0952-7915(03)00006-2
Abbreviations E day of embryonic development FGF fibroblast growth factor NCC neural crest cell RT-PCR reverse transcriptase polymerase chain reaction TEC thymic epithelial cell
Introduction The role of the thymus in T-cell development is well established. However, although fetal and adult thymocyte development has been studied in great detail, investigation of the early embryonic development of the thymus has been relatively neglected. Until recently, only a few studies of thymus organogenesis existed, and these often came to conflicting conclusions. In this article, we discuss recent studies that address lineage relationships in both the mesenchymal and epithelial cells of the thymus. In particular, we review the initial evidence for and against the two proposed models of early thymus organogenesis, and present recent evidence that overwhelmingly supports one model over the other. We www.current-opinion.com
Applying developmental principles to the study of thymus organogenesis The origins and relationships of the constituent cell types of any mature organ can only be unambiguously identified via lineage analysis; that is, the use of experimental methods to trace the embryonic origins of the cells that contribute to organ formation. Histological analysis of cell and tissue morphology or gene expression/marker studies alone are not sufficient to determine lineage, as these methods rely on properties of cells that can, and often do, change during embryonic development. In this regard, the fundamental difference between ectoderm, endoderm and mesoderm, which are designations of cell lineage or germ layer of origin, and epithelial and mesenchymal, which are descriptions of cell morphology, is of crucial importance. Just as cells can change their gene expression profile, they can change their morphology. For example, epithelial to mesenchymal transformations occur during kidney organogenesis, and neural crest cells (NCCs) transform from an epithelial cell type in the neural tube to a migratory mesenchymal cell type. However, cells cannot change their lineage — NCCs are always ectodermal, whether in an epithelial or mesenchymal morphology. Lineage analysis must therefore be based on cell labeling methods that allow the identification of specific cells and their descendents regardless of their morphology or normal patterns of gene expression (reviewed in [1]). Two of the most common methods for analyzing lineage in mice, Current Opinion in Immunology 2003, 15:225–232
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Figure 1
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(b)
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The early stages of thymus/parathyroid organogenesis in mice. (a) Diagram of a parasaggital section of an E11.5 left third pouch and associated thymus/parathyroid primordium (blue), growing into the surrounding mesenchyme (green). Each primodium contains the precursors for both one thymus lobe and one parathyroid gland [5]. (b) By E12.5, the shared primordia (blue) have separated from the pharynx. Thymus and parathyroidspecific domains are still not morphologically distinguishable at this stage. (c) E13.5 embryo with parathyroids (red) separating from thymic lobes (blue); the thyroid gland is shown in pink. The pharyngeal endoderm (a), oesophagus (b) and trachea (c) are shown in beige. (b) and (c) are frontal views of pseudo-colored 3-dimensional reconstructions generated from actual mouse embryos. The thymus develops bilaterally as a result of interactions between the third pharyngeal pouch endoderm and surrounding neural crest mesenchyme. A possible role for the pharyngeal ectoderm (not shown) remains controversial.
dye labeling and Cre-recombinase-mediated lineage analysis are outlined in Box 1. The data from such lineage tracing experiments can be combined with data from other approaches, including explant or transplantation-based assays of developmental potential, analysis of mutant phenotypes, gene expression and morphological analyses, to construct a comprehensive model of organogenesis. Lineage analysis of thymus organogenesis: the neural crest
Although controversy surrounds the exact functions of NCCs in the thymus, NCCs are widely accepted as being the embryonic source for most, if not all, of the mesenchyme in the thymus. This contribution was originally identified by classical lineage analysis of NCCs in birds [2]. These chick–quail chimera studies demonstrated that NCCs form the embryonic mesenchymal capsule and are associated with vasculature in the fetal thymus, and they further demonstrated that NCCs did not contribute to the thymic reticular epithelium. More recently, a Crebased NCC lineage analysis was performed in transgenic mice [3], in which transient and specific expression of Crerecombinase in premigratory NCCs was used to activate a lacZ reporter gene. This analysis indicated that in mice, as in birds, NCCs form the capsule of the fetal thymus. Interestingly, the number of NC-derived cells in the thymic capsule was very low in newborn and postnatal thymus in comparison to the number present in the early stages of thymus organogenesis. These results suggest that NCCs are important during fetal development, but they may be reduced or even replaced by non-NCC-derived mesenchymal cells, such as mesoderm. This intriguing possibility needs to be thoroughly tested by looking specifically at the fetal, neonatal and adult thymus at a higher resolution Current Opinion in Immunology 2003, 15:225–232
than the previous studies. Therefore, new experimental approaches have re-opened the debate on the origins of thymic mesenchyme. Furthermore, evidence of a change in the origin of mesenchymal cells with the developmental age of the thymus could have implications for the function of mesenchyme within the thymus over time. Dual-origin versus single-origin models of thymic epithelial development
Considerable controversy has surrounded the origin of thymic epithelial cells, with conflicting interpretations reaching back over decades. The thymic epithelial rudiment originates from the third pharyngeal pouch, one of a series of a transient embryonic structures formed around E9 by ‘outpocketing’ of the pharyngeal endoderm. These pouches are matched by a series of opposing ectodermal invaginations called the pharyngeal clefts (see Figure 2 and Table 1). Although the origin of the thymic rudiment in the third pharyngeal pouch endoderm is well established, two opposing views dispute the contribution of the third pharyngeal cleft ectoderm. The first and most widely accepted model suggests that the thymus has a dual origin, with the cortical epithelium deriving from ectoderm and the medullary epithelium deriving from endoderm. The second model states that the thymic epithelium has a solely endodermal origin. Both models, together with a summary of the principal evidence supporting each one, are outlined in Table 1. Although morphological and functional evidence has been used to support both models, the most convincing arguments supporting each one are drawn from a single study. The most commonly cited evidence in favor of the dualorigin model is a comparative histological analysis of www.current-opinion.com
Developmental principles in thymus organogenesis Manley and Blackburn 227
Box 1 Dye labelling.
(a)
(b)
Dye labeling uses lipid-soluble fluorescent dyes to label cells in a specific region and tissue of the embryo. The embryo is then incubated in culture for a specified time (usually 1–2 days). Labeled cells and their progeny are then detected by fluorescence microscopy, either in whole-mount or in sections. In the example shown, dye is injected in the open neural tube at the hindbrain level at about E8.5 (a). After embryo culture, the dye is present in the neural tube and in neural crest cells that have migrated out of the neural tube and into the pharyngeal arches (b). This method is limited by the specificity of injection and by the ability to culture embryos for the desired developmental time window. Recombinase-mediated lineage analysis.
X uP
Stop
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Marker gene
Cre
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lacZ
During recombinase-mediated lineage analysis, a transgenic strain carrying a tissue-specific promoter (tP) driving Cre recombinase is crossed to an indicator strain, in which a ubiquitous promoter (uP) drives expression of a marker gene only after a floxed transcriptional ‘stop’ is removed by Cre-mediated recombination. In the example shown, the Cre gene is activated only in cells at the midbrain-hindbrain boundary. The marker will be expressed in these cells as well as in all daughter cells at all subsequent stages of development. This method is limited by the availability of a very well characterized, tissue-specific promoter to drive Cre, preferably one whose expression is restricted both spatially and temporally.
thymus development in wild-type and nude mice [4,5]. This study drew two main conclusions. First, it stated that, although the medullary epithelium is derived from the endoderm of the third pharyngeal pouch, the cortical thymic epithelial compartment is derived from the pharyngeal ectoderm. Second, it concluded that the thymus defect in nude mice was due to a failure of ectodermal cells to contribute normally to the thymic rudiment; the endodermal portion of the nude thymic rudiment then fails to develop due to the absence of an ectodermderived inductive signal. This study forms the basis of the dual-origin model of thymus organogenesis currently included in most immunology textbooks [6,7]. Inherent in this model is the idea that the cortical and medullary compartments have separate embryological origins. An elegant functional study using chick–quail chimeras provides the most convincing support for a single endowww.current-opinion.com
dermal origin of the thymic epithelium [8]. In these experiments, pharyngeal endoderm was first isolated from quail embryos before development of the third pharyngeal pouch and then transplanted into the somatopleure of chick embryos. This seminal study demonstrated the extra-thymic origin of intrathymic T cells. Importantly, it also provided a clear demonstration that purified pharyngeal endoderm is sufficient for the generation of a thymus with both cortical and medullary compartments. Although the study did not directly test cell lineage, it provided a stringent assessment of the developmental potential of the pharyngeal endoderm, and therefore constitutes strong functional evidence in favor of a single origin. In our view, and that of others [9], the weight of evidence presented so far has thus favored the singleorigin over the dual-origin model, primarily because of the elegant experimental design and functional aspects Current Opinion in Immunology 2003, 15:225–232
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Figure 2
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pa1 pa2
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A model for the initial specification and patterning of the third pharyngeal pouch and the early development of the epithelial primordium. (a) Diagram of a coronal section through one half of the pharyngeal arch region of an E10.5 embryo (ectoderm is on the left, endoderm is on the right), showing the overlap in expression patterns for Hoxa3 (purple), Pax1/9, Eya 1 and Six1 (all turquoise) in the pharyngeal pouch endoderm. The third pharyngeal pouch (marked by) is the most anterior location where all of these genes are co-expressed. (b) At this stage, the third pharyngeal pouch is already at least partially patterned, as shown by the restricted expression of the parathyroid-specific marker Gcm2 (red; [14]), which is under the control of the Hox-Pax-Eya-Six pathway. Complementary expression of a thymus-specific marker has not yet been shown (blue domain), although Foxn1 can be detected in the third pouch by RT-PCR at this stage [15]. (c) At E11.25, the common primordium is beginning to develop from the pharyngeal endoderm and is detached from the ectoderm except for a very small domain (not shown). Gcm2 expression (red) remains stable, whereas Foxn1 (blue) is beginning to be expressed at high levels at the most distal aspect of the developing primordium [14]. (d) By E12, the remaining connections to the pharynx and the surface ectoderm undergo apoptosis (black), freeing the primordia to migrate ventro-medially and caudally. Condensation of the mesenchymal capsule (green) is now also apparent. The parathyroid and thymus-specific domains are clearly established by Gcm2 and Foxn1 expression, and will begin to physically separate by E13.
of the chick–quail chimera study. However, controversy has persisted because of a lack of rigorous lineage studies designed specifically to address cell lineage in the mammalian thymus.
Recent studies in mice that support the single-origin model of thymic epithelium development
Several recent papers have used new experimental approaches to investigate cell lineage relationships in
Table 1 Evidence for the two major models of thymic epithelial cell origins. Dual origin model
Type of study
References Results/conclusion
Morphological studies in pig
[41]
Morphological studies in human Morphological studies in mouse
[42]
Functional study in chick
[46]
Marker studies in mouse and human
–
[4,5]
Single origin model
Reference
– Two distinct embryological sources for epithelial elements, with inner cells of endodermal origin, outer ectodermal; this division represents primitive medulla and cortex. [43,44] Compared wild-type and nude mice; ectoderm forms cortex, endoderm forms medulla. Surgical ablation of ectoderm results in hypoplastic thymus; ectoderm is required for thymus development. –
[45]
[8]
[47–52]
Results/conclusion –
Histological analysis of human fetal development; no evidence for ectodermal contribution to thymus. Histochemical analysis of wild-type thymus development; ectoderm does not contribute to thymus. Transplanted quail endoderm ectopically into chick embryo; endoderm is sufficient to generate thymus.
Immunohistochemical analysis of human fetal thymi [47], cultured mouse thymic epithelial cells [49] and human thymic tumors [48], and ultrastructural studies [50–52]; single origin for cortical and medullary TECs.
In the figure above, red ¼ ectoderm; blue ¼ endoderm. It should be noted that this procedure can also cause damage to the underlying endoderm. ‘—’ indicates that the studies that were carried out were in favor of either the dual- or single-origin model.
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Developmental principles in thymus organogenesis Manley and Blackburn 229
the thymic epithelium of mice. A well-designed chimera analysis demonstrated that the thymic medulla initially arises as a series of clonal islets [10], apparently arguing against the derivation of the medullary compartment from a single epithelial sheet. Strong support of a single origin for all thymic epithelium is also provided by two papers that describe a discrete population of embryonic thymic epithelial cells (TECs), identified by monoclonal antibodies MTS20 and MTS24, that can differentiate into all known TEC types and, on transplantation, is sufficient to generate a thymus capable of fully supporting T-cell development [11,12]. These cells are present as early as E12.5, at which time they are homogeneous with respect to currently available markers [11], suggesting that they represent a common progenitor cell for all mature thymic epithelia. However, clonal analysis of the developmental potential of these cells has yet to be performed. Furthermore, similar to the chick–quail chimera study described above [8], these studies only address the potency of this cell population, but do not formally address lineage. To definitively address the embryonic origins of the cortical and medullary thymic epithelial compartments, we have performed a lineage analysis in mice, using dye labeling to trace the fate of ectodermal cells during early thymus organogenesis. In this experiment, pharyngeal region surface ectoderm was specifically labeled at E10.5, and the embryos cultured for a further 30 hours. Subsequent analysis found no evidence of labeled cells in the thymus at E11.5 and, importantly, demonstrated normal thymus development during the culture period, as judged by morphology and by normal initiation of Foxn1 expression (J Gordon, V Wilson, NR Manley and CC Blackburn, unpublished data). This experiment is a direct test of cell lineage, and provides clear evidence that the third pharyngeal cleft ectoderm does not contribute cells to the thymus. To complement this study we also performed a transplant-based functional analysis of pharyngeal endoderm, modeled on the chick–quail chimera studies described above [8]. At E9.0 prospective third pharyngeal pouch endoderm was isolated by enzymatic and manual dissection and transplanted under the kidney capsule of nude mice. These transplants generated a thymus positive for both cortical and medullary markers, and capable of generating both CD4þ and CD8þ T cells. These results show that the pharyngeal pouch endoderm is able to produce a functional thymic epithelium in mice. Taken together, these complimentary approaches provide compelling evidence in support of the single-origin model of thymic epithelium development. Molecular analysis of thymus organogenesis
Analysis of Foxn1, a forkhead transcription factor identified as the gene mutated in the athymic nude mouse [13], is also consistent with a single endodermal origin. Foxn1 is of special interest, as the dual-origin model is partly based on the conclusion that the morphological defect in nude www.current-opinion.com
mice is in ectodermal development [5]. Since Foxn1 was cloned, it has been shown to be highly expressed in the endodermal rudiment from E11.25, but is not detected in the surface ectoderm by in situ hybridization (NR Manley, CC Blackburn, unpublished data; [14]), and is also expressed at low levels in the third pharyngeal pouch from E10.5 [15]. These results alone do not prove an endodermal lineage for the entire rudiment, as Foxn1 expression could be turned on de novo in all cells as they become a part of the thymic primordium, regardless of their lineage. However, a chimera analysis demonstrated a cell autonomous requirement for Foxn1 for the development of all TECs, rather than for cortical epithelium alone (as predicted by Cordier and colleagues [4,5]), and further indicated a single phenotype for all presumptive TECs unable to express Foxn1 [16]. The combination of the Foxn1 expression pattern with functional analysis is therefore inconsistent with the conclusions of the earlier histological study that supported a dual origin, thereby undermining an important supporting premise for that model. The emerging model
Thus, recent evidence from our laboratories, and from other investigators, favors a model of thymus organogenesis in which the common thymus/parathyroid primordium arises solely from the endoderm of the third pharyngeal pouch (Figure 2). This model is consistent with a common progenitor giving rise to all thymic epithelial cell types, as suggested by recent studies described above [11,12]. There is significant molecular evidence pertaining to the regulation of thymus development that is consistent with the single-origin model. Analysis of mutant phenotypes suggests that the initial development of the endodermal primordium requires the action of a Hoxa3-Pax1/9-Eya1-Six1 transcription factor cascade acting in the pouch endoderm [17–20,21]. These transcription factors are required to initiate thymus organogenesis and to pattern both the pouch and the primordium into parathyroid and thymus domains by regulating the expression of the parathyroid-specific marker Gcm2, and possibly also Foxn1 [14,21,22]. The primordium is subsequently encapsulated by NCCs, which promote early growth of the rudiment, at least in part, through the expression of Fgf7/10, and may influence subsequent patterning/differentiation ([23]; see also Update). Foxn1 is then required for the differentiation [16] and proliferation [24] of all TECs.
Conclusions As we have discussed in this review, the investigation of the earliest stages of thymus organogenesis has yielded exciting new evidence regarding the origins of both mesenchymal and epithelial cells in the thymus. Because of the progress made by these studies, very specific questions concerning lineage contributions and cellular interactions in early thymus organogenesis can now be Current Opinion in Immunology 2003, 15:225–232
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addressed. Some of the key remaining questions are outlined briefly below. First, what induces the initial formation of the primordium from the third pharyngeal pouch? It is not yet clear whether the initial information required to induce formation of the thymic rudiment is entirely intrinsic to the endoderm via the Hox-Pax-Eya-Six pathway, or if it requires an additional extrinsic signal from surrounding cell types. By analogy with other organs, and consistent with results from endoderm transplantation experiments described above, we expect that the endoderm carries an intrinsic pattern or identity [25]. However, an additional external signal from the mesenchyme, or even the ectoderm, may be required to induce organ formation. Candidate signals include fibroblast growth factors (FGFs; [23,26,27]), bone morphogenic proteins (BMPs; [28,29]), wingless transcription factors (Wnts; [15]) and sonic hedgehog (Shh; [30]). Although all of these pathways have been implicated in thymus development and/or thymocyte differentiation, none have yet been linked to the initiation of organogenesis. Investigations into the source and nature of the initiating signal(s) will thus be of great interest. Second, do NCCs provide any patterning signals to the developing primordium or do they simply promote survival and/or proliferation? What, if anything, is the physical and functional contribution of NCCs to the adult thymus? Although there is general agreement that NCCs play an important role in early thymus organogenesis, the function of thymic mesenchyme remains controversial. Classic experiments in both chick [31] and mice [32], and a more recent analysis of FGF receptor2IIIb mutant mice [23] all support a crucial role for NCCs in supporting the growth of the early thymic epithelial primordium. However, controversy surrounds a potential function for mesenchyme in directly promoting thymocyte development [33–36]. In addition, the role of mesenchyme in regulating subsequent patterning of the cortical and medullary compartments, and in the differentiation and maintenance of particular TEC types remains to be addressed. Third, what is the role of other cell types in the thymus and in thymus organogenesis? Although recent studies have provided convincing evidence that hematopoietic cells are not required for thymus organogenesis or initial TEC differentiation [37], other non-bone-marrowderived stromal cell types in the thymus have been implicated in early thymus organogenesis and patterning; the contribution of endothelial cells in particular is one of the least understood. Recent data from studies of the pancreas and liver have provided the precedent, by suggesting that endothelial cells and vasculogenesis could be important regulators of patterning and differentiation [38,39]. This theory is supported by a recent study that Current Opinion in Immunology 2003, 15:225–232
provides evidence that the development of blood vessels in the thymus provides organizing signals for the medullary compartment [40]. Further work is required to investigate the initial entry of endothelial cells into the thymic primordium and their possible functions, including potential interactions with mesenchyme. Further investigation of these and other questions should permit real progress towards the ultimate goal of linking the morphological events of early thymus organogenesis with the transcription factor and signaling pathways that control these processes.
Update Recent work used a Noggin transgene to inhibit the production of BMP-dependent neural crest cells in the hindbrain, resulting in absent or ectopic hypoplastic thymus formation, among other defects [53]. Marker gene analysis also suggested that neural crest cells may have a role in inducing or maintaining Pax1 expression in the endoderm, suggesting that the expression of at least some thymus-related genes in the pharyngeal pouch endoderm may not be regulated by a genetic pathway autonomous to the endoderm.
Acknowledgements We wish to thank Julie Gordon for helpful comments and for sharing her unpublished data. CCB is supported by the Leukaemia Research Fund; NRM is supported by National Institutes of Health/National Institute of Child Health and Human Development (HD035920). The authors are also supported by a Biomedical collaboration grant from the Wellcome Trust.
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