Cellular Events in Thymus Organogenesis

2.1.1. Cellular Regulation of Early Thymus Organogenesis

Thymus organogenesis occurs in the pharyngeal region of the developing embryo. This region is composed of a number of bilateral bulges known as pha-ryngeal arches, which are separated by structures known as pharyngeal pouches and pharyngeal clefts. The pharyngeal pouches are bilateral outpocketings of pharyngeal endoderm, which form sequentially in a rostral to caudal manner and initially comprise a single layer of simple epithelium, whereas the opposing pharyngeal clefts are invaginations of surface ectoderm also consisting of a layer of simple epithelium (see Fig. 2).

During embryogenesis, the thymus arises from the endoderm of the third pha-ryngeal pouch, in a common primordium with the parathyroid gland. Overt organogenesis is apparent from approximately day 10 of embryonic development (E10) in the mouse, when third pharyngeal pouch cells begin to proliferate to form the bilateral primordia. Each primordium is surrounded by a condensing population of mesenchymal cells that will eventually form the capsule (60,61). Further outgrowth of the primordia, which results in expansion of both epithelial and mes-enchymal components, occurs until approx E12.5, at which point the primordia separate from the pharynx and begin to resolve into discrete thymus and parathyroid organs. These subsequently migrate to their final anatomical locations—at the midline and at the lateral margins of the thyroid respectively. Patterning of the common primordium into a prospective thymus domain located in the ventral aspect of the pouch, and prospective parathyroid domain located in the dorsal aspect, appears to be an early event in organogenesis, as the parathyroid domain is delineated by the transcription factor glial cells missing-2 (Gcm2) as early as E9.5. Possible mechanisms regulating the establishment/maintenance of these domains are discussed in Subheading 2.3.

The mesenchymal capsule surrounding the thymus primordium is derived from the neural crest, a transient population formed between the neural tube and

Parathyroid Organogenesis

Fig. 2. Morphology of the pharyngeal region. (A) Lateral views of an E10 mouse embryo: left panel shows whole embryo (tip of the tail is missing), box indicates pharyngeal region, arrow points to the thrid pharyngeal pouch, FLB, fore limb bud; HLB, hind limb bud. Right panel shows detail of pharyngeal region—arrows point to third and fourth pharyngeal arches; PA, pharyngeal arch; dorsal is up and ventral down. Note that the orientation of the two embryos is different. (B) Cartoon representing coronal section through the same area. In mouse there are five pharyngeal arches (PA), which consist of mesenchymal and mesodermal cells (gold) bounded by an outer layer of surface ectoderm (turquoise) and inner layer of pharyngeal endoderm (168). The ectoderm forms four invaginations, the pharyngeal clefts (PC 1-4), which separate the arches, whereas the endoderm forms four opposing outpocketings, the pharyngeal pouches (PP 1-4).

Fig. 2. Morphology of the pharyngeal region. (A) Lateral views of an E10 mouse embryo: left panel shows whole embryo (tip of the tail is missing), box indicates pharyngeal region, arrow points to the thrid pharyngeal pouch, FLB, fore limb bud; HLB, hind limb bud. Right panel shows detail of pharyngeal region—arrows point to third and fourth pharyngeal arches; PA, pharyngeal arch; dorsal is up and ventral down. Note that the orientation of the two embryos is different. (B) Cartoon representing coronal section through the same area. In mouse there are five pharyngeal arches (PA), which consist of mesenchymal and mesodermal cells (gold) bounded by an outer layer of surface ectoderm (turquoise) and inner layer of pharyngeal endoderm (168). The ectoderm forms four invaginations, the pharyngeal clefts (PC 1-4), which separate the arches, whereas the endoderm forms four opposing outpocketings, the pharyngeal pouches (PP 1-4).

the surface ectoderm. From E9.0, neural crest cells (NCC) migrate into the pharyngeal region and populate the pharyngeal arches. Studies using the chick-quail chimera system provided the first evidence that NCC are the exclusive source of mesenchymal cells in the thymus (60). This was confirmed more recently using a line of transgenic mice that expressed both a "silent LacZ" reporter from the ubiquitous Rosa26 promoter and Cre-recombinase under the control of the Wntl promoter (61), such that LacZ was expressed in all cells derived from the neural crest. As expected, this analysis demonstrated that, in the fetus, thymic mesenchyme was derived from NCC, but a reduced contribution of NCC was unexpectedly indicated in neonatal animals. These data have been interpreted to indicate that the origin of the thymic mesenchyme may change during development, however, clarification of this point is required as an alternative explanation is transgene silencing.

Lymphocytes first seed the thymus at E11.5 (62-64). As vascularization has not occurred at this stage, the first colonizing cells leave the circulation and migrate through the peri-thymic mesenchyme into the thymic epithelium. Some evidence suggests that the first progenitor cells that colonise the thymic rudiment exhibit comparatively low T-cell progenitor activity, and that a second colonizing wave which arrives between E12 and E14 displays much higher levels of T-cell potential upon in vivo transfer (65).

Post-E12.5, epithelial cells within the thymic primordium continue to proliferate, at least partly in response to factors supplied by the mesenchymal capsule. Concomitantly, TEC differentiation commences: the first cortical and medullary TEC appear by E12.5 (66) and development of the two compartments proceeds in a lymphocyte independent manner until E15.5 (67,68). The expression of MHC class II on thymic epithelial cells is first detected at E13.5, with cell surface MHC class I expression occurring at approx E16 (16,69) and expression of both these antigens is followed by the appearance of CD4+ and CD8+ SP thymocytes at E15.5 and E17.5, respectively (16,69). Although a functional thymus is present in neonates, the full organization of the stroma characteristic of the mature organ is not achieved until 2-3 wk postnatally in the mouse.

2.1.2. Origin of Thymic Epithelial Cells

The embryonic origins of the thymic epithelium have been controversial, with conflicting hypotheses suggesting that the epithelium has a dual endodermal/ ectodermal origin (63,70), or derives solely from the pharyngeal endoderm (71-74). However, a recent study has resolved this controversy by providing definitive evidence for a single endodermal origin in mice (72). This study used three approaches to address the origins of the thymic epithelium. First, detailed histological analysis indicated that although there was contact between the endoderm and ectoderm at E10.5, the germ layers subsequently separated, with apoptosis occurring in the ectoderm that had previously resided in the contact region. Second, lineage tracing of pharyngeal surface ectoderm of E10.5 mouse embryos failed to find evidence for an ectodermal contribution to the thymic primordium and third, transplantation of pharyngeal endoderm isolated from E8.5 to E9.0 embryos (i.e., prior to initiation of overt thymus organogenesis) indicated that the grafted endoderm was sufficient for complete thymus organogenesis, similar to previous results obtained using chick-quail chimeras (71). These data collectively establish that pharyngeal endoderm alone is sufficient for the generation of both cortical and medullary thymic epithelial compartments.

2.1.3. Thymic Epithelial Progenitor Cells

The formation of complex multicellular organs from the relatively simple structures found in the developing embryo relies on the capacity of specified progenitor cells to expand and differentiate into the multiple cell types that constitute the functional organ. Thus, the early thymic rudiment contains thymic epithelial progenitor cells (TEPC) capable of giving rise to the highly diverse range of epithelial subtypes that mediate T-cell differentiation.

The phenotype of TEPC has been of considerable interest. Evidence suggestive of a progenitor/stem cell activity was initially provided by analysis of a subset of human thymic epithelial tumors: these were found to contain cells that could generate both cortical and medullary subpopulations, suggesting that the tumorigenic targets were epithelial progenitor/stem cells (75). However, the first indication of a TEPC phenotype was provided by a study addressing the nature of the defect in nude mice (76). Here, MHC mismatched nude-wild type aggregation chimeras were generated and analyzed to determine whether nude-derived cells could contribute to the thymic epithelium. As nude-derived cells were unable to contribute to the major thymic epithelial subsets, this study established that the nude gene product (Foxnl) is required cell-autonomously for the development and/or maintenance of all mature TEC. However, a few nude derived cells were present in the thymi of adult chimeras, and phenotypic analysis indicated that these cells expressed determinants reactive to MAbs MTS20 (77) and MTS24, but did not express markers associated with mature TEC including MHC class II. This suggested that in the absence of Foxnl, TEC lineage cells undergo maturational arrest and persist as MTS20+24+ progenitors (76).

Further data regarding the phenotype of thymic epithelial progenitors was provided by analysis of mice with a secondary block in thymus development resulting from a primary T-cell differentiation defect. The thymi of postnatal CD3e26tg mice, in which thymocyte development is blocked at the CD44+CD25-TN1 stage (78), principally contain epithelial cells that coexpress K5 and K8 (17), which, in the normal postnatal thymus, are predominantly restricted to the medulla and cortex, respectively, and are coexpressed by only a small population of cells at the cortico-medullary junction. In this study, Klug and colleagues demonstrated that transplantation of CD3e26tg thymi into Ragl-- mice, which sustain a later block in T-cell differentiation, results in the development of K5K8+ cells, suggesting that the K5+K8+ cells are progenitors of cTEC (17).

Ontogenic analysis subsequently demonstrated that the proportion of MTS20+24+ cells in the thymic epithelium decreased from approx 50% at E12.5 to less than 1% in the postnatal thymus (66), consistent with the expression profile expected of markers of fetal tissue progenitor cells. Phenotypic analysis of the MTS20+24+ and MTS20-24-populations of the E12.5 thymus indicated that all cells in the MTS20+24+ population coexpressed K5 and K8, whereas none expressed TEC differentiation markers, including MHC class II (66). Importantly, the functional capacity of isolated MTS20+24+ cells and MTS20 24- cells was then determined via ectopic transplantation. This analysis demonstrated that the MTS20+24+ population was sufficient for establishment of a functional thymus containing both cortical and medullary TEC populations, whereas, in this assay, the MTS20 24- population fulfilled none of these functions (66,79). The MTS20+24+ population thus contains TEPC; it is unclear at present if a single MTS20+24+ cell can give rise to both cortex and medulla, or if distinct cortical and medullary progenitors are specified from an endodermal cell of wider potency (see Fig. 3). Although medullary islets in the adult thymus are clonally derived (80), this could be consistent with either of these scenarios and clarification of this issue will require clonal analysis of MTS20+24+ cells. However, the demonstration that the thymic epithelium has a single endodermal origin (72) indicates that, at some level, cortical and medullary TEC share a common endodermal progenitor. Although in the models presented, the major cortical and medullary thymic epithelial cell types arise as separate "sublineages," the best evidence supporting this model is the observation that in chimeric thymi, no correlation could be found to support a clonal origin for individual medullary islets and adjacent regions of cortical epithelium (80). However, because lineage tracing has not been reported for the adult thymic epithelium, it remains possible that a lineal relationship exists between cortical and medullary cell types.

2.1.4. Thymic Epithelial Cell Differentiation

Thymic epithelial differentiation has been described principally via expression analysis of early and late differentiation markers. These include members of the cytokeratin family of intermediate filaments and markers associated

Human Epc Differentiation

Fig. 3. Models of thymic epithelial cell differentiation. (A) A common thymic epithelial progenitor cell (TEPC) may arise from an endodermal progenitor cell (EPC). In this case TEPC will have the potential to generate both cortical thymic epithelial cells (cTEC) and medullary TEC (mTEC). (B) In the second model, an EPC gives rise directly to cortical TEPC (cTEPC) and medullary TEPC (mTEPC), which differentiate into mature cTEC and mTEC, respectively. In both cases the progenitor populations may contain self-renewing stem cells.

Fig. 3. Models of thymic epithelial cell differentiation. (A) A common thymic epithelial progenitor cell (TEPC) may arise from an endodermal progenitor cell (EPC). In this case TEPC will have the potential to generate both cortical thymic epithelial cells (cTEC) and medullary TEC (mTEC). (B) In the second model, an EPC gives rise directly to cortical TEPC (cTEPC) and medullary TEPC (mTEPC), which differentiate into mature cTEC and mTEC, respectively. In both cases the progenitor populations may contain self-renewing stem cells.

specifically with subsets of thymic epithelium, many of which are of unknown biochemical identity.

At E11.5, the MTS20 and MTS24 determinants are expressed throughout the common primordium by the majority of epithelial cells (66). K8 is also strongly expressed throughout the primordium (66), and is reported by some investigators to colocalize with K5 expression in the Foxnl expressing domain (66). At this stage of development, the expression of other known TEC differentiation markers cannot be detected (66). Thus, at E11.5, the phenotype of epithelial cells within the thymic primordium appears to be MTS20+24+K5+K8+.

By E12.5, signs of early differentiation are apparent. Approximately 50% of TEC (66) remain MTS20+24+K5+K8+, and these MTS20+24+ cells are now present as foci in the E12.5 thymic epithelium (66). K5 and K8 are also expressed in some MTS20-24- TEC, and flow cytometric analysis suggests considerable heterogeneity within this population (66). Analysis by immuno-histochemistry indicates areas of the epithelium that retain costaining for K5 and K8. However, cells displaying the highest levels of K5 expression have often downregulated expression of K8 (66,67). These cells coexpress MTS10 (67), suggesting they are differentiating into medullary epithelial cells. In more peripheral areas, cells appear K8+K5-/l°, suggesting differentiation into cortical epithelium (66,67).

As fetal development proceeds, the proportion of epithelial cells expressing MTS20 and MTS24 declines, so that by E17.5 only 1-2% of epithelial cells within the thymus express these markers (66). Similarly, coexpression of K5 and K8 declines during ontogeny. Thus, by E15.5, smaller groups of K5+K8+ TEC can be seen towards the periphery of the thymus, interspersed among K5 K8+ TEC (67). The innermost cells within the K5+K8+ clusters show strong expression of K5, whereas expression is much lower in those present at the boundary of these groups (67). By E17.5, a notable difference can be observed in the thymic architecture. A well-organized cortex is now apparent, consisting of K5 K8+ TEC (67) which also express the late cortical marker 4F1 (66). Also, presumptive K5+K8- medullary regions are present, which display strong expression of MTS10 (67). At this stage, K5+K8+ cells are still present although they no longer form central cores as they do during earlier stages of development (67).

Acquisition of the characteristic cortex-medulla structure of the thymus occurs by 3 wk of age in the mouse. At this stage, MTS20 and MTS24 expression is restricted to a small number of isolated cells in the medulla (66,76), and while the majority of epithelial cells express either K5 or K8, K5+K8+ DP cells are present in reasonably high numbers at the cortico-medullary junction (17,67), where they persist in the postnatal thymus (17,81).

2.1.5. Human Thymus Development

As in the mouse, the human thymus develops from the third pharyngeal pouch. From early week 6 of fetal development, the endoderm of the third pouch forms a hollow tube-like lateral expansion from the pharynx, which makes contact with the ectoderm of the third pharyngeal cleft (82,83). Although a single endodermal origin has not been formally demonstrated for the human thymic epithelium, it is reasonable to assume that this is the case given the demonstration of a single endodermal origin in mice and birds (71). Again, as in the mouse, the bilateral human primordia initially each comprise ventral thymic and dorsal parathyroid domains, and are encased in a neural crest derived mesenchymal capsule from the onset of development. From week 7 to mid-week 8 the thymic component of this primordium migrates ventrally, becoming stretched out in an elongated, highly lobulated cord-like structure. The upper part of this structure normally disappears, leaving the parathyroid in the location in which it will remain throughout adulthood (82). The two thymic structures meet and attach at the pericardium—their final location of the thymus into adulthood—by mid-week 8 (82). It is well documented that an accessory thymus can exist in the cervical region in humans, possibly as a result of inadequate detachment of the lobulated thymic cord from the parathyroid or development of thymic tissue left along the route of primordium migration (84), and similarly, the occurrence of cervical thymi in the mouse has recently been reported. (See Note added in proof.) The early human primordium appears to contain undifferentiated epithelial cells, as reported for the mouse (66), and some markers that are restricted to either cortical or medullary compartments in the postnatal thymus are expressed by all epithelial cells (85). Medullary development is seen from wk 8 and by wk 16, distinct cortical and medullary compartments are clearly evident. At wk 8 other cell types penetrate the thymus, including mesenchymal, vascular, and lymphoid cells. Between 14 and 16 wk, mature lymphocytes begin to leave the thymus to seed the peripheral immune tissues (84,86).

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