Department of Immunology and Microbiology, Meiji University of Oriental Medicine, Hiyoshi-cho, Funai-gun, 629-0392 Kyoto, Japan
1 Department of Immunology, Institute for Frontier Medical Sciences, Kyoto University, 606-8507 Kyoto, Japan
Correspondence to: M. Itoi
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Abstract |
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Keywords: immunohistochemistry, mesenchymal cell, nude mouse, thymic epithelial cell, thymus organogenesis
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Introduction |
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Epithelial cells, which are the major component of the thymic stromal cells, generate the three-dimensionally (3-D) organized meshwork architecture peculiar to the thymus. In contrast, in other epithelial organs, e.g. the visceral organs and skin, epithelial cells are placed on the basement membranes and make close contact with adjacent cells forming sheets. The meshwork architecture of the thymus is formed through a marked change of epithelial organization during organogenesis of the thymus anlage. Epithelial cells of the mouse thymus anlage originate in the third pharyngeal pouch endoderm and the third pharyngeal cleft ectoderm. Protrusion of both epithelial layers into the pharyngeal arch mesenchymal region on Ed911 results in formation of the thymus anlage (16). Epithelial cells of the thymus anlage develop through interactions of thymic mesenchymal cells (1720) and developing thymocytes (2125), and finally generate a 3-D organized meshwork architecture (26,27). However, it is still unclear when and how the thymus anlage begins attracting hematopoietic progenitor cells or when and how epithelial cells of the thymus anlage change their organization during organogenesis.
Analysis of the thymus anlage of mice with defective thymus development may provide valuable insight into the relationship between the development of thymic epithelial cells and migration of hematopoietic progenitor cells. Nude mice have rudimentary thymus lobes with cystic structures made up of epithelial sheets (2833) instead of the meshwork structure composed of the 3-D organized epithelial cells. The nude phenotype is caused by a loss-of-function mutation in the Whn gene (34), recently renamed Foxn1, according to a new unified nomenclature for the winged helix/forkhead transcription factors (35). The Foxn1 gene is expressed in the epithelial cells of the early thymus analge, indicating that Foxn1 gene product has an essential role in the development of epithelial cells in thymus organogenesis (36). The aberrant morphogenesis of the thymus anlage in nude mice, which is first observed around Ed11.5, is caused by failure of the ectoderm from the third pharyngeal cleft to proliferate and differentiate into a cervical vesicle (16). However, it is unclear when and how epithelial cells of the nude anlage deviate from their proper organization in the developmental process or whether hematopoietic progenitor cells can immigrate into the thymus anlage. In the present study, we comparatively investigated the organization of epithelial cells and distribution of hematopoietic progenitor cells in the early thymus anlage of normal and nude mice. Our results indicate that in the normal thymus anlage hematopoietic progenitor cells are first observed in the thymic mesenchymal layer and subsequently among epithelial cells. In nude mice, however, progenitor cells are not observed among epithelial cells even though they migrate to the mesenchymal layer of the anlage. These results indicate that immigration of progenitor cells to the early thymus anlage occurs through two steps, migration to the thymic mesenchymal layer and entrance among epithelial cells.
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Methods |
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Immunohistochemistry
Embryos were embedded in OCT compound and snap-frozen. They were cut into serial sections 5 µm thick using a cryostat and mounted onto 5% 3-amino propyltriethoxysilane (Sigma, St Louis, MO)-coated slides. Freshly cut frozen sections were fixed with acetone at room temperature for 5 min. Primary antibodies used in this study were rabbit anti-human fibronectin (FN; Cappel, West Chester, PA), rabbit anti-keratin (wide spectrum screening; Dako, Glostrup, Denmark) and rabbit anti-IKAROS antibodies (38). Staining was performed using a Vectastain ABC kit (Vector, Burlingame, CA). Peroxidase activity was developed with 0.1% 3,3'-diaminobenzidine and 0.02% H2O2 in PBS. Sections were then osmified and counterstained with hematoxylin.
Culture medium
RPMI 1640 medium (Nissui, Tokyo, Japan) supplemented with 10% FCS (Biowittaker, Walkersville, MD), sodium pyruvate (1 mM), sodium bicarbonate (2 mg/ml), non-essential amino acid solution (1mM), 2-mercaptoethanol (5x105 M), streptomycin (100 µg/ml) and penicillin (100 U/ml) was used as complete culture medium.
Co-culture of thymus anlages with deoxyguanosine (dGuo)-treated fetal thymus lobes under high oxygen submersion (HOS) conditions
Organ culture was carried out under HOS conditions as described by Watanabe et al. (39), with some modifications. Ed16 thymus lobes of B6.Ly5.1 mice were organ-cultured for 5 days in complete medium supplemented with 1.35 mM dGuo (Sigma) in microfuge tubes (Quality Scientific Plastics, Petaluma, CA). After culture, the thymus lobes were washed with complete medium to remove dGuo. Ed1113 thymus anlages were taken from embryos by fine forceps under a stereomicroscope, and blood vessels and other tissues were carefully removed. The mesenchymal layer of individual anlages remained as even as possible under microscopic inspection. Individual thymus anlages containing mesenchymal layers of B6 mice or KSN nude mice and single dGuo-treated thymus lobes of B6.Ly5.1 mice were mixed together in 0.4 ml of culture medium in microfuge tubes and centrifuged at 5000 g for 2 min. The tubes were placed into plastic bags, and air was exchanged for a gas mixture of 5% CO2, 25% N2 and 70% O2. These plastic bags were cultured at 37°C for 2 weeks. Half of the culture medium was changed every 34 days.
Flow cytometry
FITC-labeled anti-Ly5.1 mAb (A20), anti-CD8a mAb (53-6.7) and R-phycoerythrin-labeled anti-TCRß mAb (GL3), anti-CD4 mAb (L3T4), were purchased from PharMingen (San Diego, CA). For staining, thymocytes were suspended in EMEM (Nissui) supplemented with 1% FCS and reacted with the mAb for 30 min at 4°C. After washing by centrifugation, the cells were analyzed with a FACScan (Becton Dickinson, Mountain View, CA) gated to exclude non-viable cells.
Bromodeoxyuridine (BrdU) labeling and two-color immunofluorescence staining
DNA synthesizing cells were labeled at various embryonic days by i.v. injection of BrdU (250 mg/kg body wt) into the pregnant mice. Two hours later, embryos were removed, snap-frozen and cut into serial sections 5 µm thick. Primary antibodies used in this staining were mouse anti-BrdU mAb (3D4; PharMingen), anti-keratin and anti-IKAROS antibodies. Rabbit anti-mouse IgGFITC conjugate (Wako, Osaka, Japan), and goat anti-rabbit IgGTexas Red (TR) conjugate (Molecular Probes, Eugene, OR) were used as the secondary antibodies. Sections were incubated with anti-keratin or anti-IKAROS antibody at 4°C overnight and then incubated with TR-labeled secondary antibody for 2 h at room temperature. Subsequently, sections were refixed with 70% ethanol at 20°C for 10 min and treated with 0.1N HCl for 30 min at room temperature for DNA denaturation. After neutralization with 0.1 M Na2B4O7, sections were incubated with anti-BrdU mAb at 4°C overnight, followed by FITC-labeled secondary antibody for 2 h at room temperature. BrdU-incorporating cells were regarded as DNA synthesizing cells.
Statistical analysis
Data in Fig. 3 were analyzed for significance of differences using Student's t-test.
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Results |
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On Ed10, the third pharyngeal pouch and third pharyngeal cleft, both of which are keratin+, protrude into the pharyngeal arch mesenchymal region and join at the caudal side of the third pouch and at the cranial side of third cleft (Fig. 1a), as reported previously (16). The boundary of both germ layers on the connected surface cannot be distinguished. No IKAROS+ cells are found around the distal ends of these epithelial layers, which form the thymus anlage at later developmental stages (data not shown).
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On Ed12, the epithelial cells of the anlage are no longer organized in a layered structure, but form a cluster, in which the epithelial cells loose their polarized organization, showing the apicalbasal axis (Fig. 1f). At this stage, a large number of IKAROS+ cells are seen among the epithelial cells in addition to the mesenchymal layer surrounding the epithelial cells (Fig. 1g and h
). Many IKAROS+ cells are also observed in the connective tissue surrounding the anlage. On Ed13, the thymus anlage markedly increases in volume and the epithelial cells form a meshwork structure, peculiar to the thymus, with a large number of IKAROS+ cells (data not shown). These results indicate that the simple epithelia of the early thymus anlage develop a meshwork structure peculiar to the thymus through the stratified epithelial stage and the clustered epithelial stage, successively. IKAROS+ cells begin immigrating at the stratified epithelial stage on Ed11 and enter among the epithelial cells in parallel with the progress of epithelial organization from the stratified epithelia to the clustered epithelia.
Hematopoietic progenitor cells migrate to, but do not enter, the epithelial cluster of the thymus anlage in nude mice
As described in the preceding section, in normal mice a dramatic change in epithelial organization of the thymus anlage occurs during Ed1013. Accompanying this change, IKAROS+ cells start immigrating to the mesenchymal layer and then enter the epithelial cluster. However, it was previously reported that in nude mice aberrant morphogenesis of the thymus anlage is first observed around Ed11.5 (16). We next analyzed the changes in epithelial organization and distribution of IKAROS+ cells in KSN nude thymus anlage, in comparison to those in B6 mice.
While the third pharyngeal pouch and cleft fuse on Ed10 in B6 mice, this fusion occurs on Ed11 in KSN nude mice. IKAROS+ cells are not detectable in these regions (data not shown). An isolated thymus anlage is formed on Ed11.5, which shows stratified epithelia surrounded by a thin mesenchymal cell layer, with the pharyngeal cavity at the center (Fig. 2a and b). Although the size of KSN nude anlage is somewhat smaller, its morphology is similar to that of B6 mice on Ed11. At this time point, a few IKAROS+ cells are observed in the mesenchymal layer of the anlage and in the neighboring connective tissue (Fig. 2c
). On Ed12, epithelial cells of KSN nude anlage are organized in a cluster, similarly to B6 mice (Fig. 2d
). Although some IKAROS+ cells are seen in the mesenchymal layer and in the connective tissue surrounding the anlage, few are observed in the epithelial cluster (Fig. 2f
). On Ed13, a few small clusters of epithelial cells are observed (Fig. 2g
). However, epithelial cells in these clusters never form a meshwork structure. At this time point, a small number of IKAROS+ cells are observed only within the mesenchymal layer and connective tissue, but not within the epithelial clusters (Fig. 2h and i
). These results indicate that although IKAROS+ cells certainly migrate into the mesenchymal layer of KSN nude thymus anlage, they do not reside in the epithelial cluster. To examine whether the difference in genetic backgrounds between B6 mice and KSN nude mice influences the development of the thymus anlage, we performed immunohistochemical analysis of DDD mice on Ed1013, which are the background strain of KSN nude mice, and compared with those of B6 mice. In DDD mice, changes in epithelial organization and migration of hematopoietic progenitors occur almost similarly to those of B6 mice (data not shown). These results indicate that development of the thymus anlage is not affected by the differences in genetic background between these two strains of mice. Thus, in the following experiments, we used B6 mice and KSN nu/nu mice as normal and nude mice respectively.
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Hematopoietic progenitor cells migrating into the early thymus anlage generate mature T cells
The question still remains whether IKAROS+ cells that migrate into the thymus anlage are able to generate mature T cells. To examine whether the thymus anlages at various embryonic ages contain progenitor cells with T cell potential, individual thymus anlages of B6 mice and KSN nude mice (Ly5.2) were co-cultured with dGuo-treated fetal thymus lobes of B6.Ly5.1 mice. Cells were harvested from the lobes after 2 weeks of cultivation, and total cell numbers and surface molecule expression of the recovered cells were analyzed (Table 1). Generation of anlage-type T cells was observed in 10 of 14 lobes co-cultured with Ed11 thymus anlages of B6 mice. The proportion of reconstituted lobes increased with embryonic age of the co-cultured anlages. In the case of KSN nude anlages of Ed11.5 fetuses, T cell generation was observed in six of 20 lobes co-cultured. The percentage of lobes showing T cell development was increased to 57% in the case of Ed12 anlages, but decreased to 50% in the case of Ed13 anlages. The low seeding efficiency of progenitor cells into dGuo-treated lobes in these experiments may be ascribed to the co-culture of thymus anlages and dGuo-treated lobes. These results suggest that the immigration of progenitors capable of generating T cells into the thymus anlages starts on Ed11 in normal mice and Ed11.5 in nude mice. Moreover, the proportions of the lobes showing T cell development are consistent with the histological findings on the numbers of IKAROS+ cells in the thymus anlage (Figs 1 and 2
). The lower percentage of mature T cell generation from the lobes co-cultured with KSN nude thymus anlages may be attributed to the lower numbers of IKAROS+ cells migrating into the KSN nude thymus anlage, as indicated in Fig. 3
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Discussion |
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Our results show that the epithelial cells change their topographical organization gradually during the early thymus organogenesis. At the simple epithelia stage on Ed10, progenitor cells cannot be found in the third pharyngeal pouch and cleft region. At the stratified epithelial stage on Ed11, progenitor cells first appear in the mesenchymal layer surrounding the epithelial cells and many of these cells lie along the basal edge of the epithelial layer. It seems that progenitor cells that have migrated into the mesenchymal layer are retained in front of the epithelial layer. At the clustered epithelial stage on Ed12, progenitor cells are observed among the epithelial cells. These results indicate that the first step of immigration of progenitor cells into the thymus anlage starts at the stratified epithelial stage and the second step starts at the clustered epithelial stage. It is likely that during organogenesis the thymus anlage at the stratified epithelial stage acquires the ability only for induction of the first step and the anlage at the clustered epithelial stage acquires the ability for induction of the second step.
It has been reported that in organogenesis of the thymus, thymocytes play crucial roles in the development of epithelial cells (2125). These previous studies indicated that thymic epithelial cells require interactions with hematopoietic progenitor cells to form a meshwork structure in the cortex and medulla at later stages of development of thymic epithelial cells. The present results show that the change from stratified to clustered epithelial cells occurs in both normal and nude thymus anlages. Since hematopoietic progenitor cells are virtually absent in the epithelial cluster of nude thymus anlage, these results strongly suggested that the epithelial cells can change their organization from stratified to clustered epithelia without the need for interactions with progenitor cells.
IKAROS protein has been shown to be expressed by hematopoietic progenitor cells from their early stage of development (38,40,41). IKAROS+ cells are first seen on sections of the thymus anlages on Ed11 in normal mice and Ed11.5 in nude mice. The first appearance of IKAROS+ cells in the thymus anlage coincides with the appearance of progenitor cells capable of generating T cells in organ culture. The distribution pattern of IKAROS+ cells on Ed11 in normal mice was similar to that of basophilic cells previously reported by Moore and Owen (1), and that of the CD44+ cells reported by Penit and Vasseur (2). These results together with those of the present study suggest that the migration of hematopoietic progenitor cells into the thymus anlage in normal mice starts on Ed11 when the anlage has just separated from the pharynx. Using a clonal assay system, the MLP assay system (4), we showed previously that the progenitor cells restricted to the T cell lineage, which have lost the capacity to generate myeloid and B cells, but not multipotent progenitor cells, are present in the Ed12 thymus anlage (3). T cell lineage-restricted progenitors are also found in the AGM region on Ed10 (5), and in the liver and blood on Ed12 (3,4,6). Moreover, T cell lineage-restricted progenitors are found in the fetal liver of nude mice (6), indicating that the restriction occurs without any influence of the thymus anlage. These results suggest that T cell lineage-restricted progenitors in the AGM region and/or fetal liver migrate into the early thymus anlage. We show that many hematopoietic progenitor cells accumulated in the connective tissue around the thymus anlages from Ed11 to 13. The number of these cells reaches a peak on Ed12 and decreases on Ed13. Since vascularization of the thymus anlage occurs around Ed1314 (manuscript in preparation), migration of progenitor cells into the thymus anlage after Ed13 is likely to occur through blood vessels invading into the anlage. Previous reports suggested that before vascularization of the thymus anlage, progenitor cells leave the adjacent blood vessels and migrate into the thymus anlage through the surrounding mesenchymal layer (1,7). Therefore, it is likely that progenitor cells migrate into the thymus anlage through the surrounding connective tissue during the restricted period between Ed11 and 13. It has been reported that hematopoietic progenitor cells are attracted by chemoattracting factor(s) produced by the thymus anlage (815). Thus, it is likely that the thymus anlage at the stratified epithelial stage acquires the function to attract hematopoietic progenitor cells by producing chemoattracting factor(s), not only in normal mice but also in nude mice. We also show that progenitor cells are vigorously proliferating among epithelial cells. This suggests that the interaction between epithelial cells of the thymus anlage and progenitor cells is essential for proliferation of progenitor cells just after migrating into the thymus anlage.
The gene mutated in nude mice has been identified as the Foxn1 gene, encoding a new member of the winged-helix domain family of transcription factors (34). Thymic epithelial cells on Ed12 have been shown to express this gene product (36). We show that the organization of epithelial cells in nude thymus anlage develops to the cluster stage on Ed12, but degenerates thereafter without forming a meshwork structure. Moreover, in contrast to normal mice, epithelial cells in the Ed12 nude thymus anlage scarcely proliferate. Therefore, the aberration of epithelial development in the nude thymus analge occurs at the clustered epithelial stage. Since nude-derived epithelial cells cannot develop in the presence of wild-type-derived cells in the thymus of nude and wild-type chimeric mice, the nude gene product acts cell-autonomously for development of thymic epithelial cells (42). Thus, the Foxn1 gene product appears to have an essential role in the induction of proliferation and differentiation of thymic epithelial cells at the clustered epithelial stage. We also show that in nude mice the onset of migration is delayed by about half a day and the numbers of migrating cells are lower as compared with those in normal mice. The Foxn1 gene mutation may also cause retarded development and/or lower productivity in chemoattracting factor of epithelial cells. Our present results first indicate that hematopoietic progenitor cells which can develop mature T cells migrate into the mesenchymal layer of Ed11.5 nude thymus anlage. However, conflicting results that nude thymic rudiment is ineffective at mediating chemoattraction of immature precursors were reported (15,43). Wilkinson et al. (15) showed that the nude fetal thymus lobe cannot attract Ed14 thymic precursor cells using a transfilter migration assay. The discrepancy may be attributable to the Ed1415 nude thymus anlage that they used. Indeed, we found that epithelial cells of nude thymus anlage cannot develop after Ed12, but degenerate. Bleul and Boehm (43) described that CD45+ hematopoietic precursor cells were predominantly observed in close proximity to the parathyroid anlage of nude embryo located at the cranial site of the thymus anlage. We, however, clearly showed that the number of IKAROS+ hematopoietic progenitor cells migrating to the mesenchymal layer of nude thymus anlage increased on Ed12. Accordingly, progenitor cells which are moving to the thymus anlage might be found in the close vicinity of the parathyroid anlage of nude embryo. In nude mice, hematopoietic progenitor cells migrate into the mesenchymal layer of the thymus anlage, but do not enter the epithelial cluster. Since progenitor cells in the mesenchymal layer of nude thymus anlage generate mature T cells in co-culture with dGuo-treated fetal thymus lobes, impaired entrance of progenitor cells among the epithelial cells is likely to be due to a defect not of progenitor cells but of epithelial cells. The progenitor cells attracted to the mesenchymal layer may require adherence to epithelial cells to enter among them. Thus, it seems likely that between Ed11 and 12 in the normal thymus anlage the epithelial cells begin to express cell-surface molecules and/or extracellular matrix components involved in the adhesion of hematopoietic progenitor cells to epithelial cells. In the nude thymus anlage, epithelial cells may fail to express such molecules.
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Acknowledgments |
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Abbreviations |
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AGM aortagonadmesonephros |
B6 C57BL/6 |
BrdU bromodeoxyuridine |
dGuo deoxyguanosine |
Ed embryonic day |
FN fibronectin |
HOS high oxygen submersion |
MLP multilineage progenitor assay |
TR Texas Red |
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Notes |
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Received 13 February 2001, accepted 19 June 2001.
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References |
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