Skip Navigation


International Immunology Advance Access originally published online on July 11, 2007
International Immunology 2007 19(8):953-964; doi:10.1093/intimm/dxm060
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
19/8/953    most recent
dxm060v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Itoi, M.
Right arrow Articles by Amagai, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Itoi, M.
Right arrow Articles by Amagai, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Mesenchymal cells are required for functional development of thymic epithelial cells

Manami Itoi1, Noriyuki Tsukamoto1, Hisahiro Yoshida2 and Takashi Amagai1

1 Department of Immunology and Microbiology, Meiji University of Oriental Medicine, Hiyoshi-cho, Nantan, Kyoto 629-0392, Japan
2 Laboratory of Immunogenetics, RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan

Correspondence to: M. Itoi; E-mail: m_itoi{at}meiji-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Epithelial–mesenchymal interactions have essential roles in thymus organogenesis. Mesenchymal cells are known to be required for epithelial cell proliferation. However, the contribution of mesenchymal cells to thymic epithelial cell differentiation is still unclear. In the present study, we have investigated the roles of mesenchymal cells in functional development of epithelial cells in the thymus anlage in patch (ph) mutant mice, which have a primarily defect in mesenchymal cells caused by the absence of platelet-derived growth factor receptor {alpha} expression. In the ph/ph thymus anlage, T cell progenitors migrate normally among the epithelial cells, however, they are severely impaired to proliferate and differentiate to CD25-positive cells. Epithelial cells of the ph/ph thymus anlage show severely impaired proliferation and expression of functional molecules, such as SCF, Delta-like 4 and MHC class II, which have crucial roles in T cell development. Moreover, the cultured ph/ph thymus anlage fails to develop into a mature organ supporting full T cell development. Addition of intact thymic mesenchymal cells to organ culture induces development of the ph/ph thymus anlage. In the cultured lobes, added mesenchymal cells contribute to form not only the capsule but also the meshwork structure mingled with epithelial cells. Our present results strongly suggest the roles of mesenchymal cells in functional development of epithelial cells in thymus organogenesis. In addition, our data suggest that mesenchymal cells are required to create the thymic microenvironment and to maintain epithelial architecture and function.

Keywords: Delta-like 4, MHC class II, patch mutant mice, SCF, thymus organogenesis


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary data
 References
 
The thymus is the primary lymphoid organ crucial for T cell development. T cell progenitors generated in fetal liver or bone marrow colonize in the thymus (1–3) and proliferate and differentiate within the thymic microenvironment created by stromal cells (4). The major component of the thymic stromal cells is epithelial cells, which generate the three-dimensionally organized meshwork structure peculiar to the thymus (5). Thymic epithelial cells provide various signals for T cell development. Recently, the molecular basis underlying functions of thymic epithelial cells is becoming increasingly clear. Thus far, reported functional molecules produced by thymic epithelial cells are the following—chemokines: CCL21 and CCL25 for T cell progenitor migration (6) and CCL25, CXCL12 and CCL19 for thymocyte trafficking in the thymus (7–10) and Notch ligands: Delta-like (Dll) 1 and Dll4 for T lineage commitment and for early thymocyte development at CD4–CD8– double-negative (DN) stages (11–14), SCF and IL7 for thymocyte proliferation (15–21) and MHC class II molecules for positive and negative selection (22).

Epithelial cells of the mouse thymus anlage originate in the third pharyngeal pouch endoderm (23), which protrudes into the pharyngeal arch mesenchymal region on embryonic days (Eds) 9–11, resulting in formation of the thymus anlage (24, 25). Epithelial cells of the anlage on Ed11, just separated from the pharynx, show a stratified bilayer structure, converting to a clustered organization on Ed12 and into a meshwork structure on Ed13 and thereafter (26). From the early stage of thymus anlage development, epithelial cells express functional molecules essential for early T cell development, such as CCL21, CCL25 (6, 27), IL7 (28), Dll1 and Dll4 (14, 29). Initial colonization of T cell progenitors to the thymus anlage occurs around Ed11, and the earliest immigrants migrate among epithelial cells and immediately start proliferation and differentiation in the anlage on Ed12 (26, 30).

Previous reported studies have shown the importance of mesenchymal cells in thymus organogenesis (31). Aplasia of the thymus is induced in avian embryo by ablation of cephalic neural crest cells, which migrate to the pharyngeal arch and form mesenchyme of the thymus anlage (32). Mesenchymal cells are known to contribute to development of epithelial cells of the thymus anlage. Lobule formation and outgrowth of epithelial cells of the early thymus anlage require interaction with mesenchymal cells (33, 34). Recently, it has been shown that mesenchymal cells of the thymus anlage produce fibroblast growth factor (FGF) 7 and FGF10, and these FGFs induce proliferation of epithelial cells of the anlage expressing their receptor FGFR2IIIb (35, 36). These results indicate that the proliferation of epithelial cells of the thymus anlage depends on interaction with mesenchymal cells. We previously showed that mesenchymal cells are of key importance for the thymus anlage to develop into a functionally mature organ supporting full T cell development (37). Mesenchymal cells have also been reported to induce expression of MHC class II molecules on thymic epithelial cells (37, 38). These observations suggest that mesenchymal cells have a crucial role not only in proliferation but also in differentiation of epithelial cells. However, it is still not clear which steps of differentiation of epithelial cells require interactions with mesenchymal cells during thymus organogenesis.

Patch (ph/ph) homozygotes exhibit developmental abnormalities in various tissues and organs, such as a cleft face, epidermal blisters, a kinky neural tube, spina bifida and cardiac defects (39–41). These anomalies result in death before birth. ph/+ heterozygotes also exhibit defects in melanocyte development (42). The ph mutation is a deletion of a large locus including the entire coding region of platelet-derived growth factor receptor (PDGFR) {alpha} gene on chromosome 5 (43). The Ph locus carries more than one gene and the 3'-deletion end point of the locus is in juxtaposition with the c-kit gene (44). This may indicate the possibility that defects in other genes besides the PDGFR{alpha} gene are responsible for developmental abnormalities in ph/ph mutant mice. However, it has been recently shown that the phenotype of PDGFR{alpha} gene-deficient mice is nearly identical to that of ph/ph mice, except that in contrast to ph/+ mice, PDGFR{alpha}+/– heterozygotes have no defect in melanocyte migration (45). During embryogenesis, PDGFR{alpha} is expressed at high levels in the non-neuronal derivatives of the cranial neural crest cells and in the mesodermally derived mesenchyme of the trunk (46). PDGF-A and PDGF-C, the main ligands for PDGFR{alpha}, are expressed on epithelium closely adjacent to PDGFR{alpha}-expressing mesenchyme. These molecules function in a paracrine mode to regulate proliferation, survival and movement of mesenchymal cells (47–49). Thus, the ph mutation causes severe defects in mesenchyme during embryogenesis, and in consequence, defects in epithelial organ development should be caused by lack of epithelial–mesenchymal interactions in ph mutant embryos. Mesenchymal cells of the thymus anlage express PDGFR{alpha} (50). Therefore, analysis of developmental defects in epithelial cells of the ph/ph thymus anlage is valuable for elucidating the roles of mesenchymal cells in epithelial development during thymus organogenesis. So far, only a small size and fewer lobule formation have been reported as developmental defects of the ph/ph thymus anlage (40).

In this study, we show that early stages in thymocyte development in the ph/ph thymus anlagen are affected. We also show that epithelial cells of the ph/ph thymus anlage fail to express functional molecules, such as SCF, Dll4 and Ia. These results indicate that mesenchymal cells have crucial roles in the step of epithelial cell differentiation that enable these cells to express functional molecules essential for early T cell development.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary data
 References
 
Mice
BALB/c ph mutant mice and enhanced GFP transgenic (EGFP Tg) mice of C57BL/6 background (51) were used. Homozygous ph/ph or EGFP Tg embryos were obtained by mating of ph or EGFP Tg heterozygotes, respectively. Mice were mated at night and the females were examined for vaginal plugs the following morning. The day on which a vaginal plug was found was designated as Ed0. ph/ph homozygotes were identified by visual inspection. Normal looking littermates, which could be either heterozygotes or wild-type embryos, were used as controls because there was no obvious difference in thymus development between them (data not shown). Previous reports show that the patch homozygous mutation is embryonic lethal and the survival rate of ph/ph embryos varies depending on their genetic background (39–41, 52). Most of ph/ph embryos on C57BL/6 background die prior to Ed10.5 (41) and a few ph/ph embryos on BALB/c background survive until Ed14 (52) or in other case until Ed16–17 (39, 40). By breeding of BALB/c ph/+ mutant mice in our colony, only 1.3% of their offspring was ph/ph homozygotes Ed13. Therefore, we used Ed12–13 embryos for analysis.

Immunohistochemistry and immunofluorescence staining
Immunohistochemistry and immunofluorescence staining were performed as previously described (26). Primary antibodies used in this study were rabbit anti-keratin antibody (wide-spectrum screening; Dako, Glostrup, Denmark), rabbit anti-Ikaros antibody (53), rabbit anti-SCF antibody (H-189; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-human fibronectin antibody (Cappel, West Chester, PA, USA), sheep anti-keratin antibody (anti-human broad-spectrum cytokeratin, The Binding Site, Birmingham, AL, USA), anti-Ia rat mAb (ATCC-TIB120), anti-CD25 rat mAb (PC61), anti-PDGFR{alpha} rat mAb (APA5) and anti-PDGFRß rat mAb (APB5). For immunofluorescence staining, sections were stained with fluorescent-labeled or biotin-conjugated secondary antibodies. For SCF, PDGFR{alpha} and PDGFRß staining, tyramide signal amplification technology (TSA kit #25; Molecular Probes, Eugene, OR, USA) was used.

Bromodeoxyuridine labeling and two-color immunofluorescence staining
Bromodeoxyuridine (BrdU) labeling and two-color immunofluorescence staining were performed as previously described (26). BrdU-incorporating cells were regarded as DNA-synthesizing cells.

Reverse transcription–PCR analysis
mRNA was extracted from thymus anlagen of individual embryos using a QuickPrep micro mRNA purification kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s protocol and was reverse transcribed using SuperScript II (Invitrogen, Carlsbad, CA, USA) with oligo(dT) primer (Invitrogen). Reverse transcription (RT) reactions were carried out using pureTaq ready-to-go PCR beads (GE Healthcare). Semi-quantitative PCRs were performed using the same serially diluted cDNA as shown for ß-actin. The primers used for PCR were as follows: SCF, 5'-AATCTCCGAAGAGGCCAGAA-3' and 5'-CCATGGCTGTCCATTGTAGG-3'; IL7, 5'-TGAAGACCCAGCGCAAAGTAGA-3' and 5'-ACCAGTGTTTGTGTGCCTTGTG-3'; Dll1, 5'-ACCTCGGGATGACGCCTTTG-3' and 5'-AGACCACCACAGCAGCACAG-3'; Dll4, 5'-GCACCAACTCCTTCGTCGTC-3' and 5'-TCACAAAACAGACCTCCCCA-3'; Jagged1, 5'-TCTCTGACCCCTGCCATAAC-3' and 5'-TTTTACAGGGGTTGCTCTCG-3'; Jagged2, 5'-GCAAAGAAGCCGTGTGTAAA-3' and 5'-TAATAGCCGCCAATCAGGTT-3'; FGFR2IIIb, 5'-GAGGGGATGTGGAGTTTGTCT-3' and 5'-CAGCATCCATCTCCGTCACAT-3'; FGF7, 5'-GTGAGAAGACTGTTCTGTCG-3' and 5'-GCCATAGGAAGAAAATGGGC-3'; FGF10, 5'-TGAGACAATTTCCAGTGCCG-3' and 5'-TATCTCCAGGACACTGTACG-3' and ß-actin, 5'-GTGGGCCGCTCTAGGCACCAA-3' and 5'-CTCTTTGATGTCACGCACGATTTC-3'.

In situ hybridization
In situ hybridization was performed as previously described (54). Freshly cut 5-µm sections of whole embryos were used. Digoxigenin-labeled anti-sense probes for Dll1 (175-1441 and 1441-2022) and Dll4 (427-1164) were synthesized from linearized plasmid cDNA templates using T3 or T7 RNA polymerases (Roche Molecular Biochemicals, Penzberg, Germany).

Organ culture
Organ culture was carried out under high oxygen submersion (HOS) conditions as described by Watanabe and Katsura (55) with some modifications (26).

Co-culture of thymus anlagen with thymic mesenchymal cells from EGFP Tg mice
Ed13 thymus anlagen were taken from embryos from ph/ph and EGFP Tg embryos. The anlagen of EGFP Tg mice were incubated in 400 U ml–1 dispase (Godo-Shusei, Tokyo, Japan) in MEM (Nissui, Tokyo, Japan) for suspension culture supplemented with 5% FCS for 15 min at 37°C and the mesenchymal layer was carefully separated from epithelial cells with fine forceps in MEM. Individual ph/ph thymus anlagen were placed into microfuge tubes with or without thymic mesenchymal cells derived from three to five anlagen of EGFP Tg mice in 0.4 ml of culture medium and centrifuged at 5000 x g for 2 min to form pellets. Tubes were cultured under HOS condition.

Flow cytometry
FITC-labeled anti-CD8a mAb (53-6.7) and R-PE-labeled anti-CD4 mAb (L3T4) were purchased from BD Biosciences Pharmingen (Franklin Lakes, NJ, USA). For staining, cells recovered from cultured anlagen were suspended in MEM 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, USA) gated to exclude non-viable cells.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 References
 
Aberrant morphogenesis of the thymus anlagen in ph/ph mice
First, to examine whether morphological development of epithelial cells and immigration of T cell progenitors occur in the ph/ph thymus anlage, we analyzed the organization of thymic epithelial cells and the distribution of thymocytes by immunohistochemistry on serial sagittal sections of whole embryos of ph/ph mice and wild-type mice. Epithelial cells and thymocytes were identified by the expression of keratin and Ikaros, respectively. On Ed12, the thymus anlagen are comparably positioned in the upper thoracic region of wild-type embryo (Fig. 1A-a) and in ph/ph embryo (Fig. 1A-e). Although the size of the ph/ph thymus anlage is slightly smaller than that of the wild-type thymus anlage, epithelial cells of the anlagen of both types of embryos are organized in clusters (Fig. 1A-b, f) and Ikaros-positive thymocytes are observed in the epithelial region of both embryos (Fig. 1A-c, g). On Ed13, epithelial cells of the wild-type thymus anlage form a three-dimensionally organized meshwork structure (Fig. 1A-d). On the other hand, in the ph/ph thymus anlage, epithelial cells still remain in clusters in which they are closely packed with each other (Fig. 1A-h). In contrast to the wild-type anlage, which has an oval shape (Fig. 1A-d), the ph/ph thymus anlage is smaller in size and shows digitated outlines of epithelial regions in most of the embryos (Fig. 1A-h). During thymic ontogeny, the expression patterns of Keratin5 and Keratin8 dramatically change (56, 57). Although in the ph/ph thymus anlage epithelial cells showed aberrant morphology on Ed13, the expressions of Keratin5 and Keratin8 were almost similar to those of the wild type (Supplementary Fig. 1a and c, available at International Immunology Online).


Figure 1
View larger version (91K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Defects in epithelial morphogenesis of the ph/ph thymus anlage. (A) Sagittal sections of Ed12 (a–c and e–g) and Ed13 (d and h) embryos of wild type (a–d) and ph/ph embryos (e–h) were stained for keratin (a, b, d, e, f and h) and Ikaros (c and g) by immunohistochemistry. The thymus anlage forming an epithelial cluster (arrows) is located in the upper thoracic regions in wild-type (a) and ph/ph embryos (e) on Ed12. (b and f) are higher magnifications of the thymus anlagen in (a and e), respectively. Ikaros-positive cells occur among epithelial cells of the wild-type (c) and the ph/ph (g) thymus anlagen. The wild-type thymus anlage on Ed13 shows an oval shape (d). In contrast, the ph/ph thymus anlage is small in size and shows a digitating outline of epithelial cells (h). (B) Two-color immunofluorescence staining was performed on sagittal sections of Ed12 wild-type (a and b) and ph/ph (c and d) embryos to detect BrdU (green) and keratin (a and c: red) or Ikaros (b and d: red). In the wild-type thymus anlage, many epithelial cells (a) and thymocytes (b) contain BrdU in their nuclei. White arrowheads indicate BrdU-incorporated cells of outermost epithelial cells which are adjacent to the surrounding mesenchymal layer. In the ph/ph thymus anlage, only a small number of epithelial cells (c) and thymocytes (d) contains BrdU. lj, lower jaw; h, heart. Scale bars: 400 µm (A-a, e), 100 µm (A-b–d, f–h) and 50 µm (B-a–d).

 
To determine whether a small and aberrant shape of the thymus anlage in ph/ph embryo is caused by reduced cell proliferation in the anlage, we investigated proliferation of epithelial cells and thymocytes in Ed12 thymus anlagen of wild-type and ph/ph embryos by BrdU labeling (Fig. 1B). In the ph/ph thymus anlage, the number of BrdU-labeled epithelial cells was slightly reduced in comparison with that of the wild type (Fig. 1B-a, c). Especially, in the wild-type anlage, the majority of outermost epithelial cells which are adjacent to the surrounding mesenchymal layer incorporates BrdU, but in the ph/ph anlage the number of BrdU-labeled outermost epithelial cells was lower than that in the wild-type anlage. Moreover, the number of BrdU-labeled Ikaros-positive cells was also reduced in the ph/ph anlage, as compared with that in the wild-type anlage (Fig. 1B-b, d). On the other hand, the numbers of apoptotic cells detected by TUNEL assay were very small in both the wild-type and the ph/ph anlagen on Ed13 (data not shown). These results indicate that in ph/ph mice, epithelial cells normally develop to the clustered epithelial stage on Ed12. Thereafter, however, they show aberrant morphology caused by reduced proliferation of outermost epithelial cells adjacent to the mesenchymal layer of the anlage.

Impaired development of thymocytes in the ph/ph thymus anlage
The results mentioned above show reduced proliferation of thymocytes in the ph/ph anlage. In the normal thymus anlage, thymocytes which uniformly express a CD44+CD25– (DN1) surface phenotype start proliferating in between epithelial cells of the anlage on Ed12 (26) and CD44+CD25+ (DN2) cells initially develop on Ed13 (30). Therefore, we examined whether CD25-positive cells can develop in the ph/ph thymus anlagen on Ed13. In the wild-type Ed13 thymus anlage, many CD25-positive cells were observed among the epithelial cells. In contrast, in the ph/ph thymus anlage, only a few CD25-positive cells were observed (Fig. 2A). These results indicate that T cell progenitors migrate to the ph/ph thymus anlage but they fail to develop there. It is likely that the impaired thymocyte development in ph/ph embryo is caused by the aberrantly developed thymus anlage. However, it is also possible that the impaired thymocyte development is attributable to a defect in progenitor cells themselves which migrate to the ph/ph thymus anlage. Therefore, we examined surface phenotypes of the Ed13 ph/ph fetal liver cell and T cell progenitor activities among them. There were no significant differences in the number of T cell progenitors and the percentages of progenitor phenotype cells in the Ed13 fetal liver between wild-type and ph/ph embryos (Supplementary Fig. 2, available at International Immunology Online). Thus, these results indicate that in ph/ph embryo, pre-thymic T cell progenitors normally develop and migrate to the epithelial cluster of the thymus anlage. However, development of T cell progenitors among the epithelial cells is impaired. Thus, the data suggest that epithelial cells of the ph/ph thymus anlage have some failure in a function required to support T cell development on Ed13. The homozygous ph/ph mutation results in lethality of embryo, and most of ph/ph embryos die prior to Ed13 (39–41, 52; see also Methods). To examine whether Ed13 ph/ph thymus anlagen can develop further, they were organ cultured under HOS conditions for 2 weeks. After culture, thymocytes were recovered and analyzed for CD4 and CD8 expression by flow cytometric analysis (Fig. 2B). Fifty-one of 54 cultured anlagen from Ed13 wild-type embryos contained all stages of thymocyte development including CD4+CD8– and CD4–CD8+ single-positive (SP) cells. On the other hand, seven of eight cultured anlagen from ph/ph embryos failed to develop CD4+CD8+ double-positive (DP) cells or SP cells and contained only a small number of DN cells. These results indicate that the ph/ph thymus anlage cannot create functional microenvironments to support full T cell development, even after prolonged organ culture.


Figure 2
View larger version (48K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. Limited T cell development in the ph/ph thymus anlage in vivo and in vitro. (A) Serial sagittal sections of wild-type and ph/ph embryos on Ed13 were stained for keratin (green) or Ikaros (red) and CD25 (green) by immunofluorescence staining. In the wild-type thymus anlage, many Ikaros-positive cells express CD25. In the ph/ph thymus anlage, only a few CD25-positive cells are observed. Scale bar: 50 µm. (B) The thymus anlagen of wild-type and ph/ph embryos were cultured for 2 weeks. After culture, thymocytes were harvested and the expression of CD4 and CD8 was analyzed by flow cytometry. The percentages of lobes containing CD4 single-positive and CD8 single-positive cells are shown.

 
Defects in expression of functional molecules in the ph/ph thymus anlage
Next, we examined whether the impaired development of thymocytes in the ph/ph thymus anlage could be ascribed to a failure in the expression of functional molecules by epithelial cells. Since proliferation of thymocytes at the early stages of T cell development mainly depends on SCF and IL-7 (21), we performed semi-quantitative RT–PCR analysis of mRNA prepared from the thymus anlagen of individual embryos on Ed13. The level of transcripts of SCF but not IL-7 was reduced in the ph/ph thymus anlagen compared with the wild-type anlagen (Fig. 3A). To determine the distribution of SCF within the Ed13 thymus anlagen, two-color immunofluorescence staining for keratin and SCF was performed. SCF was expressed on many epithelial cells throughout the wild-type thymus anlage. However, only faint expression of SCF was observed in a small portion of the epithelial cells in the ph/ph thymus anlage (Fig. 3B-a). Moreover, Ia molecules, which are reported to be expressed on epithelial cells of Ed13 thymus anlage (58), were found broadly on epithelial cells in the wild-type anlage. In contrast, Ia expression was scarcely observed in the ph/ph thymus anlage (Fig. 3B-b). As for chemokines, the transcripts of CCL21, CCL25 and CXCL12 were comparable between the wild-type and the ph/ph Ed13 thymus anlagen (data not shown). Recently, it has been reported that signaling through Notch1 receptor plays an important role in early T cell development (13) and that Notch activation in thymic immigrants occurs as soon as they migrate into the thymic epithelial region of the fetal thymus anlage (29). Therefore, we examined whether Notch ligands were expressed on epithelial cells in the ph/ph thymus anlagen (Fig. 3A and C). RT–PCR analysis showed that the levels of expression of Dll1, Jagged1 and Jagged2 in the ph/ph thymus anlagen on Ed13 were almost comparable to those in the wild-type anlagen. In three of five ph/ph embryos, the amounts of mRNA of Dll4 in the thymus anlagen were severely reduced compared with those in the wild-type embryos (Fig. 3A). In situ hybridization analysis showed that Dll1 was expressed in a small number of the cells scattered comparably in the epithelial regions of both the wild-type (Fig. 3C-a, b) and the ph/ph Ed13 thymus anlagen (Fig. 3C-d, e). On the other hand, while Dll4 was broadly distributed in the epithelial region of the wild-type anlage (Fig. 3C-c), in the ph/ph thymus anlage, Dll4 was only faintly expressed in a small number of cells in the epithelial region (Fig. 3C-f). These results indicate that epithelial cells of the ph/ph thymus anlage have severe defects in the expression of functional molecules that are required for the early stages in T cell development. Previously, we showed lack of the expressions of Dll1, Dll4 and SCF in the epithelial cells of the nude thymus anlagen (14 and N. Tsukamoto, unpublished observation). In the ph/ph thymus anlage, however, Foxn1 expression in epithelial cells was similar to that in the wild-type thymus anlage (Supplementary Fig. 1b and d, available at International Immunology Online).


Figure 3
View larger version (62K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. Reduced expression of SCF, Ia and Dll4 on epithelial cells of the ph/ph thymus anlage. (A) RT–PCR analysis for SCF, IL7, Dll1, Dll4, Jagged (Jag) 1 and Jag2 was performed on cDNAs obtained from thymus anlagen of each of wild-type and ph/ph embryos on Ed13. Data represent 1:1 and 1:3 dilutions of template cDNA normalized by a ß-actin-specific signal. (B) Two-color immunofluorescent staining for keratin (green) and SCF (a: red) or Ia (b: red) was performed on sagittal sections of Ed13 wild-type and ph/ph embryos. SCF and Ia are broadly expressed on epithelial cells of the wild-type thymus anlage. However, in the ph/ph thymus anlage, only a few epithelial cells weakly express SCF and Ia. Scale bars: 50 µm. (C) Serial sagittal sections of wild-type (a–c) and ph/ph (d–f) embryos on Ed13 were stained for keratin by immunohistochemistry (a and d) and for Dll1 and Dll4 by in situ hybridization (b, c, e and f). Dll1 is expressed on a small number of cells in the epithelial region of both the wild-type (b) and the ph/ph thymus anlagen (e). Dll4 is broadly expressed in the epithelial region of the wild-type thymus anlage (c), but only very weakly expressed in the ph/ph thymus anlage (f). Scale bar: 100 µm.

 
Mesenchymal defects in the ph/ph thymus anlage
Previous reports show that PDGFR{alpha} is expressed on mesenchymal cells of the thymus anlage (40, 59). The ph mutation primarily affects mesenchymal cells of the ph/ph thymus anlage and may cause defects in development of epithelial cells, as indicated above, as a result of insufficient epithelial–mesenchymal interactions. To clarify the effect of the ph mutation on mesenchymal cell development, we examined the distribution of mesenchymal cells in the ph/ph thymus anlage and the expression of FGF7 and FGF10, which are reported to play roles in proliferation of epithelial cells of the thymus anlage (36, 60). By immunofluorescence staining, PDGFR{alpha} was shown to be expressed on the outer mesenchymal layer of Ed13 anlage in wild type (Fig. 4A-a), but not in ph/ph embryos (data not shown). The mesenchymal cells of the outer layer also expressed PDGFRß, and many PDGFRß-expressing mesenchymal cells invaginated into the epithelial cluster in the wild-type anlage (Fig. 4A-b, c). The outer mesenchymal layer of PDGFRß-expressing cells was thinner in the ph/ph thymus anlage than in the wild-type anlage and was discontinuous in some parts. Moreover, only a small number of mesenchymal cells were observed within the epithelial cluster (Fig. 4A-d, e). RT–PCR analysis revealed that the expression of both FGF7 and FGF10 in the ph/ph anlage was reduced compared with that in the wild-type anlage (Fig. 4B). On the other hand, FGFR2IIIb, a receptor for FGF7 and FGF10, was expressed comparably in both the wild-type and the ph/ph anlagen. These results indicate that mesenchymal cells of the ph/ph thymus anlage are deficient in their ability to (i) form the outer mesenchymal layer, (ii) provide FGFs to induce proliferation of epithelial cells and (iii) migrate into the epithelial cluster.


Figure 4
View larger version (42K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. Defects in mesenchymal development in the ph/ph thymus anlage. (A) Serial sagittal sections of wild-type (a–c) and ph/ph (d and e) embryos on Ed13 were stained for PDGFR{alpha}(a), PDGFRß (b and d) and keratin (c and e) by immunofluorescence staining. PDGFR{alpha} is expressed on the mesenchymal layer surrounding the epithelial cluster of the wild-type thymus anlage (a). PDGFRß is expressed on the outer mesenchymal layer and on many cells invaginating into the epithelial cluster in the wild-type thymus anlage (b and c). In the ph/ph thymus anlage, PDGFRß is expressed on the thinner mesenchymal layer and on a few cells in the epithelial region (d and e). Scale bar: 100 µm. (B) RT–PCR analysis for FGF7, FGF10 and FGFR2IIIb (R2IIIb) was performed on cDNAs obtained from thymus anlagen of each of wile-type and ph/ph embryos on Ed13. Data represent 1:1 and 1:3 dilutions of template cDNA normalized by a ß-actin-specific signal.

 
Addition of intact thymic mesenchymal cells enables the ph/ph thymus anlage to form proper thymic microenvironments in organ culture
It seems likely that insufficient development of epithelial cells and thymocytes in the ph/ph thymus anlage is attributable to primary defects in mesenchymal cells. Therefore, we examined whether the ph/ph thymus anlage supplemented with intact thymic mesenchymal cells develops into a mature organ supporting full T cell development in organ culture (Fig. 5). Ed13 ph/ph thymus anlagen were cultured for 2 weeks with or without mesenchymal cells isolated from Ed13 thymus anlagen of EGFP Tg mice by enzymatic treatment. Though the ph/ph anlagen cultured alone (N = 4) were small in size, all the ph/ph anlagen cultured with intact mesenchymal cells (N = 9) increased in size equivalent to the cultured wild-type anlagen and contained CD4SP and CD8SP cells (Supplementary Fig. 3, available at International Immunology Online). The ph/ph anlagen cultured alone have still tightly packed epithelial cells and some cysts (Fig. 5c and d). In contrast, in the ph/ph anlagen cultured with intact mesenchymal cells, a meshwork structure of epithelial cells were observed (Fig. 5e and h) and the organization was similar to those in the wild-type anlagen cultured alone (Fig. 5a). Added mesenchymal cells of EGFP Tg mice were found in the outer layer of the anlagen forming a capsule and also among epithelial cells forming a meshwork structure (Fig. 5g–i). These results indicate that intact thymic mesenchymal cells are required to induce complete development of the ph/ph thymus anlage and for the formation of the organized architecture of the thymic stromal cells.


Figure 5
View larger version (58K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. Requirement for mesenchymal cells for development of the ph/ph thymus anlage in organ culture. The wild-type thymus anlage, the ph/ph thymus anlage alone and the Ed13 ph/ph thymus anlage together with intact thymic mesenchymal cells of EGFP Tg embryos were cultured for 2 weeks. Serial sections of the cultured lobes were stained for keratin (a, c, e and h) and fibronectin (b, d, f and i) by immunofluorescence staining. In the ph/ph thymus anlage cultured alone, epithelial cells are tightly packed (c). The number of fibronectin-expressing cells is small and a capsule structure is not clearly formed (d). In the cultured lobe of ph/ph embryo with mesenchymal cells from the EGFP Tg thymus anlage, an organized meshwork of epithelial cells is observed (e and h). Fibronectin-expressing cells are distributed in the capsule and among the epithelial cells (f and i), as in the cultured lobes of wild-type embryo (a and b). GFP-positive mesenchymal cells are forming a capsule structure and also are distributed among epithelial cells (g–i). Scale bars: 50 µm (a–g) and 20 µm (h and i).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary data
 References
 
In the present study, we have investigated the roles of mesenchymal cells in thymus organogenesis by analyzing the developmental defects of the thymus anlagen of ph/ph embryos. These embryos show a primary defect in mesenchymal cells by lack of PDGFR{alpha} expression. Our present results show that mesenchymal cells direct differentiation of epithelial cells of the thymus anlage such that they become able to express SCF, Dll4 and Ia molecules, which have crucial roles in T cell development. Moreover, our findings suggest that mesenchymal cells as well as epithelial cells are indispensable for creation and/or maintenance of the architecture and function of thymic microenvironments.

In ph/ph embryo, pre-thymic T cell progenitors develop normally and migrate to the thymus anlage. However, we found that in the thymus anlage of the ph/ph embryo on Ed13, thymocytes among epithelial cells proliferate less and only few DN2 cells develop. Moreover, even after organ culture, neither DP cells nor SP cells develop in the ph/ph thymus anlage. Thus, it is plausible that impaired thymocyte development in the ph/ph thymus anlage is not due to defects in T cell progenitors, but to defects in microenvironments of the anlage. In wild-type embryos, initial colonization of T cell progenitors to the thymus anlage occurs around Ed11 (26). The earliest immigrants which arrive among epithelial cells of the anlage on Ed12 start to proliferate and differentiate and generate DN2 cells on Ed13 (30). Thus, the ph/ph thymus anlage can attract T cell progenitors, however, the anlage is defective to support further development of thymic immigrants. Regarding the molecular basis for those functional defects, our results show severe impairment in SCF, Dll4 and Ia expression in epithelial cells of the ph/ph thymus anlage on Ed13. SCF is one of the known growth and survival factors for DN1–DN2 cells (15, 16) and mRNA of SCF is observed in the normal thymus anlage as early as on Ed12 (N. Tsukamoto, unpublished observation). In the ph/ph embryo, mesenchymal cells surrounding the epithelial cluster of the thymus anlage ectopically express c-kit, receptor for SCF (Supplementary Fig. 2, available at International Immunology Online). Thus, it is plausible that soluble SCF produced by epithelial cells of the ph/ph thymus anlage is consumed by the reaction with aberrantly expressed c-kit on mesenchymal cells, as in the case of defective development of melanocyte in the white spotting mice; Ph/+, Wsh/+ and Rw/+ mice (42, 61). However, poor immunoreactivity of SCF in thymic epithelial cells suggests that membrane-bound form of SCF expression is actually attenuated without relation to c-kit ectopic expression. Notch ligands expressed on thymic epithelial cells have a key role in T lineage commitment and DN cell development (11–13). We recently showed that mRNA of Dll4 is also expressed on epithelial cells of Ed12 thymus anlage (14). Therefore, impairment of the expression of these molecules on epithelial cells affects development of thymocytes in the ph/ph thymus anlage. Ia molecules are essential for selection of T cells (22), and these molecules are expressed on epithelial cells of the thymus anlagen from Ed13. Ia molecule expression on epithelial cells is induced by interaction with mesenchymal cells (37, 38). As shown in Supplementary Fig. 1 (available at International Immunology Online), when epithelial cell differentiation is assessed by expression patterns of Keratin5/8 and Foxn1, epithelial cells of the Ed13 ph/ph thymus anlagen seem to differentiate similarly to those of the wild-type anlagen. Therefore, it is likely that the defects in the expressions of SCF, Dll4 and Ia on epithelial cells of the Ed13 ph/ph thymus anlagen are not ascribed to retarded organogenesis of the anlagen, but to mesenchymal defects caused by the loss of PDGFR{alpha} expression.

During thymus organogenesis, the mesenchymal cell layer surrounding the epithelial cell cluster is formed around Ed12 (26, 33). Recently, it has been reported that the cell layer produces FGF7 and FGF10, which mediates proliferation of epithelial cells in the thymus anlage (35, 36, 60). We show here that a sufficient mesenchymal layer of the ph/ph thymus anlage is not formed. Moreover, a significant decrease in the expression of FGF7 and FGF10 mRNAs was observed in the ph/ph thymus anlage. In our preliminary experiment using in situ hybridization, the expression of FGF10 mRNA was observed in the outer mesenchymal layer of the thymus anlagen of wild-type embryos, consistent with the previous report (36). However, the signal of FGF10 mRNA in thymic mesenchymal cells was extremely weak as compared with that in the other developing organs, such as the lung and the tooth. On the other hand, in the mesenchymal layer of the ph/ph thymus anlagen, FGF10 mRNA was not detected. It seems to indicate that the expression of FGF10 mRNA decreases in the individual mesenchymal cells of the ph/ph thymus anlagen. However, the possibility that the number of FGF10 mRNA-expressing mesenchymal cells decreases in the ph/ph thymus anlagen still remains. Thus, an insufficiently formed mesenchymal layer in the ph/ph thymus anlage likely fails to provide FGFs enough to support epithelial cell proliferation. Our results also show that epithelial cells of the ph/ph thymus anlage on Ed13 fail to express functional molecules such as SCF, Dll4 and Ia. This indicates defects in functional differentiation of epithelial cells in the ph/ph thymus anlagen. Moreover, we showed that the Ed13 ph/ph thymus anlage cannot develop to mature organ to support full T cell development even after organ culture. However, the ph/ph thymus anlage co-cultured with intact thymic mesenchymal cells of the wild-type thymus anlage can fully differentiate as a functional organ. Previous reports show that the thymi of FGF10- or FGFR2IIIb-deficient mice are small, but they contain a significant number of SP cells on Ed18 (35, 36). These and our present results suggest that mesenchymal cells of the ph/ph thymus anlage have defects not only in production of FGF7 and FGF10 but also in production of other factors which induce epithelial cell differentiation.

The distribution of mesenchymal cells in the thymus anlage changes dramatically around Ed13, where some mesenchymal cells surrounding the epithelial cell cluster invaginate into the epithelial cluster on Ed13 (37, 62; see also Fig. 4). Subsequently, these cells scatter among epithelial cells and form a three-dimensionally organized structure mingled with epithelial cells. In the thymus anlage of the ph/ph mice lacking PDGFR{alpha} expression, the mesenchymal layer is not well formed, failing to entirely surround the epithelial cluster. Moreover, mesenchymal cells fail to migrate in between epithelial cells. Noteworthy, upon co-culture with intact mesenchymal cells from the thymus anlagen of EGFP Tg mice, the ph/ph thymus anlage develops into a mature organ supporting full T cell development, and the added mesenchymal cells form not only a capsule but also a meshwork structure with epithelial cells in the cultured lobes. Therefore, it is likely that migration of mesenchymal cells among epithelial cells of the thymus anlage is required to form the proper three-dimensional architecture. In this context, mesenchymal cells may act as a scaffold to provide signals to adjacent epithelial cells via short-range diffusible molecules, cell-surface molecules and/or extra cellular matrix molecules (63). It is also possible that they function as a stromal component supporting thymocyte development, as reported previously (64, 65). Our present results suggest that thymic mesenchymal cells expressing PDGFR{alpha} not only provide developmental signals to epithelial cells temporally but also contribute to create and maintain thymic microenvironments able to support T cell development.

The mesenchymal layer surrounding epithelial cells of the thymus anlage expresses PDGFR{alpha} (40, 59; see also Fig. 4). Our present results show that ph/ph mice, which are completely lacking PDGFR{alpha} expression, have defect in thymic mesenchymal cells, affecting epithelial development. Mesenchymal cells of the thymus anlage are mainly originated in the neural crest-derived cells (4, 50). In ph/ph mice and PDGFR{alpha} gene-deficient mice, defects in migration and proliferation of the neural crest cells in the head and neck region and increased apoptosis in the branchial arches have been reported (40, 45, 66). Therefore, it is likely that defect in mesenchymal cells of the ph/ph thymus anlage is owing to loss of PDGFR{alpha} signals on the neural crest cells. However, a recent report shows that neural crest cell-specific ablation of PDGFR{alpha}, by using Wnt-1-Cre/loxP system, results in the craniofacial and aortic arch defects, but only in mild abnormalities in other tissues and organs, including the thymus (67). From their results and our present results, it is suggested that in thymus organogenesis at least some part of the role of neural crest-derived mesenchymal cells is alternatively compensated by mesodermally derived mesenchymal cells which also express PDGFR{alpha}. PDGFR{alpha} signals have the roles not only in migration and survival of neural crest cells but also in differentiation of them in the cranial facial and aortic arch region (67, 68). Mesodermally derived mesenchymal cells of other organs, such as lung, intestine and kidney, also express PDGFR{alpha} and their proliferation and differentiation are depend on signals through PDGFR{alpha} (49). Thus, in thymus organogenesis mesenchymal cells of the anlage which express PDGFR{alpha} could be required PDGF signaling for their migration, proliferation and differentiation. Therefore, it is suggested that PDGFR{alpha} signals on mesenchymal cells are critical for thymus organogenesis.


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary Figures 1–3 are available at International Immunology Online.


   Acknowledgements
 
We thank S. Chiba for the plasmids containing Dll1 cDNA, M. Nishikawa for the plasmids containing Dll4 cDNA, S. -I. Nishikawa for APA5 and APB5 antibodies and M. Okabe for EGFP Tg mice. We also thank W. van Ewijk for helpful discussions and for critical reading of the manuscript.


   Abbreviations
 
BrdU, bromodeoxyuridine
Dll, Delta like
DN, CD4–CD8– double negative
DN1, CD44+CD25–
DN2, CD44+CD25+
DP, CD4+CD8+ double positive
Ed, embryonic day
EGFP Tg, enhanced GFP transgenic
FGF, fibroblast growth factor
HOS, high oxygen submersion
PDGFR, platelet-derived growth factor receptor
ph, patch
RT, reverse transcription
SP, CD4+CD8– and CD4–CD8+ single positive

   Notes
 
Transmitting editor: K. Sugamura

Received 29 June 2006, accepted 15 May 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 

  1. Katsura Y. Redefinition of lymphoid progenitors. Nat. Rev. Immunol. (2002) 2:127.[CrossRef][Web of Science][Medline]
  2. Masuda K, Itoi M, Amagai T, Minato N, Katsura Y, Kawamoto H. Thymic anlage is colonized by progenitors restricted to T, NK, and dendritic cell lineages. J. Immunol. (2005) 174:2525.[Abstract/Free Full Text]
  3. Harman BC, Jenkinson WE, Parnell SM, Rossi SW, Jenkinson EJ, Anderson G. T/B lineage choice occurs prior to intrathymic notch signaling. Blood (2005) 106:886.[Abstract/Free Full Text]
  4. Petrie HT. Role of thymic organ structure and stromal composition in steady-state postnatal T-cell production. Immunol. Rev. (2002) 189:8.[CrossRef][Web of Science][Medline]
  5. van Ewijk W, Wang B-P, Holländer G, et al. Thymic microenvironments, 3-D versus 2-D? Semin. Immunol. (1999) 11:57.[CrossRef][Web of Science][Medline]
  6. Liu C, Ueno T, Kuse S, et al. The role of CCL21 in recruitment of T-precursor cells to fetal thymi. Blood (2005) 105:31.[Abstract/Free Full Text]
  7. Benz C, Heinzel K, Bleul C. Homing of immature thymocytes to the subcapsular microenvironment within the thymus is not an absolute requirement for T cell development. Eur. J. Immunol. (2004) 34:3652.[CrossRef][Web of Science][Medline]
  8. Plotkin J, Prockop SE, Lepique A, Petrie HT. Critical role for CXCR4 signaling in progenitor localization and T cell differentiation in the postnatal thymus. J. Immunol. (2003) 171:4521.[Abstract/Free Full Text]
  9. Ara T, Itoi M, Kawabata K, et al. A role of CXC chemokine ligand 12/stromal cell-derived factor-1/pre-B cell growth stimulating factor and its receptor CXCR4 in fetal and adult T cell development in vivo. J. Immunol. (2003) 170:4649.[Abstract/Free Full Text]
  10. Ueno T, Saito F, Gray DHD, et al. CCR7 signals are essential for cortex-medulla migration of developing thymocytes. J. Exp. Med. (2004) 200:493.[Abstract/Free Full Text]
  11. Schmitt TM, Zúñiga-Pflücker JC. Induction of T cell development from hematopoietic progenitor cells by Delta-like-1 in vitro. Immunity (2002) 17:749.[CrossRef][Web of Science][Medline]
  12. Hozumi K, Negishi N, Suzuki D, et al. Delta-like 1 is necessary for the generation of marginal zone B cells but not T cells in vivo. Nat. Immunol. (2004) 5:638.[CrossRef][Web of Science][Medline]
  13. Schmitt TM, Ciofani M, Petrie HT, Zúñiga-Pflücker JC. Maintenance of T cell specification and differentiation requires recurrent Notch receptor-ligand interactions. J. Exp. Med. (2004) 200:469.[Abstract/Free Full Text]
  14. Tsukamoto N, Itoi M, Nishikawa M, Amagai T. Lack of Delta like 1 and 4 expressions in nude thymus anlages. Cell. Immunol. (2005) 234:77.[CrossRef][Web of Science][Medline]
  15. Asamoto H, Mandel TE. Thymus in mice bearing the steel mutation. Morphologic studies on fetal, neonatal, organ-cultured, and grafted fetal thymus. Lab. Investig. (1981) 45:418.[Web of Science]
  16. Rodewald H-R, Kretzschmar K, Swat W, Takeda S. Intrathymically expressed c-kit ligand (stem cell factor) is a major factor driving expansion of very immature thymocytes in vivo. Immunity (1995) 3:313.[CrossRef][Web of Science][Medline]
  17. von Freeden-Jeffry U, Vieira P, Lucian LA, McNeil T, Burdach SEG, Murray R. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. (1995) 181:1519.[Abstract/Free Full Text]
  18. Peschon JJ, Morrissey PJ, Grabstein KH, et al. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J. Exp. Med. (1994) 180:1955.[Abstract/Free Full Text]
  19. DiSanto JP, Müller W, Guy-Grand D, Fischer A, Rajewsky K. Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor {gamma} chain. Proc. Natl Acad. Sci. USA. (1995) 92:377.[Abstract/Free Full Text]
  20. Ohbo K, Suda T, Hashiyama M, et al. Modulation of hematopoiesis in mice with a truncated mutant of the interleukin-2 receptor {gamma} chain. Blood (1996) 87:956.[Abstract/Free Full Text]
  21. Rodewald H-R, Ogawa M, Haller C, Waskow C, DiSant JP. Pro-thymocyte expansion by c-kit and the common cytokine receptor {gamma} chain is essential for repertoire formation. Immunity (1997) 6:265.[CrossRef][Web of Science][Medline]
  22. Starr TK, Jameson SC, Hogquist KA. Positive and negative selection of T cells. Annu. Rev. Immunol. (2003) 21:139.[CrossRef][Web of Science][Medline]
  23. Bennett AR, Farley A, Blair NF, Gordon J, Sharp L, Blackburn CC. Identification and characterization of thymic epithelial progenitor cells. Immunity (2002) 16:803.[CrossRef][Web of Science][Medline]
  24. Cordier AC, Haumont SM. Development of thymus, parathyroids, and ultimo-branchial bodies in NMRI and nude mice. Am. J. Anat. (1980) 157:227.[CrossRef][Web of Science][Medline]
  25. Manley NR. Thymus organogenesis and molecular mechanisms of thymic epithelial cell differentiation. Semin. Immunol. (2000) 12:421.[CrossRef][Web of Science][Medline]
  26. 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.[Abstract/Free Full Text]
  27. Bleul CC, Boehm T. Chemokines define distinct microenvironments in the developing thymus. Eur. J. Immunol. (2000) 30:3371.[CrossRef][Web of Science][Medline]
  28. Zamisch M, Moore-Scott B, Su D-M, Lucas PJ, Manley N, Richie ER. Ontogeny and regulation of IL-7-expressing thymic epithelial cells. J. Immunol. (2005) 174:60.[Abstract/Free Full Text]
  29. Harman BC, Jenkinson EJ, Anderson G. Entry into the thymic microenvironment triggers Notch activation in the earliest migrant T cell progenitors. J. Immunol. (2003) 170:1299.[Abstract/Free Full Text]
  30. Amagai T, Itoi M, Kondo Y. Limited development capacity of the earliest embryonic murine thymus. Eur. J. Immunol. (1995) 25:757.[Web of Science][Medline]
  31. Le Lievre CS, Le Douarin NM. Mesenchymal derivatives of the neural crest: analysis of chimeric quail and chick embryos. J. Embryol. Exp. Morphol. (1975) 34:125.[Web of Science][Medline]
  32. Bockman DE, Kirby ML. Dependence of thymus development on derivatives of the neural crest. Science (1984) 223:498.[Abstract/Free Full Text]
  33. Auerbach R. Morphogenetic interactions in the development of the mouse thymus gland. Dev. Biol. (1960) 2:271.[CrossRef][Web of Science][Medline]
  34. Shinohara T, Honjo T. Epidermal growth factor can replace thymic mesenchyme in induction of embryonic thymus morphogenesis in vitro. Eur. J. Immunol. (1996) 26:747.[Web of Science][Medline]
  35. Ohuchi H, Hori Y, Yamasaki M, et al. FGF10 acts as a major ligand for FGF receptor 2IIIb in mouse multi-organ development. Biochem. Biophys. Res. Commun. (2000) 277:643.[CrossRef][Web of Science][Medline]
  36. Revest J-M, Suniara RK, Kerr K, Owen JJT, Dickson C. Development of the thymus requires signaling through the fibroblast growth factor receptor R2-IIIb. J. Immunol. (2001) 167:1954.[Abstract/Free Full Text]
  37. Itoi M, Amagai T. Inductive role of fibroblastic cell lines in development of the mouse thymus anlage in organ culture. Cell. Immunol. (1998) 183:32.[CrossRef][Web of Science][Medline]
  38. Shinohara T, Honjo T. Studies in vitro on the mechanism of the epithelial/mesenchymal interaction in the early fetal thymus. Eur. J. Immunol. (1997) 27:522.[Web of Science][Medline]
  39. Grüneberg H, Truslove GM. Two closely linked genes in the mouse. Genet. Res. Camb. (1960) 1:69.[Web of Science]
  40. Morrison-Graham K, Schatteman GC, Bork T, Bowen-Pope DF, Weston JA. A PDGF receptor mutation in the mouse (Patch) perturbs the development of a non-neuronal subset of neural crest-derived cells. Development (1992) 115:133.[Abstract]
  41. Orr-Urtreger A, Bedford MT, Do MS, Eisenbach L, Lonai P. Developmental expression of the alpha receptor for platelet-derived growth factor, which is deleted in the embryonic lethal Patch mutation. Development (1992) 115:289.[Abstract]
  42. Duttlinger R, Manova K, Berrozpe G, et al. The Wsh and Ph mutations affect the c-kit expression profile: c-kit misexpression in embryogenesis impairs melanogenesis in Wsh and Ph mutant mice. Proc. Natl Acad. Sci. USA (1995) 92:3754.[Abstract/Free Full Text]
  43. Stephenson DA, Mercola M, Anderson E, et al. Platelet-derived growth factor receptor {alpha}-subunit gene (Pdgfra) is deleted in the mouse patch (Ph) mutation. Proc. Natl Acad. Sci. USA (1991) 88:6.[Abstract/Free Full Text]
  44. Nagle DL, Martin-DeLeon P, Hough RB, Bucan M. Structural analysis of chromosomal rearrangements associated with the developmental mutations Ph, W19H, and Rw on mouse chromosome 5. Proc. Natl Acad. Sci. USA (1994) 91:7237.[Abstract/Free Full Text]
  45. Soriano P. The PDGF{alpha} receptor is required for neural crest cell development and for normal patterning of the somites. Development (1997) 124:2691.[Abstract]
  46. Schatteman GC, Morrison-Graham K, van Koppen A, Weston JA, Bowen-Pope DF. Regulation and role of PDGF receptor {alpha}-subunit expression during embryogenesis. Development (1992) 115:123.[Abstract]
  47. Orr-Urtreger A, Lonai P. Platelet-derived growth factor-A and its receptor are expressed in separate, but adjacent cell layers of the mouse embryo. Development (1992) 115:1045.[Abstract]
  48. Li X, Pontén A, Aase K, et al. PDGF-C is a new protease-activated ligand for the PDGF{alpha}-receptor. Nat. Cell Biol. (2000) 2:302.[CrossRef][Web of Science][Medline]
  49. Betsholtz C, Karlsson L, Lindahl P. Developmental roles of platelet-derived growth factors. Bioessays (2001) 23:494.[CrossRef][Web of Science][Medline]
  50. Jiang X, Rowitch DH, Soriano P, McMahon AP, Sucov HM. Fate of the mammalian cardiac neural crest. Development (2000) 127:1607.[Abstract]
  51. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. (1997) 407:313.[CrossRef][Web of Science][Medline]
  52. Price RL, Thielen TE, Borg TK, Terracio L. Cardiac defects associated with the absence of the platelet-derived growth factor {alpha} receptor in the Patch mouse. Microsc. Microanal. (2001) 7:56.[Web of Science][Medline]
  53. Hattori N, Kawamoto H, Fujimoto S, Kuno K, Katsura Y. Involvement of transcription factors TCF-1 and GATA-3 in the initiation of the earliest step of T cell development in the thymus. J. Exp. Med. (1996) 184:1137.[Abstract/Free Full Text]
  54. Schaeren-Wiemers N, Gerfin-Moser A. A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes. Histochemistry (1993) 100:431.[CrossRef][Web of Science][Medline]
  55. Watanabe Y, Katsura Y. Development of T cell receptor {alpha}ß-bearing T cells in the submersion organ culture of murine fetal thymus at high oxygen concentration. Eur. J. Immunol. (1993) 23:200.[Web of Science][Medline]
  56. Klug DB, Carter C, Gimenez-Conti IB, Richie ER. Thymocyte-independent and thymocyte-dependent phases of epithelial patterning in the fetal thymus. J. Immunol. (2002) 169:2842.[Abstract/Free Full Text]
  57. Gill J, Malin M, Holländer GA, Boyd R. Generation of a complete thymic microenvironment by MTS24+ thymic epithelial cells. Nature (2002) 3:635.
  58. van Vliet E, Jenkinson EJ, Kingston R, Owen JJT, van Ewijk W. Stromal cell types in the developing thymus of the normal and nude mouse embryo. Eur. J. Immunol. (1985) 15:675.[Web of Science][Medline]
  59. Takakura N, Yoshida H, Ogura Y, Kataoka H, Nishikawa S, Nishikawa S. PDGFR{alpha} expression during mouse embryogenesis: immunolocalization analyzed by whole-mount immunohistostaining using the monoclonal anti-mouse PDGFR{alpha} antibody APA5. J. Histochem. Cytochem. (1997) 45:883.[Abstract/Free Full Text]
  60. Jenkinson WE, Jenkinson EJ, Anderson G. Differential requirement for mesenchyme in the proliferation and maturation of thymic epithelial progenitors. J. Exp. Med. (2003) 198:325.[Abstract/Free Full Text]
  61. Yoshida H, Kunisada T, Grimm T, Nishimura EK, Nishikawa E, Nishikawa S. Melanocyte migration and survival controlled by SCF/c-kit expression. J. Investig. Dermatol. Symp. Proc. (2001) 6:1.[CrossRef][Web of Science][Medline]
  62. Suniara RK, Jenkinson EJ, Owen JJ T. An essential role for thymic mesenchyme in early T cell development. J. Exp. Med. (2000) 191:1051.[Abstract/Free Full Text]
  63. Adams JC, Watt FM. Regulation of development and differentiation by the extracellular matrix. Development (1993) 117:1183.[Web of Science][Medline]
  64. Anderson G, Jenkinson EJ, Moore NC, Owen JJ T. MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus. Nature (1993) 362:70.[CrossRef][Medline]
  65. Anderson G, Anderson KL, Tchilian EZ, Owen JJT, Jenkinson EJ. Fibroblast dependency during early thymocyte development maps to the CD25+CD44+ stage and involves interactions with fibroblast matrix molecules. Eur. J. Immunol. (1997) 27:1200.[Web of Science][Medline]
  66. Erickson CA, Weston JA. An SEM analysis of neural crest migration in the mouse. J. Embryol. Exp. Morphol. (1983) 74:97.[Web of Science][Medline]
  67. Tallquist MD, Soriano P. Cell autonomous requirement for PDGFR{alpha} in populations of cranial and cardiac neural crest cells. Development (2003) 130:507.[Abstract/Free Full Text]
  68. Robbins JR, McGuire PG, Wehrle-Haller B, Rogers SL. Diminished matrix metalloproteinase 2 (MMP-2) in ectomesenchyme-derived tissues of the Patch mutant mouse: regulation of MMP-2 by PDGF and effects on mesenchymal cell migration. Dev. Biol. (1999) 212:255.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Histochem. Cytochem.Home page
C. Odaka
Localization of Mesenchymal Cells in Adult Mouse Thymus: Their Abnormal Distribution in Mice With Disorganization of Thymic Medullary Epithelium
J. Histochem. Cytochem., April 1, 2009; 57(4): 373 - 382.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
D. A. Sultana, S. Tomita, M. Hamada, Y. Iwanaga, Y. Kitahama, N. Van Khang, S. Hirai, I. Ohigashi, S. Nitta, T. Amagai, et al.
Gene expression profile of the third pharyngeal pouch reveals role of mesenchymal MafB in embryonic thymus development
Blood, March 26, 2009; 113(13): 2976 - 2987.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Zuklys, J. Gill, M. P. Keller, M. Hauri-Hohl, S. Zhanybekova, G. Balciunaite, K.-J. Na, L. T. Jeker, K. Hafen, N. Tsukamoto, et al.
Stabilized {beta}-Catenin in Thymic Epithelial Cells Blocks Thymus Development and Function
J. Immunol., March 1, 2009; 182(5): 2997 - 3007.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. M. Muller, C. C. Stolt, G. Terszowski, C. Blum, T. Amagai, N. Kessaris, P. Iannarelli, W. D. Richardson, M. Wegner, and H.-R. Rodewald
Neural Crest Origin of Perivascular Mesenchyme in the Adult Thymus
J. Immunol., April 15, 2008; 180(8): 5344 - 5351.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
K. Foster, J. Sheridan, H. Veiga-Fernandes, K. Roderick, V. Pachnis, R. Adams, C. Blackburn, D. Kioussis, and M. Coles
Contribution of Neural Crest-Derived Cells in the Embryonic and Adult Thymus
J. Immunol., March 1, 2008; 180(5): 3183 - 3189.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
19/8/953    most recent
dxm060v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Itoi, M.
Right arrow Articles by Amagai, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Itoi, M.
Right arrow Articles by Amagai, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?