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International Immunology Advance Access originally published online on December 6, 2006
International Immunology 2007 19(2):127-132; doi:10.1093/intimm/dxl129
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Expression of Dll4 and CCL25 in Foxn1-negative epithelial cells in the post-natal thymus

Manami Itoi*, Noriyuki Tsukamoto* and Takashi Amagai*

Department of Immunology and Microbiology, Meiji University of Oriental Medicine, Hiyoshi-cho, Nantan, Kyoto 629-0392, Japan

Correspondence to: T. Amagai; E-mail: t_amagai{at}meiji-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Foxn1 transcription factor is known to be essential for development of the thymic organ. We analyzed whether Foxn1 expression in thymic epithelial cells is necessary for the expression of functional molecules such as Delta-like 4 (Dll4) and CCL25, and whether maintenance of these molecular expressions depends on the Foxn1 transcription factor. We show that almost all thymic epithelial cells in the early thymus anlagen express Foxn1, and Dll4 and CCL25 are limitedly expressed in Foxn1-positive epithelial cells. The results are consistent with previous reports suggesting the indispensability of Foxn1 for epithelial cell differentiation which enables these cells to induce the expressions of CCL25 (Bleul, C. C. and Boehm, T. 2000. Chemokines define distinct microenvironments in the developing thymus. Eur. J. Immunol. 30:3371), Dll1 and Dll4 (Tsukamoto, N., Itoi, M., Nishikawa, M. and Amagai, T. 2005. Lack of Delta like 1 and 4 expressions in nude thymus anlages. Cell. Immunol. 234:77). On the other hand, the expression of Foxn1 was not detectable in a large number of post-natal thymic epithelial cells. Both Foxn1-positive and -negative epithelial cells seem to express Dll4 and CCL25. Therefore, the expressions of Dll4 and CCL25 are independent of Foxn1 transcription factor in the post-natal thymus. These results indicate that in the post-natal thymus, epithelial cells may maintain the expressions of those functional molecules without the aid of Foxn1 transcription factor.

Keywords: CCL25, Dll4, nude, thymus


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary data
 References
 
T cell development in the thymus depends on interactions with thymic stromal cells, especially epithelial cells originating in the pharyngeal endoderm (1). The third pharyngeal pouch endoderm protrudes into the pharyngeal arch mesenchymal region on embryonic day (Ed) 9–11, resulting in the formation of the common thymus/parathyroid anlage (2, 3). In the anlage on Ed11.5, Gcm2, a transcription factor, is expressed in the dorsal and cranial portion where the parathyroid develops. Foxn1, a transcription factor of the winged helix/forkhead class, is expressed in the caudal and ventral regions where the thymus develops (3). We showed that the thymus anlage has a stratified bilayer structure on Ed11, and then converts to a clustered organization on Ed12 and into a meshwork structure on Ed13 (4). Initial colonization of T cell progenitors to the thymus anlage occurs around Ed11 (4, 5), and the earliest immigrants migrate among epithelial cells and immediately start proliferation and differentiation in the anlage on Ed12 (4). Thymic epithelial cells produce various signaling molecules for T cell development, such as chemokines: CCL21 and CCL25 for T cell progenitor migration (6) and CXCL12, CCL19 and CCL25 for thymocyte trafficking in the thymus (7–10) and Notch ligands: Delta-like 1 (Dll1) and Dll4 for T cell lineage commitment and for early thymocyte development at CD4–CD8– double-negative stages (11–13) and stem cell factor (SCF) and IL7 for thymocyte proliferation (14–16). From the early stage of thymus anlage development, epithelial cells express functional molecules essential for early T cell development, such as CCL21, CCL25 (6, 17), IL7 (18), Dll1 and Dll4 (13, 19).

The nude phenotype of athymia and hairlessness (20) is caused by a loss-of-function point mutation in the Foxn1 gene (21). Thymic epithelial cells of the nude thymus anlage can form a cluster structure but cannot attract progenitor cells into the epithelial cluster; consequently, the colonization of lymphoid precursors into the thymus epithelial cluster does not occur (4, 22). Epithelial cells of the nude thymus anlagen do not express Dll1 and Dll4 (13) and also lack the expression of CCL25 and CXCL12 (17). These results suggest that the Foxn1 gene product has an essential role in the differentiation of epithelial cells that enable these cells to induce functional molecules for T cell development.

In this study, we addressed three questions as follows: whether the expression of Foxn1 protein changes during ontogeny, whether expressions of functional molecules, such as Dll4 and CCL25, depend on the Foxn1 transcription factor and whether thymic epithelial cells necessarily express Foxn1 protein to maintain the expression of those molecules at the single-cell level. We show that Foxn1 is expressed in almost all epithelial cells in the early thymus anlage along with Dll4 and CCL25. However, in the post-natal thymus, both Foxn1-positive and -negative epithelial cells express Dll4 or CCL25. The results indicate that Foxn1 is indispensable for the development of thymic epithelial cells but may not be necessarily required for the maintenance of functional molecule expressions, such as Dll4 and CCL25.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary data
 References
 
Mice
C57BL/6 (B6) 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.

Preparation of anti-Foxn1 antiserum
Foxn1 cDNA was inserted into the pGEX-4T-3 (Amersham Biosciences, NJ, USA) BamH1/Xho1 site. Escherichia coli, transfected with pGEX-Foxn1, were cultured in 23YT-G medium with ampicillin and induced by the addition of 0.1 mM Isopropylthio-ß-D-galactoside. The lysate of E. coli was purified with glutathione beads (Amersham Biosciences). Affinity-purified glutathione-S-transferase-tagged Foxn1 protein was electrophoresed in SDS–polyacrylamide gel, and then a 115-kDa band was excised. After washing with saline, gel containing ~1 mg of protein was emulsified with CFA and injected into Japanese White Rabbits subcutaneously, four times. Blood was taken 10 days after the final injection (23). Sera were stocked frozen at –20°C.

Western blot analysis
Tissue or cells were solubilized with SDS lysis buffer and boiled for 10 min, and then 10 µg of samples were loaded and electrophoresed in 7.5% SDS-PAGE. Proteins in gel were transferred to the polyvinylidene difluoride membrane (NEN, Boston, MA, USA). The blocked membrane was reacted with anti-Foxn1 antibodies and then HRP–anti-rabbit IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and visualized by chemiluminescence reaction with SuperSignal® West Pico kit (Pierce, IL, USA).

Cytospin specimens of thymic stromal cells
Thymi from B6 mice of various ages were removed and minced with fine scissors in MEM (Nissui, Tokyo, Japan). After pipetting, tissue fragments were sedimented in 1 x g for 15 min and floating cells were discarded, and this procedure was repeated 10 times. The sedimented fragments were then treated with Collagenase/Dispase (Boehringer Mannheim, Mannheim, Germany) for 30 min at room temperature. Digested cells were filtered through cotton layers and centrifuged at 200 x g for 10 min, and then lymphoid cells were removed by treatment with anti-CD45.1 antibody (eBioscience, San Diego, CA, USA) and CellectionTM Pan anti-mouse IgG magnetic beads (Dynal, Oslo, Norway). Cytospin specimens of these cells were prepared by centrifugation at 250 x g for 5 min in a Shandon centrifuge.

Immunohistochemistry and immunofluorescence staining
Immunohistochemistry and immunofluorescence staining were performed as previously described (4). Freshly cut frozen sections were fixed with 4% PFA in PBS at room temperature for 10 min. The primary antibodies used in this study were rabbit anti-Foxn1 antibody, sheep anti-keratin antibody (anti-human broad spectrum cytokeratin, The Binding Site, Birmingham, UK), goat anti-Dll4 antibody (R&D, Minneapolis, MN, USA) and goat anti-CCL25 antibody (R&D), and then sections were stained with fluorescent-labeled or biotin-conjugated secondary antibodies. The secondary antibodies used were horse biotinylated anti-goat IgG (Vector, Burlingame, CA, USA), donkey anti-sheep IgG Alexa Fluor 488 and donkey anti-rabbit IgG Alexa Fluor 594 (Molecular Probes, Eugene, OR, USA). Biotinylated secondary antibodies were visualized by Streptavidin Alexa Fluor 594 (Molecular Probes) and Streptavidin Alexa Fluor 488 (Molecular Probes). Thymus images were captured by Olympus Provis AX80 microscopy (Tokyo). Digital images were acquired by Coolsnap (Roper Japan, Tokyo, Japan). Images were processed using Photoshop (Adobe, San Jose, CA, USA).


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 References
 
Rabbit anti-Foxn1 antibody recognizes 85-kDa nuclear protein
First, the reactivity of anti-Foxn1 antibody was analyzed by western blot analysis and tissue distribution (Fig. 1). Anti-Foxn1 antibody specifically reacted with ~85-kDa molecule in the extract from Foxn1 cDNA-transfected COS1 cells and in the extract from the thymus anlage but not with those from the brain, heart, kidney and somite from Ed14 B6 embryos (Fig. 1a). Immunocytological staining revealed that anti-Foxn1 antibody did not react to control COS1 cells transfected with pSR{alpha}, but stained the nuclei of pSR{alpha}-Foxn1 cDNA-transfected COS1 cells (Supplementary Figure 1, available at International Immunology Online). Immunohistochemical staining of sections of Ed14 whole embryos showed that only nuclei of thymic cells were stained with this antibody (Fig. 1b).


Figure 1
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  		Fig. 1. Characterization of anti-Foxn1 antibody. (a) Western blot analysis of various tissue lysates from Ed14 fetuses of B6 mice. (b) Tissue distribution of Foxn1-positive cells in Ed14 whole embryo section. The thymus anlage is indicated by an arrow. The inset is a higher magnification of the thymus anlage.

 
Expression of Foxn1 is limited to a subset of thymic epithelial cells in post-natal mice
To clarify ontogenic changes in Foxn1 expression in thymic epithelial cells, sections of Ed11, 11.5, 12 and 16 whole embryos and sections of 1-day-old and 8-week-old thymi were stained with anti-Foxn1 (red) and anti-keratin (green) as an epithelial cell marker (Fig. 2). On Ed11, Foxn1 was not expressed in the thymus anlage (Fig. 2a). On Ed11.5, many keratin-positive cells in the anlage began to express Foxn1 (Fig. 2b) consistent with the previous report using in situ hybridization (ISH) (3). On Ed12, almost all epithelial cells in the thymus anlage expressed Foxn1 (Fig. 2c). On Ed16, the majority of keratin-positive cells were positive for Foxn1 but a few Foxn1-negative epithelial cells were distributed among them (Fig. 2d). In the newborn thymus, more epithelial cells lost Foxn1 expression (Fig. 2e), and in the adult thymus, only a small subset of keratin-positive cells expressed Foxn1 (Fig. 2f).


Figure 2
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  		Fig. 2. Expression of Foxn1 in thymic epithelial cells at various ages. Frozen sections of Ed11 (a), Ed11.5 (b), Ed12 (c), Ed16 (d) embryos, newborn (e) and 8-week-old thymi of B6 mice (f) were stained with anti-Foxn1 (red) and anti-keratin (green). M: medulla and C: cortex. (g–i) Isolated thymic stromal cells from Ed13 B6 embryos or 2- to 4-week-old B6 mice were stained with anti-Foxn1 (red) and anti-keratin (green) using cytospin preparations. Percentages of Foxn1-positive cells in keratin-positive cells are shown (g). One hundred to one thousand keratin-positive cells were examined for Foxn1 expression in a single experiment, and two or three individual experiments were performed. Representative staining of cytospin preparations of Ed13 (h) and 3-week-old (i) thymic stromal cells are shown. Scale bars: (a–e and f) 50µm; (h and i) 20 µm.

 
To quantify the proportions of Foxn1-positive cells among thymic epithelial cells of different ages, we stained Foxn1 and keratin using a cytospin preparation of thymic stromal cells from Ed13 thymus anlagen and 2- to 4-week-old thymi. The majority of epithelial cells from Ed13 thymus anlagen expressed Foxn1 and only a small subset of epithelial cells from the young thymi expressed Foxn1 (Fig. 2h and i). The proportions of Foxn1-positive cells in keratin-positive cells were 80.2% in stromal cells of Ed13 thymus anlagen and 24.0% in stromal cells of the young thymus (Fig. 2g). These histological and cytological studies may indicate that the majority of epithelial cells of the early thymus anlage but only a portion of thymic epithelial cells of post-natal mice express Foxn1. Otherwise, post-natal thymic epithelial cells express only an undetectable amount of Foxn1 protein. These results suggest that in the post-natal thymus, a large number of epithelial cells creating a thymic microenvironment do not express Foxn1 transcription factor.

Functions of thymic epithelial cells are maintained independently of Foxn1 expression
Our results show that almost all epithelial cells of the early embryonic thymus anlage expressed Foxn1 protein, whereas a limited population of post-natal thymic epithelial cells expressed Foxn1 protein. It is known that Foxn1 expression is essential for the development of epithelial cells at early stages of thymus organogenesis (24, 25). Therefore, to investigate the role of Foxn1 in the functional molecule expression in thymic epithelial cells, we performed two-color immunofluorescent staining of Foxn1 and functional molecules, such as Dll4 or CCL25, using a cytospin preparation of thymic stromal cells from Ed13 embryos and 2- to 4-week-old mice at the single-cell level. In the epithelial cells of early embryonic thymus anlage, almost all Dll4-positive cells and CCL25-positive cells expressed Foxn1 (Fig. 3a and b). The proportions of Foxn1-positive cells in Dll4-positive cells and CCL25-positive cells in Ed13 thymic stromal cells were 96.0 and 95.8%, respectively (Fig. 3e). On the other hand, in post-natal thymic epithelial cells, significant portions of Dll4-positive cells and CCL25-positive cells were shown to be Foxn1 negative or undetectable (Fig. 3c and d). The proportions of Foxn1-positive cells in Dll4-positive cells and CCL25-positive cells were 41.0 and 32.0%, respectively (Fig. 3e). These results indicate that only Foxn1-positive thymic epithelial cells in the early thymus anlage express Dll4 and CCL25 but thymic epithelial cells in post-natal mice can express these molecules without a detectable amount of Foxn1 transcription factor.


Figure 3
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  		Fig. 3. Expressions of Foxn1 and functional molecules at the single-cell level in cytospin preparations of thymic stromal cells. Thymic stromal cells from Ed13 embryos (a and b) and 3-week-old B6 mice (c and d) were stained for Foxn1 (red) and Dll4 (green) (a and c) and for Foxn1 (red) and CCL25 (green) (b and d). (e) Percentages of Foxn1-positive cells among Dll4- and CCL25-positive cells using cytospin preparations of stromal cells. One hundred to one thousand Dll4- or CCL25-positive cells were examined for Foxn1 expression in a single experiment, and two or three individual experiments were performed. Scale bar: (a–d) 20 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary data
 References
 
Nude mice have loss-of-function mutations in the gene encoding the transcription factor Foxn1 (21). 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 the development of thymic epithelial cells (26). Our previous study showed that early epithelial cells of the normal thymus anlage can attract T progenitor cells but epithelial cells of the nude thymus anlage cannot (4). The nude thymus anlage lacks the expression of CCL25 and CXCL12 (17). Recently, we reported that mRNAs of Dll1 and Dll4 are expressed in epithelial cells of the Ed12 thymus anlage of normal mice but not in those of the nude thymus anlage (13). Moreover, thymic epithelial cells of the nude thymus anlage also have defects in the expression of SCF and FGFR2IIIb (N. Tsukamoto, unpublished observation). Our results indicate that Foxn1 protein is detectable from Ed11.5, consistent with a previous report using ISH (3) in the thymus anlage, and Foxn1 expression is found in almost all epithelial cells of the early thymus anlage of normal mice on Ed12. On Ed13, epithelial cells of the thymus anlage express Foxn1 along with functional molecules, Dll4 and CCL25, at the single-cell level. These results indicate that the expression of Foxn1 in thymic epithelial cells is required to induce the expressions of functional molecules such as Dll4 and CCL25. Therefore, it is suggested that Foxn1 is indispensable for the functional differentiation of thymic epithelial cells.

In this study, we first showed that in post-natal thymi only a small subset of epithelial cells express Foxn1 protein, and both Foxn1-positive and -negative epithelial cells express functional molecules, such as Dll4 and CCL25, which have essential roles in T cell development. Our results and previous studies (13, 17) indicate that Foxn1 transcription factor functions to induce functional epithelial cell development. However, in the post-natal thymus, a large number of epithelial cells which create a thymic microenvironment maintain their function without Foxn1 or with an undetectable amount of Foxn1. Thus, these results suggest that the expression of functional molecules, Dll4 and CCL25, may not be directly regulated by Foxn1 transcription factor.

Since Foxn1-positive epithelial cells are scattered throughout the cortex and medulla, the distribution pattern of Foxn1-positive cells differs from that of MTS24-positive epithelial cells localized in the medulla, which are shown as putative epithelial progenitor cells (27). The N-terminal domain of Foxn1 is required for crosstalk-dependent thymic epithelial cell differentiation to form cortical and medullary architecture (28). Recently, Mohtashami et al. (29) reported that a monolayer of cultured fetal thymic epithelial cells lost expressions of Foxn1 as well as Dll1 and Dll4. Therefore, three-dimensional thymic architecture is essential for the maintenance of thymic epithelial cell function (30). Furthermore, in the course of preparing the manuscript, Bleul et al. (31) reported that post-natal thymic epithelial cells of normal mice contain bipotent progenitor cells which can give rise to cortical and medullary epithelial cells, and epithelial cells of the post-natal thymic rudiment in Foxn1 mutant mice can develop to functionally mature epithelial cells to support full T cell development after the supply of Foxn1 protein. Therefore, the expression of Foxn1 is essential for progenitor activity in thymic epithelial cells. Collectively, these and our results suggest that Foxn1-positive epithelial cells in the post-natal thymus may serve as progenitor cells essential for the maintenance of functional epithelial cells. We are presently analyzing the role of Foxn1-positive epithelial cells in the post-natal thymus.


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary figure 1 is available at International Immunology Online.


   Acknowledgements
 
We thank Dr. Hiroyuki Kishi for providing the plasmid and Ms. Yuko Sakai for technical help. This work was supported by a grant from the Ministry of Education, Culture, Sports, Sciences and Technology, Japan.


   Abbreviations
 
B6, C57BL/6
Dll, Delta like
Ed, embryonic day
ISH, in situ hybridization
SCF, stem cell factor

   Notes
 
* These authors contributed equally to this study. Back

Transmitting editor: T. Saito

Received 4 July 2006, accepted 9 November 2006.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 

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