HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mohtashami, M. Articles by Zúñiga-Pflücker, J. C. Search for Related Content PubMed PubMed Citation Articles by Mohtashami, M. Articles by Zúñiga-Pflücker, J. C.

Cutting Edge: Three-Dimensional Architecture of the Thymus Is Required to Maintain Delta-Like Expression Necessary for Inducing T Cell Development¹

Department of Immunology, University of Toronto, and Sunnybrook & Women’s Research Institute, Toronto, Ontario, Canada

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The three-dimensional microarchitecture of the thymus plays a unique role in directing T cell lineage commitment and development. This is supported by the fact that, in contrast to fetal thymic organ cultures, thymic stromal cell monolayer cultures (TSMC) fail to support T lymphopoiesis. Nevertheless, OP9-DL1 cell monolayer cultures induce T lineage commitment and differentiation. Thus, the inability of TSMC to support T lymphopoiesis may be due to a loss of Notch ligand expression and/or function during culture. In this study, we report that, in contrast to fetal thymic organ cultures, TSMC fail to maintain expression of the Notch ligands, Delta-like (Dll) 1 and Dll4, and concomitantly lose the ability to support T lymphopoiesis. Importantly, ectopic re-expression of Dll1 or Dll4 is sufficient to restore the ability of TSMC to support T lymphopoiesis. These findings demonstrate that maintenance of endogenous Dll1 or Dll4 expression by thymic stromal cells is required for the commitment and differentiation of T cells in the absence of a three-dimensional microenvironment.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The thymus plays a central role in inducing and supporting the differentiation of hemopoietic progenitor cells (HPCs)³ into T cells. An important feature of the thymic microenvironment is its three-dimensional (3-D) organization, consisting of an ordered architecture of thymic stromal cells (TSCs), which may be epithelial or mesenchymal in origin, through which the developing thymocytes migrate and mature (1). This 3-D configuration maximizes the interaction of developing thymocytes with the supporting stromal cells, allowing intercellular cross-talk integral to the development of both T cells and TSCs (2). With this in mind, in vitro models for the generation of T cells were thought to require an intact 3-D thymic architecture, such as that maintained by fetal thymic organ cultures (FTOCs) (3) or reaggregate thymic organ cultures (RTOCs) (4). Both FTOCs and RTOCs support the generation of all T cell subsets. In contrast to RTOCs, in which TSCs and HPCs are assembled to form a 3-D structure, two-dimensional monolayers composed of freshly isolated TSCs or cell lines derived from TSCs were shown to be incapable of replicating a functional thymic microenvironment (5).

The requirement for a 3-D-supporting stroma appears to be unique to T cell development, as the in vitro development of other hemopoietic lineages, including B cells and NK cells, does not require a 3-D structure (6). However, this paradigm had to be revised with the advent of the OP9-DL1 coculture system (7, 8), in which T cell development progresses unimpeded on heterologous bone marrow stromal cell monolayers. Nevertheless, the reason behind the seemingly paradoxical finding that fresh ex vivo thymic stromal cell monolayer cultures (TSMCs) fail to support T lymphopoiesis remained unresolved.

To elucidate the mechanism behind the failure of TSMCs to support T cell development, we examined the expression levels of Notch ligands in TSCs in both monolayer culture and in FTOCs. Strikingly, expression of Delta-like (Dll) 1 and its closely related homologue, Dll4, were extinguished in TSMCs, while both transcripts were maintained in FTOCs and RTOCs. In contrast, Jagged 1 (Jag1) expression remained similar in both TSMCs and FTOCs, whereas Jag2 expression was down-regulated in TSMCs. Importantly, TSCs transduced to re-express Dll1 or Dll4 in monolayer culture regained the ability to support T lineage commitment and differentiation. This indicates that the loss of Dll expression (Dll1 and Dll4) by TSCs in monolayer culture is a causal mechanism behind the erosion of their ability to support T lymphopoiesis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Cells and cell lines

HPCs were isolated as previously described (7). Briefly, day 14–15 fetal livers obtained from timed-pregnant CD-1 mice (Charles River Breeding Laboratories) were depleted of mature cells using anti-CD24 mAb (J11d.2) plus complement (9), and viable cells were subsequently flow cytometrically sorted for CD117⁺Sca-1⁺ cells. NIH3T3 cells were provided by Dr. M. Julius (Sunnybrook & Women’s Research Institute). GP+E.86-GFP, GP+E.86-DL1, GP+E.86-DL4 viral packaging cell lines and OP9-DL1 cells were generated in our laboratory by T. M. Schmitt (7). All animal procedures were approved by Sunnybrook & Women’s Research Institute’s Animal Care Committee.

FTOCs

Fetal thymic lobes isolated on days 14 and15 of gestation were cultured as previously described (3, 10). FTOCs were either treated with 1.1 mM 2-deoxyguanosine (dG) (Sigma-Aldrich) or left untreated for 5 days (3). dG-FTOCs were reconstituted with 2000 Sca-1⁺CD117⁺ HPCs and incubated as previously described (10).

RTOCs, TSMCs, and retroviral transduction

To establish RTOCs and TSMCs, dG-FTOCs were incubated with serum-free medium containing 0.05% trypsin and 2 mM EDTA for 20–25 min at 37°C. Lobes were then disaggregated into a single-cell suspension and filtered through 70-µm nylon mesh. For RTOCs,10⁶ TSCs were resuspended in 20 µl of FTOC medium and placed on FTOC rafts as previously described (10). For TSMCs, 60,000 TSCs were added to each well of a 96-well flat-bottom plate to form monolayers. After overnight incubation, TSMCs were retrovirally transduced by incubating the cultures with fresh retroviral supernatants from GP+E.86 packaging lines each day for 4 days, then cultured for 2 days in medium. Two hundred HPCs were added to each confluent well of TSMCs supplemented with 5 ng/ml Flt-3 ligand (PeproTech) and 1 ng/ml IL-7 (PeproTech).

Similar protocols were used to transduce NIH3T3 cells. After transduction, NIH3T3 cells were sorted for GFP expression. Before coculture, transduced NIH3T3 cells were gamma-irradiated (55 Gy) and plated at 40,000 cells/well in 96-well flat-bottom plates overnight. Cultures were seeded with 200 HPCs the next day as above. Hemopoietic cells were transferred to a fresh plate of gamma-irradiated NIH3T3 cells every 5 days and supplemented with Flt-3 ligand and IL-7 as above, with 25 ng/ml stem cell factor (PeproTech) included only in the first 5-day culture.

RT-PCR

Total RNA was isolated from TSMCs and FTOCs using TRIzol (Invitrogen Life Technologies). cDNA was prepared using Superscript III (Invitrogen Life Technologies). Semiquantitative PCR was performed using serially diluted cDNA, normalized based on -actin signal. PCR primers are as follows: -actin (11); Jag1, Jag2, and Dll1 (12); keratin 8 (13) and keratin-5F (5'-CGACCCCACCATCCAGCG), keratin-5R (5'-TCTCCCCCAGAACCCCGT); Dll4F (5'-GGTGACCTGGCGAACAGACGAGCAAAAT) and Dll4R (5'-GGATCCGGGGAAGCTGGGTGGCAA); Foxn1F (5'-TTTGGCTTTGAGGAGGGC) and Foxn1R (5'-GTTGCAAGGGAGGCTGGTA). All PCR products were resolved by agarose gel electrophoresis and corresponded to the predicted molecular mass.

Flow cytometry and cell sorting

All mAbs were purchased from BD Biosciences: PE-conjugated Sca-1, CD8, CD44, and allophycocyanin-conjugated CD117, CD4, CD25, and CD19. Cells were stained and sorted as previously described (9). All sorts yielded 99% cell purity, as determined by post-sort analysis.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
TSMCs fail to support T cell development

To analyze the role of the thymic architecture in supporting T cell development, we first depleted FTOCs of endogenous thymocytes by treatment with dG (3). The resulting dG-FTOCs were either left intact or trypsinized and plated as a monolayer culture (TSMC). Both intact dG-FTOCs and TSMCs were cocultured with CD117⁺Sca-1⁺ HPCs obtained from day 14 fetal livers (Fig. 1). Flow cytometric analysis at days 7–13 of coculture showed that TSMCs failed to support differentiation of HPCs past the initial CD4^–CD8^– (double-negative, DN) stage of T cell development (DN1, CD44⁺ CD25^–). In contrast, intact FTOCs promoted T lymphopoiesis through all DN stages and yielded CD4⁺CD8⁺ double-positive (DP) and single-positive (SP) cells (Fig. 1). These results indicate that interactions between TSCs and HPCs necessary for the induction of T cell development are only maintained in FTOCs, but not in TSMCs.

View larger version (47K): [in this window] [in a new window]   FIGURE 1. T cell development in FTOCs vs TSMCs. dG-treated FTOCs either intact or trypsinized and plated as a monolayer (TSMC) were seeded with HPCs, then harvested at day 7 or day 13 and analyzed by flow cytometry.

To elucidate the intrinsic deficiency between TSMCs and FTOCs, we examined expression of characteristic thymic stromal genes under various culture conditions by RT-PCR. RNA samples were obtained from unmanipulated FTOCs, dG-FTOCs, and dG-FTOCs cultured for an additional 5 days either intact or dissociated as TSMCs. Fig. 2A shows that expression of keratin-8 transcripts was similar under all conditions analyzed, demonstrating that the abundance of thymic epithelial cells (TECs) was equivalent in TSMC vs intact FTOCs. In contrast, keratin-5 expression was diminished in TSMCs compared with intact FTOCs. Since keratin-8 is normally expressed in all developing TECs, while keratin-5 is present only in more mature TECs (14), this suggests that the conditions used for TSMCs were not fully conducive to the normal developmental maturation of TECs.

View larger version (68K): [in this window] [in a new window]   FIGURE 2. RT-PCR analysis of fetal TSCs. Expression of Notch ligands and epithelial markers in TSMCs were compared with transcripts from FTOCs cultured under different conditions using semiquantitative RT-PCR. A, Conditions are as follows: intact FTOCs, dG-treated FTOCs for 5 days; dG-FTOC cultured for an additional 5 days in normal medium either intact or as TSMCs (left to right, respectively). Comparisons were based on equalization of -actin transcripts by RT-PCR of 3-fold serial dilutions of template cDNA. Data are representative of at least three independent experiments. B, Expression analysis using RT-PCR was performed as in A with the addition of 5-day RTOCs generated exclusively from TSCs. Data are representative of two independent experiments.

To address whether Dll expression by TSCs is critically dependent on a 3-D stromal structure, TSCs were used in RTOCs in the absence of thymocytes (Fig. 2B). In contrast to TSMCs, TSCs placed in RTOCs maintain expression of both Dll1 and Dll4. Additionally, we also observed that the expression of Foxn1 (2) is down-regulated in TMSCs, but not in FTOCs or RTOCs (Fig. 2B). Although there is no evidence for direct regulation of Dll transcription by Foxn1, mutations in foxn1 responsible for the nude phenotype in mice result in the loss of both Dll1 and Dll4 expression in the fetal thymic anlage (16).

Effect of forced Dll1 and Dll4 expression in TSC cultures

We explored whether restoration of either Dll1 or Dll4 expression in TSMCs would be sufficient to re-establish T lymphopoietic function. To this end, TSMCs were retrovirally transduced to express either Dll1 or Dll4 plus GFP, or GFP alone as control. Following a transduction period of 4 and 6 days in normal medium, we consistently achieved 60–80% transduction efficiency, as indicated by GFP expression (Fig. 3A). We next verified that Dll1 and Dll4 were expressed in the transduced TSMCs. As shown in Fig. 3B, comparison of Dll expression levels in TSMCs relative to freshly dissociated TSCs reveals that TSMCs transduced to express Dll1 or Dll4 regained expression of each of the respective Notch ligands. In contrast, expression levels of Dll transcripts in TSMCs transduced with GFP alone were undetectable after 10 days of culture, consistent with our previous results (see Fig. 2).

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Ectopic expression of Dll1 and Dll4 in fetal TSMCs. TSCs derived from dG-treated FTOCs were plated overnight and retrovirally transduced for 4 days with either GFP alone or GFP with Dll1 or GFP with Dll4. A, Transduction efficiency was determined by GFP expression by flow cytometry after 6 days. B, RT-PCR analysis of Dll1 and Dll4 transcripts in transduced TSMCs vs freshly dissociated TSCs. Data represent 3-fold serial dilutions of cDNA. Results are representative of two independent experiments.

To ascertain whether TSMCs with forced Dll1 or Dll4 expression would regain their ability to support T cell development, HPCs were cocultured with the transduced TSMCs. As expected, TSMC-GFP cocultures failed to generate a significant population of DN CD25⁺ T lineage cells, and DP cells were not present at later time points (Fig. 4A). In striking contrast, >20% of hemopoietic cells expressed CD25 in TSMC-DL1 and TSMC-DL4 cocultures after 10 days. Furthermore, nearly 50% of the cells had advanced to the DP stage and 10% reached the CD8 SP stage (Fig. 4A). These results demonstrate that forced expression of either Dll1 or Dll4 alone in TSMC cells is sufficient to confer the ability to induce and support T cell development. Moreover, at least one Dll ligand is necessary for T lymphopoiesis in TSMC culture, but each Dll ligand is functionally redundant and can compensate for the loss of the other. In addition, these findings indicate that the lack of T lymphopoietic potential in TSMCs is due to a loss of Dll expression following disruption of the 3-D stromal architecture. This suggests that 3-D interactions between TSCs are necessary to maintain normal Notch ligand expression in the thymus.

View larger version (33K): [in this window] [in a new window]   FIGURE 4. Forced expression of Dll1 or Dll4 in TSMC supports T cell development. HPCs were cocultured with OP9-DL1 cells and transduced TSMCs as indicated. A, Analysis was performed on day 10 (for DN subsets) and day 17 (for DP and SP cells). Numbers in each quadrant indicate the percentage of gated cells. Data are representative of five independent experiments. B, Cells harvested from day 17 HPC/OP9-DL1 and HPC/TSMC cocultures were analyzed for surface expression of CD19 by flow cytometry. Results are representative of four independent experiments.

Ectopic expression of Dll1 or Dll4 in fibroblasts fails to support T lymphopoiesis

To address whether expression of specific Dll family members is the only critical requirement for HPCs to generate T lineage cells in vitro, we investigated whether expression of Dll1 or Dll4 by NIH3T3 fibroblast cells is sufficient to support T cell development. To this end, retrovirally transduced NIH3T3 cells expressing Dll1, Dll4, or GFP alone were cocultured with HPCs, under similar conditions to TSMC, and analyzed on days 10 and 17 for evidence of lymphopoiesis. Notably, none of the NIH3T3 cocultures was capable of generating T lineage cells, suggesting that Dll-Notch signals alone are not sufficient to support T lymphopoiesis (Fig. 5). Importantly, although NIH3T3-GFP cells gave rise to B lineage cells under the conditions tested, NIH3T3-DL1 or NIH3T3-DL4 cells did not support the generation of B lineage cells from HPCs (Fig. 5). Thus, in addition to the requirement for appropriate Dll expression in stromal cells, T lymphopoiesis also requires critical interactions provided in TSMCs that are lacking in NIH3T3 cells. Thus, Dll-Notch signaling alone may be sufficient to suppress B lymphopoiesis without inducing T lymphopoiesis.

View larger version (33K): [in this window] [in a new window]   FIGURE 5. Dll-transduced NIH3T3 cells do not support T cell development or B cell differentiation. Dll-transduced NIH3T3 fibroblasts were seeded with HPCs. Hemopoietic cells were harvested at days 10 and 17 for flow cytometric analysis as indicated. Data are representative of four independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Both Dll1 and Dll4 are expressed in fetal thymus and appear to have redundant functions in their ability to induce T cell generation. This redundancy can explain results from a recent report showing that conditional deletion of Dll1 in vivo was dispensable for T cell development (17). We show here that the dramatic down-regulation of both Dll1 and Dll4 expression in TSMC, to an extent that it may mimic the effect of a double Dll1/Dll4 deletion in the thymus, results in a complete block in T cell development. This block is not compensated for by the residual Jag1 and Jag2 expression that remain unchanged or reduced, respectively. Further, other reports demonstrated that Jag1 and Jag2 overexpression in bone marrow derived cell lines failed to induce T-lymphopoiesis from HPCs (18, 19, 20). Although Jag2 has been shown to influence NK cell development (20), the evidence so far suggests that it is unlikely that the loss of Jag2 expression in TSMCs is responsible for the loss in T lymphopoiesis.

The renewed ability of TSMCs with forced expression of Dll1 or Dll4 to support T cell development clearly establishes that a critical missing feature of cultured TSCs is the expression of the required Dll family members. Nevertheless, overexpression of Dll1 or Dll4 does not de facto transform any stromal cell monolayer into one capable of supporting T lymphopoiesis. Several possibilities exist for the failure of NIH3T3-Dll cocultures to generate T cells. These include possible expression of inhibitory factors (e.g., TGF-) (21), insufficient expression of other regulatory factors (e.g., Hedgehog or WNTs) (22, 23), or an inability of NIH3T3 cells to support lymphocyte lineage commitment to a T cell fate (despite supporting B cell development in the presence of exogenous IL-7). In an attempt to uncover possible inhibitory signals, we performed cocultures using mixed OP9-DL1 and NIH3T3 cells; however, these conditions could efficiently generate DP T cells (data not shown), indicating that NIH3T3 cells did not possess any intrinsic inhibitory properties. In any case, our results point to a requirement for additional factors necessary for the generation of T cells from HPCs.

Our results imply that normal expression levels of Dll1 and Dll4 are maintained through close intercellular interactions between TSCs in a 3-D arrangement. In this study, we show that FTOC and RTOC matrices devoid of lymphocytes are still able to maintain Dll expression for at least 5 days despite thymocyte depletion (Fig. 2); this suggests that the key stimulatory intercellular interactions missing in TSMCs may involve TECs and mesenchymal cells (24). Candidate molecules for these interactions include fibroblast growth factors derived from mesenchymal cells interacting with TECs expressing fibroblast growth factor R2-IIIb (25) and homotypic interactions of E-cadherin between neighboring TECs (26). Although the downstream effects of these specific signals on Dll expression are unknown, concomitant loss of that Foxn1 and Dll transcripts in TSMCs (Fig. 2B) merits examining whether Foxn1 mediates an event regulating the expression of Dll molecules (16). Identifying factors involved in stromal cell interactions will be an important additional step in outlining the signaling pathways responsible for maintaining Dll expression in an intact thymic microenvironment, thus in turn conferring the unique ability of the thymus to generate T cells.

	Acknowledgments

 
We thank H. T. Petrie and J. R. Carlyle for critical review of this manuscript and G. Knowles for expert assistance in cell sorting.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by a grant from Canadian Institutes of Health Research. J.C.Z.-P. is a Canada Research Chair in Developmental Immunology.

² Address correspondence and reprint requests to Dr. Juan Carlos Zúñiga-Pflücker, Department of Immunology, University of Toronto, Sunnybrook & Women’s Research Institute, Room A-331, 2075 Bayview Avenue, Toronto, Ontario, M4N 3M5, Canada. E-mail address: jczp{at}swri.ca

³ Abbreviations used in this paper: HPC, hematopoietic progenitor cell; 3-D, three-dimensional; dG, 2'-deoxyguanosine; Dll, Delta-like; DN, double negative; DP, double positive; FTOC, fetal thymic organ culture; RTOC, reaggregate thymic organ culture; SP, single positive; TEC, thymic epithelial cell; TSC, thymic stromal cell; TSMC, thymic stromal cell monolayer culture.

Received for publication September 2, 2005. Accepted for publication November 5, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Petrie, H. T.. 2002. Role of thymic organ structure and stromal composition in steady-state postnatal T-cell production. Immunol. Rev. 189: 8-19. [Medline]
2. Manley, N. R., C. C. Blackburn. 2003. A developmental look at thymus organogenesis: where do the non-hematopoietic cells in the thymus come from?. Curr. Opin. Immunol. 15: 225-232. [Medline]
3. Jenkinson, E. J., L. L. Franchi, R. Kingston, J. J. Owen. 1982. Effect of deoxyguanosine on lymphopoiesis in the developing thymus rudiment in vitro: application in the production of chimeric thymus rudiments. Eur. J. Immunol. 12: 583-587. [Medline]
4. Jenkinson, E. J., G. Anderson, J. J. Owen. 1992. Studies on T cell maturation on defined thymic stromal cell populations in vitro. J. Exp. Med. 176: 845-853. [Abstract/Free Full Text]
5. Montecino-Rodriguez, E., A. Johnson, K. Dorshkind. 1996. Thymic stromal cells can support B cell differentiation from intrathymic precursors. J. Immunol. 156: 963-967. [Abstract]
6. Dorshkind, K.. 1990. Regulation of hemopoiesis by bone marrow stromal cells and their products. Annu. Rev. Immunol. 8: 111-137. [Medline]
7. Schmitt, T. M., J. C. Zúñiga-Pflücker. 2002. Induction of T cell development from hematopoietic progenitor cells by Delta-like-1 in vitro. Immunity. 17: 749-756. [Medline]
8. Zúñiga-Pflücker, J. C.. 2004. T-cell development made simple. Nat. Rev. Immunol. 4: 67-72. [Medline]
9. Carlyle, J. R., J. C. Zúñiga-Pflücker. 1998. Requirement for the thymus in Ab T lymphocyte lineage commitment. Immunity 9: 187-197. [Medline]
10. de Pooter, R. F., S. K. Cho, J. R. Carlyle, J. C. Zúñiga-Pflücker. 2003. In vitro generation of T lymphocytes from embryonic stem cell-derived prehematopoietic progenitors. Blood 102: 1649-1653. [Abstract/Free Full Text]
11. Ciofani, M., T. M. Schmitt, A. Ciofani, A. M. Michie, N. Cuburu, A. Aublin, J. L. Maryanski, J. C. Zúñiga-Pflücker. 2004. Obligatory role for cooperative signaling by pre-TCR and Notch during thymocyte differentiation. J. Immunol. 172: 5230-5239. [Abstract/Free Full Text]
12. Hoflinger, S., K. Kesavan, M. Fuxa, C. Hutter, B. Heavey, F. Radtke, M. Busslinger. 2004. Analysis of Notch1 function by in vitro T cell differentiation of Pax5 mutant lymphoid progenitors. J. Immunol. 173: 3935-3944. [Abstract/Free Full Text]
13. Tao, G. Z., D. M. Toivola, B. Zhong, S. A. Michie, E. Z. Resurreccion, Y. Tamai, M. M. Taketo, M. B. Omary. 2003. Keratin-8 null mice have different gallbladder and liver susceptibility to lithogenic diet-induced injury. J. Cell Sci. 116: 4629-4638. [Abstract/Free Full Text]
14. Klug, D. B., C. Carter, I. B. Gimenez-Conti, E. R. Richie. 2002. Thymocyte-independent and thymocyte-dependent phases of epithelial patterning in the fetal thymus. J. Immunol. 169: 2842-2845. [Abstract/Free Full Text]
15. Anderson, G., E. J. Jenkinson. 2001. Lymphostromal interactions in thymic development and function. Nat. Rev. Immunol. 1: 31-40. [Medline]
16. Tsukamoto, N., M. Itoi, M. Nishikawa, T. Amagai. 2005. Lack of Delta like 1 and 4 expressions in nude thymus anlages. Cell. Immunol. 234: 77-80. [Medline]
17. Hozumi, K., N. Negishi, D. Suzuki, N. Abe, Y. Sotomaru, N. Tamaoki, C. Mailhos, D. Ish-Horowicz, S. Habu, M. J. Owen. 2004. Delta-Like 1 is necessary for the generation of marginal zone B cells but not T cells in vivo. Nat. Immunol. 5: 638-644. [Medline]
18. Lehar, S. M., J. Dooley, A. G. Farr, M. J. Bevan. 2005. Notch ligands Delta1 and Jagged1 transmit distinct signals to T-cell precursors. Blood 105: 1440-1447. [Abstract/Free Full Text]
19. Jaleco, A. C., H. Neves, E. Hooijberg, P. Gameiro, N. Clode, M. Haury, D. Henrique, L. Parreira. 2001. Differential effects of Notch ligands Delta-1 and Jagged-1 in human lymphoid differentiation. J. Exp. Med. 194: 991-1002. [Abstract/Free Full Text]
20. DeHart, S. L., M. J. Heikens, S. Tsai. 2005. Jagged2 promotes the development of natural killer cells and the establishment of functional natural killer cell lines. Blood 105: 3521-3527. [Abstract/Free Full Text]
21. Gorelik, L., R. A. Flavell. 2002. Transforming growth factor- in T-cell biology. Nat. Rev. Immunol. 2: 46-53. [Medline]
22. Shah, D. K., A. L. Hager-Theodorides, S. V. Outram, S. E. Ross, A. Varas, T. Crompton. 2004. Reduced thymocyte development in sonic hedgehog knockout embryos. J. Immunol. 172: 2296-2306. [Abstract/Free Full Text]
23. Staal, F. J., J. Meeldijk, P. Moerer, P. Jay, B. C. van de Weerdt, S. Vainio, G. P. Nolan, H. Clevers. 2001. Wnt signaling is required for thymocyte development and activates Tcf-1 mediated transcription. Eur. J. Immunol. 31: 285-293. [Medline]
24. Jenkinson, W. E., E. J. Jenkinson, G. Anderson. 2003. Differential requirement for mesenchyme in the proliferation and maturation of thymic epithelial progenitors. J. Exp. Med. 198: 325-332. [Abstract/Free Full Text]
25. Revest, J. M., R. K. Suniara, K. Kerr, J. J. Owen, C. Dickson. 2001. Development of the thymus requires signaling through the fibroblast growth factor receptor R2-IIIb. J. Immunol. 167: 1954-1961. [Abstract/Free Full Text]
26. Muller, K. M., C. J. Luedecker, M. C. Udey, A. G. Farr. 1997. Involvement of E-cadherin in thymus organogenesis and thymocyte maturation. Immunity 6: 257-264. [Medline]

This article has been cited by other articles:

				V. Besseyrias, E. Fiorini, L. J. Strobl, U. Zimber-Strobl, A. Dumortier, U. Koch, M.-L. Arcangeli, S. Ezine, H. R. MacDonald, and F. Radtke Hierarchy of Notch-Delta interactions promoting T cell lineage commitment and maturation J. Exp. Med., February 19, 2007; 204(2): 331 - 343. [Abstract] [Full Text] [PDF]

				M. Itoi, N. Tsukamoto, and T. Amagai Expression of Dll4 and CCL25 in Foxn1-negative epithelial cells in the post-natal thymus Int. Immunol., February 1, 2007; 19(2): 127 - 132. [Abstract] [Full Text] [PDF]

				M. J. Malecki, C. Sanchez-Irizarry, J. L. Mitchell, G. Histen, M. L. Xu, J. C. Aster, and S. C. Blacklow Leukemia-Associated Mutations within the NOTCH1 Heterodimerization Domain Fall into at Least Two Distinct Mechanistic Classes Mol. Cell. Biol., June 15, 2006; 26(12): 4642 - 4651. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mohtashami, M. Articles by Zúñiga-Pflücker, J. C. Search for Related Content PubMed PubMed Citation Articles by Mohtashami, M. Articles by Zúñiga-Pflücker, J. C.