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Delta-like 4 is indispensable in thymic environment specific for T cell development.

Hozumi K, Mailhos C, Negishi N, Hirano K, Yahata T, Ando K, Zuklys S, Holländer GA, Shima DT, Habu S - J. Exp. Med. (2008)

Bottom Line: Further analysis showed that the double-negative cell fraction was lacking T cell progenitors.The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4.These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Research Center for Embryogenesis and Organogenesis, Tokai University School of Medicine, Isehara 259-1193, Japan. hozumi@is.icc.u-tokai.ac.jp

ABSTRACT
The thymic microenvironment is required for T cell development in vivo. However, in vitro studies have shown that when hematopoietic progenitors acquire Notch signaling via Delta-like (Dll)1 or Dll4, they differentiate into the T cell lineage in the absence of a thymic microenvironment. It is not clear, however, whether the thymus supports T cell development specifically by providing Notch signaling. To address this issue, we generated mice with a loxP-flanked allele of Dll4 and induced gene deletion specifically in thymic epithelial cells (TECs). In the thymus of mutant mice, the expression of Dll4 was abrogated on the epithelium, and the proportion of hematopoietic cells bearing the intracellular fragment of Notch1 (ICN1) was markedly decreased. Corresponding to this, CD4 CD8 double-positive or single-positive T cells were not detected in the thymus. Further analysis showed that the double-negative cell fraction was lacking T cell progenitors. The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4. These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

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Notch signaling was decreased in the thymus with Dll4- epithelial cells. (A) The cleaved Notch1 fragment was found in fetal thymocytes of E15.5 Dll4lox/lox (Dll4-floxed) embryos. The cells with cleaved Notch1 are indicated with arrowheads. Bar, 10 μm. (B) Frequencies of cells with cleaved Notch1 (ICN1+; mean percentage from five fields in a slide with >100 cells from each embryo ± SD) were counted in E15.5 WT (n = 3), Dll4lox/loxFoxN1Cre (FoxN1Cre:Dll4-floxed, n = 6), or Dll4lox/lox (Dll4-floxed, n = 6) mice. The cultured DN cells were prepared after the FTOC of E14.5 WT fetal thymus for 4 d with γ-secretase inhibitor (GSI, n = 3) or without γ-secretase inhibitor (DMSO, n = 3). Asterisk indicates unpaired Student's t test; P < 0.001.
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fig3: Notch signaling was decreased in the thymus with Dll4- epithelial cells. (A) The cleaved Notch1 fragment was found in fetal thymocytes of E15.5 Dll4lox/lox (Dll4-floxed) embryos. The cells with cleaved Notch1 are indicated with arrowheads. Bar, 10 μm. (B) Frequencies of cells with cleaved Notch1 (ICN1+; mean percentage from five fields in a slide with >100 cells from each embryo ± SD) were counted in E15.5 WT (n = 3), Dll4lox/loxFoxN1Cre (FoxN1Cre:Dll4-floxed, n = 6), or Dll4lox/lox (Dll4-floxed, n = 6) mice. The cultured DN cells were prepared after the FTOC of E14.5 WT fetal thymus for 4 d with γ-secretase inhibitor (GSI, n = 3) or without γ-secretase inhibitor (DMSO, n = 3). Asterisk indicates unpaired Student's t test; P < 0.001.

Mentions: In the thymus of the Dll4-floxed fetus, as well as the WT fetus, substantial thymocytes (∼30%) bore the ICN1, which was generated by the proteolysis of Notch1 as a signal transducer of Notch signaling, in cytoplasm or nucleus (Fig. 3 A, arrowheads). However, this proportion of thymocytes with ICN1 was markedly decreased in the mutant mice without Dll4 (Fig. 3 B). The specificity of this intracellular staining was confirmed by the reduction of the ratio of double-negative (DN) cells possessing the intracellular fragment after the fetal thymus organ culture (FTOC) with γ-secretase inhibitor (GSI; Fig. 3 B), which blocks the proteolysis of Notch1. These results indicated that the intrathymic immigrants are provided Notch signaling after migrating into the thymus, which is dependent on the Dll4 expressed on epithelial cells.


Delta-like 4 is indispensable in thymic environment specific for T cell development.

Hozumi K, Mailhos C, Negishi N, Hirano K, Yahata T, Ando K, Zuklys S, Holländer GA, Shima DT, Habu S - J. Exp. Med. (2008)

Notch signaling was decreased in the thymus with Dll4- epithelial cells. (A) The cleaved Notch1 fragment was found in fetal thymocytes of E15.5 Dll4lox/lox (Dll4-floxed) embryos. The cells with cleaved Notch1 are indicated with arrowheads. Bar, 10 μm. (B) Frequencies of cells with cleaved Notch1 (ICN1+; mean percentage from five fields in a slide with >100 cells from each embryo ± SD) were counted in E15.5 WT (n = 3), Dll4lox/loxFoxN1Cre (FoxN1Cre:Dll4-floxed, n = 6), or Dll4lox/lox (Dll4-floxed, n = 6) mice. The cultured DN cells were prepared after the FTOC of E14.5 WT fetal thymus for 4 d with γ-secretase inhibitor (GSI, n = 3) or without γ-secretase inhibitor (DMSO, n = 3). Asterisk indicates unpaired Student's t test; P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2571926&req=5

fig3: Notch signaling was decreased in the thymus with Dll4- epithelial cells. (A) The cleaved Notch1 fragment was found in fetal thymocytes of E15.5 Dll4lox/lox (Dll4-floxed) embryos. The cells with cleaved Notch1 are indicated with arrowheads. Bar, 10 μm. (B) Frequencies of cells with cleaved Notch1 (ICN1+; mean percentage from five fields in a slide with >100 cells from each embryo ± SD) were counted in E15.5 WT (n = 3), Dll4lox/loxFoxN1Cre (FoxN1Cre:Dll4-floxed, n = 6), or Dll4lox/lox (Dll4-floxed, n = 6) mice. The cultured DN cells were prepared after the FTOC of E14.5 WT fetal thymus for 4 d with γ-secretase inhibitor (GSI, n = 3) or without γ-secretase inhibitor (DMSO, n = 3). Asterisk indicates unpaired Student's t test; P < 0.001.
Mentions: In the thymus of the Dll4-floxed fetus, as well as the WT fetus, substantial thymocytes (∼30%) bore the ICN1, which was generated by the proteolysis of Notch1 as a signal transducer of Notch signaling, in cytoplasm or nucleus (Fig. 3 A, arrowheads). However, this proportion of thymocytes with ICN1 was markedly decreased in the mutant mice without Dll4 (Fig. 3 B). The specificity of this intracellular staining was confirmed by the reduction of the ratio of double-negative (DN) cells possessing the intracellular fragment after the fetal thymus organ culture (FTOC) with γ-secretase inhibitor (GSI; Fig. 3 B), which blocks the proteolysis of Notch1. These results indicated that the intrathymic immigrants are provided Notch signaling after migrating into the thymus, which is dependent on the Dll4 expressed on epithelial cells.

Bottom Line: Further analysis showed that the double-negative cell fraction was lacking T cell progenitors.The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4.These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Research Center for Embryogenesis and Organogenesis, Tokai University School of Medicine, Isehara 259-1193, Japan. hozumi@is.icc.u-tokai.ac.jp

ABSTRACT
The thymic microenvironment is required for T cell development in vivo. However, in vitro studies have shown that when hematopoietic progenitors acquire Notch signaling via Delta-like (Dll)1 or Dll4, they differentiate into the T cell lineage in the absence of a thymic microenvironment. It is not clear, however, whether the thymus supports T cell development specifically by providing Notch signaling. To address this issue, we generated mice with a loxP-flanked allele of Dll4 and induced gene deletion specifically in thymic epithelial cells (TECs). In the thymus of mutant mice, the expression of Dll4 was abrogated on the epithelium, and the proportion of hematopoietic cells bearing the intracellular fragment of Notch1 (ICN1) was markedly decreased. Corresponding to this, CD4 CD8 double-positive or single-positive T cells were not detected in the thymus. Further analysis showed that the double-negative cell fraction was lacking T cell progenitors. The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4. These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

Show MeSH
Related in: MedlinePlus