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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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Hematopoietic progenitors with active form of Notch1 give rise to T cell lineage in Dll4-deficient thymus. Hematopoietic progenitors (Lin.−c-kit+ cells) derived from E15.5 fetal liver were infected with the retrovirus encoding the intracellular region of Notch1 (ICN1) or empty vector (Mock). Infected cells were monitored by expression of GFP. These cells were cultured into deoxyguanosine-treated thymic lobes of Dll4lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed) or Dll4lox/lox (Dll4-floxed) fetuses for 13 d as FTOC and analyzed for the expression of CD4, CD8, CD25, and CD19 (A). Alternatively, these cells were injected intravenously into irradiated Dll4lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed) or Dll4lox/lox (Dll4-floxed) neonate mice. After 3 wk, thymocytes were analyzed for expression of CD4, CD8, and CD19 (B). Profiles are shown with the gate (GFP+ or GFP+CD4−CD8−, DN-gated in A). Numbers in the profiles indicate the relative percentages for each corresponding quadrant.
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fig5: Hematopoietic progenitors with active form of Notch1 give rise to T cell lineage in Dll4-deficient thymus. Hematopoietic progenitors (Lin.−c-kit+ cells) derived from E15.5 fetal liver were infected with the retrovirus encoding the intracellular region of Notch1 (ICN1) or empty vector (Mock). Infected cells were monitored by expression of GFP. These cells were cultured into deoxyguanosine-treated thymic lobes of Dll4lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed) or Dll4lox/lox (Dll4-floxed) fetuses for 13 d as FTOC and analyzed for the expression of CD4, CD8, CD25, and CD19 (A). Alternatively, these cells were injected intravenously into irradiated Dll4lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed) or Dll4lox/lox (Dll4-floxed) neonate mice. After 3 wk, thymocytes were analyzed for expression of CD4, CD8, and CD19 (B). Profiles are shown with the gate (GFP+ or GFP+CD4−CD8−, DN-gated in A). Numbers in the profiles indicate the relative percentages for each corresponding quadrant.

Mentions: Next, we examined whether the Dll4-deficient thymus retains any functions for a T cell development niche other than Dll4. For this, ICN1, which bypasses Notch signaling without any Notch–NotchL interaction, was expressed into WT HPCs of fetal liver by gene introduction with retrovirus vector, and these cells were cultured in thymic lobes obtained from Dll4-floxed mice with or without FoxN1-Cre transgene for FTOC. After a 13-d culture, all mock- or ICN1-introduced cells differentiated into T lineage cells, in part to DP stage or DN stage with CD25 expression, and they also bore Thy1 (unpublished data) in the lobe derived from Dll4-floxed fetus (Fig. 5 A, top). On the other hand, the mock-introduced cells mainly differentiated into CD19-positive B lineage cells in the lobe from FoxN1Cre:Dll4-floxed fetus (Fig. 5 A, bottom left), but even when without Dll4 on the epithelium, the ICN1-introduced cells differentiated into T lineage cells (Fig. 5 A, bottom right), as seen in the lobe with intact Dll4. Also, the ICN1-introduced HPCs could migrate and differentiate into DP cells in the thymus after intravenous injection into the irradiated FoxN1Cre:Dll4-floxed mice (Fig. 5 B), although the ICN1-transduced cells stopped their differentiation at DP stage (18). These results indicated that the depletion of Dll4 on the epithelium does not deprive the thymus of environment potential for T cell development other than the Dll4, and, if the intrathymic immigrants already have Notch signaling, the subsequent differentiation for the T cell lineage is possible in the thymus without Dll4.


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)

Hematopoietic progenitors with active form of Notch1 give rise to T cell lineage in Dll4-deficient thymus. Hematopoietic progenitors (Lin.−c-kit+ cells) derived from E15.5 fetal liver were infected with the retrovirus encoding the intracellular region of Notch1 (ICN1) or empty vector (Mock). Infected cells were monitored by expression of GFP. These cells were cultured into deoxyguanosine-treated thymic lobes of Dll4lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed) or Dll4lox/lox (Dll4-floxed) fetuses for 13 d as FTOC and analyzed for the expression of CD4, CD8, CD25, and CD19 (A). Alternatively, these cells were injected intravenously into irradiated Dll4lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed) or Dll4lox/lox (Dll4-floxed) neonate mice. After 3 wk, thymocytes were analyzed for expression of CD4, CD8, and CD19 (B). Profiles are shown with the gate (GFP+ or GFP+CD4−CD8−, DN-gated in A). Numbers in the profiles indicate the relative percentages for each corresponding quadrant.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Hematopoietic progenitors with active form of Notch1 give rise to T cell lineage in Dll4-deficient thymus. Hematopoietic progenitors (Lin.−c-kit+ cells) derived from E15.5 fetal liver were infected with the retrovirus encoding the intracellular region of Notch1 (ICN1) or empty vector (Mock). Infected cells were monitored by expression of GFP. These cells were cultured into deoxyguanosine-treated thymic lobes of Dll4lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed) or Dll4lox/lox (Dll4-floxed) fetuses for 13 d as FTOC and analyzed for the expression of CD4, CD8, CD25, and CD19 (A). Alternatively, these cells were injected intravenously into irradiated Dll4lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed) or Dll4lox/lox (Dll4-floxed) neonate mice. After 3 wk, thymocytes were analyzed for expression of CD4, CD8, and CD19 (B). Profiles are shown with the gate (GFP+ or GFP+CD4−CD8−, DN-gated in A). Numbers in the profiles indicate the relative percentages for each corresponding quadrant.
Mentions: Next, we examined whether the Dll4-deficient thymus retains any functions for a T cell development niche other than Dll4. For this, ICN1, which bypasses Notch signaling without any Notch–NotchL interaction, was expressed into WT HPCs of fetal liver by gene introduction with retrovirus vector, and these cells were cultured in thymic lobes obtained from Dll4-floxed mice with or without FoxN1-Cre transgene for FTOC. After a 13-d culture, all mock- or ICN1-introduced cells differentiated into T lineage cells, in part to DP stage or DN stage with CD25 expression, and they also bore Thy1 (unpublished data) in the lobe derived from Dll4-floxed fetus (Fig. 5 A, top). On the other hand, the mock-introduced cells mainly differentiated into CD19-positive B lineage cells in the lobe from FoxN1Cre:Dll4-floxed fetus (Fig. 5 A, bottom left), but even when without Dll4 on the epithelium, the ICN1-introduced cells differentiated into T lineage cells (Fig. 5 A, bottom right), as seen in the lobe with intact Dll4. Also, the ICN1-introduced HPCs could migrate and differentiate into DP cells in the thymus after intravenous injection into the irradiated FoxN1Cre:Dll4-floxed mice (Fig. 5 B), although the ICN1-transduced cells stopped their differentiation at DP stage (18). These results indicated that the depletion of Dll4 on the epithelium does not deprive the thymus of environment potential for T cell development other than the Dll4, and, if the intrathymic immigrants already have Notch signaling, the subsequent differentiation for the T cell lineage is possible in the thymus without Dll4.

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