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Delta-like 4 is the essential, nonredundant ligand for Notch1 during thymic T cell lineage commitment.

Koch U, Fiorini E, Benedito R, Besseyrias V, Schuster-Gossler K, Pierres M, Manley NR, Duarte A, Macdonald HR, Radtke F - J. Exp. Med. (2008)

Bottom Line: Although the physiological ligands that interact with N1 expressed on thymic precursors are currently unknown, in vitro culture systems point to Delta-like 1 (DL1) and DL4 as prime candidates.Although loss of DL4 in hematopoietic progenitors did not perturb thymus development, inactivation of DL4 in TECs led to a complete block in T cell development coupled with the ectopic appearance of immature B cells in the thymus.Collectively, our results demonstrate that DL4 is the essential and nonredundant N1 ligand responsible for T cell lineage commitment.

View Article: PubMed Central - PubMed

Affiliation: Ecole Polytechnique Fédérale de Lausanne, Swiss Institute for Experimental Cancer Research, 1066 Epalinges, Switzerland.

ABSTRACT
Thymic T cell lineage commitment is dependent on Notch1 (N1) receptor-mediated signaling. Although the physiological ligands that interact with N1 expressed on thymic precursors are currently unknown, in vitro culture systems point to Delta-like 1 (DL1) and DL4 as prime candidates. Using DL1- and DL4-lacZ reporter knock-in mice and novel monoclonal antibodies to DL1 and DL4, we show that DL4 is expressed on thymic epithelial cells (TECs), whereas DL1 is not detected. The function of DL4 was further explored in vivo by generating mice in which DL4 could be specifically inactivated in TECs or in hematopoietic progenitors. Although loss of DL4 in hematopoietic progenitors did not perturb thymus development, inactivation of DL4 in TECs led to a complete block in T cell development coupled with the ectopic appearance of immature B cells in the thymus. These immature B cells were phenotypically indistinguishable from those developing in the thymus of conditional N1 mutant mice. Collectively, our results demonstrate that DL4 is the essential and nonredundant N1 ligand responsible for T cell lineage commitment. Moreover, they strongly suggest that N1-expressing thymic progenitors interact with DL4-expressing TECs to suppress B lineage potential and to induce the first steps of intrathymic T cell development.

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Complete block in T cell development and accumulation of thymic B cells in DL4ΔFoxn1 mice. (A) Representative flow cytometric analysis of thymocytes derived from Ctrl and DL4ΔFoxn1 14-d-old mice. Total thymocytes were stained with anti-CD4 and -CD8 antibodies. Absolute cell numbers for total thymocytes and indicated subsets are shown as bar diagrams on a logarithmic scale. The bar diagrams represent mean values ± SD (n = 5 for Ctrl and 6 for DL4ΔFoxn1). (B) Representative FACS analysis of CD4−CD8−lin− thymocytes stained with anti-CD44 and -CD25 antibodies. (C) Representative histograms of B220 staining gated on lin−CD44+CD25− cells. Data are representative of three independent experiments. Percentages of positively stained cells are indicated within the contour plots and histograms.
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fig3: Complete block in T cell development and accumulation of thymic B cells in DL4ΔFoxn1 mice. (A) Representative flow cytometric analysis of thymocytes derived from Ctrl and DL4ΔFoxn1 14-d-old mice. Total thymocytes were stained with anti-CD4 and -CD8 antibodies. Absolute cell numbers for total thymocytes and indicated subsets are shown as bar diagrams on a logarithmic scale. The bar diagrams represent mean values ± SD (n = 5 for Ctrl and 6 for DL4ΔFoxn1). (B) Representative FACS analysis of CD4−CD8−lin− thymocytes stained with anti-CD44 and -CD25 antibodies. (C) Representative histograms of B220 staining gated on lin−CD44+CD25− cells. Data are representative of three independent experiments. Percentages of positively stained cells are indicated within the contour plots and histograms.

Mentions: Side-by-side analysis of littermate thymi derived from Ctrl and DL4ΔFoxn1 mice revealed a 14-fold decrease in absolute thymocyte numbers in DL4ΔFoxn1 compared with Ctrl mice (Fig. 3 A). Flow cytometric analysis of CD4 and CD8 expression on thymocytes derived from DL4ΔFoxn1 mice shows nearly complete loss (<1%) of the CD4SP, the CD8SP, and the DP thymocyte subsets, whereas all of the major subsets are present in the expected frequency in Ctrl mice. DL4ΔFoxn1-derived thymi were almost exclusively populated with cells falling into the DN gate (Fig. 3 A). In absolute numbers, DP, CD4SP, and CD8SP subsets were reduced by 180–3,000-fold in DL4ΔFoxn1 mice, whereas DN cells were slightly increased (twofold). Whether the small residual population of T lineage cells in the thymus of DL4ΔFoxn1 mice results from a failure to delete DL4 efficiently in a subset of TECs (which stain below the threshold of detection with our anti-DL4 mAb) or, alternatively, represents a minor pathway of DL4-independent (and hence, presumably Notch-independent) T cell development remains to be investigated.


Delta-like 4 is the essential, nonredundant ligand for Notch1 during thymic T cell lineage commitment.

Koch U, Fiorini E, Benedito R, Besseyrias V, Schuster-Gossler K, Pierres M, Manley NR, Duarte A, Macdonald HR, Radtke F - J. Exp. Med. (2008)

Complete block in T cell development and accumulation of thymic B cells in DL4ΔFoxn1 mice. (A) Representative flow cytometric analysis of thymocytes derived from Ctrl and DL4ΔFoxn1 14-d-old mice. Total thymocytes were stained with anti-CD4 and -CD8 antibodies. Absolute cell numbers for total thymocytes and indicated subsets are shown as bar diagrams on a logarithmic scale. The bar diagrams represent mean values ± SD (n = 5 for Ctrl and 6 for DL4ΔFoxn1). (B) Representative FACS analysis of CD4−CD8−lin− thymocytes stained with anti-CD44 and -CD25 antibodies. (C) Representative histograms of B220 staining gated on lin−CD44+CD25− cells. Data are representative of three independent experiments. Percentages of positively stained cells are indicated within the contour plots and histograms.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2571927&req=5

fig3: Complete block in T cell development and accumulation of thymic B cells in DL4ΔFoxn1 mice. (A) Representative flow cytometric analysis of thymocytes derived from Ctrl and DL4ΔFoxn1 14-d-old mice. Total thymocytes were stained with anti-CD4 and -CD8 antibodies. Absolute cell numbers for total thymocytes and indicated subsets are shown as bar diagrams on a logarithmic scale. The bar diagrams represent mean values ± SD (n = 5 for Ctrl and 6 for DL4ΔFoxn1). (B) Representative FACS analysis of CD4−CD8−lin− thymocytes stained with anti-CD44 and -CD25 antibodies. (C) Representative histograms of B220 staining gated on lin−CD44+CD25− cells. Data are representative of three independent experiments. Percentages of positively stained cells are indicated within the contour plots and histograms.
Mentions: Side-by-side analysis of littermate thymi derived from Ctrl and DL4ΔFoxn1 mice revealed a 14-fold decrease in absolute thymocyte numbers in DL4ΔFoxn1 compared with Ctrl mice (Fig. 3 A). Flow cytometric analysis of CD4 and CD8 expression on thymocytes derived from DL4ΔFoxn1 mice shows nearly complete loss (<1%) of the CD4SP, the CD8SP, and the DP thymocyte subsets, whereas all of the major subsets are present in the expected frequency in Ctrl mice. DL4ΔFoxn1-derived thymi were almost exclusively populated with cells falling into the DN gate (Fig. 3 A). In absolute numbers, DP, CD4SP, and CD8SP subsets were reduced by 180–3,000-fold in DL4ΔFoxn1 mice, whereas DN cells were slightly increased (twofold). Whether the small residual population of T lineage cells in the thymus of DL4ΔFoxn1 mice results from a failure to delete DL4 efficiently in a subset of TECs (which stain below the threshold of detection with our anti-DL4 mAb) or, alternatively, represents a minor pathway of DL4-independent (and hence, presumably Notch-independent) T cell development remains to be investigated.

Bottom Line: Although the physiological ligands that interact with N1 expressed on thymic precursors are currently unknown, in vitro culture systems point to Delta-like 1 (DL1) and DL4 as prime candidates.Although loss of DL4 in hematopoietic progenitors did not perturb thymus development, inactivation of DL4 in TECs led to a complete block in T cell development coupled with the ectopic appearance of immature B cells in the thymus.Collectively, our results demonstrate that DL4 is the essential and nonredundant N1 ligand responsible for T cell lineage commitment.

View Article: PubMed Central - PubMed

Affiliation: Ecole Polytechnique Fédérale de Lausanne, Swiss Institute for Experimental Cancer Research, 1066 Epalinges, Switzerland.

ABSTRACT
Thymic T cell lineage commitment is dependent on Notch1 (N1) receptor-mediated signaling. Although the physiological ligands that interact with N1 expressed on thymic precursors are currently unknown, in vitro culture systems point to Delta-like 1 (DL1) and DL4 as prime candidates. Using DL1- and DL4-lacZ reporter knock-in mice and novel monoclonal antibodies to DL1 and DL4, we show that DL4 is expressed on thymic epithelial cells (TECs), whereas DL1 is not detected. The function of DL4 was further explored in vivo by generating mice in which DL4 could be specifically inactivated in TECs or in hematopoietic progenitors. Although loss of DL4 in hematopoietic progenitors did not perturb thymus development, inactivation of DL4 in TECs led to a complete block in T cell development coupled with the ectopic appearance of immature B cells in the thymus. These immature B cells were phenotypically indistinguishable from those developing in the thymus of conditional N1 mutant mice. Collectively, our results demonstrate that DL4 is the essential and nonredundant N1 ligand responsible for T cell lineage commitment. Moreover, they strongly suggest that N1-expressing thymic progenitors interact with DL4-expressing TECs to suppress B lineage potential and to induce the first steps of intrathymic T cell development.

Show MeSH
Related in: MedlinePlus