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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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Specific targeting of the DL4 gene in TECs by the Foxn1-Cre recombinase. (A) A schematic representation of the genomic organization of the DL4lox/lox locus is shown. Exons 1–3 are flanked by loxP sequences (black triangles). DL4lox/lox mice were crossed to mice in which the Cre recombinase was knocked into the Foxn1 locus (Foxn1-Cre) to obtain DL4lox/loxFoxn1-Cre mice (DL4ΔFoxn1). (B) Histograms of total TECs (PanCyt+CD45−), cTECs (PanCyt+CD45−BP1+), and mTECs (PanCyt+CD45−BP1−) from Ctrl (DL4lox/lox) or DL4ΔFoxn1 mice stained for DL1 (shaded) and DL4 (continuous lines). DL1 staining was indistinguishable from Ctrl isotype staining (not depicted). Data are representative of five Ctrl and three DL4ΔFoxn1 mice aged 2–3 wk from three independent experiments. Percentages of cells positively staining for DL4 are indicated in the histograms.
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fig2: Specific targeting of the DL4 gene in TECs by the Foxn1-Cre recombinase. (A) A schematic representation of the genomic organization of the DL4lox/lox locus is shown. Exons 1–3 are flanked by loxP sequences (black triangles). DL4lox/lox mice were crossed to mice in which the Cre recombinase was knocked into the Foxn1 locus (Foxn1-Cre) to obtain DL4lox/loxFoxn1-Cre mice (DL4ΔFoxn1). (B) Histograms of total TECs (PanCyt+CD45−), cTECs (PanCyt+CD45−BP1+), and mTECs (PanCyt+CD45−BP1−) from Ctrl (DL4lox/lox) or DL4ΔFoxn1 mice stained for DL1 (shaded) and DL4 (continuous lines). DL1 staining was indistinguishable from Ctrl isotype staining (not depicted). Data are representative of five Ctrl and three DL4ΔFoxn1 mice aged 2–3 wk from three independent experiments. Percentages of cells positively staining for DL4 are indicated in the histograms.

Mentions: Studying the role of DL4 in hematopoiesis and, more specifically, during T lineage commitment by a conventional loss-of-function approach is hampered by the fact that DL4 gene-targeted mice are embryonic lethal (24, 30). We therefore generated conditional gene-targeted mice for DL4. These mice carry loxP sites flanking the first three coding exons of the DL4 gene (DL4lox/lox). Because the gene expression and antibody studies suggested that DL4 expression in TECs might be critical for T cell lineage commitment, we crossed the DL4lox/lox (Ctrl) mice to knock-in mice expressing the Cre recombinase under the control of the Foxn1 gene (31), which is highly expressed in TECs, to generate DL4lox/lox&Foxn1-Cre (DL4ΔFoxn1) mice (Fig. 2 A). Staining of DL4ΔFoxn1 TECs with anti-DL4 and -DL1 mAbs confirmed the complete loss of DL4 protein in the gene-targeted mice compared with Ctrl animals (Fig. 2 B). Interestingly, DL1 expression on TECs derived from DL4ΔFoxn1 mice was not detectable, indicating that there is no functional compensation by DL1 as a consequence of loss of DL4 (Fig. 2 B).


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)

Specific targeting of the DL4 gene in TECs by the Foxn1-Cre recombinase. (A) A schematic representation of the genomic organization of the DL4lox/lox locus is shown. Exons 1–3 are flanked by loxP sequences (black triangles). DL4lox/lox mice were crossed to mice in which the Cre recombinase was knocked into the Foxn1 locus (Foxn1-Cre) to obtain DL4lox/loxFoxn1-Cre mice (DL4ΔFoxn1). (B) Histograms of total TECs (PanCyt+CD45−), cTECs (PanCyt+CD45−BP1+), and mTECs (PanCyt+CD45−BP1−) from Ctrl (DL4lox/lox) or DL4ΔFoxn1 mice stained for DL1 (shaded) and DL4 (continuous lines). DL1 staining was indistinguishable from Ctrl isotype staining (not depicted). Data are representative of five Ctrl and three DL4ΔFoxn1 mice aged 2–3 wk from three independent experiments. Percentages of cells positively staining for DL4 are indicated in the histograms.
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fig2: Specific targeting of the DL4 gene in TECs by the Foxn1-Cre recombinase. (A) A schematic representation of the genomic organization of the DL4lox/lox locus is shown. Exons 1–3 are flanked by loxP sequences (black triangles). DL4lox/lox mice were crossed to mice in which the Cre recombinase was knocked into the Foxn1 locus (Foxn1-Cre) to obtain DL4lox/loxFoxn1-Cre mice (DL4ΔFoxn1). (B) Histograms of total TECs (PanCyt+CD45−), cTECs (PanCyt+CD45−BP1+), and mTECs (PanCyt+CD45−BP1−) from Ctrl (DL4lox/lox) or DL4ΔFoxn1 mice stained for DL1 (shaded) and DL4 (continuous lines). DL1 staining was indistinguishable from Ctrl isotype staining (not depicted). Data are representative of five Ctrl and three DL4ΔFoxn1 mice aged 2–3 wk from three independent experiments. Percentages of cells positively staining for DL4 are indicated in the histograms.
Mentions: Studying the role of DL4 in hematopoiesis and, more specifically, during T lineage commitment by a conventional loss-of-function approach is hampered by the fact that DL4 gene-targeted mice are embryonic lethal (24, 30). We therefore generated conditional gene-targeted mice for DL4. These mice carry loxP sites flanking the first three coding exons of the DL4 gene (DL4lox/lox). Because the gene expression and antibody studies suggested that DL4 expression in TECs might be critical for T cell lineage commitment, we crossed the DL4lox/lox (Ctrl) mice to knock-in mice expressing the Cre recombinase under the control of the Foxn1 gene (31), which is highly expressed in TECs, to generate DL4lox/lox&Foxn1-Cre (DL4ΔFoxn1) mice (Fig. 2 A). Staining of DL4ΔFoxn1 TECs with anti-DL4 and -DL1 mAbs confirmed the complete loss of DL4 protein in the gene-targeted mice compared with Ctrl animals (Fig. 2 B). Interestingly, DL1 expression on TECs derived from DL4ΔFoxn1 mice was not detectable, indicating that there is no functional compensation by DL1 as a consequence of loss of DL4 (Fig. 2 B).

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