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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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DL4 but not DL1 expression is detected on TECs. (A) LacZ staining on thymic sections derived from DL1 and DL4 lacZ knock-in mice. mRNA expression of the DL1-driven lacZ gene is confined to blood vessels within the thymus, whereas DL4 drives expression preferentially within the cortical epithelium. The dashed lines indicate cortical–medullary boundaries. Bars, 100 μm. (B) Anti-DL1 and -DL4 antibodies specifically bind their corresponding ligands without exhibiting cross-reactivity; OP-9–DL1–EGFP (top) and OP-9–DL4–EGFP (bottom) were stained with isotype Ctrl (shaded), anti-DL1 (dashed line), or anti-DL4 antibodies (continuous line; dilution, 1:100). The analysis was performed on gated EGFP-positive cells, and representative histograms are shown. (C) Anti-DL1 and -DL4 antibody staining on TECs. Enriched TECs extracted from wild-type thymi were stained for BP1 (3C6), CD45, and isotype Ctrl, anti-DL1, or anti-DL4 antibodies, followed by intracellular staining for PanCyt (C11) and flow cytometric analysis. Total TECs were gated as PanCyt+CD45− (top left), and cTECs and mTECs were further defined as BP1+ and BP1−, respectively (bottom left); isotype Ctrl (shaded), anti-DL1 (dashed line; top right), and anti-DL4 (continuous line; bottom right) are shown. Percentages of DL4+ TECs represent the mean ± SD of five mice. (D) DL1 and DL4 are not detectably expressed on thymocytes. (left) A representative flow cytometric analysis of CD4 versus CD8 of wild-type thymocytes. (right) DL1 and DL4 expression are shown as representative histograms (shaded, isotype Ctrl; dashed line, DL1; continuous line, DL4) after gating on the indicated thymic subpopulations: DN (Lin−, CD45+, CD4−, CD8−), DP (Lin−, CD45+, CD4+, CD8+), CD4SP (Lin−, CD45+, CD4+, CD8−), and CD8SP (Lin−, CD45+, CD4−, CD8+) thymocytes. Percentages of individual thymic subpopulations are indicated within the plots. Data are representative of three independent experiments.
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fig1: DL4 but not DL1 expression is detected on TECs. (A) LacZ staining on thymic sections derived from DL1 and DL4 lacZ knock-in mice. mRNA expression of the DL1-driven lacZ gene is confined to blood vessels within the thymus, whereas DL4 drives expression preferentially within the cortical epithelium. The dashed lines indicate cortical–medullary boundaries. Bars, 100 μm. (B) Anti-DL1 and -DL4 antibodies specifically bind their corresponding ligands without exhibiting cross-reactivity; OP-9–DL1–EGFP (top) and OP-9–DL4–EGFP (bottom) were stained with isotype Ctrl (shaded), anti-DL1 (dashed line), or anti-DL4 antibodies (continuous line; dilution, 1:100). The analysis was performed on gated EGFP-positive cells, and representative histograms are shown. (C) Anti-DL1 and -DL4 antibody staining on TECs. Enriched TECs extracted from wild-type thymi were stained for BP1 (3C6), CD45, and isotype Ctrl, anti-DL1, or anti-DL4 antibodies, followed by intracellular staining for PanCyt (C11) and flow cytometric analysis. Total TECs were gated as PanCyt+CD45− (top left), and cTECs and mTECs were further defined as BP1+ and BP1−, respectively (bottom left); isotype Ctrl (shaded), anti-DL1 (dashed line; top right), and anti-DL4 (continuous line; bottom right) are shown. Percentages of DL4+ TECs represent the mean ± SD of five mice. (D) DL1 and DL4 are not detectably expressed on thymocytes. (left) A representative flow cytometric analysis of CD4 versus CD8 of wild-type thymocytes. (right) DL1 and DL4 expression are shown as representative histograms (shaded, isotype Ctrl; dashed line, DL1; continuous line, DL4) after gating on the indicated thymic subpopulations: DN (Lin−, CD45+, CD4−, CD8−), DP (Lin−, CD45+, CD4+, CD8+), CD4SP (Lin−, CD45+, CD4+, CD8−), and CD8SP (Lin−, CD45+, CD4−, CD8+) thymocytes. Percentages of individual thymic subpopulations are indicated within the plots. Data are representative of three independent experiments.

Mentions: Because DL1- and DL4-expressing OP-9 stromal cells (10, 16, 17) or normal thymic stroma (22) can both support the complete development of mature functional T cells from BM precursors in vitro, it is conceivable that either or both of these proteins function as physiological ligands for the N1 receptor during thymic T cell lineage commitment. To gain further insight into the expression pattern of DL1 and DL4 in the thymus, we analyzed thymi of mice in which the lacZ gene was knocked into either the DL1 or DL4 locus (23, 24). LacZ staining on thymic sections derived from DL1lacZ/+ mice was only positive for rare endothelial cells within the thymus, whereas DL4LacZ/+ mice exhibited a strong reticular expression pattern within the outer cortex (characteristic of epithelial cells) and a weaker more punctuated staining within the medulla of the thymus (Fig. 1 A). This expression pattern was confirmed at the protein level using novel mAbs generated against the extracellular domains of DL1 and DL4. The specificity of the anti-DL1 and -DL4 mAbs was tested on DL1- and DL4-expressing OP-9 cells. Anti-DL1 mAbs bound specifically to DL1- but not to DL4-expressing OP-9 cells, whereas the anti-DL4 mAbs showed a reciprocal binding pattern (Fig. 1 B). These results indicate that both mAbs specifically bind to the corresponding Notch ligands and that they do not exhibit any cross-reactivity. These mAbs were then used to stain cell preparations enriched for TECs and analyzed by flow cytometry. TECs were identified as CD45−PanCytokeratin (PanCyt)+ (25) and were further subdivided into cortical TECs (cTECs) and medullary TECs (mTECs) based on BP1 expression (26). Anti-DL1 mAbs did not stain cTECs or mTECs above background. In contrast anti-DL4 mAbs showed significant staining of TECs, with brighter staining for cortical- (BP1+) versus medullary- (BP1−) derived populations (Fig. 1 C). Analysis of thymocyte subpopulations for the expression of DL1 and DL4 with these mAbs was negative (Fig. 1 D). These expression data are in general agreement with previous studies (18, 27, 28), with the notable exception of one report in which TECs were broadly stained by a commercial polyclonal antibody against DL1 (12). The specificity of this polyclonal reagent has already been challenged by others (29). In conclusion, the thymic expression pattern of DL1 and DL4 revealed by our novel mAbs confirms the results obtained with the lacZ knock-in mice, strongly suggesting that DL4 might be the physiological ligand expressed by TECs that interacts with N1-expressing T cell precursors to specify the T cell lineage.


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)

DL4 but not DL1 expression is detected on TECs. (A) LacZ staining on thymic sections derived from DL1 and DL4 lacZ knock-in mice. mRNA expression of the DL1-driven lacZ gene is confined to blood vessels within the thymus, whereas DL4 drives expression preferentially within the cortical epithelium. The dashed lines indicate cortical–medullary boundaries. Bars, 100 μm. (B) Anti-DL1 and -DL4 antibodies specifically bind their corresponding ligands without exhibiting cross-reactivity; OP-9–DL1–EGFP (top) and OP-9–DL4–EGFP (bottom) were stained with isotype Ctrl (shaded), anti-DL1 (dashed line), or anti-DL4 antibodies (continuous line; dilution, 1:100). The analysis was performed on gated EGFP-positive cells, and representative histograms are shown. (C) Anti-DL1 and -DL4 antibody staining on TECs. Enriched TECs extracted from wild-type thymi were stained for BP1 (3C6), CD45, and isotype Ctrl, anti-DL1, or anti-DL4 antibodies, followed by intracellular staining for PanCyt (C11) and flow cytometric analysis. Total TECs were gated as PanCyt+CD45− (top left), and cTECs and mTECs were further defined as BP1+ and BP1−, respectively (bottom left); isotype Ctrl (shaded), anti-DL1 (dashed line; top right), and anti-DL4 (continuous line; bottom right) are shown. Percentages of DL4+ TECs represent the mean ± SD of five mice. (D) DL1 and DL4 are not detectably expressed on thymocytes. (left) A representative flow cytometric analysis of CD4 versus CD8 of wild-type thymocytes. (right) DL1 and DL4 expression are shown as representative histograms (shaded, isotype Ctrl; dashed line, DL1; continuous line, DL4) after gating on the indicated thymic subpopulations: DN (Lin−, CD45+, CD4−, CD8−), DP (Lin−, CD45+, CD4+, CD8+), CD4SP (Lin−, CD45+, CD4+, CD8−), and CD8SP (Lin−, CD45+, CD4−, CD8+) thymocytes. Percentages of individual thymic subpopulations are indicated within the plots. Data are representative of three independent experiments.
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fig1: DL4 but not DL1 expression is detected on TECs. (A) LacZ staining on thymic sections derived from DL1 and DL4 lacZ knock-in mice. mRNA expression of the DL1-driven lacZ gene is confined to blood vessels within the thymus, whereas DL4 drives expression preferentially within the cortical epithelium. The dashed lines indicate cortical–medullary boundaries. Bars, 100 μm. (B) Anti-DL1 and -DL4 antibodies specifically bind their corresponding ligands without exhibiting cross-reactivity; OP-9–DL1–EGFP (top) and OP-9–DL4–EGFP (bottom) were stained with isotype Ctrl (shaded), anti-DL1 (dashed line), or anti-DL4 antibodies (continuous line; dilution, 1:100). The analysis was performed on gated EGFP-positive cells, and representative histograms are shown. (C) Anti-DL1 and -DL4 antibody staining on TECs. Enriched TECs extracted from wild-type thymi were stained for BP1 (3C6), CD45, and isotype Ctrl, anti-DL1, or anti-DL4 antibodies, followed by intracellular staining for PanCyt (C11) and flow cytometric analysis. Total TECs were gated as PanCyt+CD45− (top left), and cTECs and mTECs were further defined as BP1+ and BP1−, respectively (bottom left); isotype Ctrl (shaded), anti-DL1 (dashed line; top right), and anti-DL4 (continuous line; bottom right) are shown. Percentages of DL4+ TECs represent the mean ± SD of five mice. (D) DL1 and DL4 are not detectably expressed on thymocytes. (left) A representative flow cytometric analysis of CD4 versus CD8 of wild-type thymocytes. (right) DL1 and DL4 expression are shown as representative histograms (shaded, isotype Ctrl; dashed line, DL1; continuous line, DL4) after gating on the indicated thymic subpopulations: DN (Lin−, CD45+, CD4−, CD8−), DP (Lin−, CD45+, CD4+, CD8+), CD4SP (Lin−, CD45+, CD4+, CD8−), and CD8SP (Lin−, CD45+, CD4−, CD8+) thymocytes. Percentages of individual thymic subpopulations are indicated within the plots. Data are representative of three independent experiments.
Mentions: Because DL1- and DL4-expressing OP-9 stromal cells (10, 16, 17) or normal thymic stroma (22) can both support the complete development of mature functional T cells from BM precursors in vitro, it is conceivable that either or both of these proteins function as physiological ligands for the N1 receptor during thymic T cell lineage commitment. To gain further insight into the expression pattern of DL1 and DL4 in the thymus, we analyzed thymi of mice in which the lacZ gene was knocked into either the DL1 or DL4 locus (23, 24). LacZ staining on thymic sections derived from DL1lacZ/+ mice was only positive for rare endothelial cells within the thymus, whereas DL4LacZ/+ mice exhibited a strong reticular expression pattern within the outer cortex (characteristic of epithelial cells) and a weaker more punctuated staining within the medulla of the thymus (Fig. 1 A). This expression pattern was confirmed at the protein level using novel mAbs generated against the extracellular domains of DL1 and DL4. The specificity of the anti-DL1 and -DL4 mAbs was tested on DL1- and DL4-expressing OP-9 cells. Anti-DL1 mAbs bound specifically to DL1- but not to DL4-expressing OP-9 cells, whereas the anti-DL4 mAbs showed a reciprocal binding pattern (Fig. 1 B). These results indicate that both mAbs specifically bind to the corresponding Notch ligands and that they do not exhibit any cross-reactivity. These mAbs were then used to stain cell preparations enriched for TECs and analyzed by flow cytometry. TECs were identified as CD45−PanCytokeratin (PanCyt)+ (25) and were further subdivided into cortical TECs (cTECs) and medullary TECs (mTECs) based on BP1 expression (26). Anti-DL1 mAbs did not stain cTECs or mTECs above background. In contrast anti-DL4 mAbs showed significant staining of TECs, with brighter staining for cortical- (BP1+) versus medullary- (BP1−) derived populations (Fig. 1 C). Analysis of thymocyte subpopulations for the expression of DL1 and DL4 with these mAbs was negative (Fig. 1 D). These expression data are in general agreement with previous studies (18, 27, 28), with the notable exception of one report in which TECs were broadly stained by a commercial polyclonal antibody against DL1 (12). The specificity of this polyclonal reagent has already been challenged by others (29). In conclusion, the thymic expression pattern of DL1 and DL4 revealed by our novel mAbs confirms the results obtained with the lacZ knock-in mice, strongly suggesting that DL4 might be the physiological ligand expressed by TECs that interacts with N1-expressing T cell precursors to specify the T cell lineage.

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