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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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Normal T cell development and absence of immature thymic B cells in BM chimeras reconstituted with DL4ΔMx BM. Ctrl or DL4ΔMx CD45.2+ BM was transplanted into lethally irradiated CD45.1+ hosts and analyzed 8 wk after reconstitution. The reconstitution efficiency for both Ctrl and DL4ΔMx BM chimeras was >95% (not depicted). (A) Representative flow cytometric analysis of CD45.2+ thymocytes stained with anti-CD4 and -CD8. (B) CD45.2+ lineage-negative DN thymocytes were analyzed for the expression of CD44 and CD25. (C and D) Lineage-negative thymocytes were analyzed for the presence of B cells expressing B220 and IgM, or BP1 and CD93. Data in A–D are representative of three individual chimeras per group with virtually identical results, and relative percentages are indicated in the contour plots. Two independent experiments were performed. (E) Southern blot analysis of ScaI-digested genomic DNA derived from BM cells of Ctrl and DL4ΔMx chimeras showing the floxed alleles in Ctrl chimeras and the completely deleted DL4 locus in DL4ΔMx chimeras. The targeting strategy and size of the restriction fragments are as described in Fig. 2 A.
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fig5: Normal T cell development and absence of immature thymic B cells in BM chimeras reconstituted with DL4ΔMx BM. Ctrl or DL4ΔMx CD45.2+ BM was transplanted into lethally irradiated CD45.1+ hosts and analyzed 8 wk after reconstitution. The reconstitution efficiency for both Ctrl and DL4ΔMx BM chimeras was >95% (not depicted). (A) Representative flow cytometric analysis of CD45.2+ thymocytes stained with anti-CD4 and -CD8. (B) CD45.2+ lineage-negative DN thymocytes were analyzed for the expression of CD44 and CD25. (C and D) Lineage-negative thymocytes were analyzed for the presence of B cells expressing B220 and IgM, or BP1 and CD93. Data in A–D are representative of three individual chimeras per group with virtually identical results, and relative percentages are indicated in the contour plots. Two independent experiments were performed. (E) Southern blot analysis of ScaI-digested genomic DNA derived from BM cells of Ctrl and DL4ΔMx chimeras showing the floxed alleles in Ctrl chimeras and the completely deleted DL4 locus in DL4ΔMx chimeras. The targeting strategy and size of the restriction fragments are as described in Fig. 2 A.

Mentions: Although Foxn1-Cre expression within the thymus should be restricted to TECs (31) and wild-type thymocytes do not stain detectably with anti-DL4 mAbs (Fig. 1 D), it remains formally possible that rare DL4-expressing hematopoietic cells might be involved in T cell lineage commitment in the thymus. In this scenario, loss of DL4 expression on these cells could theoretically contribute to the generation of immature B cells in the DL4ΔFoxn1 thymus. To exclude this possibility, we generated DL4lox/loxMx-Cre (DL4ΔMx) mice to inducibly inactivate DL4 in BM progenitors. Because the Mx-Cre transgene is also active to some extent in TECs (16), BM from DL4ΔMx or Ctrl mice (both CD45.2+) was transplanted into CD45.1+ wild-type hosts to critically evaluate whether DL4ΔMx hematopoietic cells could contribute to the phenotype. Ctrl and DL4ΔMx BM chimeras were analyzed 8 wk after transplantation. The reconstitution efficiency of CD45.2+ cells for both Ctrl and DL4ΔMx chimeras was >95%. A portion of the BM cells of the corresponding BM chimeras was analyzed by Southern blot analysis to ensure that reconstitution was not caused by rare cells that might have escaped deletion of the DL4 locus (Fig. 5 E). As expected, Ctrl and DL4ΔMx BM progenitors generated all major blood lineages in the BM and spleen (unpublished data). More importantly, all major mature and immature thymocyte subsets were generated normally from Ctrl and DL4ΔMx BM progenitors (Fig. 5, A and B), and only very small numbers of immature B cells were found in the thymus (Fig. 5, C and D). These data exclude a significant role for putative DL4-expressing hematopoietic cells in thymic T cell lineage commitment.


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)

Normal T cell development and absence of immature thymic B cells in BM chimeras reconstituted with DL4ΔMx BM. Ctrl or DL4ΔMx CD45.2+ BM was transplanted into lethally irradiated CD45.1+ hosts and analyzed 8 wk after reconstitution. The reconstitution efficiency for both Ctrl and DL4ΔMx BM chimeras was >95% (not depicted). (A) Representative flow cytometric analysis of CD45.2+ thymocytes stained with anti-CD4 and -CD8. (B) CD45.2+ lineage-negative DN thymocytes were analyzed for the expression of CD44 and CD25. (C and D) Lineage-negative thymocytes were analyzed for the presence of B cells expressing B220 and IgM, or BP1 and CD93. Data in A–D are representative of three individual chimeras per group with virtually identical results, and relative percentages are indicated in the contour plots. Two independent experiments were performed. (E) Southern blot analysis of ScaI-digested genomic DNA derived from BM cells of Ctrl and DL4ΔMx chimeras showing the floxed alleles in Ctrl chimeras and the completely deleted DL4 locus in DL4ΔMx chimeras. The targeting strategy and size of the restriction fragments are as described in Fig. 2 A.
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fig5: Normal T cell development and absence of immature thymic B cells in BM chimeras reconstituted with DL4ΔMx BM. Ctrl or DL4ΔMx CD45.2+ BM was transplanted into lethally irradiated CD45.1+ hosts and analyzed 8 wk after reconstitution. The reconstitution efficiency for both Ctrl and DL4ΔMx BM chimeras was >95% (not depicted). (A) Representative flow cytometric analysis of CD45.2+ thymocytes stained with anti-CD4 and -CD8. (B) CD45.2+ lineage-negative DN thymocytes were analyzed for the expression of CD44 and CD25. (C and D) Lineage-negative thymocytes were analyzed for the presence of B cells expressing B220 and IgM, or BP1 and CD93. Data in A–D are representative of three individual chimeras per group with virtually identical results, and relative percentages are indicated in the contour plots. Two independent experiments were performed. (E) Southern blot analysis of ScaI-digested genomic DNA derived from BM cells of Ctrl and DL4ΔMx chimeras showing the floxed alleles in Ctrl chimeras and the completely deleted DL4 locus in DL4ΔMx chimeras. The targeting strategy and size of the restriction fragments are as described in Fig. 2 A.
Mentions: Although Foxn1-Cre expression within the thymus should be restricted to TECs (31) and wild-type thymocytes do not stain detectably with anti-DL4 mAbs (Fig. 1 D), it remains formally possible that rare DL4-expressing hematopoietic cells might be involved in T cell lineage commitment in the thymus. In this scenario, loss of DL4 expression on these cells could theoretically contribute to the generation of immature B cells in the DL4ΔFoxn1 thymus. To exclude this possibility, we generated DL4lox/loxMx-Cre (DL4ΔMx) mice to inducibly inactivate DL4 in BM progenitors. Because the Mx-Cre transgene is also active to some extent in TECs (16), BM from DL4ΔMx or Ctrl mice (both CD45.2+) was transplanted into CD45.1+ wild-type hosts to critically evaluate whether DL4ΔMx hematopoietic cells could contribute to the phenotype. Ctrl and DL4ΔMx BM chimeras were analyzed 8 wk after transplantation. The reconstitution efficiency of CD45.2+ cells for both Ctrl and DL4ΔMx chimeras was >95%. A portion of the BM cells of the corresponding BM chimeras was analyzed by Southern blot analysis to ensure that reconstitution was not caused by rare cells that might have escaped deletion of the DL4 locus (Fig. 5 E). As expected, Ctrl and DL4ΔMx BM progenitors generated all major blood lineages in the BM and spleen (unpublished data). More importantly, all major mature and immature thymocyte subsets were generated normally from Ctrl and DL4ΔMx BM progenitors (Fig. 5, A and B), and only very small numbers of immature B cells were found in the thymus (Fig. 5, C and D). These data exclude a significant role for putative DL4-expressing hematopoietic cells in thymic T cell lineage commitment.

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