Delta-like 4 is the essential, nonredundant ligand for Notch1 during thymic T cell lineage commitment - PubMed (original) (raw)

Comparative Study

. 2008 Oct 27;205(11):2515-23.

doi: 10.1084/jem.20080829. Epub 2008 Sep 29.

Affiliations

Comparative Study

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

Ute Koch et al. J Exp Med. 2008.

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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Figures

Figure 1.

Figure 1.

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.

Figure 2.

Figure 2.

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.

Figure 3.

Figure 3.

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.

Figure 4.

Figure 4.

Comparative phenotypic analysis of B cells within thymi of DL4ΔFoxn1 and N1ΔMx mice. (A) Representative flow cytometric analysis of B cells in the DN thymus compartment from Ctrl, N1ΔMx, and DL4ΔFoxn1 mice stained for B220 and IgM (top) or BP1 and CD93 (bottom). B220/IgM staining is electronically gated on lineage-negative CD44+CD25− cells, whereas BP1/CD93 staining is gated on lineage-negative B220+ cells. (left) A comparative analysis of normal BM B cells to highlight the phenotypic similarity. Percentages of populations staining positively for the indicated markers are shown in the contour plots. (B) Bar graphs show the absolute numbers ± SD of immature (B220+IgM−) B cells per thymus derived from Ctrl, Notch1ΔMx, and DL4ΔFoxn1 mice (n = 6 mice per sample group). Note the logarithmic scale. All mice used were between 2–3 wk old, and data are representative of three independent experiments.

Figure 5.

Figure 5.

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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