Delta-like 4 is indispensable in thymic environment specific for T cell development - PubMed (original) (raw)

Comparative Study

. 2008 Oct 27;205(11):2507-13.

doi: 10.1084/jem.20080134. Epub 2008 Sep 29.

Affiliations

Comparative Study

Delta-like 4 is indispensable in thymic environment specific for T cell development

Katsuto Hozumi et al. J Exp Med. 2008.

Abstract

The thymic microenvironment is required for T cell development in vivo. However, in vitro studies have shown that when hematopoietic progenitors acquire Notch signaling via Delta-like (Dll)1 or Dll4, they differentiate into the T cell lineage in the absence of a thymic microenvironment. It is not clear, however, whether the thymus supports T cell development specifically by providing Notch signaling. To address this issue, we generated mice with a loxP-flanked allele of Dll4 and induced gene deletion specifically in thymic epithelial cells (TECs). In the thymus of mutant mice, the expression of Dll4 was abrogated on the epithelium, and the proportion of hematopoietic cells bearing the intracellular fragment of Notch1 (ICN1) was markedly decreased. Corresponding to this, CD4 CD8 double-positive or single-positive T cells were not detected in the thymus. Further analysis showed that the double-negative cell fraction was lacking T cell progenitors. The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4. These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

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Figures

Figure 1.

Figure 1.

Dll4 was expressed on thymic epithelium. The expression of Dll4 on the epithelium was detected in thymus (A and B, Thymus), but not gut (C, Gut) or salivary gland (C, Sal. G.) by immunofluorescence histochemistry. Staining was done with anti–pan-cytokeratin (CK; A, green), anti-K5 (K5; B, green), anti-K8 (K8; B, red), anti-Dll4 (Dll4; A, red; B, blue), or control IgG (IgG; A, red; B, blue) antibodies and analyzed by confocal laser microscopy. (A and B, right) High-magnification images of the red square in the middle images. (B, left) Hemotoxylin and eosin staining of serial section. Bars: (A, left and middle) 50 μm; (A, right) 10 μm; (B, left) 200 μm; (B, right) 10 μm; (C) 20 μm.

Figure 2.

Figure 2.

Specific abrogation of Dll4 on thymic epithelium. (A) Targeted insertion of loxP sequences flanking a part of exon 1 and whole exons 2 and 3 of the Dll4 gene. Numbers indicate exon number. 1*, first exon modified with loxP sequence; 1**, part of exon 1 after gene deletion; ATG, translational initiation codon of Dll4; triangles, loxP sequences; open boxes, Dll4 exons; B, BglII; C, ClaI; RI, EcoRI; Xb, XbaI; Xh, XhoI. (B) Cre activity was targeted to thymic epithelia using mice, designated FoxN1-Cre, in which this recombinase is under the transcriptional control of the FoxN1 locus. The timing and specificity of Cre-mediated recombination was visualized by the expression of enhanced GFP (eGFP) in thymus tissue sections of E12.5 (left) and 6-wk-old (right) crosses of FoxN1-Cre mice with the transgenic Z/EG reporter mice. The thymus anlage in the left image is outlined with a dashed line. C, cortex; M, medulla. (C) The expression of Dll4 on thymic epithelial, but not endothelial, cells was abrogated in _Dll4_lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed) mice. Epithelial and endothelial cells in the thymus were identified by the expression of cytokeratin (CK, green, left and middle) and CD31 (red, right), respectively, with Dll4 (red, middle; green, right). Bars: (B and C) 50 μm.

Figure 3.

Figure 3.

Notch signaling was decreased in the thymus with Dll4-null epithelial cells. (A) The cleaved Notch1 fragment was found in fetal thymocytes of E15.5 _Dll4_lox/lox (Dll4-floxed) embryos. The cells with cleaved Notch1 are indicated with arrowheads. Bar, 10 μm. (B) Frequencies of cells with cleaved Notch1 (ICN1+; mean percentage from five fields in a slide with >100 cells from each embryo ± SD) were counted in E15.5 WT (n = 3), _Dll4_lox/loxFoxN1Cre (FoxN1Cre:Dll4-floxed, n = 6), or _Dll4_lox/lox (Dll4-floxed, n = 6) mice. The cultured DN cells were prepared after the FTOC of E14.5 WT fetal thymus for 4 d with γ-secretase inhibitor (GSI, n = 3) or without γ-secretase inhibitor (DMSO, n = 3). Asterisk indicates unpaired Student's t test; P < 0.001.

Figure 4.

Figure 4.

No T cells, but substantial B cells, appear in the thymus with Dll4-null epithelium. (A) Thymic cellularity (mean ± SD) of fetus (E15.5; left, n = 3; right, n = 5), neonate (N.B.; left, n = 5; right, n = 3), 4-wk-old (4W, n = 4), or 8-wk-old (8W, n = 3) _Dll4_lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed, shaded columns) mice compared with _Dll4_lox/lox (Dll4-floxed, open columns) mice. Asterisks indicate unpaired Student's t test; P < 0.001. (B) Flow cytometry of thymocytes from young adult (4W) _Dll4_lox/lox mice with (bottom, FoxN1Cre:Dll4-floxed) or without (top, Dll4-floxed) FoxN1-Cre transgene. Thymocytes were stained with monoclonal antibodies to surface molecules as shown. Numbers in the profiles indicate the relative percentages, in CD4−CD8− cells (right, CD4+CD8 vs. Thy1.2), for each corresponding quadrant or fraction. (C) The ETP population was not observed in _Dll4_lox/loxFoxN1-Cre (4W) mice. Thymocytes from _Dll4_lox/lox (top) or _Dll4_lox/loxFoxN1-Cre mice (bottom) were stained for lineage markers (Lin.: CD4, CD8, CD3, B220, CD19, Gr1, CD11b, TER119), CD44, CD25, CD24, and c-kit. Profiles are shown with the gate (Lin.−, middle; Lin.−CD44+CD25− as DN1, right). Numbers on the plots represent the frequency of cells lying in the indicated regions within the gate. The ETP population is identified as Lin.−CD44+CD25−CD24− or lowc-kit+ cells (DN1a+b). DN1c and DN1–3 populations are also shown in the plots. Data are representative of three experiments.

Figure 5.

Figure 5.

Hematopoietic progenitors with active form of Notch1 give rise to T cell lineage in Dll4-deficient thymus. Hematopoietic progenitors (Lin.−c-kit+ cells) derived from E15.5 fetal liver were infected with the retrovirus encoding the intracellular region of Notch1 (ICN1) or empty vector (Mock). Infected cells were monitored by expression of GFP. These cells were cultured into deoxyguanosine-treated thymic lobes of _Dll4_lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed) or _Dll4_lox/lox (Dll4-floxed) fetuses for 13 d as FTOC and analyzed for the expression of CD4, CD8, CD25, and CD19 (A). Alternatively, these cells were injected intravenously into irradiated _Dll4_lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed) or _Dll4_lox/lox (Dll4-floxed) neonate mice. After 3 wk, thymocytes were analyzed for expression of CD4, CD8, and CD19 (B). Profiles are shown with the gate (GFP+ or GFP+CD4−CD8−, DN-gated in A). Numbers in the profiles indicate the relative percentages for each corresponding quadrant.

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