The bHLH gene Hes1 is essential for expansion of early T cell precursors - PubMed (original) (raw)

The bHLH gene Hes1 is essential for expansion of early T cell precursors

K Tomita et al. Genes Dev. 1999.

Abstract

Mice mutant for the bHLH gene Hes1, which is known to keep cells in a proliferative state, mostly lack thymus. Transfer of Hes1-null fetal liver cells into RAG2-null host mice normally reconstitutes B cells but fails to generate mature T cells in the thymus. In the reconstituted thymus, T cell differentiation is arrested at the CD4(-)CD8(-) double negative (DN) stage. Both the initial T cell receptor (TCR)-independent and the subsequent TCR-dependent selective expansion during the DN stage are severely affected. Thus, Hes1 is essential for the earliest thymocyte expansion in a cell-autonomous manner.

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Figures

Figure 1

Hes1 expression in the developing thymus and the thymic defects of mice mutant for Hes1. (A) Hes1 expression in the thymus was determined by Northern blot analysis. Hes1 was already expressed at a high level at E15, but expression decreased gradually afterward. Hes1 was expressed by both thymocytes and stroma while CD3 was expressed only by thymocytes. G3PDH expression was examined as a control. (B_–_K) Thymic sections were prepared from wild-type (B,D,F,H,J) and _Hes1_-null (C,E,G,I,K) embryos and subjected to HE staining (B_–_G) and immunohistochemistry with anti-TCRβ (H,I) and anti-Thy1 antibodies (J,K). _Hes1_-null embryos lacked thymus (C) or had a very small thymus (E) compared to wild type (D). (F,G) A higher magnification of D and E. (H,I) Even when the thymus existed, αβ TCR+ thymocytes were absent in _Hes1_-null embryos (I), whereas they were present in wild-type thymus (H). (J,K) In the _Hes1_-null thymus, there were Thy1+ cells (K) as in the wild-type thymus (J). Scale bar, 150 μm (B_–_E); 25 μm (F_–_K). (H) Heart; (Th) thymus; (Thr) thyroid.

Figure 1

Figure 2

Flow cytometric analysis of fetal liver cells. Flow cytometric analysis was performed to determine the surface expression of TER-119 (A), Gr-1 (B), and Mac-1 (C). For B cell lineage analysis, fetal liver cells were cultured on PA6 stromal cells in the presence of IL-7 and examined for expression of IgM (D) and B220 (E). Cells were analyzed after incubation in the presence (shaded area) or absence (bold line only) of FITC-conjugated antibodies. No abnormality was detected in _Hes1_-null cells.

Figure 3

Reconstitution of the lymphoid system of RAG2 mutant mice by transfer of wild-type and _Hes1_-null fetal liver cells. Either wild-type or _Hes1_-null fetal liver cells (1 × 106 or 1 × 107, indicated in parentheses above the panel) were transferred intravenously into RAG2 mutant mice to reconstitute the lymphoid system, and after 4–6 weeks of transfer, the lymphoid cells were examined. (A) The absolute numbers of total thymocytes. From the reconstituted thymuses, only Ly5.2+ cells were counted. The average thymocyte number with standard error is _RAG2_−/−, (3.2 ± 0.6) × 106 (n = 10); _RAG2_−/−/WT(1 × 106 cells injected), (1.6 ± 0.3) × 108 (n = 13); _RAG2_−/−/WT(1 × 107 cells injected), (1.0 ± 0.2) × 108 (n = 6); _RAG2_−/−/_Hes1_−/−(1 × 106 cells injected), (1.7 ± 0.7) × 105 (n = 9); _RAG2_−/−/_Hes1_−/−(1 × 107 cells injected), (4.1 ± 1.0) × 105 (n = 8). The number of _Hes1_-null thymocytes averaged 200- to 1000-fold less than that of wild-type thymocytes and 10- to 20-fold less than that of irradiated and nonreconstituted _RAG2_-null thymocytes. (B) Flow cytometric analysis of the reconstituted thymus with α-Ly5.2 and α-Thy1. The absolute number of total cells is indicated above each panel; the number of Ly5.2+Thy1+ cells is shown above the gated region. (C) The absolute numbers of B cells in the spleen. The B cell number of _Hes1_-null origin was normal. (D) Flow cytometric analysis of the reconstituted spleen with α-IgM and α-B220. B cells developed normally in the absence of Hes1.

Figure 3

Figure 4

Flow cytometric analysis of reconstituted T-lineage cells. Flow cytometric analysis of reconstituted thymocytes (A_–_D) and spleen cells (E) and nonreconstituted host spleen cells (F) was performed to determine the surface expression of αβ and γδ TCR (A,E,F), CD4 and CD8 (B), HSA and CD3 (C), and CD44 and CD25 (D). The absolute number of Ly5.2+ thymocytes is indicated above each panel (A_–_C). For analysis of surface expression of CD44 and CD25, DN thymocytes from donor origin were analyzed (D), and the number is shown above each panel (D). In the absence of Hes1, αβ and γδ TCR+ cells, CD4+CD8+ DP cells, and CD3+ cells were virtually absent in the thymus (A_–_C). In the spleen reconstituted with _Hes1_-null cells, γδ T cells were present at a comparable level to the wild type, whereas αβ T cells were not detected (E).

Figure 4

Figure 5

Rearrangement of TCR genes. Either 1 × 106 (A,C,E,G) or 1 × 107 (B,D,F,H) fetal liver cells of wild-type (+/+) and _Hes1_-null origin (−/−) were transferred intravenously into RAG2 mutant mice, and rearrangements of TCR genes were examined by PCR followed by Southern blot analysis with 32P-labeled oligonucleotide probes. In the thymus reconstituted with 1 × 106 _Hes1_-null cells, no apparent rearrangement was detected in the TCRα and TCRβ loci (A,E), although only a low level of D_–_J recombination could be detected (C). Whereas TCRγ gene rearrangement was not detected in the thymus reconstituted with 1 × 106 _Hes1_-null cells, TCRγ2 and TCRγ4 genes (Maki et al. 1996) were rearranged in the spleen in the absence of Hes1 (G). When 1 × 107 _Hes1_-null cells were injected, TCR gene rearrangement was detected (B,D,F,H). As an internal control for PCR, the RAG2 gene was amplified. The RAG2 signal was derived only from the donor cells.

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The bHLH gene Hes1 is essential for expansion of early T cell precursors - PubMed (original) (raw)