T cell development requires constraint of the myeloid regulator C/EBP-α by the Notch target and transcriptional repressor Hes1

Abstract

Notch signaling induces gene expression of the T cell lineage and discourages alternative fate outcomes. Hematopoietic deficiency in the Notch target Hes1 results in severe T cell lineage defects; however, the underlying mechanism is unknown. We found here that Hes1 constrained myeloid gene-expression programs in T cell progenitor cells, as deletion of the myeloid regulator C/EBP-α restored the development of T cells from Hes1-deficient progenitor cells. Repression of Cebpa by Hes1 required its DNA-binding and Groucho-recruitment domains. Hes1-deficient multipotent progenitor cells showed a developmental bias toward myeloid cells and dendritic cells after Notch signaling, whereas Hes1-deficient lymphoid progenitor cells required additional cytokine signaling for diversion into the myeloid lineage. Our findings establish the importance of constraining developmental programs of the myeloid lineage early in T cell development.

Figure 1

Hes1 expression is upregulated in the thymus and is reciprocal to Cebpa expression. (a) Quantitative PCR analysis of Hes1, Dtx1 and Cebpa mRNA in adult bone marrow (BM) LSK cells, lymphoid-primed multipotential progenitor cells (LMPP), common lymphoid progenitor cells (CLP) and thymic ETP, DN2, DN3 and CD4⁺CD8⁺ double-positive (DP) progenitor cells isolated from C57/BL6 mice by cell sorting; results are relative to those of the control gene Gapdh. ND, not determined. (b) Quantitative PCR analysis of Hes1, Dtx1 and Cebpa mRNA in fetal liver (FL) Lin⁻Kit⁺Flt3⁻ cells, MPPs (Lin⁻Kit⁺Flt3⁺L-7Ra⁻), lymphoid progenitor cells (Lin⁻Kit⁺Flt3⁺L-7Ra⁺), GMP, Mac-1⁺ cells and DN2 fetal thymocytes collected from C57/BL6 embryos at E12.5–E13.5 (presented as in a). (c) Flow cytometry of cells from FL of Hes1^−/− mice and their Hes1^+/+ littermates at E12.5–E13.5. Numbers adjacent to outlined areas indicate percent c-Kit⁺Lin⁻ cells (left) or IL-7Ra⁺Flt3⁺ cells (right, top) or IL-7Ra⁻Flt3⁺ cells (right, bottom). (d) Quantitative PCR analysis of Hes1 and Cebpa mRNA in progenitor populations isolated by cell sorting as in (c) from Hes1^−/− embryos (n = 2) and a Hes1^+/+ littermate control embryo (n = 1) at E12.5–E13.5; Hes1^+/+ fetal thymocyte progenitor cells (E13.5) (ETP or DN3) serve as a comparator population (presented as in a). UD, undetected; ND, not determined. Data are representative of three independent experiments (a–c; error bars, s.e.m. of triplicates in a and b) or are representative of one experiment (d; error bars, s.e.m.).

Figure 2

Hes1-deficient multipotent progenitor cells, but not lymphoid progenitor cells, divert to myeloid fates in T cell–inductive conditions. Flow cytometry of FL progenitor cells (Lin⁻c-Kit⁺Flt3⁺ IL-7Ra⁻ or IL-7Ra⁺) isolated by cell sorting from Hes1^−/− embryos and their Hes1^+/+ littermates at E12.5–E13.5 and cultured for 6 d on OP9 stromal cells (a) or OP9-DL4 stromal cells (b,c) (250 cells per well, in triplicate). Numbers adjacent to outlined areas indicate percent Mac-1⁺CD19⁻ cells (a,c, top left) or Mac-1⁻CD19⁺ cells (a,c, bottom right), or percent Mac-1⁺CD25⁻ cells (b, top left) or Mac-1⁻CD25⁺ cells (b, bottom right). Right (b), Thy-1⁺CD25⁺ cells (top) and Mac-1⁺Gr-1⁺ cells (bottom). *P < 0.05 and **P < 0.01 (Student’s t-test) Data are from one experiment representative of three experiments with similar results (average and s.e.m. in b, right).

Figure 3

Hes1-deficient lymphoid progenitor cells undergo apoptosis in T cell–inductive conditions lacking myeloid cytokines. (a) Flow cytometry of FL multipotent progenitor cells (Lin⁻c-Kit⁺Flt3⁺IL-7Ra⁻) or lymphoid progenitor cells (Lin⁻c-Kit⁺Flt3⁺IL-7Ra⁺) isolated by cell sorting from Hes1^−/− embryos and their Hes1^+/+ littermates at E12.5–E13.5 and cultured for 4 d on OP9-DL4 stroma (500 cells per well in triplicate). Right, frequency of annexin V–positive (AnnV⁺) cells (left) or Mac-1⁻Thy-1⁻ cells (right) among DAPI⁻ cells. *P < 0.05 and **P < 0.01 (Student’s t-test). (b) Flow cytometry of FL multipotent progenitor cells (Lin⁻c-Kit⁺Flt3⁺IL-7Ra⁻) or lymphoid progenitor cells (Lin⁻c-Kit⁺Flt3⁺IL-7Ra⁺) isolated by cell sorting from embryos as in a and cultured for 6 d on OP9-DL1 stroma (250 cells per well in triplicate) with IL-7, Flt3L and supplemented with myeloid cytokines (IL-3, SCF, additional Flt3L, M-CSF, GM-CSF and G-CSF). Right, Mac-1⁺CD11c⁺ cells analyzed per well. *P < 0.01 (Student’s t-test). (c) Flow cytometry (left) and quantitative PCR analysis of Hes1, Gata3 and Cebpa mRNA (right) of FL multipotent progenitor cells (Lin⁻c-Kit⁺Flt3⁺IL-7Ra⁻) or lymphoid progenitor cells (Lin⁻c-Kit⁺Flt3⁺IL-7Ra⁺) isolated by cell sorting from embryos as in a and cultured for 6 d on OP9-DL4 stromal cells, followed by sorting of the Mac-1⁻Thy-1⁻CD25⁻ population for quantitative PCR analysis. *P < 0.001 (Student’s t-test). Numbers in quadrants or adjacent to outlined areas indicate percent cells in each throughout. Data are representative of two experiments with similar results (a,b,c; error bars, s.e.m.).

Figure 4

Hes1 inhibits myeloid development and Cebpa expression. (a) Quantitative PCR analysis of Hes1 and Cebpa mRNA in BM LSK cells from mice reconstituted 8 weeks earlier with 5-fluorouracil-conditioned BM that was transduced with empty vector (EV) or retroviral vector encoding human Hes1 (hHes1) before transfer; results are presented relative to those of 18S RNA. (b) Immunoblot analysis of C/EBPa (isoforms p42 and p30) and a-actin (loading control) in 32D cells 48 h after transduction as in a, followed by isolation of GFP⁺ cells by cell sorting before lysis. (c) Quantitative PCR analysis of Hes1, Sfpi1 and Cdkn1b mRNA in Hes1^+/+ FL (E15.5) progenitor cells (Lin⁻c-Kit⁺Flt3⁺) 48 h after transduction of empty vector or with vector encoding wild-type human Hes1 (hHes1(WT)) or human Hes1 lacking the DNA-binding domain (hHes1(DDNA)) or the WRPW domain (hHes1(DGroucho)); results are presented relative to those of Gapdh. (d,e) Flow cytometry of Hes1^−/− FL (E15.5) cells (Lin⁻c-Kit⁺Flt3⁺) transduced as in c and cultured for 8 d on OP9-DL4 cells (gated on GFP⁺ cells). Numbers adjacent to outlined areas indicate percent Mac-1⁺CD25⁻ cells (top left) or Mac-1⁻CD25⁺ cells (bottom right). (f) ChIP analysis of the binding of Hes1 to sites 1.2 kilobases (–1.2 kb) and 165 base pairs (–165 bp) upstream of the transcriptional start site of the mouse Cebpa locus (top) and to the Hes1 promoter (prom; positive control) and Nanog promoter (negative control) in DN3 thymocytes isolated from Rag1-deficient mice. (g) Luciferase activity in 293T cells cotransfected with a luciferase reporter construct containing the Cebpa promoter sequence including one Hes1-binding site (Cebpa prom) or a mutated Hes1-binding site (Cebpa prom(DHes1)); results are normalized to empty vector and are presented relative to the activity of renilla luciferase. NS, not significant; *P < 0.001, Student’s t-test. Data are representative of two experiments (a), one experiment (b), two experiments (c), two experiments (d), two experiments (e) or three experiments (f), three experiments (g; error bars (a,c,g), s.e.m.) or (error bars (d),s.d. of triplicate wells).

Figure 5

Deletion of C/EBPa restores T cell development from Hes1-deficient progenitor cells in vitro. (a) Flow cytometry of FL (E15.5) multipotent progenitor cells (Lin⁻c-Kit⁺Flt3⁺) isolated by cell sorting from Hes1^−/−Cebpa^fl/fl or Hes1^+/−Cebpa^fl/fl (control) embryos and transduced with retroviral vector encoding Cre-GFP and cultured for 7 d on OP9-DL4 cells. Numbers adjacent to outlined areas indicate percent Cre-GFP⁺CD45⁺ (transduced) cells (left, top) or Cre-GFP⁻CD45⁺ (untransduced) cells (left, bottom), or percent Mac-1⁺CD25⁻ cells (right; top left) or Mac-1⁻ CD25⁺ cells (right, bottom right). (b) Flow cytometry of Hes1^−/−Cebpa^fl/fl FL (E15.5) progenitor cells (Lin⁻c-Kit⁺Flt3⁺) transduced with empty retroviral vector (left) or retroviral vector encoding Cre-GFP (right) and sorted for GFP expression, then cultured for 7 d on OP9-DL4 cells (numbers adjacent to outlined areas as in a, right). Below, quantification of CD25⁺ T cells, normalized to GFP⁺ cells plated. *P < 0.05 (Student’s t-test). (c) Limiting-dilution analysis of the frequency of T cell progenitors among Hes1^−/−Cebpa^fl/fl or Hes1^+/+Cebpa^fl/fl FL (E15.5) progenitor cells (Lin⁻c-Kit⁺Flt3⁺) transduced and sorted as in b, then cultured for 6 d on OP9-DL4 cells and analyzed by flow cytometry. Data are representative of three experiments (a), two experiments (b; average and s.e.m.) or one experiment (c).

Figure 6

Deletion of C/EBPa restores T cell development from Hes1-deficient progenitor cells in vivo. (a) Flow cytometry of cells from irradiated CD45.1⁺ mice 10–12 weeks after intravenous injection of a mixture of FL from CD45.2⁺ Hes1^−/−Cebpa^fl/flVav1-Cre, Hes1^−/−Cebpa^fl/+Vav1-Cre or Hes1^+/−Cebpa^fl/flVav1-Cre embryos (at E15.5) and BM from CD45.1⁺ mice. Numbers adjacent to outlined areas indicate percent CD45.2⁺ (FL donor) cells. (b) Quantitative PCR analysis of Hes1 mRNA and Tcf7 mRNA (which encodes TCF-1) in CD45.2⁺ FL-derived DN3 thymocytes sorted from mice reconstituted with Hes1^−/−Cebpa^fl/flVav1-Cre FL, or Hes1^+/+ DN3 cells (positive control), to verify absence of Hes1 mRNA. (c) Quantification of CD45.2⁺ donor–derived thymocytes in chimeras generated as in a (n = 3 with Hes1^−/−Cebpa^fl/flVav1-Cre or Hes1^+/−Cebpa^fl/flVav1-Cre FL and 1 with Hes1^−/− Cebpa^fl/+Vav1-Cre FL). (d) Frequency of CD45.2⁺ donor chimerism in various populations (horizontal axis) of chimeras generated as in a (n = 6 with Hes1^−/−Cebpa^fl/flVav1-Cre FL, 5 with Hes1^+/− Cebpa^fl/+Vav1-Cre FL and 2 with Hes1^−/−Cebpa^fl/+Vav1-Cre FL); BM chimerism was inferred from CD19⁺ splenic B cell chimerism; mice reconstituted with Hes1^+/− Cebpa^fl/+Vav1-Cre or Hes1^+/−Cebpa^fl/flVav1-Cre cells had equivalent T cell development, so those groups were pooled. (e) Flow cytometry of FL multipotent progenitor cells (Lin⁻c-Kit⁺Flt3⁺IL-7Ra⁻) or lymphoid progenitor cells (Lin⁻c-Kit⁺Flt3⁺IL-7Ra⁺) obtained from Hes1^−/−Cebpa^fl/flVav1-Cre or Hes1^+/− Cebpa^fl/flVav1-Cre embryos (E13.5) and cultured for 6 d on OP9-DL1 cells. Numbers adjacent to outlined areas indicate percent Mac-1⁻CD25⁺ cells. Right, quantification of Thy-1⁺CD25⁺ cells among those at left. Data are from one experiment representative of two experiments with similar results (a,b,c,e; average and s.e.m.) or represent two independent experiments (d) (d; average and s.e.m.).