The expression of Sox17 identifies and regulates haemogenic endothelium

Abstract

Although it is well recognized that haematopoietic stem cells (HSCs) develop from a specialized population of endothelial cells known as haemogenic endothelium, the regulatory pathways that control this transition are not well defined. Here we identify Sox17 as a key regulator of haemogenic endothelial development. Analysis of Sox17-GFP reporter mice revealed that Sox17 is expressed in haemogenic endothelium and emerging HSCs and that it is required for HSC development. Using the mouse embryonic stem cell differentiation model, we show that Sox17 is also expressed in haemogenic endothelium generated in vitro and that it plays a pivotal role in the development and/or expansion of haemogenic endothelium through the Notch signalling pathway. Taken together, these findings position Sox17 as a key regulator of haemogenic endothelial and haematopoietic development.

Figure 1

Expression of Sox17 identifies the emergence of HE and hematopoietic progenitors in vitro. (a) Schematic representation of differentiation protocol for murine ESCs. (b) Left: Representative experiment depicting the expression of Flk-1 and Sox17-mCherry (Sox17-mC) in days (D) 3.25 and 5.25 EBs by flow cytometry. Right: Analysis of D5.25 EBs for co-expression of endothelial (CD31, AA4.1, CXCR4), HE (VEC, Sca-1), hematopoietic (CD41) markers with Sox17-mC (c) D5.25 EBs were dissociated and isolated for the following fractions F⁺S⁺, F⁺S⁻ and F⁻S⁻. The cells were cultured for 2 or 4 days as indicated in (a) and analyzed for the expression of Sox17-mC, VEC and CD45. Flow cytometry plots of D7 and D9 cells showing the 3 fractions isolated for hematopoietic analyses demonstrate that CD45⁺ only emerge from fractions that aquired expression of Sox17-mC. (d) Hematopoietic progenitor potential of D5.25 sorted fractions depicted in (c) cultured for 4 days. D9 cells were dissociated and plated in methylcellulose cultures and scored for definitive erythroid (D-Ery), macrophage/monocyte (Mac) or granulocyte/erythrocyte/macrophage/megakaryocyte (Mixed) colonies after 8 days in culture. Bars represent standard deviation of the mean of 3 independent experiments; P=0.003. (e) T lymphoid potential of the D5.25 sorted fractions cultures for 4 days. D9 cells were dissociated and plated on irradiated OP9-DL1 stromal cells in limiting dilution for 21 days. Positive wells were those that contained more than 500 cells of which more than 5% expressed the T cell signature CD45⁺TCRβ⁺CD4⁺/CD8⁺. Bars represent standard deviation of the mean of 3 independent experiments P=0.0001.

Figure 2

Sox17 expression marks HE in vivo and is required for the generation of long-term repopulating HSCs. (a) Representative flow cytometric profiles of AGM regions isolated from E11.5 Sox17^GFP/+ embryos stained with endothelial (Flk-1, CD31, Tie2) HE (VEC, CD34, Sca-1) and hematopoietic (CD41, CD45, CD11b) markers. (b) Whole-mount immunofluorescence of an intra-arterial hematopoietic cluster from the dorsal aorta of a 36-39 somite pair Sox17^GFP/+ embryo (20x) following staining with antibodies recognizing GFP (Sox17), cKIT and CD31. Scale bars = 50μm. (c) Representative flow cytometric profiles of AGM regions isolated from E11.5 Sox17^GFP/+ heterozygote and Sox17^GFP/GFP null embryos demonstrates the absence of the VEC⁺CD45⁺ population that contains HSCs. (d) Proportion of donor-derived hematopoietic cells detected in the peripheral blood of recipients 4 months following transplantation of the indicated populations. Three embryo equivalents were used for transplantation. Each dot represents an individual transplant recipient. Error bars represent SEM. (e) Progeny derived from mating VEC-Cre⁺Sox17^fl/+ males with Sox17^fl/fl females: conditional deletion of Sox17 using VEC-Cre was lethal by E13.5. (f) Compared to control embryos the VEC-Cre⁺Sox17^fl/fl embryos were growth retarded, pale and lacked visible hematopoiesis. (g) Representative flow cytometric profiles of AGM regions isolated from VEC-Cre⁺Sox17^fl/+and VEC-Cre⁺Sox17^fl/fl embryos depicting the total decrease in VEC and CD45 staining and the absence of the VEC⁺CD45⁺ population that contains HSCs.

Figure 3

Expression of Sox17 is required for the endothelial to hematopoietic transition and definitive hematopoiesis in mESC differentiation cultures. (a) Flow cytometric analyses showing the proportion of Flk-1⁺ cells in D3.23 and D5.25 EBs generated from Sox17-mC and Sox17^−/− ESCs. (b) Primitive erythroid (EryP) and myeloid (Myeloid) progenitor potential of D3.25 Flk-1⁺ cells that were aggregated for 24 hours. There is no significant change in primitive hematopoietic output with the loss of Sox17 expression. Bars represent standard deviation of the mean of 3 independent experiments. (c) Flow cytometric analyses showing the proportion of VEC⁺, CD45⁺, CD31⁺ and CXCR4⁺ cells in D7 from Sox17-mC and Sox17^−/− EBs. To generate D9 cultures the D7 VEC⁺CD45⁻ population was isolated by FACS and reaggregated as depicted in Fig 1a (d) qRT-PCR based analyses showing expression of indicate genes in D7 and D9 aggregates generated from SOX17-mC and Sox17^−/− EBs. Values shown are relative to Actβ. For comparison of the 2 populations, the values for the expression levels in the Sox17-mC cells are set to 1. Bars represent standard deviation of the mean of 3 independent experiments. D7: Sox7 P=0.01. D9: Sox7 P=0.003, Sox18 P=0.014, EphrinB2 P=0.0004, Notch1 P=0.01, Jagged1 P=0.003. (e) Hematopoietic progenitor potential of the Sox17-mC and Sox17^−/− derived D9 aggregates. Bars represent standard deviation of the mean of 3 independent experiments. For mixed colonies P=0.001. (f) T-lymphoid progenitors measured on OP9-DL1 stromal cells. Bars represent standard deviation of the mean of 3 independent experiments. P=0.0001.

Figure 4

Effects of enforced Sox17 expression on HE and hematopoietic development. (a) Flow cytometric analyses showing the proportion of CD31⁺CXCR4⁺ and VEC⁺ CD45⁺ cells in D5.25 Flk-1⁺ derived monolayers cultured for 2 days in the presence (+dox) or absence (−dox) of 1μg/ml doxycycline. Far right: Flow cytometric analysis showing the proportion of VEC⁺ and CD45⁺ cells in aggregates generated from the D7 induced and non-induced VEC⁺CD45⁻ population cultured for 2 days without doxycycline in the presence of hematopoietic cytokines. (b) Myeloid/erythroid and (top) and T-lymphoid (bottom) progenitor potential of the D7 induced and non-induced monolayers. Bars represent standard deviation of the mean of 3 independent experiments, for myeloid/erythroid P=0.003 and for T lymphoid P=0.0006 (c) Myeloid/erythroid and (top) and T-lymphoid (bottom) progenitor potential of aggregates generated from VEC⁺CD45⁻ cells isolated by FACS from the day 7 induced and non-induced monolayer populations. Cells were cultures as aggregates for 2 days (D9) in the absence of doxycycline prior to analyses. Bars represent standard deviation of the mean of 3 independent experiments, For T lymphoid progenitors P=0.0004 (d) qRT-PCR analyses of indicated genes in D7 induced and non-induced monolayer and in D9 aggregates generated from them, VEC⁺ cells isolated from the AGM of an E11.5 wildtype embryo is shown as a control. Values shown are relative to Actβ. Bars represent standard deviation of the mean of 3 independent experiments Aml1c P=0.012, Notch1 P=0.03, Jagged1 P=0.038, EphrinB2 P=0.006, COUP-TFII P=0.006.

Figure 5

The effects of enforced Sox17 expression are mediated through Notch signaling. (a) Flow cytometric analyses of D5.25 Flk-1⁺derived monolayer cultures for 2 days (D7) with (+dox) or without (−dox) 1μg/ml doxycycline in the presence or absence of γ-secretase inhibitor (γSI, L-685458; 10 μM, Tocris). Cells cultured in the vehicle DMSO represent the control. (b) qRT-PCR-based analysis of EphrinB2 and COUP-TFII expression in the different D7 populations described in (a). Values shown are relative to Actβ, Bars represent standard deviation of the mean of 3 independent experiments, COUP-TFII P=0.002, EphrinB2 P=0.003. (c) Flow cytometric analyses showing the proportion of VEC⁺ and CD45⁺ in aggregates generated from VEC⁺CD45⁻ cells isolated by FACS from the different D7 monolayer populations indicated in (a). The sorted cells were cultured for 2 days as aggregates in the absence of doxycycline and γ-secretase inhibitor. (d) T-lymphoid progenitor potential in the D9 aggregates generated from the D7 monolayer populations grown under the indicated conditions. Bars represent standard deviation of the mean of 3 independent experiments, P=0.0008 (e) Schematic diagram depicting the wildtype and mutated version of the BS1 and BS2 Sox17 binding sites in the Notch1 promoter. Numbers indicate the position relative to the transcriptional start site. (f) Luciferase assays using U2OS cells co-transfected with pGL3 control vector or the indicated Notch1 promoter constructs and increasing concentrations of the pCAG expression plasmids. Bars represent mean luciferase intensity relative to pGL3-empty ±s.d., n=3, P=0.001.