Eomesodermin induces Mesp1 expression and cardiac differentiation from embryonic stem cells in the absence of Activin

Abstract

The transcription factor Eomesodermin (Eomes) is involved in early embryonic patterning, but the range of cell fates that it controls as well as its mechanisms of action remain unclear. Here we show that transient expression of Eomes promotes cardiovascular fate during embryonic stem cell differentiation. Eomes also rapidly induces the expression of Mesp1, a key regulator of cardiovascular differentiation, and directly binds to regulatory sequences of Mesp1. Eomes effects are strikingly modulated by Activin signalling: high levels of Activin inhibit the promotion of cardiac mesoderm by Eomes, while they enhance Eomes-dependent endodermal specification. These results place Eomes upstream of the Mesp1-dependent programme of cardiogenesis, and at the intersection of mesodermal and endodermal specification, depending on the levels of Activin/Nodal signalling.

Figure 1

Induction of Eomesodermin (Eomes) during embryonic stem cell (ESC) differentiation promotes development of cardiovascular mesoderm. (A) Schematic representation of the differentiation protocol. (B–G) ESCs were cultured in defined default medium (DDM) in the presence or absence of doxycyclin (Dox) at days 2–3, and immunostained for β-tubulin III and a cardiac-specific isoform of Troponin T (cTnT; B,C), VE-cadherin (D,E) or Sox17 and smooth muscle actin (SMA; F,G). Nuclei are stained with Hoechst dye. Scale bar, 100 μm. (H–L) Quantification of the cells expressing β-tubulin III (H), cTnT (I), CD31 (J), SMA (K) or Sox17 (L). Quantification was performed by FACS (I,J), counting after cytospin (H,K) or counting of cells cultivated on the coverslip (L). Data are presented as mean percentage of all cells+s.e.m. (M,N) Quantitative reverse transcription–PCR analysis for Tubb3 (M) and Sox1 (N) at day 10 with or without Dox. Data are presented as mean expression normalized to TBP+s.e.m. (O) Flow cytometric analysis of Flk1–Pdgfrα double-positive cells at day 4 (48 h after addition of Dox). Data are presented as mean percentage of all cells+s.e.m. (P) Higher magnification shows cTnT-positive striated myofibrils. Scale bar, 10 μm.

Figure 2

High concentrations of Activin prevent Eomesodermin (Eomes)-stimulated cells from differentiating into cardiovascular mesoderm. Embryonic stem cells (ESCs) were cultured in defined default medium (DDM) for 10 days, with or without doxycyclin (Dox) at days 2–3 and increasing concentrations of Activin (0, 1, 10 and 100 ng/ml) from day 0 to day 4, or 2% serum from day 0 to day 10. (A,B) The percentage of cells positive for cardiac-specific isoform of Troponin T (cTnT; A) or CD31 (B) was determined by FACS. Data are presented as mean percentage of positive cells+s.e.m. There is a statistically significant interaction between effects of Activin and Eomes induction on the percentage of cTnT and CD31-expressing cells; P=0.013 and 0.002, respectively. (C) Immunostaining for cTnT and β-tubulin III at day 10 with or without Activin (100 ng/ml) and/or Dox. Scale bar, 100 μm. (D) Quantitative reverse transcription–PCR analysis for Tnnt2, CD31, Sox17 and Afp at day 10 with or without Dox and with increasing concentrations of Activin (0, 10 and 100 ng/ml). Data are presented as mean expression normalized to TBP+s.e.m.

Figure 3

Eomesodermin (Eomes) promotes Mesp1 expression in the absence of Activin. (A–C) Quantitative reverse transcription (qRT)–PCR for Mesp1 (A), Sox17 (B) and Gsc (C) at day 3 in defined default medium (DDM) with increasing concentrations of Activin (0, 10 and 100 ng/ml), 24 h after addition of doxycyclin (+Dox). (D) Immunofluorescence for Sox17 at day 3 of differentiation, with or without Dox or Activin (10 ng/ml). Scale bar, 100 μm. (E) Quantification of the percentage of Sox17-positive cells at day 3. (F) FACS quantification of Cxcr4–Epcam double-positive cells at day 4 with increasing concentrations of Activin (0, 10 and 100 ng/ml), 48 h after addition of Dox. Data from quantifications are presented as mean percentage+s.e.m. qRT–PCR data are presented as mean expression normalized to TBP+s.e.m. (A–C), or as mean fold change+s.e.m. of Dox-treated cells compared with non-treated cells at the same time (A′). For Mesp1 qRT–PCR and Cxcr4–Epcam FACS, there is a statistically significant interaction between effects of Activin and Eomes induction; P=0.042 and 0.016, respectively.

Figure 4

Eomesodermin (Eomes) directly binds to the Mesp1 promoter in vitro and in vivo. (A) Representation of the genomic region of Mesp1, showing the exons (red), putative Eomes-binding sites (yellow), and a control negative region (−) located 6.9 kb upstream of the transcription start site. (B,C) Quantification as measured by quantitative PCR of DNA fragment enrichment by chromatin immunoprecipitation (ChIP) on differentiating embryonic stem cells (ESCs) at day 3, with or without Activin 10 ng/ml from day 0 to 3 and/or doxycyclin (Dox) for 24 h (B) or on E7 embryos (C) using anti-Eomes antibody and primers encompassing the indicated regions of the Mesp1 promoter (T1, T2, T3 or (−)). Data are presented as percentage of the input. In vitro data are presented as mean+s.e.m. (B). (D,E) FACS quantification of the percentage of cTnT expression (D) and immunostaining for cTnT (E) at day 10 following induction at days 2–3 of either MycEomes alone or MycEomes combined with Flag–Mesp1–Engrailed in defined default medium without Activin. Scale bar, 100 μm.