Hes1 regulates embryonic stem cell differentiation by suppressing Notch signaling

Abstract

Embryonic stem (ES) cells display heterogeneous responses upon induction of differentiation. Recent analysis has shown that Hes1 expression oscillates with a period of about 3-5 h in mouse ES cells and that this oscillating expression contributes to the heterogeneous responses: Hes1-high ES cells are prone to the mesodermal fate, while Hes1-low ES cells are prone to the neural fate. These outcomes of Hes1-high and Hes1-low ES cells are very similar to those of inactivation and activation of Notch signaling, respectively. These results suggest that Hes1 and Notch signaling lead to opposite outcomes in ES cell differentiation, although they work in the same direction in most other cell types. Here, we found that Hes1 acts as an inhibitor but not as an effector of Notch signaling in ES cell differentiation. Our results indicate that sustained Hes1 expression delays the differentiation of ES cells and promotes the preference for the mesodermal rather than the neural fate by suppression of Notch signaling.

Figure 1

Hes1 protein expression in Hes1-sustained embryonic stem (ES) cells. (A) The structure for sustained Hes1 expression. The Hes1 cDNA with the IRES-EGFP sequence was knocked-in into the Rosa26 locus, so that Hes1 and EGFP were constitutively expressed from the Rosa26 promoter (Kobayashi et al. 2009). (B) Hes1 and Oct3/4 expression in the wild-type (WT) and Hes1-sustained (R5, R6) ES cell lines. Actin is a loading control. (C) Immunostaining of Hes1 (red) with DAPI staining (blue) in ES cell lines.

Figure 2

Kinetics of marker gene expression after the removal of LIF and feeder cells. After the removal of feeder cells, embryonic stem (ES) cells were cultured on gelatin-coated plate with LIF-removed ES cell medium (day 0). mRNA levels of marker genes, Mash1, Brachyury and Gata4, in the control (WT; blue) and Hes1-sustained ES cells (R5 and R6; red) were analyzed by quantitative real-time PCR. Each value was given in the ratio to the wild-type cells (WT) on day 0.

Figure 4

Comparison of marker protein expression under a neural differentiation condition. The control (WT, upper panels) and Hes1-sustained cells (R6, lower panels) cultured in N2B27 medium were analyzed on days 4 and 6 by immunocytochemistry using anti-Oct3/4, anti-Nestin and anti-Tuj-1 antibodies (red) with DAPI staining (blue). Scale bars, 100 μm.

Figure 3

Comparison of the cell morphology and the growth under a neural differentiation condition. Phase contrast view of the wild-type (WT, upper panel) and Hes1-sustained embryonic stem cells (R6, lower panels). Cells were cultured in the N2B27 medium for 2, 4 and 6 days. Scale bar; 100 μm.

Figure 5

Kinetics of marker gene expression under a neural differentiation condition. mRNA levels of marker genes in the control (WT; blue) and Hes1-sustained embryonic stem cells (R5 and R6; red) under a neural differentiation condition were analyzed by quantitative real-time PCR. Each value was given in the ratio to the wild-type cells (WT) on day 0. (A) mRNA levels of marker genes, Oct3/4, Fgf5, Brachyury and Gata4. (B) mRNA levels of neural marker genes, Mash1, Nestin and Tuj-1.

Figure 6

Hes1 knockdown promotes further mesodermal differentiation in Hes1-sustained cells. mRNA kinetics of marker genes, Oct3/4, Gata4 and Brachyury (A), neural marker genes, Mash1, Sox1 and Nestin (B), and mesodermal lineage marker genes, Nkx-2.5 and Flk-1 (C) were measured by quantitative real-time PCR. Arrows show the infection of lentivirus vectors carrying shRNA of scramble (blue) or Hes1 knockdown (Hes1-KD, red) into Hes1-sustained cells on day 6. Cells without virus infection were also analyzed (green).

Figure 7

Hes1 regulates expression of Notch signaling and cell cycle factors during differentiation. (A) mRNA levels of Notch signaling genes, Dll1, Jag1, Hes5 and Notch1. (B) Protein levels of Notch1 receptor, Notch intracellular domain (NICD), p57, Oct3/4 and Hes1 in the wild-type cells (WT; lanes 1, 4, 7 and 10) and in Hes1-sustained cells (R5; lanes 2, 5, 8 and 11, R6; lanes 3, 6, 9 and 12) under a neural differentiation condition.

Figure 8

Proposed model. In embryonic stem (ES) cells, Hes1 expression is controlled by LIF and BMP. Hes1 suppresses Notch signaling by repressing Dll1 and Jag1 expression and thereby inhibiting neural differentiation. Sustained Hes1 expression leads ES cells to the mesodermal differentiation but inhibits the following commitment probably at the mesodermal stem/progenitor state.