Ultradian Oscillations in Notch Signaling Regulate Dynamic Biological Events

Abstract

Notch signaling regulates many dynamic processes; accordingly, expression of genes in this pathway is also dynamic. In mouse embryos, one dynamic process regulated by Notch is somite segmentation, which occurs with a 2-h periodicity. This periodic event is regulated by a biological clock called the segmentation clock, which involves cyclic expression of the Notch effector gene Hes7. Loss of Hes7 expression and sustained expression of Hes7 result in identical and severe somite defects, suggesting that Hes7 oscillation is required for proper somite segmentation. Mathematical models of this oscillator have been used to generate and test hypothesis, helping to uncover the role of negative feedback in regulating the oscillator. Oscillations of another Notch effector gene, Hes1, plays an important role in maintenance of neural stem cells. Hes1 expression oscillates with a period of about 2–3 h in neural stem cells, whereas sustained Hes1 expression inhibits proliferation and differentiation of these cells, suggesting that Hes1 oscillations are important for their proper activities. Hes1 inhibits its own expression as well as the expression of the proneural gene Neurogenin2 and the Notch ligand Delta1, driving oscillations of these two genes. Delta1 oscillations in turn maintain neural stem cells by mutual activation of Notch signaling, which re-activates Hes1 to close the cycle. Hes1 expression also oscillates in embryonic stem (ES) cells. Cells expressing low and high levels of Hes1 tend to differentiate into neural and mesodermal cells, respectively. Furthermore, Hes1-null ES cells display early and uniform neural differentiation, indicating that Hes1 oscillations act to promote multipotency by generating heterogeneity in both the differentiation timing and the fate choice. Taken together, these results suggest that Notch signaling can drive short-period oscillatory expression of Hes7 and Hes1 (ultradian oscillation) and that ultradian oscillations are important for many biological events.

Introduction

Upon activation of Notch signaling, the Notch intracellular domain (NICD) is released from the membrane region and is transferred to the nucleus, where NICD forms a complex with the DNA-binding protein RBPj (Fig. 10.1A) (Honjo, 1996, Kopan and Ilagan, 2009). The NICD–RBPj complex recruits additional transcriptional activators and induces downstream genes such as Hes and Hey genes, forming the Notch–RBPj–Hes axis, often called the canonical pathway (Fig. 10.1A). Hes genes are mammalian homologues of Drosophila hairy and Enhancer of split and encode basic helix-loop-helix (bHLH)-type transcriptional repressors (Fig. 10.1B) (Sasai et al., 1992, Kageyama et al., 2007). There are seven members in the Hes family (Hes1 to Hes7), although Hes4 is not present in the mouse genome. Through their bHLH domain, Hes factors form homodimers or heterodimers with Hes-related bHLH factors such as Hey1 and bind to the DNA sequences called the N box (CACNAG) or the class C site (CACG(C/A)G) (Iso et al., 2003, Kageyama et al., 2007). In addition, Hes factors contain an orange domain located just C-terminal to the bHLH domain followed by the WRPW sequence at the carboxyl terminus, and both the orange domain and the WRPW sequence recruit several co-repressors. Thus, Hes proteins act as transcriptional repressors by binding to the target sequences. The orange domain also regulates the selection of bHLH heterodimer partners (Dawson et al., 1995, Taelman et al., 2004), while the WRPW sequence acts as a polyubiquitination signal, controlling the half-life of Hes protein by promotion of proteasome-mediated degradation (Kang et al., 2005).

It is well established that the Notch–RBPj–Hes pathway regulates many biological events by repressing target gene expression (Honjo, 1996, Kageyama et al., 2007. Major Hes target genes include proneural genes such as Mash1 and Neurogenin2 (Ngn2), themselves bHLH proteins acting as transcriptional activators (Fig. 10.1A) (Bertrand et al., 2002, Ross et al., 2003). Proneural genes promote differentiation of neural stem cells into neurons, while Hes genes repress proneural gene expression and maintain neural stem cells (Kageyama et al., 2007). Other major Hes target genes are Hes genes themselves (Fig. 10.1A) (Takebayashi et al., 1994, Bessho et al., 2003). For example, Hes1 and Hes7 can repress their own expression by directly binding to their own promoters, thereby forming negative feedback loops. This negative feedback is sufficient to produce oscillating gene expression (Hirata et al., 2002, Bessho et al., 2003). In this chapter, we describe the mechanism and significance of oscillating Hes expression in somite formation, neural development, and embryonic stem (ES) cell differentiation and discuss that not only the expression level but also the expression mode (oscillating versus sustained) is very important for many biological events.