Notch signalling is required for the survival of epithelial stem cells in the continuously growing mouse incisor

Abstract

The Notch pathway regulates the renewal and fate decisions of stem cells in multiple tissues. Notch1, -2, as well as the Notch target gene Hes1 are expressed in the putative stem cells in the continuously growing mouse incisors, but so far there has not been any evidence for a function of the Notch pathway in the regulation of the incisor stem cells. We have analysed the effects of the Notch pathway inhibitor DAPT on the maintenance, proliferation, and differentiation of the epithelial stem cells in explant cultures of the mouse incisor. The proximal part of the incisor containing the cervical loop stem cell niche was dissected from newborn mice and cultured for 2–6 days in vitro. DAPT inhibited the expression of Notch target gene Hes1 in the cervical loop indicating that Notch signalling was inhibited in the putative stem cells. The most striking effect of DAPT was a significant reduction in the size of the cervical loop. DAPT caused a marked but partially reversible decrease in cell proliferation, as well as massive apoptosis in the epithelial stem cell niche. Interestingly, restricted apoptosis was detected within the Notch expressing putative stem cells also in the control cultures as well as in incisors in vivo, suggesting that apoptosis may be a mechanism regulating the size of the epithelial stem cell pool in the incisor. The differentiation of the epithelial cells into enamel-forming ameloblasts was not affected by DAPT but the number of preameloblasts was progressively decreased during culture period reflecting the depletion of stem and progenitor cells. Our results indicate that Notch signalling is required for epithelial stem cell survival and enamel formation in the continuously growing mouse incisor.

Introduction

Continuously growing teeth such as rodent incisors, erupt throughout the lifetime of the animal and rely upon stem cells to maintain their regenerative capacity. The epithelial stem cell compartments have been located in the proximal parts of the teeth both in the continuously growing incisors of mice and in molars of the vole (Harada et al., 1999, Tummers and Thesleff, 2003). The stem cells reside within the cervical loop composed of loosely arranged epithelial cells, i.e. stellate reticulum, surrounded by a single layer of basal epithelial cells i.e. enamel epithelium. The exact location of the stem cells is not known, but it has been suggested that they reside in the stellate reticulum compartment from where they are incorporated into the enamel epithelium and migrate apically as they proliferate and differentiate into enamel secreting ameloblasts (Fig. 1), (Tummers and Thesleff, 2003).

Tooth development is regulated by epithelial–mesenchymal interactions which are mediated by signals belonging to several signalling families including fibroblast growth factor (FGF), bone morphogenetic protein (BMP), transforming growth factor β (TGFβ), sonic hedgehog (SHH), and Wnt (Harada et al., 1999, Tummers and Thesleff, 2003, Thesleff and Mikkola, 2002, Tummers and Thesleff, 2009). Epithelial–mesenchymal interactions also regulate the maintenance, proliferation, and differentiation of the epithelial stem cells in the mouse incisors, and a delicate balance of stimulatory and inhibitory signals have been implicated in these processes (Tummers and Thesleff, 2009). In particular, the FGF, Activin, and BMP signalling pathways have been implicated in the incisor stem cell regulation, and their antagonists for example Sprouty and Follistatin, have been shown to finetune the signalling. Notably these inhibitors play central roles in the generation of the labial–lingual asymmetry of the incisor (Mitsiadis et al., 1995, Mitsiadis et al., 2005, Klein et al., 2008, Wang et al., 2004, Wang et al., 2007).

Notch signalling regulates the maintenance, fate decision, and proliferation of the stem and progenitor cells in several tissues including bone marrow, intestinal epithelium, and various neuronal tissues (Sakata-Yanagimoto et al., 2008, Fan et al., 2006, Breunig et al., 2007, Fre et al., 2005). Active Notch signalling involves the interaction of Notch transmembrane glycoprotein receptors (Notch 1–4) with their ligands Delta1, -2, and -4 and Jagged1 and -2 (Borkosky et al., 2008, Zanotti and Canalis, 2010). The Notch ligand binding induces proteolysis of the receptor by a metalloproteinase and γ-secretase complex to release the Notch intracellular domain (NICD) (Tagami et al., 2008). In the nucleus, NICD forms a complex with RBP-Jκ (also called CBF1) and transcriptional coactivators of the Mastermind family promoting the transcription of downstream target genes such as the basic helix-loop-helix genes Hes1, Herp1, and Herp2, which are thought to form transcriptional repression complexes (Bray, 2006, Schwanbeck et al., 2008, Kopan and Ilagan, 2009, Carlén et al., 2009, Mitsiadis et al., 1998). The expression patterns of several Notch ligands, receptors, and key target genes have been reported during mouse tooth development (Harada et al., 1999, Mitsiadis et al., 1998, Mustonen et al., 2002). Notch1, -2, and -3 as well as Hes1 are expressed in the stellate reticulum cells, both in the molars and incisors, and BMP as well as FGF signalling were shown to regulate the Notch pathway in these cells (Harada et al., 1999, Mitsiadis et al., 1995, Mitsiadis et al., 1998, Mitsiadis et al., 2005, Mustonen et al., 2002). Using a mouse incisor cell line Harada and co-workers showed that the fate of stratum intermedium cells depends on Notch1/Jagged signalling (Harada et al., 2006).

We have examined the role of Notch signalling in the regulation of epithelial stem cells in the developing mouse incisor. We used an organ culture model system and cultured the proximal end of the continuously growing mouse lower incisor in the presence of DAPT, which is an inhibitor of the γ-secretase complex and frequently used as an inhibitor of the Notch pathway. The results indicate that Notch signalling promotes the survival of the epithelial stem cells and plays a key role in the continuous regeneration of the mouse incisor.