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

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.