Root or crown: a developmental choice orchestrated by the differential regulation of the epithelial stem cell niche in the tooth of two rodent species

0 Institute of Biotechnology, Viikki Biocenter, FIN-00014 University of Helsinki , Finland regulation of the epithelial stem cell niche in the tooth of two rodent species - The rodent incisor grows continuously throughout its lifetime. The epithelial stem cell niche is located at the apical end of the tooth and its progeny gives rise to the ameloblasts that form the hard enamel. Previously, mesenchymal FGF10 was shown to support the niche, in conjunction with epithelial Notch signaling. Here we show that in a different continuously growing tooth type, the molar of the sibling vole, a similar regulatory system is in place. Moreover, the identical expression pattern of Bmp4 compared to Fgf10 suggests that BMP4 could also be involved in the regulation of the epithelial stem cell niche. Notch and FGF10 signaling is mainly absent in the mouse molar, which stops growing and develops roots. The regulation of the epithelial stem cell niche seems to be flexible allowing for the existence of different tooth types, such as continuously growing teeth, and high and low crowned molars. INTRODUCTION Adult stem cells require a special environment and are therefore usually found in the so-called stem cell niche (Spradling et al., 2001; Watt and Hogan, 2000; Nishimura et al., 2002). The niche can be defined as the environment, which allows the stem cells to function; i.e. it houses the stem cells, allows for self-renewal of the stem cells and is instructive in the generation of the differentiating progeny. Many types of stem cells have been shown to have a high degree of plasticity when transplanted, emphasizing the importance of the local environment in the regulation of differentiation (Bjornson et al., 1999; Anderson et al., 2001). The incisor of rodents represents a special tooth type since it grows continuously throughout the lifetime of the animal. Therefore, it must possess adult mesenchymal and epithelial stem cells. The exact location of the mesenchymal stem cells in teeth is not clear, although dental mesenchymal cells have been isolated from the pulp of adult human teeth (Gronthos et al., 2000). The cervical loop area located opposite of the distal tip of the tooth, and more specifically the epithelial tissue named stellate reticulum, has been put forward as the putative site of the epithelial stem cell compartment in the mouse incisor (Harada et al., 1999). It was also suggested that Notch signaling and FGF10 are involved in the regulation of this epithelial stem cell compartment (Harada et al., 2002). In the incisor the stem cells migrate from the stellate reticulum to the inner enamel epithelium and contribute to a pool of proliferating cells, also known as transit-amplifying cells. These cells then move towards the distal tip and show increasingly higher states of differentiation of the ameloblast cell lineage. The ameloblasts produce and deposit the enamel matrix responsible for the hardness of the tooth. The enamel is then constantly worn down at the distal tip of the incisor. In contrast, in mouse molars and all human teeth for instance, the stellate reticulum is lost after crown formation and a double layer of root sheath epithelium is left that directs root formation (Fig. 1). The rodent incisor is not the only tooth type that shows continuous growth. Also molars of certain species grow continuously. One such species is the rabbit. Tritium labeling studies suggested that also in the rabbit molar the cervical loop area is the origin of the epithelial cell lineage (Starkey, 1963). It was concluded that the pool of transit-amplifying cells in the inner enamel epithelium cannot be sustained by itself, but probably originates from the stratum intermedium, which is the denser layer of the stellate reticulum closest to the inner enamel epithelium. A cell that is recruited into the basal layer of proliferating cells must therefore change epithelial compartments and actually re-laminate itself into the inner enamel epithelium, since the opposite side of the inner enamel epithelium faces the dental mesenchyme and therefore also borders with a basal lamina (Ten Cate, 1961). Here we focus on a less well-known tooth system; the continuously growing molar of a vole species known as the sibling vole (Microtus rossiaemeridionalis). The vole and mouse are both rodents and are closely related species. Previously, the earlier stages of the vole and mouse molar were compared until the late bell stage of development (E17), and the basic aspects of morphogenesis and the distribution of Vole and Mouse molar development The vole molar Fig. 1. Coronal and sagittal sections of the vole molar show a distinct and complex morphology. The general appearance of the sagittal section is dependent on the position it is taken, unlike most of the coronal sections. Here we used the particular sagittal section as shown that runs through the middle of the molar. The developmental histories of the vole and mouse molar start similarly and are almost identical until cap stage. After this, the actions of the enamel knots result in a different folding pattern of the epithelium. This results in the intercuspal folds or loops in the vole molar that reach almost down to the base. In the mouse molar the cervical loop epithelium loses its crown fate, i.e. it loses the stellate reticulum, and switches to root. In the vole molar most cervical loop epithelium retains the crown fate except for three small areas that are converted to root fate. After completion of root formation the mouse molar has no functional cervical loop epithelium left (i.e. it is missing stellate reticulum) unlike the vole molar, where most of the cervical loop continues to generate crown. ERM, epithelial cell rests of Malassez; HERS, Hertwigs epithelial root sheath; bl, basal lamina; icl, intercuspal loop; iee, inner enamel epithelium; oee, outer enamel epithelium; si, stratum intermedium; sr, stellate reticulum; dashed circles indicate the cervical loop area. oee sr crown iee important developmental regulatory molecules were found to be almost identical (Kernen et al., 1998). Nature, therefore, provided for us the experimental setup. We have two tooth systems, the molar of the vole and the molar of the mouse, whose early development and morphogenesis are remarkably similar, but later venture on different developmental paths. The mouse molar develops roots and stops growing, the vole molar maintains the crown fate and will grow continuously. Therefore, a regulatory difference must be present and through comparison of the two phylogenetically closely related systems the developmental mechanism responsible for this divergence might be discovered. From the incisor it is known that FGF10 and Notch signaling is important for the maintenance of the stem cell niche and the continuous growth of the mouse incisor (Harada et al., 1999; Harada et al., 2002). We compared the expression patterns of these genes between mouse and vole molar. Simil (...truncated)