Follistatin regulates enamel patterning in mouse incisors by asymmetrically inhibiting BMP signaling and ameloblast differentiation

Rodent incisors are covered by enamel only on their labial side. This asymmetric distribution of enamel is instrumental to making the cutting edge sharp. Enamel matrix is secreted by ameloblasts derived from dental epithelium. Here we show that overexpression of follistatin in the dental epithelium inhibits ameloblast differentiation in transgenic mouse incisors, whereas in follistatin knockout mice, ameloblasts differentiate ectopically on the lingual enamel-free surface. Consistent with this, in wild-type mice, follistatin was continuously expressed in the lingual dental epithelium but downregulated in the labial epithelium. Experiments on cultured tooth explants indicated that follistatin inhibits the ameloblast-inducing activity of BMP4 from the underlying mesenchymal odontoblasts and that follistatin expression is induced by activin from the surrounding dental follicle. Hence, ameloblast differentiation is regulated by antagonistic actions of BMP4 and activin A from two mesenchymal cell layers flanking the dental epithelium, and asymmetrically expressed follistatin regulates the labial-lingual patterning of enamel formation.     

Scoring mitotic activity in longitudinal sections of mouse embryonic incisors: significant differences exist for labial and lingual inner dental epithelia.

The incisors of rodents (e.g., mice and rats) have an enamel-free lingual surface. It is not known how position specific differential evolution of labial versus lingual inner dental epithelium (IDE) is regulated. The patterns of mitotic activity of lingual and labial preodontoblasts and IDE were determined on sagittal sections of embryonic mouse incisors. No statistical differences were observed for the preodontoblasts. On the other hand the lingual IDE withdraw from the cell cycle before the corresponding labial cells. The incapacity of lingual IDE to differentiate into functional ameloblasts might be a consequence of a failure of cell kinetic dependent acquisition of competence to respond to specific epigenetic control mechanisms.     

Regional ultrastructural and cytochemical comparisons of the epithelial-mesenchymal interface during rat incisor development

A major theme in understanding epithelial-mesenchymal interactions during development focuses upon regional mesenchyme specification of epithelial differentiation. One particularly useful epidermal organ system for studying this issue is the rodent continuously growing and erupting incisor tooth organ. One advantage of this particular system resides in the regional features of the rodent incisor tooth organ. Along the labial surface, inner dental epithelial cells differentiate into ameloblasts that produce enamel extracellular matrix, whereas the epithelia along the lingual surface do not become ameloblasts and do not produce enamel matrix. This study has been designed to compare ultrastructural features of labial versus lingual surfaces, with particular emphasis upon mesenchymal cell shape, the orientation of extracellular matrix collagen, the basal lamina, and the distribution of sulfated glycoconjugates. Critical analyses of the data indicated that different microenvironments exist between epithelia and mesenchyme in the labial versus the lingual surfaces of the developing rodent incisor tooth organ.     

An integrated gene regulatory network controls stem cell proliferation in teeth

Epithelial stem cells reside in specific niches that regulate their self-renewal and differentiation, and are responsible for the continuous regeneration of tissues such as hair, skin, and gut. Although the regenerative potential of mammalian teeth is limited, mouse incisors grow continuously throughout life and contain stem cells at their proximal ends in the cervical loops. In the labial cervical loop, the epithelial stem cells proliferate and migrate along the labial surface, differentiating into enamel-forming ameloblasts. In contrast, the lingual cervical loop contains fewer proliferating stem cells, and the lingual incisor surface lacks ameloblasts and enamel. Here we have used a combination of mouse mutant analyses, organ culture experiments, and expression studies to identify the key signaling molecules that regulate stem cell proliferation in the rodent incisor stem cell niche, and to elucidate their role in the generation of the intrinsic asymmetry of the incisors. We show that epithelial stem cell proliferation in the cervical loops is controlled by an integrated gene regulatory network consisting of Activin, bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Follistatin within the incisor stem cell niche. Mesenchymal FGF3 stimulates epithelial stem cell proliferation, and BMP4 represses Fgf3 expression. In turn, Activin, which is strongly expressed in labial mesenchyme, inhibits the repressive effect of BMP4 and restricts Fgf3 expression to labial dental mesenchyme, resulting in increased stem cell proliferation and a large, labial stem cell niche. Follistatin limits the number of lingual stem cells, further contributing to the characteristic asymmetry of mouse incisors, and on the basis of our findings, we suggest a model in which Follistatin antagonizes the activity of Activin. These results show how the spatially restricted and balanced effects of specific components of a signaling network can regulate stem cell proliferation in the niche and account for asymmetric organogenesis. Subtle variations in this or related regulatory networks may explain the different regenerative capacities of various organs and animal species.     

Tooth development in a scincid lizard, Chalcides viridanus (Squamata), with particular attention to enamel formation

Comparative analysis of tooth development in the main vertebrate lineages is needed to determine the various evolutionary routes leading to current dentition in living vertebrates. We have used light, scanning and transmission electron microscopy to study tooth morphology and the main stages of tooth development in the scincid lizard, Chalcides viridanus, viz., from late embryos to 6-year-old specimens of a laboratory-bred colony, and from early initiation stages to complete differentiation and attachment, including resorption and enamel formation. In C. viridanus, all teeth of a jaw have a similar morphology but tooth shape, size and orientation change during ontogeny, with a constant number of tooth positions. Tooth morphology changes from a simple smooth cone in the late embryo to the typical adult aspect of two cusps and several ridges via successive tooth replacement at every position. First-generation teeth are initiated by interaction between the oral epithelium and subjacent mesenchyme. The dental lamina of these teeth directly branches from the basal layer of the oral epithelium. On replacement-tooth initiation, the dental lamina spreads from the enamel organ of the previous tooth. The epithelial cell population, at the dental lamina extremity and near the bone support surface, proliferates and differentiates into the enamel organ, the inner (IDE) and outer dental epithelium being separated by stellate reticulum. IDE differentiates into ameloblasts, which produce enamel matrix components. In the region facing differentiating IDE, mesenchymal cells differentiate into dental papilla and give rise to odontoblasts, which first deposit a layer of predentin matrix. The first elements of the enamel matrix are then synthesised by ameloblasts. Matrix mineralisation starts in the upper region of the tooth (dentin then enamel). Enamel maturation begins once the enamel matrix layer is complete. Concomitantly, dental matrices are deposited towards the base of the dentin cone. Maturation of the enamel matrix progresses from top to base; dentin mineralisation proceeds centripetally from the dentin–enamel junction towards the pulp cavity. Tooth attachment is pleurodont and tooth replacement occurs from the lingual side from which the dentin cone of the functional teeth is resorbed. Resorption starts from a deeper region in adults than in juveniles. Our results lead us to conclude that tooth morphogenesis and differentiation in this lizard are similar to those described for mammalian teeth. However, Tomes processes and enamel prisms are absent.     

Constitutive activation of β-catenin in ameloblasts leads to incisor enamel hypomineralization

Enamel is the hardest tissue with the highest degree of mineralization protecting the dental pulp from injury in vertebrates. The ameloblasts differentiated from ectoderm-derived epithelial cells are a single cell layer and are important for the enamel formation and mineralization. Wnt/β-catenin signaling has been proven to exert an important role in the mineralization of bone, dentin and cementum. Little was known about the regulatory mechanism of Wnt/β-catenin signaling pathway in ameloblasts during amelogenesis, especially in the mineralization of enamel. To investigate the role of β-catenin in ameloblasts, we established Amelx-Cre; β-catenin^?ex3fl/fl (CA-β-catenin) mice, which could constitutive activate β-catenin in ameloblasts. It showed the delayed mineralization and eventual hypomineralization in the incisor enamel of CA-β-catenin mice. Meanwhile, the amelogenesis-related proteinases Mmp20 and Klk4 were decreased in the incisors of CA-β-catenin mice. These data indicated that β-catenin plays an essential role in differentiation and function of ameloblasts during amelogenesis.     

Signaling by FGFR2b controls the regenerative capacity of adult mouse incisors

Rodent incisors regenerate throughout the lifetime of the animal owing to the presence of epithelial and mesenchymal stem cells in the proximal region of the tooth. Enamel, the hardest component of the tooth, is continuously deposited by stem cell-derived ameloblasts exclusively on the labial, or outer, surface of the tooth. The epithelial stem cells that are the ameloblast progenitors reside in structures called cervical loops at the base of the incisors. Previous studies have suggested that FGF10, acting mainly through fibroblast growth factor receptor 2b (FGFR2b), is crucial for development of the epithelial stem cell population in mouse incisors. To explore the role of FGFR2b signaling during development and adult life, we used an rtTA transactivator/tetracycline promoter approach that allows inducible and reversible attenuation of FGFR2b signaling. Downregulation of FGFR2b signaling during embryonic stages led to abnormal development of the labial cervical loop and of the inner enamel epithelial layer. In addition, postnatal attenuation of signaling resulted in impaired incisor growth, characterized by failure of enamel formation and degradation of the incisors. At a cellular level, these changes were accompanied by decreased proliferation of the transit-amplifying cells that are progenitors of the ameloblasts. Upon release of the signaling blockade, the incisors resumed growth and reformed an enamel layer, demonstrating that survival of the stem cells was not compromised by transient postnatal attenuation of FGFR2b signaling. Taken together, our results demonstrate that FGFR2b signaling regulates both the establishment of the incisor stem cell niches in the embryo and the regenerative capacity of incisors in the adult.     

Amelogenesis: Transformation of a protein-mineral matrix into tooth enamel

During enamel formation, the organic enamel protein matrix interacts with calcium phosphate minerals to form elongated, parallel, and bundled enamel apatite crystals of extraordinary hardness and biomechanical resilience. The enamel protein matrix consists of unique enamel proteins such as amelogenin, ameloblastin, and enamelin, which are secreted by highly specialized cells called ameloblasts. The ameloblasts also facilitate calcium and phosphate ion transport toward the enamel layer. Within ameloblasts, enamel proteins are transported as a polygonal matrix with 5 nm subunits in secretory vesicles. Upon expulsion from the ameloblasts, the enamel protein matrix is re-organized into 20 nm subunit compartments. Enamel matrix subunit compartment assembly and expansion coincide with C-terminal cleavage by the MMP20 enamel protease and N-terminal amelogenin self-assembly. Upon enamel crystal precipitation, the enamel protein phase is reconfigured to surround the elongating enamel crystals and facilitate their elongation in C-axis direction. At this stage of development, and upon further amelogenin cleavage, central and polyproline-rich fragments of the amelogenin molecule associate with the growing mineral crystals through a process termed “shedding”, while hexagonal apatite crystals fuse in longitudinal direction. Enamel protein sheath-coated enamel “dahlite” crystals continue to elongate until a dense bundle of parallel apatite crystals is formed, while the enamel matrix is continuously degraded by proteolytic enzymes. Together, these insights portrait enamel mineral nucleation and growth as a complex and dynamic set of interactions between enamel proteins and mineral ions that facilitate regularly seeded apatite growth and parallel enamel crystal elongation.     

New insight into dental epithelial stem cells: Identification,regulation, and function in tooth homeostasis and repair

Tooth enamel, a highly mineralized tissue covering the outermost area of teeth, is always damaged by dental caries or trauma. Tooth enamel rarely repairs or renews itself, due to the loss of ameloblasts and dental epithelial stem cells (DESCs) once the tooth erupts. Unlike human teeth, mouse incisors grow continuously due to the presence of DESCs that generate enamel-producing ameloblasts and other supporting dental epithelial lineages. The ready accessibility of mouse DESCs and wide availability of related transgenic mouse lines make mouse incisors an excellent model to examine the identity and heterogeneity of dental epithelial stem/progenitor cells; explore the regulatory mechanisms underlying enamel formation; and help answer the open question regarding the therapeutic development of enamel engineering. In the present review, we update the current understanding about the identification of DESCs in mouse incisors and summarize the regulatory mechanisms of enamel formation driven by DESCs. The roles of DESCs during homeostasis and repair are also discussed, which should improve our knowledge regarding enamel tissue engineering.     

Physiological implications of DLX homeoproteins in enamel formation

Tooth development is a complex process including successive stages of initiation, morphogenesis, and histogenesis. The role of the Dlx family of homeobox genes during the early stages of tooth development has been widely analyzed, while little data has been reported on their role in dental histogenesis. The expression pattern of Dlx2 has been described in the mouse incisor; an inverse linear relationship exists between the level of Dlx2 expression and enamel thickness, suggesting a role for Dlx2 in regulation of ameloblast differentiation and activity. In vitro data have revealed that DLX homeoproteins are able to regulate the expression of matrix proteins such as osteocalcin. The aim of the present study was to analyze the expression and function of Dlx genes during amelogenesis. Analysis of Dlx2/LacZ transgenic reporter mice, Dlx2 and Dlx1/Dlx2 null mutant mice, identified spatial variations in Dlx2 expression within molar tooth germs and suggests a role for Dlx2 in the organization of preameloblastic cells as a palisade in the labial region of molars. Later, during the secretory and maturation stages of amelogenesis, the expression pattern in molars was found to be similar to that described in incisors. The expression patterns of the other Dlx genes were examined in incisors and compared to Dlx2. Within the ameloblasts Dlx3 and Dlx6 are expressed constantly throughout presecretory, secretory, and maturation stages; during the secretory phase when Dlx2 is transitorily switched off, Dlx1 expression is upregulated. These data suggest a role for DLX homeoproteins in the morphological control of enamel. Sequence analysis of the amelogenin gene promoter revealed five potential responsive elements for DLX proteins that are shown to be functional for DLX2. Regulation of amelogenin in ameloblasts may be one method by which DLX homeoproteins may control enamel formation. To conclude, this study establishes supplementary functions of Dlx family members during tooth development: the participation in establishment of dental epithelial functional organization and the control of enamel morphogenesis via regulation of amelogenin expression.     

Ameloblast differentiation in the human developing tooth: Effects of extracellular matrices

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.     

A mouse model expressing a truncated form of ameloblastin exhibits dental and junctional epithelium defects

Ameloblastin (AMBN) is the second most abundant extracellular matrix protein produced by the epithelial cells called ameloblasts and is found mainly in forming dental enamel. Inactivation of its expression by gene knockout results in absence of the enamel layer and its replacement by a thin layer of dysplastic mineralized matrix. The objective of this study was to further characterize the enamel organ and mineralized matrix produced in the AMBN knockout mouse. However, in the course of our study, we unexpectedly found that this mouse is in fact a mutant that does not express the full-length protein but that produces a truncated form of AMBN. Mandibles from wild type and mutant mice were processed for morphological analyses and immunolabeling. Microdissected enamel organs and associated matrix were also prepared for molecular and biochemical analyses. In incisors from mutants, ameloblasts lost their polarized organization and the enamel organ detached from the tooth surface and became disorganized. A thin layer of dysplastic mineralized material was deposited onto dentin, and mineralized masses were present within the enamel organ. These mineralized materials generated lower backscattered electron contrast than normal enamel, and immunocytochemistry with colloidal gold revealed the presence of amelogenin, bone sialoprotein and osteopontin. In addition, the height of the alveolar bone was reduced, and the junctional epithelium lost its integrity. Immunochemical and RT–PCR results revealed that the altered enamel organ in the mutant mice produced a shorter AMBN protein that is translated from truncated RNA missing exons 5 and 6. These results indicate that absence of full-length protein and/or expression of an incomplete protein have direct/indirect effects beyond structuring of mineral during enamel formation, and highlight potential functional regions on the AMBN molecule.     

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest^1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal''s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.     

Unicuspid and bicuspid tooth crown formation in squamates

The molecular and developmental factors that regulate tooth morphogenesis in nonmammalian species, such as snakes and lizards, have received relatively little attention compared to mammals. Here we describe the development of unicuspid and bicuspid teeth in squamate species. The simple, cone-shaped tooth crown of the bearded dragon and ball python is established at cap stage and fixed in shape by the differentiation of cells and the secretion of dental matrices. Enamel production, as demonstrated by amelogenin expression, occurs relatively earlier in squamate teeth than in mouse molars. We suggest that the early differentiation in squamate unicuspid teeth at cap stage correlates with a more rudimentary tooth crown shape. The leopard gecko can form a bicuspid tooth crown despite the early onset of differentiation. Cusp formation in the gecko does not occur by the folding of the inner enamel epithelium, as in the mouse molar, but by the differential secretion of enamel. Ameloblasts forming the enamel epithelial bulge, a central swelling of cells in the inner enamel epithelium, secrete amelogenin at cap stage, but cease to do so by bell stage. Meanwhile, other ameloblasts in the inner enamel epithelium continue to secrete enamel, forming cusp tips on either side of the bulge. Bulge cells specifically express the gene Bmp2, which we suggest serves as a pro-differentiation signal for cells of the gecko enamel organ. In this regard, the enamel epithelial bulge of the gecko may be more functionally analogous to the secondary enamel knot of mammals than the primary enamel knot.