Over- and ectopic expression of Wnt3 causes progressive loss of ameloblasts in postnatal mouse incisor teeth

Intercellular signaling is essential for the development of teeth during embryogenesis and in maintenance of the continuously growing incisor teeth in postnatal rodents. WNT intercellular signaling molecules have been implicated in the regulation of tooth development, and the Wnt3 gene shows specific expression in the enamel knot at the cap stage. We demonstrate here that Wnt3 also is expressed in specific epithelial cell layers in postnatal incisor teeth. To begin to delineate the functions of Wnt3 in developing and postnatal teeth, we determined the effects of over- and ectopic expression of Wnt3 in the tooth epithelium of mice carrying a keratin 14-Wnt3 transgene. Expression of the transgene caused a progressive loss of ameloblasts from postnatal lower incisor teeth. Loss of ameloblasts may be due to defective proliferation or differentiation of ameloblast precursors, progressive apoptosis of ameloblasts, or loss of ameloblast stem cells.     

Distribution of Amelotin in Mouse Tooth Development

Amelotin is expressed and secreted by ameloblasts in tooth development, but amelotin distribution during enamel development is not clear. In this report, we first investigated amelotin expression in developing teeth by immunohistochemistry. Amelotin was detected in the enamel matrix at the secretion and maturation stages of enamel development. Amelotin was also observed at Tomes' processes on the apical ends of secretory ameloblasts. We then compared amelotin gene expression with those of amelogenin, enamelin, and ameloblastin in the mandibles of postnatal mice by RT‐PCR. The expression of amelotin was detected as early as in postnatal day 0 mandibles and amelotin was coexpressed with amelogenin, ameloblastin, and enamelin during tooth development. These data strongly suggest that amelotin is an enamel matrix protein expressed at the secretion and maturation stages of enamel development. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

Gonadotropin-releasing hormone-1 (GnRH-1) is involved in tooth maturation and biomineralization.

Gonadotropin releasing-hormone-1 (GnRH-1) is expressed in mouse incisors during development. In this report, we identify (1) cell type(s) that express GnRH-1 throughout tooth development, (2) the GnRH-1 receptor, and (3) the role of GnRH-1/GnRH-1 receptor signaling in tooth maturation. Results show that GnRH-1-positive cells in dental epithelium differentiate and populate multiple tooth structures including ameloblast and papillary layers that are involved in enamel formation and mineralization. The GnRH-1 receptor was present, and in vitro a GnRH-1 antagonist attenuated incisor GnRH-1 cell expression. In vivo, in mice lacking GnRH-1 (-/-), the incisors were discolored, longer, and more curved compared to wildtype. Elemental analysis of calcium, phosphorus, and iron revealed changes in -/- incisors consistent with GnRH-1 affecting movement of minerals into the dental matrix. In sum, in tooth development a signal transduction pathway exists for GnRH-1 via the GnRH-1 receptor and disruption of such signaling affects incisor growth and biomineralization.     

Cellular renewal in the enamel organ and the odontoblast layer of the rat incisor as followed by radioautography using 3H-thymidine

Renewal of the cell populations of the incisor was studied in 100 gm male rats injected with a single dose of ³H-thymidine and sacrificed at various times from one hour to 32 days after injection. Radioautographs showed that a cohort of labeled cells within the enamel organ, odontoblast layer, and pulp was carried passively with the erupting incisor from the apical end toward the gingival margin where the life cycle of these cells was terminated. Labeled cells in the upper and lower incisor, although traversing different absolute lengths, were found in approximately the same functional stage of their life cycle at similar times after the injection. Thus, by one and one-half days labeled ameloblasts began inner enamel secretion. By 32 days labeled ameloblasts had traversed the entire maturation zone and were located at the gingival margin. Labeled odontoblasts followed closely the movement of labeled ameloblast. The mean rate of ameloblast migration was 567 μm/day on the upper incisor and 651 μm/day on the lower. For the odontoblasts this rate was 500 μm/day (upper) and 631 μm/day (lower). Finally, it was found that as the rat aged, the duration of the life cycle for epithelial and pulp cell populations of the incisor increased because of growth within the longitudinal axis of the tooth. It was concluded that the apical end of the incisor literally “grows backward” in the bony socket, and hence, the duration of the life cycle becomes greater simply because it takes cells longer to physically reach the gingival margin.     

Postnatal epithelium and mesenchyme stem/progenitor cells in bioengineered amelogenesis and dentinogenesis

Rodent incisors provide a classic model for studying epithelial–mesenchymal interactions in development. However, postnatal stem/progenitor cells in rodent incisors have not been exploited for tooth regeneration. Here, we characterized postnatal rat incisor epithelium and mesenchyme stem/progenitor cells and found that they formed enamel- and dentin-like tissues in vivo. Epithelium and mesenchyme cells were harvested separately from the apical region of postnatal 4–5 day rat incisors. Epithelial and mesenchymal phenotypes were confirmed by immunocytochemistry, CFU assay and/or multi-lineage differentiation. CK14+, Sox2+ and Lgr5+ epithelium stem cells from the cervical loop enhanced amelogenin and ameloblastin expression upon BMP4 or FGF3 stimulation, signifying their differentiation towards ameloblast-like cells, whereas mesenchyme stem/progenitor cells upon BMP4, BMP7 and Wnt3a treatment robustly expressed Dspp, a hallmark of odontoblastic differentiation. We then control-released microencapsulated BMP4, BMP7 and Wnt3a in transplants of epithelium and mesenchyme stem/progenitor cells in the renal capsule of athymic mice in vivo. Enamel and dentin-like tissues were generated in two integrated layers with specific expression of amelogenin and ameloblastin in the newly formed, de novo enamel-like tissue, and DSP in dentin-like tissue. These findings suggest that postnatal epithelium and mesenchyme stem/progenitor cells can be primed towards bioengineered tooth regeneration.     

Distinct roles of MicroRNAs in epithelium and mesenchyme during tooth development

Background: Tooth development is known to be mediated by the cross‐talk between signaling pathways, including Shh, Fgf, Bmp, and Wnt. MicroRNAs (miRNAs) are 19‐ to 25‐nt noncoding small single‐stranded RNAs that negatively regulate gene expression by binding target mRNAs, which is believed to be important for the fine‐tuning signaling pathways in development. To investigate the role of miRNAs in tooth development, we examined mice with either mesenchymal (Wnt1Cre/Dicer^fl/fl) or epithelial (ShhCre/Dicer^fl/fl) conditional deletion of Dicer, which is essential for miRNA processing. Results: By using a CD1 genetic background for Wnt1Cre/Dicer^fl/fl, we were able to examine tooth development, because the mutants retained mandible and maxilla primordia. Wnt1Cre/Dicer^fl/fl mice showed an arrest or absence of teeth development, which varied in frequency between incisors and molars. Extra incisor tooth formation was found in ShhCre/Dicer^fl/fl mice, whereas molars showed no significant anomalies. Microarray and in situ hybridization analysis identified several miRNAs that showed differential expression between incisors and molars. Conclusion: In tooth development, miRNAs thus play different roles in epithelium and mesenchyme, and in incisors and molars. Developmental Dynamics 241:1465–1472, 2012. © 2012 Wiley Periodicals, Inc.     

Cellular renewal in the enamel organ and the odontoblast layer of the rat incisor as followed by radioautography using 3H-thymidine.

Renewal of the cell populations of the incisor was studied in 100 gm male rats injected with a single dose of 3H-thymidine and sacrificed at various times from one hour to 32 days after injection. Radioautographs showed that a cohort of labeled cells within the enamel organ, odontoblast layer, and pulp was carried passively with the erupting incisor from the apical end towards the gingival margin where the life cycle of these cells was terminated. Labeled cells in the upper and lower incisor, although traversing different absolute lengths, were found in approximately the same functional stage of their life cycle at similar times after the injection. Thus, by one and on-half days labeled ameloblasts began inner enamel secretion and, by eight days (upper) or nine days (lower), complement outer enamel secretion. By 32 days labeled ameloblasts had traversed the entire enamel maturation zone and were located at the gingival margin. Labeled odontoblasts followed closely the movement of labeled ameloblasts. The mean rate of ameloblast migration was 567 mum/day on the upper incisor and 651 mim/day on the lower. For the odontoblasts this rate was 55 mum/day (upper) and 631 mum/day (lower). Finally, it was found that as the rat age, the duration of the life cycle for epithelial and pulp cell populations of the incisor increased because of growth within the lonitudinal axis of the tooth. It was concluded that the apical end of the incisor literally "grows backward" in the bony socket, and hence, the duration of the life cycle becomes greater simply because it takes cells longer to physically reach the gingival margin.     

Wnt5a regulates growth,patterning, and odontoblast differentiation of developing mouse tooth

Wnt/β‐catenin signaling is essential for tooth development beyond the bud stage, but little is known about the role of non‐canonical Wnt signaling in odontogenesis. Here we compared the expression of Wnt5a, a representative of noncanonical Wnts, with that of Ror2, the Wnt5a receptor for non‐canonical signaling, in the developing tooth, and analyzed tooth phenotype in Wnt5a mutants. Wnt5a‐deficient mice exhibit retarded tooth development beginning from E16.5, leading to the formation of smaller and abnormally patterned teeth with a delayed odontoblast differentiation at birth. These defects are associated with upregulated Axin2 and Shh expression in the dental epithelium and reduced levels of cell proliferation in the dental epithelium and mesenchyme. Retarded tooth development and defective odontoblast differentiation were also observed in Ror2 mutant mice. Our results suggest that Wnt5a regulates growth, patterning, and odontoblast differentiation during odontogenesis, at least partially by modulating Wnt/β‐catenin canonical signaling. Developmental Dynamics 240:432–440, 2011. © 2011 Wiley‐Liss, Inc.     

Delayed tooth eruption in membrane type-1 matrix metalloproteinase deficient mice

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is expressed highly in mineralizing tissues including bones and teeth. Mice deficient in MT1-MMP (-/-) display severe defects in skeletal development including dwarfism, osteopenia, and craniofacial abnormalities. Death occurs in these mice by about 3 weeks of age. Since MT1-MMP is expressed by the ameloblasts of the enamel organ and by the odontoblasts of the dental papilla, we asked if the developing teeth were adversely affected in the knockout animals. Molars from MT1-MMP -/- mice and controls were examined by histological, X-ray, and SEM analysis at 4, 18-20, and 25 days of postnatal development. At 4 days of development the molars from the -/- mice appeared histologically normal. At 18-20 days of development, the first molars of the -/- mice had apparently normal tooth crowns with normal dentin and enamel; however, the roots were truncated and the teeth had not yet erupted. In contrast to the -/- mice, the first molars of the 18-20-day control animals had erupted. SEM analysis of a -/- first molar and incisor revealed a normal enamel prism pattern. However, X-ray analysis demonstrated that tooth eruption was delayed by approximately 5 days and that the tooth roots were abnormally short in the knockout animals. Since MT1-MMP-deficient mice have been demonstrated to display a generalized increase in bone resorption, these data suggest that inefficient growth of bone surrounding the tooth root complex causes a delay in tooth eruption.     

Quantitative analysis of cell turnover in the enamel organ of the rat incisor. Evidence for ameloblast death immediately after enamel matrix secretion

During renewal of the enamel organ in the rat incisor cohorts of epithelial cells are transported sequentially through presecretory, secretory and maturation zones to the gingival margin where the life cycles of these cells terminate. This process was examined kinetically by determining the absolute flux of cells within each of these zones of amelogenesis. It was found that the efflux of ameloblasts, stratum intermedium and papillary layer cells from the presecretory zone was about equal to the efflux plus expected growth within the secretory zone. However, between the secretory and maturation zones about 50% more ameloblasts entered the maturation zone than were required to account for the egress at the gingival margin and the expected growth. Since there was no similar imbalance between these zones for papillary layer cells, it was concluded that this discrepancy must represent a 50% reduction in the size of the ameloblast population during the maturation stage of amelogenesis. It was calculated that a little over 25% of the loss occurred immediately at the start of maturation within the region of postsecretory transition and the remaining 25% of the loss occurred throughout the subsequent regions of the maturation zone. In addition to the kinetic analysis graphic reconstructions, or surface maps, of ameloblast nuclei were prepared. These maps illustrated the characteristics of ameloblast nuclear packing within the three zones of amelogenesis and they provided quantitative confirmation that as ameloblasts progress through the maturation zone, there is a loss of cells in an amount predicted by the kinetic analysis.     

Tooth patterning and enamel formation can be manipulated by misexpression of TNF receptor Edar.

Signaling by Edar, a tumor necrosis factor receptor, is required for the development of ectodermal organs. Mutations in Edar or other molecules of the same signaling pathway cause ectodermal dysplasias in humans and mice. In these diseases, teeth are missing or malformed, and the development of hairs and several glands is hypoplastic. During tooth and hair development, Edar expression becomes patterned to ectodermal placodes and signaling centers. This localization has been suggested to be required for organogenesis. We have expressed Edar throughout the ectoderm using the keratin 14 promoter and show that this misexpression disrupts tooth patterning and differentiation. Tooth shape and cusp number are differentially affected, depending on the amount of transgene expression. In addition, tooth enamel formation is defective in a dose-dependent manner. We speculate that the tooth patterning defects are caused by ectopic Edar activity outside the signaling centers.     

Immunolocalization of Alk8 during replacement tooth development in zebrafish

The novel type I transforming growth factor-beta (TGF-beta) family member receptor Alk8 was previously identified in a degenerate RT-PCR screen for zebrafish type I and II TGF-beta family member receptors. Functional analyses revealed that Alk8 acts through Bmp signaling pathways in early embryonic dorsoventral patterning, in neural crest cell specification, and in patterning and differentiation of neural crest cell-derived pharyngeal arch cartilages. In addition, Alk8 forms active signaling complexes with TGF-beta1 and the TGF-beta RII receptor, suggesting that Alk8 mediates cross talk between Bmp and TGF-beta subfamily members. In this study, immunohistochemical analysis was performed on zebrafish aged 2 days postfertilization to 1 year, revealing immunolocalization of Alk8 to tissues of the tooth-bearing ceratobranchial 5 (cb5) arch including dental epithelial and mesenchymal tooth tissues of developing primary and replacement teeth, mucous-producing crypt epithelium, keratinized bite plate, and developing taste buds. These results suggest roles for Alk8 in patterning tooth-bearing pharyngeal epithelium, in the initiation of tooth development, in odontoblast and ameloblast differentiation, and in osteoblast maturation. The ability for zebrafish to continuously form teeth throughout their lives allows for the comparison of Alk8 expression in both primary and replacement tooth development, revealing identical Alk8 expression profiles. This study advances our current understanding of the functions of Alk8, particularly with respect to primary and replacement tooth formation, reveals additional roles for Alk8 in dental epithelial patterning and in odontoblast, ameloblast and osteoblast differentiation, and demonstrates the utility of the zebrafish as a model for primary and replacement tooth development.     

Essential roles of ameloblastin in maintaining ameloblast differentiation and enamel formation

During tooth development, dental epithelial cells interact with extracellular matrix components, such as the basement membrane and enamel matrix. Ameloblastin, an enamel matrix protein, plays a crucial role in maintaining the ameloblast differentiation state and is essential for enamel formation. Ameloblastin-null mice developed severe enamel hypoplasia. In mutant mice, dental epithelial cells started to differentiate into ameloblasts, but ameloblasts soon lost cell polarity, proliferated, and formed multiple cell layers, indicative of some aspects of preameloblast phenotypes. In addition, the expression of amelogenin, another component of the enamel matrix, was specifically reduced in mutant ameloblasts. More than 20% of amelobastin-null mice developed odontogenic tumors. We also found that recombinant ameloblastin specifically bound to ameloblasts and inhibited proliferation of dental epithelial cells. These results suggest that ameloblastin is an important regulator to maintain the differentiation state of ameloblasts.     

Differential expression of calbindin-D 28 kDa in rat incisor ameloblasts throughout enamel development

Calbindin-D 28 kDa (CaBP 28 kDa), a vitamin D-dependent calcium-binding protein, has been associated with calcium handling by cells. We have investigated the expression of this protein in the rat incisor enamel organ, an epithelium interposed between a mineralizing matrix and connective tissue rich in blood vessels, by radioimmunoassay (RIA), Western blotting, and quantitative protein A-gold immunocytochemistry with antibodies to rat kidney CaBP 28 kDa. RIA of cytosolic extracts showed that enamel organs contained relatively high concentrations of CaBP 28 kDa (compared to kidney; see review by Christakos S., C. Gabrielides, and W.B. Rhoten 1989 Endocr. Rev., 10:3-25). Immunoblotting of proteins extracted from enamel organ strips revealed an intensely-stained band near 28 kDa throughout amelogenesis following ameloblast differentiation. Immunocytochemically, CaBP 28 kDa was localized exclusively within ameloblasts. The density of labelling increased from the presecretory stage to the secretory stage and fluctuated across the maturation stage in relation to ameloblast modulation. Ruffle-ended ameloblasts consistently showed the most intense immunoreaction. Gold particles were present throughout the cytoplasm and nuclei of ameloblasts but regions rich in rough endoplasmic reticulum or cell webs showed a higher immunolabelling. Some gold particles were also associated with the external face of the rough endoplasmic reticulum. Multivesicular bodies in maturation stage ameloblasts were occasionally immunoreactive. These data suggest that the intracellular concentration of CaBP 28 kDa is regulated throughout amelogenesis reflecting a stage-specific control of calcium homeostasis in ameloblasts.     

Possible role of heat shock protein (Hsp) 25 in the enamel organ during amelogenesis in the rat molar.

The postnatal expression of heat shock protein (Hsp) 25 during the amelogenesis of rat molars was investigated by immunocytochemistry and confocal microscopy. The localization pattern of Hsp 25-immunoreactivity in the inner enamel epithelium and ameloblast cell layer of the rat molars was almost identical to that in the rat incisors which we have previously reported: an intense Hsp25-immunoreactivity, which first appeared in the preameloblasts, was recognized in secretory ameloblasts and ruffle-ended ameloblasts with stage-specific immunointensity. Confocal microscopy with Hsp 25-antibody and rhodamine-labeled phalloidin clearly demonstrated the co-localization of Hsp 25 and actin filaments in the ameloblast layer, supporting our hypothesis that this molecule might serve to reinforce the ameloblast layer during enamel formation as well as the formation and maintenance of the ruffled border in ruffle-ended ameloblasts. Interestingly, the enamel free area cells, which essentially lack the ability for enamel formation, showed the Hsp 25-immunoreactivity during 4-11 days when they developed a ruffled border, but decreased in that immunoreactivity after postnatal 15 days following apoptosis. Since Hsp 25 has been shown to be a specific inhibitor of apoptosis, the enamel-free area cells contribute to determine the outline of dentin at the cusped area. These data support our previous hypothesis on the diverse functions of Hsp 25 in amelogenesis.     

Cyclic localization of actin and its relationship to junctional complexes in maturation ameloblasts of the rat incisor

The patterns of fluorescence associated with maturation ameloblasts of mandibular incisors labeled with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin) for the detection of F-actin were investigated in normal and fluoride-treated rats. In normal rats, bands of smooth-ended ameloblasts (SA) exhibited intense fluorescence at their proximal ends only. Bands of ruffle-ended ameloblasts (RA) exhibited strong fluorescence at their distal ends as well as at their proximal ends. Regional differences in degree of intensity within the bands and between bands were displayed. In the apical part of the RA bands the proximal fluorescence was intense; it then decreased in an incisal direction; and it finally was absent close to the adjacent SA band. The incisal extension of strong proximal fluorescence in RA bands was short in early maturation and long in late maturation. The fluorescence pattern at both ends of the ameloblasts was cyclically repeated throughout the region of ameloblast modulation corresponding to the numbers of SA bands. In rats receiving 113 ppm fluoride in their drinking water for 2 months the number of fluorescence and ameloblast modulation cycles was reduced equally indicating that the cyclic F-actin localization is a phenomenon related to ameloblast modulation. Electron microscopy revealed that areas of strong fluorescence contained filament bundles, presumably actin filaments, in relation to continuous junctions occluding the interameloblast spaces. Areas of weak or no fluorescence were related to discontinuous macular junctions. The results suggest that the changes in F-actin distribution correlate well with junctional complex development, and therefore, possible functions related to the intermeloblast spaces within the RA bands may be redistributed as the ameloblasts are carried incisally by the erupting incisor.     

Activin is an essential early mesenchymal signal in tooth development that is required for patterning of the murine dentition

Development of the mammalian tooth has been intensively studied as a model system for epithelial/mesenchymal interactions during organogenesis, and progress has been made in identifying key molecules involved in this signaling. We show that activin βA is expressed in presumptive tooth-germ mesenchyme and is thus a candidate for a signaling molecule in tooth development. Analysis of tooth development in activin βA mutant embryos shows that incisor and mandibular molar teeth fail to develop beyond the bud stage. Activin βA is thus an essential component of tooth development. Development of maxillary molars, however, is unaffected in the mutants. Using tissue recombination experiments we show that activin is required in the mesenchyme prior to bud formation and that although activin signaling from mesenchyme to epithelium takes place, mutant epithelium retains its ability to support tooth development. Implantation of beads soaked in activin A, into developing mandibles, is able to completely rescue tooth development from E11.5, but not E12.5 or E13.5, confirming that activin is an early, essential mesenchyme signal required before tooth bud formation. Normal development of maxillary molars in the absence of activin shows a position specific role for this pathway in development of dentition. Functional redundancy with activin B or other TGFβ family members that bind to activin receptors cannot explain development of maxillary molars in the mutants since the activin-signaling pathway appears not to be active in these tooth germs. The early requirement for activin signaling in the mesenchyme in incisor and mandibular molar tooth germs must be carried-out in maxillary molar mesenchyme by other independent signaling pathways.