Expression of planar cell polarity genes during mouse tooth development.

OBJECTIVE: Planar cell polarity (PCP) refers to the cell polarity across the tissue plane and controls various cell behaviors and structures. Although the expression of several PCP signaling components has been detected in tooth germs, knowledge of the gene expression patterns of these PCP components during tooth development remains incomplete. The aim of this study is to characterize the temporal and spatial changes in PCP gene expression during tooth development. DESIGN: Expression of Celsr1 and 2, Fzd3 and 6, Vangl1 and 2, and Dvl1-3 genes was analyzed in mouse molar germs from the bud to the bell stage using in situ hybridization. RESULTS: At the bud stage, all target genes were expressed in all areas of the tooth bud. In the enamel organ at the cap stage, expression of Fzd3 was suppressed in the enamel knot, whereas Fzd6 was strongly expressed there. Expression of Vangl2 was strongly expressed in the inner dental epithelium from the cap stage onwards. In the inner dental epithelium, strong expression of Fzd3, Dvl2 and Vangl2 was noted at the early bell stage, and of Celsr1, Fzd3, Fzd6, Vangl2 and Dvl2 at the bell stage. Furthermore, differentiated odontoblasts strongly expressed Celsr1, Vangl2, and Dvl2. CONCLUSION: The gene expression patterns delineated in this study improve our understanding of the role(s) of PCP components during tooth development.

Effects of tooth storage media on periodontal ligament preservation.

BACKGROUND/AIMS: An easily available tooth storage medium is required to preserve a tooth after avulsion. Milk and Hank's balanced salt solution (HBSS) are recommended as tooth storage media, and egg white is also reported to be comparable with milk. The aim of this study was to histologically and immunohistochemically evaluate the effect of different tooth storage media on the periodontal ligament (PDL) of extracted teeth. MATERIALS AND METHODS: This experiment used HBSS, milk, and egg white as tooth storage media. A total of ninety-six 6-week-old male Sprague-Dawley rats were used in these experiments. In each experiment, six rats were used for each medium and for the control group. Extracted rat molar teeth were immersed in these three different storage media for 1 hour. In each medium, six samples (n=18) were fixed immediately, and the remaining samples (n=54) were subcutaneously transplanted. In the control group (n=24), the extracted teeth were fixed or transplanted immediately after extraction. At day 4, 1 and 2 weeks after transplantation, the teeth were examined by radiographic, histological, and immunohistochemical methods. The number of PDL cells in the storage media was also counted. RESULTS: Teeth immersed for 1 hour in milk showed the thinnest PDL. Immunohistochemistry of periostin and CD68 labeling suggested degradation of the extracellular matrix in the PDL. In the media used for immersion, more PDL cells were observed in milk than in the other solutions. After transplantation, the HBSS and egg white groups maintained adequate thickness of PDL but in the milk group, thinner PDL and ankylosis were observed. CONCLUSION: Adequate thickness of PDL was maintained in the egg white group, whereas the milk group showed disturbance in the PDL, which may lead to ankylosis.

CXCR4/CXCL12 signaling impacts enamel progenitor cell proliferation and motility in the dental stem cell niche.

Dental stem cells are located at the proximal ends of rodent incisors. These stem cells reside in the dental epithelial stem cell niche, termed the apical bud. We focused on identifying critical features of a chemotactic signal in the niche. Here, we report that CXCR4/CXCL12 signaling impacts enamel progenitor cell proliferation and motility in dental stem cell niche cells. We report cells in the apical bud express CXCR4 mRNA at high levels while expression is restricted in the basal epithelium (BE) and transit-amplifying (TA) cell regions. Furthermore, the CXCL12 ligand is present in mesenchymal cells adjacent to the apical bud. We then performed gain- and loss-of-function analyses to better elucidate the role of CXCR4 and CXCL12. CXCR4-deficient mice contain epithelial cell aggregates, while cell proliferation in mutant incisors was also significantly reduced. We demonstrate in vitro that dental epithelial cells migrate toward sources of CXCL12, whereas knocking down CXCR4 impaired motility and resulted in formation of dense cell colonies. These results suggest that CXCR4 expression may be critical for activation of enamel progenitor cell division and that CXCR4/CXCL12 signaling may control movement of epithelial progenitors from the dental stem cell niche.

Expression, localisation and synthesis of versican by the enamel organ of developing mouse molar tooth germ: an in vivo and in vitro study.

OBJECTIVE: Versican is a large, aggregating chondroitin sulphate proteoglycan. In dental tissue, versican expression occurs primarily in mesenchymal tissue but rarely in epithelial tissue. We investigated the expression, localisation and synthesis of versican in the enamel organ of the developing tooth germ. DESIGN: To elucidate versican localisation in vivo, in situ hybridisation and immunohistochemistry were conducted in foetal ICR mice at E11.5-E18.5. Epithelium and mesenchyme from the lower first molars at E16.0 were enzymatically separated and versican mRNA expression was investigated by semi-quantitative RT-PCR. Organ culture of the separated samples combined with metabolic labelling with [(35)S], followed by gel filtration, was performed to analyse secreted proteoglycans. RESULTS: Versican mRNA was first expressed in the thickened dental epithelium at E12.0 and continued to be expressed in the enamel organ until the bell stage. Versican immunostaining was detected in the stellate reticulum areas from the bud stage to the apposition stage. The enamel organ at E16.0 expressed versican mRNA at a level comparable to that in dental mesenchyme. Furthermore, when compared to dental mesenchyme, about 1/2-3/4 of the [(35)S]-labelled versican-like large proteoglycan was synthesised and released into tissue explants by the enamel organ. CONCLUSIONS: The dental epithelium of developing tooth germ is able to synthesise significant amounts of versican.

Asymmetrical growth, differential cell proliferation, and dynamic cell rearrangement underlie epithelial morphogenesis in mouse molar development.

During molar development from the cap to bell stage, the morphology of the enamel knots, inner dental epithelium, and epithelial-mesenchymal junction dynamically changes, leading to the formation of multiple cusps. To study the basic histological features of this morphogenetic change, we have investigated the cell arrangement, mitosis, and apoptosis simultaneously in the developing first lower molar of the mouse by means of BrdU injection and immunostaining for P-cadherin, BrdU, and single-stranded DNA. At the typical cap stage, the primary enamel knot shows a characteristic cell arrangement, absence of mitosis, and abundant apoptosis, but also actively dividing cells at its lateral margins. Two secondary enamel knots then appear in the anterior part of the tooth germ. One is completely non-proliferating, whereas the other contains dividing cells, indicating asymmetrical growth of the inner dental epithelium. From this transitional stage to the early bell stage, additional minor BrdU-negative domains appear, and at the same time, the cell arrangement in the inner dental epithelium rapidly changes to show regional differences. Comparisons between the histology and the distribution of BrdU-positive cells have revealed that both the regionally different cell rearrangement and the differential cell proliferation in the enamel knots and inner dental epithelium probably play a significant role in multiple cusp formation.

Simultaneous immunofluorescence detection of cadherin and beta-catenin in mouse tissue sections.

Both cell-cell adhesion and secreted signaling molecules are involved in the regulation of normal tissue development and maintenance. beta-catenin seems to link cadherin-mediated cell-cell adhesion and Wnt signaling. Thus, its activity in particular cells or tissues, either during normal development or in tumorigenesis, has become a fascinating topic of investigation. Because the multiple functions of beta-catenin intimately relate to its amount and localization within a cell, immunohistochemical detection of this molecule simultaneously with the cell adhesion molecule cadherin should provide helpful information on its potential activities. This detection can be conducted on tissue sections by using appropriate tissue preparation procedures and primary antibodies from different species.

Gene expression of beta-catenin is up-regulated in inner dental epithelium and enamel knots during molar tooth morphogenesis in the mouse.

Beta-catenin is a multi-functional molecule that is involved in both cell-cell adhesion and signaling. We analyzed changes in beta-catenin gene expression during mouse molar tooth development by in situ hybridization. Prominent up-regulation of the expression of this gene was evident exclusively in the enamel knot at the early cap stage. During the cap and bell stages, the enamel knot, inner dental epithelium, and differentiating stratum intermedium expressed the beta-catenin gene more strongly than other parts of the enamel organ. During these stages, the strength of the gene expression changed heterogeneously within the inner dental epithelium and stratum intermedium. However, the heterogeneity was not evident at the late bell stage, when the cells in the inner dental epithelium had differentiated into ameloblasts at the cusp tip. No spatiotemporal change in beta-catenin gene expression was apparent in the dental papilla except for the cells that differentiated into odontoblasts, which became negative for the expression of the gene after their differentiation. Thus, the up-regulated expression of the beta-catenin gene was strongly associated with epithelial morphogenesis. These findings raise the possibility that the up-regulation of the gene expression and the stabilization of the protein by Wnt signaling play a role in the regulation of the activities of beta-catenin in tooth morphogenesis.

Expression of glial cell line-derived neurotrophic factor (GDNF) and GDNF family receptor alpha1 in mouse taste bud cells after denervation.

Glial cell line-derived neurotrophic' factor (GDNF) has been isolated as a neurotrophic factor that affects the survival and maintenance of central and peripheral neurons. Using immunocytochemical methods, we examined whether the taste bud cells in mouse circumvallate papillae after transection of the glossopharyngeal nerves expressed GDNF and its receptor, GDNF family receptor alpha1 (GFRalpha1). By 5 and 10 days after denervation, the number of taste buds had decreased markedly; however, the remaining taste bud cells still expressed GDNF and GFRalpha1. By 14 days after denervation, most of the taste buds had disappeared and GDNF- and GFRalpha1-immunoreactive cells were not seen. By 4 weeks after denervation, numerous TrkB-immunoreactive nerve fibers had invaded the papilla and a few taste buds expressing GDNF and GFRalpha1 had regenerated. Thus, GDNF- and GFRalpha1-immunoreactive taste bud cells after denervation vanished following the disappearance of the taste buds and reappeared at the same time as the taste buds reappeared.

Immunohistochemical detection of neurotrophin-3 and -4, and their receptors in mouse taste bud cells.

Neurotrophin-3 (NT3) and neurotrophin-4 (NT4) affect the survival and maintenance of central and peripheral neurons. Using an immunohistochemical method, we examined whether the taste bud cells in the circumvallate papillae of normal mice expressed NT3, NT4, and their respective receptors TrkC and TrkB, and if so, what type of cells in the taste buds expressed them. Double immunostaining for either of them and PGP 9.5, NCAM, or gustducin was used to determine which cell types expressed which neurotrophins and receptors. Normal taste bud cells expressed NT3, NT4, and the TrkB receptor, but not TrkC. The percentage of NT3-immunoreactive cells among all taste bud cells was 89.0%, that of NT4-immunoreactive cells, 58.6%, and that of TrkB-immunoreactive cells, 80.8%. Almost none of the NT4-immunoreactive cells were reactive with anti-PGP 9.5 or the anti-NCAM antibody, but they could be stained with anti-gustducin, revealing that NT4-immunoreactive cells were contained only in the type-II--and possibly type-I--cell population. On the other hand, NT3-, and TrkB-immunoreactive cells included type-III cells, together with type-II, -I, and basal cells, because they were positive for PGP 9.5 and gustducin. We conclude that NT4 may exert trophic actions on all types of taste bud cells by binding to their TrkB receptors, and NT3 may also have a similar, though negligible role.

Expression of GDNF and GFR alpha 1 in mouse taste bud cells.

GDNF (glial cell line-derived neurotrophic factor) affects the survival and maintenance of central and peripheral neurons. Using an immunocytochemical method, we examined whether the taste bud cells in the circumvallate papillae of normal mice expressed GDNF and its GFR alpha 1 receptor. Using double immunostaining for either of them and NCAM, PGP 9.5, or alpha-gustducin, we additionally sought to determine what type of taste bud cells expressed GDNF or GFR alpha 1, because NCAM is reported to be expressed in type-III cells, PGP 9.5, in type-III and some type-II cells, and alpha-gustducin, in some type-II cells. Normal taste bud cells expressed both GDNF and GFR alpha 1. The percentage of GDNF-immunoreactive cells among all taste bud cells was 31.63%, and that of GFR alpha 1-immunoreactive cells, 83.21%. Confocal laser scanning microscopic observations after double immunostaining showed that almost none of the GDNF-immunoreactive cells in the taste buds were reactive with anti-NCAM or anti-PGP 9.5 antibody, but could be stained with anti-alpha-gustducin antibody. On the other hand, almost all anti-PGP 9.5- or anti-alpha-gustducin-immunoreactive cells were positive for GFR alpha 1. Thus, GDNF-immunoreactive cells did not include type-III cells, but type-II cells, which are alpha-gustducin-immunoreactive; on the other hand, GFR alpha 1-immunoreactive cells included type-II and -III cells, and perhaps type-I cells. We conclude that GDNF in the type-II cells may exert trophic actions on type-I, -II, and -III taste bud cells by binding to their GFR alpha 1 receptors.

Subcellular localization of beta-catenin and cadherin expression in the cap-stage enamel organ of the mouse molar.

We analyzed the subcellular distribution of beta-catenin in the cap-stage enamel organ and compared it with the expression of E- and P-cadherin by using confocal laser microscopy. The amounts of the molecules in the cytoplasm and the nucleus showed regional variations in the enamel organ, whereas cell surface-associated beta-catenin was ubiquitous. In both the enamel knot and the inner dental epithelium, beta-catenin was detected in the cytoplasm and in the nucleus. However, the amount of nuclear beta-catenin was apparently higher in the enamel knot than in the inner dental epithelium. P-cadherin also gave a stronger signal in the enamel knot than in other parts of the enamel organ. In the stellate reticulum, where E-cadherin was preferentially expressed, as well as in the cervical loop and outer dental epithelium, beta-catenin was localized in the cytoplasm but not in the nucleus. The nuclear localization of beta-catenin in the enamel knot suggests a specific activation of the canonical Wnt signaling pathway. A coincident upregulation of P-cadherin was observed in this area. Altogether, these observations suggest the possibility of a linkage between cell adhesion and Wnt signaling in the enamel knot.

Expression of Hes6 and NeuroD in the olfactory epithelium, vomeronasal organ and non-sensory patches.

Basic helix-loop-helix transcription factors NeuroD and Hes6 promote neuronal differentiation. The expression of their genes in the olfactory epithelium (OE), vomeronasal organ (VNO) and the non-sensory patches of the posterior nasal cavity of mice was examined. As detected by in situ hybridization, Hes6 was expressed in a basal progenitor layer of the embryonic OE. After birth, the expression of Hes6 was detected in a cell layer above the basement membrane, globose basal cells (GBCs). Expression of NeuroD in the embryonic OE was in agreement with that previously described; and in the postnatal OE, it was detected in cells of GBC layer and cells upper to GBCs. In the VNO, Hes6 was expressed throughout the sensory epithelium (S-VNO) at embryonic day 12, and later became restricted to a single layer of cells in the basal region of the S-VNO, where Hes5-expressing undifferentiated cells were present. NeuroD was expressed throughout the S-VNO during the embryonic stage. After birth, Hes6 and NeuroD expressions were observed in the border between the S-VNO and non-sensory VNO. Immunohistochemistry using anti-NeuroD antibody revealed that NeuroD-positive cells were still present not only at the edges but also in the center of the S-VNO until P3. These findings suggest that Hes6 and NeuroD are expressed in progenitors of chemoreceptor neurons and that the expression of Hes6 precedes that of NEUROD: Moreover, in the regenerating VNO of bulbectomized mice, NeuroD-positive cells were observed both at the edges and in the center of the S-VNO, suggesting that neuronal turnover occurred in both regions. Moreover, in the dorsal fossa of the posterior nasal cavity, several non-sensory patches are formed between postnatal (P) days 10 and 21 because of programmed death of ORNs and GBCs. During embryonic stages, the expression of Hes6 and NeuroD in the OE showed no regional differences. At P3-P7, expression of NeuroD and Hes6 disappeared in the region corresponding to the presumptive non-sensory patches. The loss of these genes may stop the differentiation and may cause apoptosis of GBCs and ORNs.

Expression of the neural cell adhesion molecule during mouse tooth development.

The neural cell adhesion molecule (NCAM) is a molecule that mediates calcium-independent cell-cell adhesion, and is expressed during development in a variety of tissues including nonneuronal ones. Expression of NCAM during molar tooth development was analyzed in this present study. In the enamel organ, NCAM expression was detected from the embryonic day 13 onward in restricted areas, including a part of the stratum intermedium of the tooth germ at the bell stage. Mesenchymal cells also expressed NCAM from the bud stage of the first molar onward. In addition to the strong expression in the dental follicle, weak and transient expression was detected in the dental papilla. The second and the third molars appeared to initiate their development by the interaction between the epithelial cells and NCAM-expressing mesenchymal cells. Comparison of the expression patterns of NCAM and transcription factor Barx1 revealed a possibility that Barx1 negatively regulates NCAM expression.

Expression of neural cell-adhesion molecule mRNA during mouse molar tooth development.

This study employed in situ hybridisation using a probe recognising all isoforms of the molecule. Expression of the molecule in tooth germs started at embryonic day 13, when they were at the bud stage. Both inner cells of the epithelial bud and peripheral cells of the dental mesenchyme were positive. At the cap stage, positive cells were found in the inner part of the enamel organ but only in a limited area near the outer enamel epithelium. In the mesenchyme at the cap stage, expression was weak in the dental papilla and strong in the follicle. From the bell stage onward, epithelial cells in the enamel organ were negative except for the cells of the stratum intermedium, which were transiently positive at early and late bell stages. In the dental papilla, expression had mostly ceased during and after the bell stage, although transient expression was found in cuspal areas at the early bell stage. The dental follicle strongly expressed neural cell-adhesion molecule (NCAM) to the end of the experimental period, at post-natal day 4. In contrast to the first molar at its earliest stage of appearance, in which both the thickened epithelium and surrounding mesenchyme were negative for the expression of the molecule, the second molar appeared as a combination of extending epithelial thickenings and mesenchymal cells strongly positive for its expression. This study newly identifies the dental papilla and the stratum intermedium as NCAM-expressing sites.

Expression of NeuroD in the mouse taste buds.

NeuroD, a basic helix-loop-helix transcription factor, has been shown to play a role in the differentiation of neurons, olfactory cells, and neuroendocrine tissues. Since the taste buds have characteristics of typical paraneurons, we examined the expression of NeuroD in the taste buds of mice. By RT-PCR analysis, NeuroD mRNA was found to be expressed in the epithelium of circumvallate papillae-containing taste buds, but not in the lingual epithelium lacking them. NeuroD immunoreactivity was detected in a subset of taste bud cells in the circumvallate, foliate, and fungiform papillae and in the soft palate. NeuroD-expressing cells had a spindle-like shape, first appeared at postnatal day 3, and increased in number during postnatal development. After bisection of the glossopharyngeal nerves, NeuroD-expressing cells decreased in number at day 4 and disappeared from the trench wall of the circumvallate papillae by day 14. A few NeuroD-expressing taste buds reappeared at postoperative day 28. Denervation and regeneration experiments showed that expression of NeuroD in the taste buds was dependent upon gustatory innervation. Double immunolabeling with gustducin or with neural cell adhesion molecule (NCAM) showed that NeuroD-expressing cells did not express NCAM, but did express gustducin. These results suggest that NeuroD is expressed in a mature cell type, type-II cells, but not in type-III cells.