BMP7 and EREG Contribute to the Inductive Potential of Dental Mesenchyme.

Odontogenesis is accomplished by reciprocal signaling between the epithelial and mesenchymal compartments. It is generally accepted that the inductive mesenchyme is capable of inducing the odontogenic commitment of both dental and non-dental epithelial cells. However, the duration of this signal in the developing dental mesenchyme and whether adult dental pulp tissue maintains its inductive capability remain unclear. This study investigated the contribution of growth factors to regulating the inductive potential of the dental mesenchyme. Human oral epithelial cells (OEs) were co-cultured with either human dental mesenchymal/papilla cells (FDPCs) or human dental pulp cells (ADPCs) under 2-dimensional or 3-dimensional conditions. Odontogenic-associated genes and proteins were detected by qPCR and immunofluorescence, respectively, and significant differences were observed between the two co-culture systems. The BMP7 and EREG expression levels in FDPCs were significantly higher than in ADPCs, as indicated by human growth factor PCR arrays and immunofluorescence analyses. OEs co-cultured with ADPCs supplemented with BMP7 and EREG expressed ameloblastic differentiation genes. Our study suggests that BMP7 and EREG expression in late bell-stage human dental papilla contributes to the inductive potential of dental mesenchyme. Furthermore, adult dental pulp cells supplemented with these two growth factors re-established the inductive potential of postnatal dental pulp tissue.

c-kit positive cells and networks in tooth germs of human midterm fetuses.

Numerous studies have attempted to characterize the dental pulp stem cells. However, studies performed on prenatal human tissues have not been performed to evaluate the in situ characterization and topography of progenitor cells. We aimed to perform such a study using of antibodies for CD117/c-kit and multiplex antibody for Ki67+ caspase 3. Antibodies were applied on samples dissected from five human midterm fetuses. Positive CD117/c-kit labeling was found in mesenchymal derived tissues, such as the dental follicle and the dental papilla. The epithelial tissues, that is, dental lamina, enamel organ and oral epithelia, also displayed isolated progenitor cells which were CD117/c-kit positive. Interestingly, CD117/c-kit positive cells of mesenchymal derived tissues extended multiple prolongations building networks; the most consistent of such networks were those of the dental follicle and the perivascular networks of the dental papilla. However, the mantle of the dental papilla was also positive for CD117/c-kit positive stromal networks. The CD117/c-kit cell populations building networks appeared mostly with a Ki67 negative phenotype. The results suggest that CD117/c-kit progenitor cells of the prenatal tooth germ tissues might be involved in intercellular signaling.

Expression patterns of ABCG2, Bmi-1, Oct-3/4, and Yap in the developing mouse incisor.

Recent studies have demonstrated the existence of dental stem cells in the continuously growing tooth. However, much remains to be learned about the complex mechanism involving stem cells during tooth development. We determined the expression patterns of four stem cell markers ABCG2, Bmi-1, Oct-3/4, and Yap in the developing mouse incisors between embryonic day (E) 11 and postnatal day (PN) 20. ABCG2 was localized strongly in the perivascular region of the incisor mesenchyme from E11 to PN20, and in the odontoblasts from E18 to PN20. Bmi-1 was expressed in both the dental epithelium and mesenchyme from E11 to E14. The expression of Bmi-1 was noticeably reduced at E16, and was restricted to the apical bud from E16 to PN20. Oct-3/4 was localized in the nucleus of the cells in the superficial layer and stellate reticulum within the dental epithelium from E11 to E14 and in the apical bud from E16 to PN20. Meanwhile, once the ameloblasts and odontoblasts began to appear at E16, they expressed Oct-3/4 in the cytoplasm. Yap was expressed in most of the basal cells of the incisor dental epithelium from E11 to E14, but was expressed mainly in the transit-amplifying (TA) cells within the basal cell layer from E16 to PN20. The unique and overlapping expression patterns of ABCG2, Bmi-1, Oct-3/4, and Yap suggest the independent and interactive functions of the four stem cell markers in the developing mouse incisor.

Spatial and temporal distribution of Ki-67 proliferation marker, Bcl-2 and Bax proteins in the developing human tooth.

OBJECTIVE: To investigate the spatial and temporal expression of proliferation Ki-67 marker, pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins during early development of the human tooth. MATERIALS AND METHODS: Histological sections of eight human conceptuses, 5-10 postovulatory weeks old, were used for immunolocalization for Ki-67, Bax and Bcl-2 markers. Quantification was performed by calculating the fraction of Ki-67 positive cells, expressed as a mean ± SD, and analysed by Mann-Whitney test, Kruskal-Wallis and Dunn's post hoc test. RESULTS: In 6th-7th developmental weeks, the tooth germ and dental crest contained 37% of proliferating cells, which increased to 40% in the 8th week, and then decreased to 15% in the 10th week, whilst the proliferation in the ectomesenchyme subsequently dropped from 37% to 23%. Epithelial parts of the enamel organ displayed similar proliferation activity (31-36%), dental crest 10%, whilst enamel knot showed no proliferating activity. The tooth ectomesenchyme contained more proliferating cells (50%) than the jaw ectomesenchyme (35%), and both dropped to 28% in the 10th week. Ectomesenchyme between the tooth germs contained 23%, whilst the jaw ectomesenchyme contained 15% of proliferating cells. Bcl-2 expression had following pattern: strong in proliferating cells, moderate in tooth germs and dental crest, and weak in the ectomesenchyme. Bax co-expressed with Bcl-2 in the tooth germ and dental crest. In the reticulum and inner enamel epithelium Bcl-2 had prevalent expression, whilst Bax prevailed in the outer enamel epithelium and tooth ectomesenchyme. CONCLUSIONS: Proliferating cells most likely influence growth of the tooth germ, Bcl-2 affects proliferation and differentiation of specific cell lineages, whilst Bax influences process of cell death.

Down-regulated genes in mouse dental papillae and pulp.

Important factors involved in odontogenesis in mouse dental papillae disappear between the pre- and post-natal stages of development. Therefore, we hypothesized that certain genes involved in odontogenesis in dental papillae were subject to pre-/post-natal down-regulation. Our goal was to identify, by microarray analysis, which genes were down-regulated. Dental papillae were isolated from embryonic 16-day-, 18-day- (E16, E18), and post-natal 3-day-old (P3) murine first mandibular molar germs and analyzed by microarray. The number of down-regulated genes was 2269 between E16 and E18, and 3130 between E18 and P3. Drastic down-regulation (fold change > 10.0) of Adamts4, Aldha1a2, and Lef1 was observed at both E16 and E18, and quantitative RT-PCR revealed a post-natal reduction in their expression (Adamts4, 1/3; Aldh1a2, 1/13; and Lef1, 1/37). These results suggest that down-regulation of these three genes is an important factor in normal odontogenesis in dental papillae.

In situ expression of heat shock proteins, Hsc73, Hsj2 and Hsp86 in the developing tooth germ of mouse lower first molar.

This study examined the detailed gene expression pattern of three different heat shock proteins (HSPs), Hsc73, Hsj2, and Hsp86, by means of an in situ hybridization method. Hsc73, Hsj2, and Hsp86 were shown in our previous study to be differentially expressed in the mouse embryonic mandible at day 10.5 (E10.5) gestational age. These HSP genes showed similar expression patterns during development of the mouse lower first molar. HSPs-expressing cells were widely distributed in both the epithelial and underlying ectomesenchymal cells at E10.5, and then were slightly localized at E12 in an area where the tooth germ of the lower first molar is estimated to be formed. A strong expression of HSPs was observed in the tooth germ at E13.5. At the cap stage, HSPs were expressed in the enamel organ and dental papilla. At the bell stage, HSPs were distinctly expressed in the inner enamel epithelium and dental papilla cells facing the inner enamel epithelial layer, which later differentiate into ameloblasts and odontoblasts, respectively. This study is the first report in which Hsc73, Hsj2, and Hsp86 were distinctly expressed in the developing tooth germ, thus suggesting these HSPs are related to the development and differentiation of odontogenic cells.

Development and maturation of taste buds of the palatal epithelium of the rat: histological and immunohistochemical study.

Palatal taste buds are intriguing partners in the mediation of taste behavior and their spatial distribution is functionally important for suckling behavior, especially in the neonatal life. Their prenatal development has not been previously elucidated in the rat, and the onset of their maturation remains rather controversial. We delineated the development and frequency distribution of the taste buds as well as the immunohistochemical expression of alpha-gustducin, a G protein closely related to the transduction of taste stimuli, in the nasoincisor papilla (NIP) and soft palate (SP) from the embryonic day 17 (E17) till the postnatal day 70 (PN70). The main findings in the present study were the development of a substantial number of taste pores in the SP of fetal rats (60.3 +/- 1.7 out of 122.8 +/- 5.5; mean +/- SD/animal at E19) and NIP of neonatal rats (9.8 +/- 1.0 out of 44.8 +/- 2.2 at PN4). alpha-gustducin-like immunoreactivity (-LI) was not expressed in the pored taste buds of either prenatal or newborn rats. The earliest expression of alpha-gustducin-LI was demonstrated at PN1 in the SP (1.5 +/- 0.5 cells/taste bud; mean +/- SD) and at PN4 in the NIP (1.4 +/- 0.5). By age the total counts of pored taste buds continuously increased and their morphological features became quite discernible. They became pear in shape, characterized by distinct pores, long subporal space, and longitudinally oriented cells. Around the second week, a remarkable transient decrease in the total number of taste buds was recorded in the oral epithelium of NIP and SP, which might be correlated with the changes of ingestive behaviors. The total counts of cells showing alpha-gustducin-LI per taste bud gradually increased till the end of our investigation (14.1 +/- 2.7 in NIP and 12.4 +/- 2.5 in SP at PN70). We conclude that substantial development of taste buds began prenatally in the SP, whereas most developed entirely postnatal in the NIP. The present study provides evidence that the existence of a taste pore which is considered an important criterion for the morphological maturation of taste buds is not enough for the onset of the taste transduction, which necessitates also mature taste cells. Moreover, the earlier maturation of palatal taste buds compared with the contiguous populations in the oral cavity evokes an evidence of their significant role in the transmission of gustatory information, especially in the early life of rat.

Prenatal fluoride for growth and development: Part X.

Examinations of prenatal fluoride supplemented (PNF) teeth in an animal model and in a five-month human fetus find these teeth to be more developed than the non-supplemented controls. The fact that PNF allows teeth to develop to their full potential suggests that PNF could be an essential nutrient for the entire human and this could be demonstrated most easily during rapid fetal growth. A review of the recent literature, including trials by NIH and The World Health Organization, provide evidence that fluoride (F) does allow the fetus to grow and develop to its full potential. The authors conclude that PNF must be supplied in at least a 2 mg/day pulse dose, and then F must be given from shortly after birth in a daily amount appropriate for the weight of the child with some consideration for the amount of F water utilized.

Distribution of the neural cell adhesion molecule (NCAM) during pre- and postnatal development of mouse incisors.

Developmental changes in the distribution of the neural cell adhesion molecule (NCAM) were investigated in mouse incisors by means of the indirect immunofluorescence method. During the prenatal stages of development, NCAM was predominantly found in the dental follicle, but not in the dental papilla; the results were analogous to the distribution of NCAM during molar development. After birth, the expression of NCAM continued in the tissue between the enamel organ and the alveolar bone on the labial aspect. In contrast, the follicular tissue covering the lingual aspect of the incisor gradually lost NCAM immunoreactivity from its outer zone as it differentiated into the highly organized periodontal ligament. The intermediate zone of the ligament continued to express NCAM-immunoreactivity even in mice of 6 weeks of age. This pattern of NCAM expression was different from that found in molar teeth, where the organized peridontal ligament was NCAM-negative. The dental pulp, in which we previously reported that an NCAM-positive area appeared at later stages of molar tooth development, did not express NCAM immunoreactivity even at the latest stage of development covered in this study. These differences in the distribution of NCAM between the incisors and the molars might be related to the fact that rodent incisors continue to grow throughout the life of the animal.

Development and cell fate in interspecific (Mus musculus/Mus caroli) intraocular transplants of mouse molar tooth-germ tissues detected by in situ hybridization.

Mandibular first molar tooth germs were dissected from Mus musculus (CDI) and Mus caroli (age range: 14-day embryo to 1-day postnatal). Most of the tooth germs were separated enzymically into epithelial and mesenchymal components. Interspecific tissue recombinations and intact M. caroli tooth germs were grown in the anterior chamber of the eye of adult CDI mice for 24 weeks. Recombinations of M. caroli enamel-organ epithelium with M. musculus, dental papilla and follicle mesenchyme developed into normal teeth with advanced root, periodontal ligament and bone formation, thereby confirming extensive epithelial-mesenchymal interactions across the species barrier. Labelling sections by in situ hybridization with a M. musculus-specific DNA probe (pMSat5) showed that almost all cells in the pulp, periodontal ligament and bone were M. musculus, including cementoblasts. Reduced enamel epithelium and epithelial cell rests derived from donor M. caroli enamel organ were unlabelled. This indicates that any cementogenic role of Hertwig's epithelial root sheath must be short-lived. The immunological privilege of the intraocular transplantation site in M. musculus CDI mice did not extend to grafts including xenogeneic M. caroli dental mesenchyme. Thus, intact M. caroli tooth germs and recombinations of M. musculus enamel organ with M. caroli dental papilla and follicle showed limited development, with no root formation, and were populated almost exclusively with labelled host M. musculus lymphocytes.

Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development.

The Msx1 homeobox gene is expressed at diverse sites of epithelial-mesenchymal interaction during vertebrate embryogenesis, and has been implicated in signalling processes between tissue layers. To determine the phenotypic consequences of its deficiency, we prepared mice lacking Msx1 function. All Msx1- homozygotes manifest a cleft secondary palate, a deficiency of alveolar mandible and maxilla and a failure of tooth development. These mice also exhibit abnormalities of the nasal, frontal and parietal bones, and of the malleus in the middle ear. Msx1 thus has a critical role in mediating epithelial-mesenchymal interactions during craniofacial bone and tooth development. The Msx1-/Msx1- phenotype is similar to human cleft palate, and provides a genetic model for cleft palate and oligodontia in which the defective gene is known.

[Recombinant study of mouse enamel organ and dental papilla by renal transplantation: effects of discrepancy in number of these two components].

Interactions between the epithelium and mesenchyme were very important in tooth development. When these relationships changed qualitatively or quantitatively, developmental tooth abnormalities or odontogenic lesions might develop. In order to clarify this hypothesis, we examined the effects of discrepancy in the number of enamel organs and dental papilla on histo- and morpho-differentiation of the recombinants by renal subcapsular transplantation. Five kinds of recombinants were prepared from the enamel organs(E) and dental papilla (M) of the mandibular first molars of 17-gestational-day C3H mice: group 1 (E/M = 2/6), group 2(E/M = 3/5), group 3(E/M = 4/4), group 4 (E/M = 5/3) and group 5 (E/M = 6/2). After 28 days of transplantation, all the grafts were harvested and examined roentogenographically and histopathologically. All the grafts developed into teeth. The teeth of groups 1 and 5 were independent from each other and the teeth of groups 2, 3 and 4 fused with each other by dentin. The number and size of the teeth depend on the number of M and the differences between E and M of the recombinants, respectively. The smaller the difference, smaller was the teeth size. The cysts lined by the keratinizing squamous epithelium developed from E-rich recombinants. These could be comparative to the compound odontoma, fused teeth and odontogenic keratocyst of the human being respectively. It might be speculated that the histogenesis of these lesions might be related to the quantitative change in the epithelial-mesenchymal interactions of odontogenesis.

An investigation of S-100 protein in embryonic dental papillae of rats.

We attempted to identify S-100 protein, a marker of neural crest tissue, within cells of the developing dental papillae of 18-day rat fetuses by means of the immunoperoxidase technique. Although there is experimental evidence that dental papillae are derived from neural crest, no marker protein was identified in this study.