Amelogenins as potential buffers during secretory-stage amelogenesis.

Amelogenins are the most abundant protein species in forming dental enamel, taken to regulate crystal shape and crystal growth. Unprotonated amelogenins can bind protons, suggesting that amelogenins could regulate the pH in enamel in situ. We hypothesized that without amelogenins the enamel would acidify unless ameloblasts were buffered by alternative ways. To investigate this, we measured the mineral and chloride content in incisor enamel of amelogenin-knockout (AmelX(-/-)) mice and determined the pH of enamel by staining with methyl-red. Ameloblasts were immunostained for anion exchanger-2 (Ae2), a transmembrane pH regulator sensitive for acid that secretes bicarbonate in exchange for chloride. The enamel of AmelX(-/-) mice was 10-fold thinner, mineralized in the secretory stage 1.8-fold more than wild-type enamel and containing less chloride (suggesting more bicarbonate secretion). Enamel of AmelX(-/-) mice stained with methyl-red contained no acidic bands in the maturation stage as seen in wild-type enamel. Secretory ameloblasts of AmelX(-/-) mice, but not wild-type mice, were immunopositive for Ae2, and stained more intensely in the maturation stage compared with wild-type mice. Exposure of AmelX(-/-) mice to fluoride enhanced the mineral content in the secretory stage, lowered chloride, and intensified Ae2 immunostaining in the enamel organ in comparison with non-fluorotic mutant teeth. The results suggest that unprotonated amelogenins may regulate the pH of forming enamel in situ. Without amelogenins, Ae2 could compensate for the pH drop associated with crystal formation.

Podoplanin, E-cadherin, ß-catenin, and CD44v6 in recurrent ameloblastoma: their distribution patterns and relevance.

BACKGROUND: Ameloblastoma is a benign but locally infiltrative odontogenic epithelial neoplasm with a high risk for recurrence. Podoplanin, a lymphatic endothelium marker, putatively promotes collective cell migration and invasiveness in this neoplasm. However, its role in the recurrent ameloblastoma (RA) remains unclear. As morphological, signaling, and genetic differences may exist between primary and recurrent tumors, clarification of their distribution patterns is of relevance. MATERIALS AND METHODS: Podoplanin was examined immunohistochemically in conjunction with E-cadherin, ß-catenin, and CD44v6 in 25 RA. Immunostaining according to tumor area, cellular type, and location, and relationship of these proteins were analyzed. Findings were compared with 25 unrelated primary ameloblastomas (UPA). RESULTS: All four proteins were detected in RA and UPA samples. Expression rates for each protein were not significantly different between these two groups. RA demonstrated significant upregulation of podoplanin at the invasive front (P < 0.05), whereas upregulation of ß-catenin and CD44v6 and downregulation of E-cadherin at this site were not statistically significant (P > 0.05). Immunolocalization for all four proteins was predominantly membranous and less frequently cytoplasmic. Pre-ameloblast-like cells were podoplanin(+) /CD44v6(-), while stellate reticulum-like cells were podoplanin(-)/CD44v6(+). Acanthomatous, granular cell, and desmoplastic variants in both RA and UPA were podoplanin(-/low) but stained weak-to-moderate for E-cadherin, ß-catenin, and CD44v6. Stromal fibroblasts and lymph channels were variably podoplanin-positive. CONCLUSIONS: Podoplanin, ß-catenin, and CD44v6 upregulation at the tumor invasive fronts in RA and UPA supports a differential regulatory role by these molecules in mediating collective cell migration and local invasiveness. E-cadherin downregulation suggests altered cell adhesion function during tumor progression.

A post-classical theory of enamel biomineralization and why we need one.

Enamel crystals are unique in shape, orientation and organization. They are hundreds of thousands times longer than they are wide, run parallel to each other, are oriented with respect to the ameloblast membrane at the mineralization front and are organized into rod or interrod enamel. The classical theory of amelogenesis postulates that extracellular matrix proteins shape crystallites by specifically inhibiting ion deposition on the crystal sides, orient them by binding multiple crystallites and establish higher levels of crystal organization. Elements of the classical theory are supported in principle by in vitro studies; however, the classical theory does not explain how enamel forms in vivo. In this review, we describe how amelogenesis is highly integrated with ameloblast cell activities and how the shape, orientation and organization of enamel mineral ribbons are established by a mineralization front apparatus along the secretory surface of the ameloblast cell membrane.

Amelogenin and enamelysin localization in human dental germs.

Odontogenesis is extensively studied in animal models but less understood in human. In early amelogenesis, amelogenin constitutes 90% of enamel organic matrix, which is degraded by enamelysin and replaced by hydroxyapatite crystals. Here, amelogenin and enamelysin distribution changes during amelogenesis were shown by co-localization experiments by confocal microscopy. Early bell stage showed more amelogenin labeling than enamelysin, as free immune-reactive granular patches towards basal membrane between ameloblast and odontoblast. Increased amelogenin expression and secretion towards extracellular matrix formation region was found. Enamelysin distribution was perinuclear in early bell stage. During late bell stage, a decreasing amelogenin labeling in contrast with enamelysin increasing along the cells was found, suggesting specific temporal amelogenin degradation. Enamelysin was located initially around nuclei and later was found in all the ameloblast and stellate reticulum cytoplasm. Amelogenin was observed inside ameloblast, stellate reticulum, and intermediate stratum cells in the enamel as well as in the newly formed dentin extracellular matrix. In contrast, in dentin more amelogenin than enamelysin was found located close to the periphery.

Consequences for enamel development and mineralization resulting from loss of function of ameloblastin or enamelin.

Although the nonamelogenin proteins, ameloblastin and enamelin, are both low-abundance and rapidly degrading components of forming enamel, they seem to serve essential developmental functions, as suggested by findings that an enamel layer fails to appear on teeth of mice genetically engineered to produce either a truncated form of ameloblastin (exons 5 and 6 deleted) or no enamelin at all (null). The purpose of this study was to characterize, by direct micro weighing, changes in enamel mineralization occurring on maxillary and mandibular incisors of mice bred for these alterations in nonamelogenin function (Ambn(+/+, +/-5,6, -5,6/-5,6), Enam(+/+, +/- ,-/-)). The results indicated similar changes to enamel-mineralization patterns within the altered genotypes, including significant decreases by as much as 50% in the mineral content of maturing enamel from heterozygous mice and the formation of a thin, crusty, and disorganized mineralized layer, rather than true enamel, on the labial (occlusal) surfaces of incisors and molars along with ectopic calcifications within enamel organ cells in Ambn(-5,6/-5,6) and Enam(-/-) homozygous mice. These findings confirm that both ameloblastin and enamelin are required by ameloblasts to create an enamel layer by appositional growth as well as to assist in achieving its unique high level of mineralization.

Immunocytochemistry of matrix proteins in calcified tissues: functional biochemistry on section.

The organic matrix of calcified tissues comprises collagenous and/or noncollagenous matrix proteins (NCPs). Identification and precise mapping of these matrix components is essential for determining their function, formulating coherent hypotheses on their mechanism(s) of action, and developing novel therapeutic approaches based on biologics. Fibrillar collagen can be readily identified by its conspicuous structure, however, NCPs, in general, do not individually exhibit characteristic structural features that permit to identify them and morphologically determine their localization. To address this limitation, we have used immunocytochemistry, a form of "biochemistry on section", to correlate composition with structure. For cytochemical characterizations, including immunolabeling, our laboratory has opted for colloidal gold labelings and pioneered their application to calcified tissues because they yield high spatial resolution and are quantitative. Over the years, this approach has been applied to identify and map various NCPs in bone and teeth and, in this review of our work, we will emphasize some selected studies that highlight it application to also achieve functional information.

Subcultured odontogenic epithelial cells in combination with dental mesenchymal cells produce enamel-dentin-like complex structures.

We showed in a previous study that odontogenic epithelial cells can be selectively cultured from the enamel organ in serum-free medium and expanded using feeder layers of 3T3-J2 cells. The subcultured odontogenic epithelial cells retain the capacity for ameloblast-related gene expression, as shown by semiquantitative RT-PCR. The purpose of the present study was to evaluate the potential of subcultured odontogenic epithelial cells to form tooth structures in cell-polymer constructs maintained in vivo. Enamel organs from 6-month-old porcine third molars were dissociated into single odontogenic epithelial cells and subcultured on feeder layers of 3T3-J2 cells. Amelogenin expression was detected in the subcultured odontogenic epithelial cells by immunostaining and Western blotting. The subcultured odontogenic epithelial cells were seeded onto collagen sponge scaffolds in combination with fresh dental mesenchymal cells, and transplanted into athymic rats. After 4 weeks, enamel-dentin-like complex structures were present in the implanted constructs. These results show that our culture system produced differentiating ameloblast-like cells that were able to secrete amelogenin proteins and form enamel-like tissues in vivo. This application of the subculturing technique provides a foundation for further tooth-tissue engineering and for improving our understanding of ameloblast biology.

Towards second-generation proteome analysis of murine enamel-forming cells.

Proteome analysis of rat enamel-forming cells, initiated over a decade ago, has provided valuable insights to enamel biology. In preparation for a more comprehensive, second-generation proteomic exploration, we evaluated an updated microsample-profiling strategy that comprises sequential extraction of enamel epithelium, parallel one- and two-dimensional gel electrophoresis, and mass spectrometric sequence analysis. The results indicated that several hundred proteins, representing various cellular compartments (including membranes), are amenable to identification with a starting tissue volume of <10 microl. With its increased proteomic depth and breadth, this straightforward approach constitutes a major advance from the first-generation work (10-fold increased proteome coverage), although care was needed to ensure a comparably high stringency of protein identification. Expression proteomics has an exciting potential to elucidate the inner workings of murine enamel epithelial cells, leading to an improved understanding of enamel in health and disease.

Altering biomineralization by protein design.

To create a bioceramic with unique materials properties, biomineralization exploits cells to create a tissue-specific protein matrix to control the crystal habit, timing, and position of the mineral phase. The biomineralized covering of vertebrate teeth is enamel, a distinctive tissue of ectodermal origin that is collagen-free. In forming enamel, amelogenin is the abundant protein that undergoes self-assembly to contribute to a matrix that guides its own replacement by mineral. Conserved domains in amelogenin suggest their importance to biomineralization. We used gene targeting in mice to replace native amelogenin with one of two engineered amelogenins. Replacement changed enamel organization by altering protein-to-crystallite interactions and crystallite stacking while diminishing the ability of the ameloblast to interact with the matrix. These data demonstrate that ameloblasts must continuously interact with the developing matrix to provide amelogenin-specific protein to protein, protein to mineral, and protein to membrane interactions critical to biomineralization and enamel architecture while suggesting that mutations within conserved amelogenin domains could account for enamel variations preserved in the fossil record.

Immunohistochemical localization of transcription factor Sp3 during dental enamel development in rat tooth germ.

Sp3, a member of the Sp family of transcription factors, has previously been thought to be ubiquitously expressed, and its expression pattern in tooth development is not clear. This study was carried out to investigate the immunolocalization of Sp3 during the development of rat tooth germs. Sprague-Dawley rats at ages of 1, 3, 7, 10, and 14 d were used to represent different stages of tooth development. First mandibular molar tooth germs were sectioned and studied by immunohistochemistry. Sp3 was found to be localized within the nuclei of cells in developing tooth germs; however, ameloblast nuclei showed variable intensities at different developmental stages. At the same time, the positive signals in odontoblast nuclei remained stable. The results suggest that Sp3 may play a role in the development of teeth, specifically in the transcription of enamel-specific genes.

The dentin sialoprotein (DSP) expression in rat tooth germs following fluoride treatment: an immunohistochemical study.

UNLABELLED: Fluoride is known to alter expression of dentin matrix proteins and affect their posttranslational modifications. OBJECTIVE: The objective of our study was to examine dentin sialoprotein (DSP) expression in the early and late bell stages of development of the first molar tooth germs in rats treated with fluoride. DESIGN AND METHODS: Pregnant dumps were divided into three groups. They were fed a standard diet and from the fifth day of pregnancy, each group received either tap water (with trace amounts of fluoride), tap water with a low concentration of fluoride, or tap water with a high concentration of fluoride. Changes in DSP expression and distribution were visualized by immunohistochemistry. RESULTS: Immunoreactivity for DSP was detected in the cervical regions of the early bell stage in tooth germs of the 1-day-old animals. The earliest reaction was visible in the control group and the group supplemented with the low fluoride concentration (F(L)) but not in the group supplemented with the high fluoride concentration (F(H)). In early bell stages across all experimental groups, the immunoreactivity to DSP was observed in the cusp tip regions and was localized to preameloblasts, young and mature odontoblasts, dental pulp cells, predentin, and dentin. Generally, more intense positive staining for DSP was detected in animals supplemented with the high fluoride concentration. In the late bell stage found in the 4-day-old control group and the group supplemented with the low fluoride concentration, immunoreactivity for DSP was less intense compared with younger animals. However, immunoreactivity was greater in the group treated with the high dose of fluoride. In this group, the positive immunostaining for DSP, especially in young ameloblasts, was prolonged and relatively strong. CONCLUSIONS: Fluoride supplementation causes changes in the developmental pattern of DSP expression and its distribution in rat tooth germs.

Expression pattern of Dlx3 during cell differentiation in mineralized tissues.

The present study was designed to compare the expression pattern of Dlx3 in four different mineralized tissues because of: 1-its role in skeleton patterning, 2-its expression in dental epithelium and mesenchyme during morphogenesis, 3-the membranous and endochondral bone and tooth phenotype of tricho-dento-osseous syndrome related to Dlx3 gene mutation and 4-recently emerging knowledge on Dlx family members in the bone field. Ameloblasts, odontoblasts, osteoblasts and chondrocytes were analyzed in vitro and in vivo. Dlx3 transcripts were detected by RT-PCR in established model systems (microdissected dental epithelium and mesenchyme; primary cultures of rat chondrocytes), as recently performed in osteoblasts in vitro. A human 414-bp Dlx3 probe was generated. A 4.5-kb human Dlx3 sense RNA was identified in maxillo-facial samples by Northern blotting. Immunolabeling and in situ hybridization were performed in mice from Theiler stage E 14.5 until birth. In teeth, although Dlx3 was still expressed in differentiated ameloblasts, it was down regulated during odontoblast polarization. During endochondral bone formation, Dlx3 protein was detected in chondrocytes and was most strongly expressed in the prehypertrophic cartilage zone and in differentiating and differentiated osteoblasts of metaphyseal periosteum. In vitro, real-time PCR studies supported this upregulation in prehypertrophic chondrocytes, closely correlated with Ihh variations. In membranous bone, Dlx3 was present in preosteoblasts, osteoblasts and osteoid-osteocytes. The present data on Dlx3 and recently published functional studies show that this transcription factor may be instrumental during growth in the control of matrix deposition and biomineralization in the entire skeleton.

Histological and immunohistochemical studies of tissue engineered odontogenesis.

The successful regeneration of complex tooth structures based on tissue-engineering principles was recently reported. The process of this regeneration, however, remains poorly characterized. In this study, we have used histochemistry to examine the regeneration process of tissue engineered teeth in order to determine the cell types that give rise to these engineered tooth structures. Porcine third molar tooth buds were dissociated into single-cell suspensions and seeded onto a biodegradable polyglycolic acid polymer scaffold. Following varying periods of growth in rat hosts, the specimens were evaluated by histology and immunohistochemistry. Aggregates of epithelial cells were first observed 4-6 weeks after implantation. These aggregates assumed three different shapes: a natural tooth germ-like shape, a circular shape, or a bilayer-bundle. Based on the structure of the stellate reticulum in the dental epithelium, the circular and bilayer-bundle aggregates could be clearly classified into two types: one with extensively developed stellate reticulum, and the other with negligible stellate reticulum. The epithelial cells in the circular aggregates differentiated into ameloblasts. The continuous bilayer bundles eventually formed the epithelial sheath, and dentin tissue was evident at the apex of these bundles. Finally, enamel-covered dentin and cementum-covered dentin formed, a process most likely mediated by epithelial-mesenchymal interaction. These results suggest that the development of these engineered teeth closely parallels that of natural odontogenesis derived from the immature epithelial and mesenchymal cells.

Characteristic distribution of immunoreaction for estrogen receptor alpha in rat ameloblasts.

Estrogen has a diverse function, including cell proliferation and differentiation via estrogen receptors (ER), which have been reported to be the case in various tissues in addition to female reproductive organs. A recent immunocytochemical study has reported the expression of ERalpha, a subtype of ER, in rat odontoblasts, suggesting an involvement of estrogen in the differentiation of tooth-forming cells. However, there is no information on the ERalpha immunoexpression in ameloblasts. The present study was therefore undertaken to examine the localization of ERalpha immunoreaction in rat ameloblasts during amelogenesis. A computer-assisted quantitative analysis under a confocal laser scanning microscope was employed to demonstrate the stage-specific localization pattern of ERalpha immunoreaction. Immunohistochemistry of the rat enamel organ revealed ERalpha expression as nuclear localization in ameloblasts, stratum intermedium, stellate reticulum, and papillary layer, in addition to mature and immature odontoblasts. The ratio of immunopositive nuclei to total nuclei (immunopositive ratio) in ameloblasts was high at the apical loop region and gradually declined at the presecretory stage to zero at the secretory stage with statistically significant difference. The ERalpha immunolabeling pattern exhibited a periodic change at the maturation stage proper with constant higher labeling in ruffle-ended ameloblasts than in smooth-ended ameloblasts. The positive ratio was then followed by a statistically significant increase in immunolabeling thereafter. This stage-specific immunolabeling pattern during amelogenesis suggests a possible role of ERalpha in ameloblast proliferation and differentiation.

Rat enamel contains RP59: a new context for a protein from osteogenic and haematopoietic precursor cells.

We have recently identified a protein, RP59, in bone marrow cells and young osteoblasts, in cells involved in bone repair and in young erythroblasts and megakaryocytes. Here, we report immunohistochemical data at the light- and electron-microscope level indicating that RP59 is also present in newly secreted tooth enamel of the rat and in ameloblasts, the formative cells. In enamel matrix, RP59 was located proximal to secretory ameloblasts only, i.e. in newly secreted material. Distal enamel and enamel in association with maturation stage ameloblasts were unlabelled. Secretory ameloblasts contained RP59 in the matrix-proximal region including Tomes' processes, post-secretory ameloblasts in the cell-matrix interface. Western blotting of proteins from tooth germs identified RP59 as a band at 90 kD, co-migrating with RP59 from bone marrow and spleen. Antisera versus a chemically synthesised RP59 peptide and versus a bacteria-synthesised protein fragment reacted in the same manner. In situ hybridisation of tooth tissue revealed RP59 RNA specifically in ameloblasts. The reverse transcription/polymerase chain reaction method identified tooth RNA coding for RP59. Sequence analysis indicated that RP59 RNA from tooth and marrow had the same sequence. An internal sequence motif was found in rat RP59 resembling a signal implicated in secretion of the chicken "engrailed" gene product. The findings indicate that RP59 is a genuine product of ameloblasts and that it is secreted in the course of enamel formation together with other matrix components.

Immunohistochemical evidence for proteolipid protein and nestin expression in the late bell stage of developing rodent teeth.

In this study, the expression of proteolipid protein (PLP) and nestin is studied in the late bell stage of developing rodent teeth in neonatal rats. By using immunohistochemistry, it was shown that odontoblasts, ameloblasts and the stratum intermedium are positive for PLP in regions of active matrix deposition. Reactivity for nestin could be detected in the odontoblasts, stratum intermedium and in some of the apical processes of the ameloblasts. The fact that mutations in the PLP gene can cause disturbances in tooth form, number and eruption taken together with the presence of PLP reactivity in odontoblasts and ameloblasts of healthy animals, suggests a crucial role for PLP in developing teeth because of its structural supportive characteristics. These results also imply the possible use of PLP antibody as a new marker for, respectively, dentin and enamel-secreting odontoblasts and ameloblasts. PLP and nestin expression could point to a possible similarity in function between the oligodendrocyte and the odontoblast, both derived from the neural crest. To compare with the situation in human tissue, PLP and nestin expression were preliminarily tested on human dental pulp. The odontoblasts were positive for both PLP and nestin.

The expression pattern of hyaluronan synthase during human tooth development.

In previous studies, hyaluronan (HA) and its major cell surface receptor CD44 have been suggested to play an important role during tooth development. HA synthases (HASs) are the enzymes that polymerize hyaluronan. Data on the expression pattern of HASs during tooth development is lacking and the aim of the present study was to investigate the localisation of HAS by immunohistochemistry in human tooth germs from different developmental stages. The distribution pattern of HAS in the various tissues of the "bell stage" tooth primordia corresponded to that of hyaluronan in most locations: positive HAS immunoreactivity was observed in the dental lamina cells, inner- and outer-enamel epithelium. On the stellate reticulum cells, moderate HAS signal was observed, similar to the layers of the oral epithelium, where faint HAS immunoreactivity was detected. At the early phase of dental hard tissues mineralization, strong HAS immunoreactivity was detected in the odontoblasts and their processes, as well as in the secretory ameloblasts and their apical processes and also, the pulpal mesenchymal cells. The HAS signals observed in odontoblasts and ameloblasts gradually decreased with age. Our results demonstrate that hyaluronan synthesised locally by different dental cells and these results provide additional indirect support to the suggestion that HA may contribute both to the regulation of tooth morphogenesis and dental hard tissue formation.

Overexpression of TRAP in the enamel matrix does not alter the enamel structural hierarchy.

The secreted, full-length amelogenin is the dominant protein of the forming enamel organ. As enamel mineralization progresses, amelogenin is quickly subjected to proteolytic activity, and eliminated from the enamel environment. Mature enamel contains only traces of structural proteins, including enamelin and the sheath protein ameloblastin. In addition, a proteolytic fragment of amelogenin, known as the tyrosine-rich amelogenin peptide or TRAP, is present in low but isolatable quantities. By overexpressing TRAP during enamel development we sought to determine if such overexpression would result in structural alterations to the mature enamel. We reasoned that overexpressing a protein associated with enamel maturation, at an inappropriate developmental stage, would result in alterations to the enamel protein assembly and hence, alterations in enamel structure and morphology. As judged by transmission and scanning electron microscopy, the enamel formed by overexpressing TRAP showed little morphological differences when compared to the enamel of normal nontransgenic animals. Based on scanning electron-microscopic images, there was modest hypomineralization evident in the interrod enamel of the TRAP-overexpressing animals. However, this finding was inconsistent and inconsequential from a structural and functional perspective. From these results it appears that additional amounts of TRAP protein in the immature enamel matrix are not sufficient to alter the properties of the enamel extracellular matrix to an extent that the hierarchical structure of mature enamel is altered.

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.

New insight into progenitor/stem cells in dental pulp using Col1a1-GFP transgenes.

In recent years there has been increasing progress in identifying stem cells from adult tissues and their potential application in tissue engineering. These advances provide a promising future for tooth replacement/regeneration. Essential for this approach is the identification of donor stem cells for various components of the teeth. Our studies show that pOBCol3.6GFPtpz and pOBCol2.3GFPemd transgenic animals provide a unique model to gain insight into stem cells in the dental pulp. Our in vivo studies of the developing teeth of these transgenic lines show both Col1a1-GFP transgenes are expressed in functional and fully differentiated odontoblasts. The patterns of expression of Col1a1-GFP transgenes during odontoblast differentiation correlates with the expression of DSPP. In the developing craniofacial bones both Col1a1-GFP transgenes are also expressed in osteoblasts and osteocytes of alveolar and calvarial bones. In the alveolar bones, the expression of Col1a1-GFP in osteocytes correlates with the expression of DMP1. Col1a1-3.6-GFP is expressed in the entire layer of the periosteum and in suture mesenchyme containing osteoprogenitor cells. On the other hand, Col1a1-2.3- GFP expression was limited to the osteoblastic layer of the periosteum and was not detected in the fibroblastic layer of the periosteum or in the suture mesenchyme. These observations indicate that Col1a1-3.6-GFP and Col1a1-2.3-GFP transgenes identify different subpopulations of cells during intramembranous ossification. By using the coronal portion of dental pulps isolated from postnatal transgenic mice our observations also provide direct evidence that the dental pulp contains progenitor/stem cells capable of giving rise to a new generation of odontoblast-like cells, as well as osteoblast-like cells.