Fate map of the dental mesenchyme: dynamic development of the dental papilla and follicle.

At the bud stage of tooth development the neural crest derived mesenchyme condenses around the dental epithelium. As the tooth germ develops and proceeds to the cap stage, the epithelial cervical loops grow and appear to wrap around the condensed mesenchyme, enclosing the cells of the forming dental papilla. We have fate mapped the dental mesenchyme, using in vitro tissue culture combined with vital cell labelling and tissue grafting, and show that the dental mesenchyme is a much more dynamic population then previously suggested. At the bud stage the mesenchymal cells adjacent to the tip of the bud form both the dental papilla and dental follicle. At the early cap stage a small population of highly proliferative mesenchymal cells in close proximity to the inner dental epithelium and primary enamel knot provide the major contribution to the dental papilla. These cells are located between the cervical loops, within a region we have called the body of the enamel organ, and proliferate in concert with the epithelium to create the dental papilla. The condensed dental mesenchymal cells that are not located between the body of the enamel organ, and therefore are at a distance from the primary enamel knot, contribute to the dental follicle, and also the apical part of the papilla, where the roots will ultimately develop. Some cells in the presumptive dental papilla at the cap stage contribute to the follicle at the bell stage, indicating that the dental papilla and dental follicle are still not defined populations at this stage. These lineage-tracing experiments highlight the difficulty of targeting the papilla and presumptive odontoblasts at early stages of tooth development. We show that at the cap stage, cells destined to form the follicle are still competent to form dental papilla specific cell types, such as odontoblasts, and produce dentin, if placed in contact with the inner dental epithelium. Cell fate of the dental mesenchyme at this stage is therefore determined by the epithelium.

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.

Epiprofin/Sp6 regulates Wnt-BMP signaling and the establishment of cellular junctions during the bell stage of tooth development.

Epiprofin/Specificity Protein 6 (Epfn) is a Krüppel-like family (KLF) transcription factor that is critically involved in tooth morphogenesis and dental cell differentiation. However, its mechanism of action is still not fully understood. We have employed both loss-of-function and gain-of-function approaches to address the role of Epfn in the formation of cell junctions in dental cells and in the regulation of junction-associated signal transduction pathways. We have evaluated the expression of junction proteins in bell-stage incisor and molar tooth sections from Epfn(-/-) mice and in dental pulp MDPC-23 cells overexpressing Epfn. In Epfn(-/-) mice, a dramatic reduction occurs in the expression of tight junction and adherens junction proteins and of the adherens-junction-associated ß-catenin protein, a major effector of canonical Wnt signaling. Loss of cell junctions and ß-catenin in Epfn(-/-) mice is correlated with a clear decrease in bone morphogenetic protein 4 (BMP-4) expression, a decrease in nestin in the tooth mesenchyme, altered cell proliferation, and failure of ameloblast cell differentiation. Overexpression of Epfn in MDPC-23 cells results in an increased cellular accumulation of ß-catenin protein, indicative of upregulation of canonical Wnt signaling. Together, these results suggest that Epfn enhances canonical Wnt/ß-catenin signaling in the developing dental pulp mesenchyme, a condition that promotes the activity of other downstream signaling pathways, such as BMP, which are fundamental for cellular induction and ameloblast differentiation. These altered signaling events might underlie some of the most prominent dental defects observed in Epfn(-/-) mice, such as the absence of ameloblasts and enamel, and might throw light on developmental malformations of the tooth, including hyperdontia.

Developmentally regulated expression of intracellular Fgf11-13, hormone-like Fgf15 and canonical Fgf16, -17 and -20 mRNAs in the developing mouse molar tooth.

OBJECTIVE: To investigate and compare the cellular expression of non-secreted Fgf11-14 and secreted Fgf15-18 and -20 mRNAs during tooth formation. MATERIALS AND METHODS: mRNA expression was analyzed from the morphological initiation of the mouse mandibular first molar development to the onset of crown calcification using sectional in situ hybridization. RESULTS: This study found distinct, differentially regulated expression patterns for the Fgf11-13, -15-17 and -20, in particular in the epithelial-mesenchymal interface, whereas Fgf14 and 18 mRNAs were not detected. Fgf11, -15, -16, -17 and -20 were seen in the epithelium, whereas Fgf12 and -13 signals were restricted to the mesenchymal tissue component of the tooth. Fgf11 was observed in the putative epithelial signaling areas, the tertiary enamel knots and enamel free areas of the calcifying crown. Fgf15, Fgf17 and -20 were transiently colocalized in the thickened dental epithelium at E11.5. Later Fgf15 and -20 were exclusively expressed in the epithelial enamel knot signaling centers. In contrast, Fgf13 was present in the dental mesenchyme including odontoblasts cell lineage, whereas Fgf12 appeared transiently in the preodontoblasts. CONCLUSIONS: The expression of the Fgf11-13, -15, -17 and -20 in the epithelial signaling centers and/or epithelial-mesenchymal interfaces at key stages of the tooth formation suggest important functions in odontogenesis. Future analyses of the transgenic mice will help elucidate in vivo functions of the studied Fgfs during odontogenesis and whether any of the functions of the tooth expressed epithelial and mesenchymal Fgfs of different sub-families are redundant.

Expression of prion gene and presence of prion protein during development of mouse molar tooth germ.

In order to gain insight into possible cellular functions of the prion protein (PrP) during normal development, the expression of Prnp (encoding the PrP) and the distribution of the PrP were studied in murine tooth germs. Expression of Prnp in the mouse first molar tooth germ was highly dynamic, increasing several-fold during the secretory phase of odontogenesis, exhibiting a time-course of expression similar to that of genes coding for other extracellular proteins [e.g. enamel matrix proteins (Amelx, Ambn, Enam), Aplp1, Clstn1, and Clu]. Western blot analysis suggested that the amounts of PrP and amyloid beta (A4) precursor-like protein 1 (APLP1) in the tooth germ followed time-courses similar to those of the corresponding mRNAs. Immunohistochemical studies of the distribution of PrP in murine molar and incisor tooth germs at embryonic day (E)18.5 suggested that this protein was located in the cervical loop, outer enamel epithelium, pre-ameloblasts, and dental papilla. Different degrees of immunolabelling of pre-ameloblasts on the mesial and distal aspects of a lower molar cusp may be related to different enamel configurations on the two aspects. It is concluded that the dynamic patterns of expression of Prnp, and of distribution of PrP, suggest that PrP may have functions during secretory odontogenesis, perhaps in relation to amelogenesis.

Synthesis of collagen type I, type I trimer and type III by embryonic mouse dental epithelial and mesenchymal cells in vitro.

Epithelial and mesenchymal dental cells were grown in primary monolayer culture and the ability of both cell types to synthesize interstitial collagens was investigated. Pepsin-solubilized collagens were analyzed by CM-cellulose chromatography and both cell types were found to synthesize collagen type I, type III and type I trimer. The collagen phenotype of mesenchymal cells (type I: 82.4%, type III: 8.5%, type I trimer: 9.1%) was different from that of epithelial cells (type I: 71.8%, type III: 9.5%, type I trimer: 18.7%). The radioactivity incorporated into collagen molecules by mesenchymal cells was 34-times greater than the radioactivity incorporated by epithelial cells. This result agreed with previous observations obtained from tissue culture experiments (Lesot, H. and Ruch, J.V. (1979) Biol. Cell. 34, 23--37) which indicated a low synthesis of interstitial collagens by isolated dental epithelia when compared to isolated dental mesenchymes.

Slit1 is specifically expressed in the primary and secondary enamel knots during molar tooth cusp formation.

The shape and diversity of the mammalian molar teeth is suggested to be regulated by the primary and secondary enamel knots, which are putative epithelial signaling centers of the tooth. In search of novel molecules involved in tooth morphogenesis, we analyzed mRNA expression of Slit1, -2 and -3, earlier characterized as secreted signals needed for axonal pathfinding and their two receptors Robo1 and -2 (Roundabout1 and -2) in the developing mouse first molar. In situ hybridization analysis showed that Slit1 mRNAs were expressed in the primary enamel knot of the bud and cap stage tooth germ and later the expression continued in the secondary enamel knots of the late cap and bell stage tooth. In contrast, expression of Slit2 and -3 as well Robo1, and -2 was largely restricted to mesenchymal tissue components of the tooth until the bell stage. At the late bud stage, however, Robo1 transcripts were evident in the primary enamel knot, and at the cap stage a pronounced expression was noted in the middle of the tooth germ covering the primary enamel knot and dental papilla mesenchyme. During the bell stage, Robo1 and Slit2 expression became restricted to the dental epithelia, while Slit3 continued in the dental mesenchyme. Prior to birth, Robo1 and -2 were co-localized in the predontoblasts. These results indicate that Slits and Robos display distinct, developmentally regulated expression patterns during tooth morphogenesis. In addition, our results show that Slit1 is the second known gene specifically located in the primary and secondary enamel knots.

The early distribution and possible role of nerves during odontogenesis.

Neural crest cells migrate along specific pathways to reach the mandibular and maxillary arches where they condense under specific areas of the ectoderm which will give rise to the primary and permanent dentition. In the mouse, the trigeminal ganglion becomes evident on E9 and the superior cervical sympathetic ganglion E13. Several studies have suggested that nerves in the vicinity of the developing teeth could influence the surrounding tissues and initiate tooth development, whereas other investigators have suggested that tooth development will proceed without an intact innervation. Innervation of the dental papilla has been reported as early as the cap stage in human teeth using an antibody to PGP 9.5. A large variety of putative neurotransmitters have been localized in the nerves of the dental pulp. Many of the putative neurotransmitters function in vasoregulation while others have unknown functions. A hypothesis is presented describing a possible signal transduction pathway between odontoblasts and nerve terminals.

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.

Analysis of expression patterns of IGF-1, caspase-3 and HSP-70 in developing human tooth germs.

AIMS: To analyze expression patterns of IGF-1, caspase-3 and HSP-70 in human incisor and canine tooth germs during the late bud, cap and bell stages of odontogenesis. MATERIALS AND METHODS: Head areas or parts of jaw containing teeth from 10 human fetuses aged between 9th and 20th developmental weeks were immunohistochemically analyzed using IGF-1, active caspase-3 and HSP-70 markers. Semi-quantitative analysis of each marker's expression pattern was also performed. RESULTS: During the analyzed period, IGF-1 and HSP-70 were mostly expressed in enamel organ. As development progressed, expression of IGF-1 and HSP-70 became more confined to differentiating tissues in the future cusp tip area, as well as in highly proliferating cervical loops. Few apoptotic bodies highly positive to active caspase-3 were observed in enamel organ and dental papilla from the cap stage onward. However, both enamel epithelia moderately expressed active caspase-3 throughout the investigated period. CONCLUSIONS: Expression patterns of IGF-1, active caspase-3 and HSP-70 imply importance of these factors for early human tooth development. IGF-1 and HSP-70 have versatile functions in control of proliferation, differentiation and anti-apoptotic protection of epithelial parts of human enamel organ. Active caspase-3 is partially involved in formation and apoptotic removal of primary enamel knot, although present findings might reflect its ability to perform other non-death functions such as differentiation of hard dental tissues secreting cells and guidance of ingrowth of proliferating cervical loops.

Xenografts of recombined bovine odontogenic tissues and cultured cells to hypothymic mice.

It was demonstrated in this study that recombined tissues of bovine dental papilla and reduced enamel epithelium, when transplanted to the subcapsular kidney site of hypothymic mice, elaborated tissues resembling osteodentin and dentin matrices with which latter tissues were closely associated cells resembling odontoblasts. In xenografts of cultured cell populations of these tissues, osteodentin-like matrices occurred in the absence of dentin-like matrices.

Crown morphology and pattern of odontoblast differentiation in lower molars of tabby mice.

The Tabby mutation leads to abnormal crown morphology in the developing molars. To identify cusps which were altered in number, size, and position in the first lower molars of mutant mice, we analyzed the patterning of odontoblast differentiation using morphological criteria on serial sections and 3D reconstructions. In wildtype mice, polarized and functional odontoblasts were first observed in the median L2 and B2 cusps, then in the distal cusps L3 and B3, and finally in L1, B1, and 4. In Tabby mice, terminal differentiation of odontoblasts was retarded by 24-36 hours compared with wild-type mice. Polarized odontoblasts first appeared in the most mesial part of the tooth and progressively extended distally. The mesial part of the M1 in Tabby fetuses may correspond to the L2, B2 area from wild-type mice. The ante-molar dental primordium observed in some samples would thus represent remnants of cusps L1 and B1.

Behavior of odontoblasts and basal lamina of trypsin or EDTA-isolated mouse dental papillae in short-term culture.

Embryonic mouse first mandibular molars (day 18), containing the first overtly differentiated odontoblasts, were treated with EDTA or trypsin, to obtain isolated dental papillae. Trypsin dissociation was accompanied by disappearance of the basal lamina. EDTA-treatment removed the basal lamina from the basal surface of the epithelium, but left it associated with the dental papillae. EDTA- or trypsin-isolated dental papillae were grown for three to 24 h at the top of a plasma clot. Trypsin-isolated dental papillae rapidly lost their typical morphology, and the post-mitotic odontoblasts never became functional. EDTA-isolated dental papillae remained covered by the basal lamina for 15 to 18 h. During this period, the typical morphology was maintained, and post-mitotic odontoblasts secreted predentin. Preodontoblasts and odontoblasts went through the basal lamina and migrated at the outer surface of the basal lamina (i.e., at the side facing away from the enamel organ).

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 of innervation in primary incisors in the foetal period.

Sections from the frontal part of the mandible of 43 human foetuses from 9 to 39 weeks of prenatal age, which contained two, three and sometimes four lower incisors were immunohistochemically examined using protein gene product and neuron specific enolase (NSE) antibodies in order to establish the time of appearance of nerve fibres in the developing tooth germ and to define their topography. Nerve fibres were first detected in the dental follicle in the 11th week of intrauterine life. Their presence in the dental papilla was confirmed in the 18th week when the first layers of dentine and enamel were deposited. In the 24th week of intrauterine life, the nerve fibres first reached the subodontoblastic region. In the subsequent weeks, an increase in the number of nerve fibres accompanying blood vessels in the central portion of the dental papilla resulted in the formation of neuro-vascular bundles. Moreover, the progressive deposition of enamel and dentine was accompanied by branching of papillary nerves, which thereby formed a fan-pattern. In the foetal period, no evidence was found for the formation of a subodontoblastic plexus. However, we did observe single nerve fibres in close proximity to the odontoblast layer at the end of intrauterine life. Nerve fibres were not detected in either predentine or dentine throughout foetal life.

Transforming growth factor-beta 1 mRNA in neonatal ovine molars visualized by in situ hybridization: potential role for the stratum intermedium.

Human dentine contains relatively large amounts of transforming growth factor-beta (TGF-beta), which might originate from odontoblasts. The expression of the TGF-beta 1 message in developing teeth was examined by in situ hybridization. The analysis was made on 5-microns serial sections of mandibular third molars of neonatal sheep cut from tissues that had been fixed in glutaraldehyde and paraffin-embedded. A 35S-labelled cRNA probe, complementary to TGF-beta 1 mRNA, was constructed from human TGF-beta 1 cDNA. Northern analysis of total RNA from sheep placenta and neonatal third molars demonstrated hybridization to a single 2.4 kb TGF-beta 1 transcript from both tissues, indicating cross-reactivity of the human probe in the sheep. In the neonatal molars, in situ hybridization was observed in cells of the inner enamel epithelium, mature ameloblasts and mature odontoblasts, but not within preodontoblasts before dentine matrix formation. TGF-beta 1 mRNA expression was also evident in the cells of the dental papilla but scarcely so in the stellate reticulum. The most striking feature was the appearance of hybridization signal in the cells of the stratum intermedium before hybridization was evident in the inner enamel epithelium. Control sections incubated with RNAase before incubation with probe did not show evidence of hybridization. These findings suggest that TGF-beta 1 may have an important regulatory role in the differentiation of ameloblasts and odontoblasts, perhaps by modulating matrix formation during amelogenesis or odontogenesis. They also suggest a potential novel regulatory role for the cells of the stratum intermedium.

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.

Immunohistochemical localization of nerve fibres during development of embryonic rat molar using peripherin and protein gene product 9.5 antibodies.

Nerve fibres were localized during the initiation and early morphogenesis of the first molar tooth in rat embryos by immunoperoxidase detection of the intermediate-filament protein peripherin and protein gene product 9.5 (PGP 9.5). Nerve fibres from the trigeminal ganglion were detected in the developing first branchial arch of E12-14 embryos. Nerves were not seen in the vicinity of the developing tooth germ before the buid stage (E15), when they were seen around the condensed dental mesenchyme. During transition from the bud to the cap stage (E15), nerve fibres were detected not only in the area of the future dental follicle but also in the mesenchyme next to dental epithelium on the buccal side of the tooth germ. During later cap and bell stages nerve fibres persisted in the dental follicle, but they were not seen in the epithelial dental organ or dental papilla mesenchyme. Absence of trigeminal nerve fibres from the presumptive tooth-bearing area indicates that they are not involved in the initiation of rat tooth development. In addition, the localization of nerve fibres shows that there are some differences in the innervation of rat teeth compared with human and mouse teeth. These results provide data for further studies on the regulation of embryonic rat tooth innervation.

Bone morphogenetic protein-2 and -4 expression during murine orofacial development.

In the developing orofacial region, epithelial-mesenchymal interactions induce a differentiation cascade leading to bone and cartilage formation. Although the nature of this interaction is unknown, bone morphogenetic proteins (BMP)-2 and -4 have been suggested as putative signalling molecules. Using 35S-labelled cDNA probes, the expression patterns of BMP-2 and -4 mRNA were examined in murine perioral tissues preceding, during and following the time of the epithelial-mesenchymal interaction leading to mandibular formation. At embryonic age (e) 9.5 days, a restricted pattern of BMP-4 mRNA was expressed in the epithelium of the developing facial processes. This decreased rapidly, with little or no signal on E10.5 or E11.5. By E13.5, BMP-4 signal was restricted to the dental lamina, follicle and papilla. BMP-2 expression was not prominent in the developing face until E13.5. At this stage, signal was widespread throughout mesenchyme of neural-crest, but not somatic origin. Different domains of expression were present in the developing epithelium: for example, there was strong signal in the floor of the mouth and the ventral tongue, in contrast to that of the dorsum of the tongue and primary palate, which were negative. These results support the role of BMP-2 and -4 as regulators of orofacial development and demonstrates different fields of BMP-2 expression in developing oral mucosal epithelium.

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.