Tooth morphogenesis and cell differentiation.

The tooth is one of the vertebrate organs in which development at the molecular level is beginning to be understood. Secreted signaling molecules have been identified that mediate sequential and reciprocal inductive interactions between the dental epithelium and mesenchyme. Transcription factors have been found that participate in these signaling cascades. A signaling or organizing center was recently discovered in the dental enamel knot that expresses the same signals as other organizing centers in the embryo, and which presumably regulates tooth shape. It has recently become evident that the signaling networks that operate in the development of mammalian teeth are similar to those that are involved in the development of other vertebrate organs.

A lipophilic iron chelator can replace transferrin as a stimulator of cell proliferation and differentiation.

Of the different growth supplements used in chemically defined media, only transferrin is required for differentiation of tubules in the embryonic mouse metanephros. Since transferrin is an iron-carrying protein, we asked whether iron is crucial for tubulogenesis. Differentiation of metanephric tubules both in whole embryonic kidneys and in a transfilter system was studied. The tissues were grown in chemically defined media containing transferrin, apotransferrin, the metal-chelator complex ferric pyridoxal isonicotinoyl hydrazone (FePIH), and excesses of ferric ion. Although we found that apotransferrin was not as effective as iron-loaded transferrin in promoting proliferation in the differentiating kidneys, excess ferric ion at up to 100 microM, five times the normal serum concentration, could not promote differentiation or proliferation. However, iron coupled to the nonphysiological, lipophilic iron chelator, pyridoxal isonicotinoyl hydrazone, to form FePIH, could sustain levels of cell proliferation and tubulogenesis similar to those attained by transferrin. Thus, the role of transferrin in cell proliferation during tubulogenesis is solely to provide iron. Since FePIH apparently bypasses the receptor-mediated route of iron intake, the use of FePIH as a tool for investigating cell proliferation and its regulation is suggested.

Syndecan and tenascin expression is induced by epithelial-mesenchymal interactions in embryonic tooth mesenchyme.

Morphogenesis of embryonic organs is regulated by epithelial-mesenchymal interactions associating with changes in the extracellular matrix (ECM). The response of the cells to the changes in the ECM must involve integral cell surface molecules that recognize their matrix ligand and initiate transmission of signal intracellularly. We have studied the expression of the cell surface proteoglycan, syndecan, which is a matrix receptor for epithelial cells (Saunders, S., M. Jalkanen, S. O'Farrell, and M. Bernfield. J. Cell Biol. In press.), and the matrix glycoprotein, tenascin, which has been proposed to be involved in epithelial-mesenchymal interactions (Chiquet-Ehrismann, R., E. J. Mackie, C. A. Pearson, and T. Sakakura. 1986. Cell. 47:131-139) in experimental tissue recombinations of dental epithelium and mesenchyme. Our earlier studies have shown that in mouse embryos both syndecan and tenascin are intensely expressed in the condensing dental mesenchyme surrounding the epithelial bud (Thesleff, I., M. Jalkanen, S. Vainio, and M. Bernfield. 1988. Dev. Biol. 129:565-572; Thesleff, I., E. Mackie, S. Vainio, and R. Chiquet-Ehrismann. 1987. Development. 101:289-296). Analysis of rat-mouse tissue recombinants by a monoclonal antibody against the murine syndecan showed that the presumptive dental epithelium induces the expression of syndecan in the underlying mesenchyme. The expression of tenascin was induced in the dental mesenchyme in the same area as syndecan. The syndecan and tenascin positive areas increased with time of epithelial-mesenchymal contact. Other ECM molecules, laminin, type III collagen, and fibronectin, did not show a staining pattern similar to that of syndecan and tenascin. Oral epithelium from older embryos had lost its ability to induce syndecan expression but the presumptive dental epithelium induced syndecan expression even in oral mesenchyme of older embryos. Our results indicate that the expression of syndecan and tenascin in the tooth mesenchyme is regulated by epithelial-mesenchymal interactions. Because of their early appearance, syndecan and tenascin may be used to study the molecular regulation of this interaction. The similar distribution patterns of syndecan and tenascin in vivo and in vitro and their early appearance as a result of epithelial-mesenchymal interaction suggest that these molecules may be involved in the condensation and differentiation of dental mesenchymal cells.

Tenascin is associated with chondrogenic and osteogenic differentiation in vivo and promotes chondrogenesis in vitro.

The tissue distribution of the extracellular matrix glycoprotein, tenascin, during cartilage and bone development in rodents has been investigated by immunohistochemistry. Tenascin was present in condensing mesenchyme of cartilage anlagen, but not in the surrounding mesenchyme. In fully differentiated cartilages, tenascin was only present in the perichondrium. In bones that form by endochondral ossification, tenascin reappeared around the osteogenic cells invading the cartilage model. Tenascin was also present in the condensing mesenchyme of developing bones that form by intramembranous ossification and later was present around the spicules of forming bone. Tenascin was absent from mature bone matrix but persisted on periosteal and endosteal surfaces. Immunofluorescent staining of wing bud cultures from chick embryos showed large amounts of tenascin in the forming cartilage nodules. Cultures grown on a substrate of tenascin produced more cartilage nodules than cultures grown on tissue culture plastic. Tenascin in the culture medium inhibited the attachment of wing bud cells to fibronectin-coated substrates. We propose that tenascin plays an important role in chondrogenesis by modulating fibronectin-cell interactions and causing cell rounding and condensation.

Expression of Notch 1, 2 and 3 is regulated by epithelial-mesenchymal interactions and retinoic acid in the developing mouse tooth and associated with determination of ameloblast cell fate.

Notch 1, Notch 2, and Notch 3 are three highly conserved mammalian homologues of the Drosophila Notch gene, which encodes a transmembrane protein important for various cell fate decisions during development. Little is yet known about regulation of mammalian Notch gene expression, and this issue has been addressed in the developing rodent tooth during normal morphogenesis and after experimental manipulation. Notch 1, 2, and 3 genes show distinct cell-type specific expression patterns. Most notably, Notch expression is absent in epithelial cells in close contact with mesenchyme, which may be important for acquisition of the ameloblast fate. This reveals a previously unknown prepatterning of dental epithelium at early stages, and suggests that mesenchyme negatively regulates Notch expression in epithelium. This hypothesis has been tested in homo- and heterotypic explant experiments in vitro. The data show that Notch expression is downregulated in dental epithelial cells juxtaposed to mesenchyme, indicating that dental epithelium needs a mesenchyme-derived signal in order to maintain the downregulation of Notch. Finally, Notch expression in dental mesenchyme is upregulated in a region surrounding beads soaked in retinoic acid (50-100 micrograms/ml) but not in fibroblast growth factor-2 (100-250 micrograms/ml). The response to retinoic acid was seen in explants of 11-12-d old mouse embryos but not in older embryos. These data suggest that Notch genes may be involved in mediating some of the biological effects of retinoic acid during normal development and after teratogenic exposure.

Midkine (MK), a heparin-binding growth/differentiation factor, is regulated by retinoic acid and epithelial-mesenchymal interactions in the developing mouse tooth, and affects cell proliferation and morphogenesis.

Midkine (MK) is the first cloned gene in a new family of heparin-binding growth/differentiation factors involved in the regulation of growth and differentiation. We have analyzed the expression of MK mRNA and protein during tooth development in mouse embryos and studied the regulation of MK expression and the biological effects of MK protein in organ cultures. MK expression was restricted and preferential in the tooth area as compared to the rest of the developing maxillary and mandibular processes suggesting specific functions for MK during tooth morphogenesis. MK mRNA and protein were expressed during all stages of tooth formation (initiation, morphogenesis, and cell differentiation), and shifts of expression were observed between the epithelial and mesenchymal tissue components. However, the expression of mRNA and protein showed marked differences at some stages suggesting paracrine functions for MK. Tissue recombination experiments showed that MK gene and protein expression are regulated by epithelial-mesenchymal interactions, and, moreover, that dental tissue induces the ectopic expression of MK protein in non-dental tissue. The expression of MK gene and protein in the mandibular arch mesenchyme from the tooth region were stimulated by local application of retinoic acid in beads. Cell proliferation was inhibited in dental mesenchyme around the beads releasing MK, but this effect was modulated by simultaneous application of FGF-2. Morphogenesis and cell differentiation were inhibited in tooth germs cultured in the presence of neutralizing antibodies for MK, whereas the development of other organs (e.g., salivary gland, kidney) was unaffected. These results suggest important roles for MK in the molecular cascade that regulates tooth development.

High expression of 92-kD type IV collagenase (gelatinase B) in the osteoclast lineage during mouse development.

cDNA clones for murine 92 kD type IV collagenase (gelatinase B) were generated for the determination of its primary structure and for analysis of temporal and spatial expression in vivo. The mouse enzyme has 72% sequence identity with the human counterpart, the major difference being the presence of a 16-residue segment absent from the human enzyme. In situ hybridization analyses of embryonic and postnatal mouse tissues revealed intense signals in cells of the osteoclast cell lineage. Clear expression above background was not observed in macrophages, polymorphonuclear leukocytes, monocytes, or epithelial cells which have been shown to express the gene in vitro in cell cultures. Expression of the gene was first observed at early stage of cartilage and tooth development at E13, where signals were seen transiently in surrounding mesenchymal cells. At later developmental stages and postnatally strong expression was seen in large cells at the surface of bones. These cells were presumably osteoclasts as their location correlated with that of TRAP positive cells. Signals above background were not observed in a number of other tissues studied. The results represent the first demonstration of a highly osteoclast specific extracellular proteinase. The results suggest that during normal development of embryonic organs the 92-kD type IV collagenase does not have a major role in basement membrane degradation, but is rather mainly used for the turnover of bone matrix, possibly as a gelatinase required for the removal of denatured collagen fragments (gelatin) generated by interstitial collagenase.

Induced expression of syndecan in healing wounds.

We have studied the expression of an integral cell surface proteoglycan, syndecan, during the healing of cutaneous wounds, using immunohistochemical and in situ hybridization methods. In normal mouse skin, both syndecan antigen and mRNA were found to be expressed exclusively by epidermal and hair follicle cells. After incision and subsequent suturing, remarkably increased amounts of syndecan on the cell surfaces of migrating and proliferating epidermal cells and on hair follicle cells adjacent to wound margins were noted. This increased syndecan expression was shown to be a consequence of greater amounts of syndecan mRNA. Induction was observed already 1 d after wounding, was most significant at the time of intense cell proliferation, and was still observable 14 d after incision. The migrating cells of the leading edge of the epithelium also showed enhanced syndecan expression, although clearly less than that seen in the proliferating epithelium. The merging epithelial cells at the site of incision showed little or no syndecan expression; increased syndecan expression, however, was detected during later epithelial stratification. When wounds were left unsutured, in situ hybridization experiments also revealed scattered syndecan-positive signals in the granulation tissue near the migrating epidermal sheet. By immunohistochemical analysis, positive staining in granulation tissue was observed around vascular endothelial cells in a subpopulation of growing capillaries. Induction of syndecan in granulation tissue both at the protein and mRNA levels was temporally and spatially highly restricted. Granulation tissue, which formed in viscose cellulose sponge cylinders placed under the skin of rats, was also found to produce 3.4 and 2.6 kb mRNA species of syndecan similar to that observed in the normal murine mammary epithelial cell line, NMuMG. These results suggest that syndecan may have a unique and important role as a cell adhesion and a growth factor-binding molecule not only during embryogenesis but also during tissue regeneration in mature tissues.

Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling.

The continuously growing mouse incisor is an excellent model to analyze the mechanisms for stem cell lineage. We designed an organ culture method for the apical end of the incisor and analyzed the epithelial cell lineage by 5-bromo-2'-deoxyuridine and DiI labeling. Our results indicate that stem cells reside in the cervical loop epithelium consisting of a central core of stellate reticulum cells surrounded by a layer of basal epithelial cells, and that they give rise to transit-amplifying progeny differentiating into enamel forming ameloblasts. We identified slowly dividing cells among the Notch1-expressing stellate reticulum cells in specific locations near the basal epithelial cells expressing lunatic fringe, a secretory molecule modulating Notch signaling. It is known from tissue recombination studies that in the mouse incisor the mesenchyme regulates the continuous growth of epithelium. Expression of Fgf-3 and Fgf-10 were restricted to the mesenchyme underlying the basal epithelial cells and the transit-amplifying cells expressing their receptors Fgfr1b and Fgfr2b. When FGF-10 protein was applied with beads on the cultured cervical loop epithelium it stimulated cell proliferation as well as expression of lunatic fringe. We present a model in which FGF signaling from the mesenchyme regulates the Notch pathway in dental epithelial stem cells via stimulation of lunatic fringe expression and, thereby, has a central role in coupling the mitogenesis and fate decision of stem cells.

Dependence of kidney morphogenesis on the expression of nerve growth factor receptor.

Nerve growth factor receptor (NGFR) serves as the binding site for the neurotrophic growth factors. Although NGFR has been found in several embryonic tissues outside the nervous system, the function of NGFR in embryogenesis of non-neuronal organs remains unknown. NGFR is transiently synthesized by embryonic rat kidney and disappears from nephrons upon their terminal differentiation. Anti-sense oligonucleotide inhibition of NGFR expression inhibits kidney morphogenesis. Therefore, NGFR is required not only for development of the nervous system, but also for differentiation of the kidney tubules.

Modulation of epithelial cell fate of the root in vitro.

Mouse molars are normally not capable of continuous growth. We hypothesized that the mouse molar has intrinsic potential to maintain the epithelial stem cell niche and assessed this potential by growth in vitro. Although the tooth germs flattened considerably, they developed a mineralized crown and a root. However, histologically, the root surface was composed of 3 structurally different regions affecting the fate of the dental epithelium. The anterior and posterior aspects maintained the morphological and molecular characteristics of the cervical loop of a continuously growing incisor, with a continuous layer of ameloblasts. The epithelium making contact with the supporting filter resembled Hertwig's epithelial root sheath. The top of the cultured molar exposed to air lacked epithelium altogether. We conclude that the fate of the epithelium is regulated by external cues influenced by culture conditions, and that the molar has the intrinsic capacity to grow continuously.

Inhibition of morphogenesis and stimulation of vascular proliferation in embryonic tooth cultures by a sarcoma growth factor preparation.

Sarcoma growth factor (SGF) induces proliferation and anchorage-independent growth of nonmalignant cells. It competes with epidermal growth factor (EGF) for the EGF-receptors at the cell surface. SGF-like factors have recently been isolated from embryos, suggesting that SGFs may represent embryonic forms of EGF. Therefore, we have tested whether SGF preparations affect organogenesis and differentiation of cultured embryonic tissues. The embryonic tooth rudiments were cultivated in the presence of SGF and EGF. Stimulation of vascularization was seen in both of these organ cultures. Therefore, we propose that endothelial cells may be target cells for SGF, and SGF may be involved in the control of vascularization during embryogenesis. SGF and also, to a certain extent, EGF profoundly inhibited morphogenesis and differentiation of the tooth germ, with concomitant stimulation of vascularization. Analysis of cell proliferation revealed that some cell types of the tooth germ did not respond to SGF by proliferation, while a stimulation by EGF was observed. Nevertheless, tooth morphogenesis was also slightly inhibited by EGF, suggesting that growth factors which enhance proliferation do not necessarily stimulate morphogenesis and differentiation. Since the SGF preparations contain several factors, the effects observed could be due to the action of one or more factors.

Signalling networks regulating dental development.

There has been rapid progress recently in the identification of signalling pathways regulating tooth development. It has become apparent that signalling networks involved in Drosophila development and development of mammalian organs such as the limb are also used in tooth development. Teeth are epithelial appendages formed in the oral region of vertebrates and their early developmental anatomy resembles that of other appendages, such as hairs and glands. The neural crest origin of tooth mesenchyme has been confirmed and recent evidence suggests that specific combinations of homeobox genes expressed in the neural crest cells may regulate the types of teeth and their patterning. Signalling molecules in the Shh, FGF, BMP and Wnt families appear to regulate the early steps of tooth morphogenesis and some transcription factors associated with these pathways have been shown to be necessary for tooth development. Several of the conserved signals are also transiently expressed in the enamel knots in the dental epithelium. The enamel knots are associated with the characteristic epithelial folding morphogenesis which is responsible for the development of tooth shape and it is currently believed that the enamel knots function as signalling centres regulating tooth shape development. The developing tooth has proven to be an excellent model in studies of the molecular basis of patterning and morphogenesis of organs and it can be expected that continuing studies will rapidly increase the understanding of these mechanisms.

Reiterative signaling and patterning during mammalian tooth morphogenesis.

Mammalian dentition consists of teeth that develop as discrete organs. From anterior to posterior, the dentition is divided into regions of incisor, canine, premolar and molar tooth types. Particularly teeth in the molar region are very diverse in shape. The development of individual teeth involves epithelial-mesenchymal interactions that are mediated by signals shared with other organs. Parts of the molecular details of signaling networks have been established, particularly in the signal families BMP, FGF, Hh and Wnt, mostly by the analysis of gene expression and signaling responses in knockout mice with arrested tooth development. Recent evidence suggests that largely the same signaling cascade is used reiteratively throughout tooth development. The successional determination of tooth region, tooth type, tooth crown base and individual cusps involves signals that regulate tissue growth and differentiation. Tooth type appears to be determined by epithelial signals and to involve differential activation of homeobox genes in the mesenchyme. This differential signaling could have allowed the evolutionary divergence of tooth shapes among the four tooth types. The advancing tooth morphogenesis is punctuated by transient signaling centers in the epithelium corresponding to the initiation of tooth buds, tooth crowns and individual cusps. The latter two signaling centers, the primary enamel knot and the secondary enamel knot, have been well characterized and are thought to direct the differential growth and subsequent folding of the dental epithelium. Several members of the FGF signal family have been implicated in the control of cell proliferation around the non-dividing enamel knots. Spatiotemporal induction of the secondary enamel knots determines the cusp patterns of individual teeth and is likely to involve repeated activation and inhibition of signaling as suggested for patterning of other epithelial organs.

Transient and recurrent expression of the Egr-1 gene in epithelial and mesenchymal cells during tooth morphogenesis suggests involvement in tissue interactions and in determination of cell fate.

We have analyzed the expression of early growth response gene (Egr-1) by mRNA in situ hybridization during mouse embryonic tooth development and in experimental recombinations of dental epithelium and mesenchyme. Egr-1 was transiently and recurrently expressed both in epithelial and mesenchymal cells starting from day 13 of gestation and up to 4 days after birth. The expression correlated with developmental transition points of dental mesenchymal and epithelial cells suggesting a role for Egr-1 in sequential determination and differentiation of cells. In recombination cultures of early dental epithelium and mesenchyme Egr-1 RNA was localized at the epithelial-mesenchymal interface in mesenchymal cells, and in two cases also in epithelial cells. These data indicate that Egr-1 expression may be regulated by epithelial-mesenchymal interactions when they are specific enough to initiate differentiation. We have also analyzed by in situ hybridization whether Wilms' tumour-1 gene (wt-1) is expressed in the developing tooth as it was proposed on the bases of in vitro studies that it may inhibit Egr-1 expression. No wt-1 expression was detected at any stage of tooth development showing that wt-1 is not obligatory for regulation of Egr-1 expression.

Shh, Bmp-2, Bmp-4 and Fgf-8 are associated with initiation and patterning of mouse tongue papillae.

Spacing patterns are of fundamental importance in various repeated structures which develop at regular intervals such as feathers, teeth and insect ommatidia. The mouse tongue develops a regular papilla pattern and provides a good model to study pattern formation. We examined the expression patterns of the signalling molecules, sonic hedgehog (Shh), bone morphogenetic proteins -2 and -4 (Bmp-2 and Bmp-4), and fibroblast growth factor-8 (Fgf-8) in mouse embryos between E 10.5 and 15. We show that all four genes are expressed uniformly in the tongue epithelium between E 10.5 and 11. At E 13, before morphologically detectable gustatory papillae initiation, Shh, Bmp-2 and Bmp-4 expression segregates into discrete spots, whereas, Fgf-8 is downregulated. At E 14, small eminences in the anterior part of the tongue are the first morphological indications of fungiform papillae, and they express Shh and Bmp-2, whereas, Bmp-4 is almost absent in the tongue. We conclude that these conserved signalling molecules are associated with the initiation and early morphogenesis of the tongue papillae.

The enamel knot as a signaling center in the developing mouse tooth.

Mammalian tooth forms are produced during development by folding of the enamel epithelium but the molecular mechanisms involved in the formation and patterning of tooth cusps are not understood. We now report that several key signaling molecules found in well-known vertebrate signaling tissues such as the node, the notochord, the apical ectodermal ridge, and the zone of polarizing activity in the limb bud are specifically expressed in cells of the enamel knot, which is a transient cluster of dental epithelial cells. By comparing three-dimensional reconstructions of serial sections following in situ hybridization we localized Sonic hedgehog, Bone morphogenetic proteins-2, -4 and -7, as well as Fibroblast growth factor-4 in nested domains within the enamel knot. We suggest that the enamel knot acts as a signaling or organizing center, which provides positional information for tooth morphogenesis and regulates the growth of tooth cusps.

Ectodysplasin, a protein required for epithelial morphogenesis, is a novel TNF homologue and promotes cell-matrix adhesion.

In the mouse Tabby (Ta) mutant and human X-linked anhidrotic ectodermal dysplasia (EDA) syndrome development of several ectodermal organs such as hair, teeth, and sweat glands is impaired. The gene behind Tabby and EDA has been cloned, and several alternative transcripts have been isolated. The protein product named ectodysplasin had no obvious function or prominent homology to other known gene products apart from a short collagen-like sequence. We have isolated two novel Ta transcripts which are variants of the longest isoform of Tabby, named Ta-A. In situ hybridizations revealed Ta-A to be the major transcript in the developing embryo. It was detected in the endoderm of early embryos and subsequently in specific locations in the neuroepithelium and ectoderm. Unexpectedly, sequence analysis of the most C-terminal domain of Ta revealed that ectodysplasin is a novel member of the tumor necrosis factor (TNF) ligand superfamily. Mouse ectodysplasin was biochemically and functionally characterized, and shown to be a glycosylated, oligomeric type II membrane protein (N-terminus inside), all characteristics typical to TNF-like proteins. Members of the TNF family are critically involved in host defence and immune response often mediating either apoptosis or cell survival. Expression of Ta in several epithelial cell lines did not result in prominent changes in cell morphology and did not promote apoptosis. Instead, it was shown to promote cell adhesion to extracellular matrix, a function consistent with its postulated role in epithelial-mesenchymal interactions regulating the development of ectodermal appendages. Ectodysplasin is the first TNF-like signaling molecule described known to be required for epithelial morphogenesis.

Runx2 (Cbfa1) inhibits Shh signaling in the lower but not upper molars of mouse embryos and prevents the budding of putative successional teeth.

Heterozygous mutations in the RUNX2 (CBFA1) gene cause cleidocranial dysplasia, characterized by multiple supernumerary teeth. This suggests that Runx2 inhibits successional tooth formation. However, in Runx2 knockout mice, molar development arrests at the late bud stage, and lower molars are more severely affected than upper ones. We have proposed that compensation by Runx3 may be involved. We compared the molar phenotypes of Runx2/Runx3 double-knockouts with those of Runx2 knockouts, but found no indication of such compensation. Shh and its mediators Ptc1, Ptc2, and Gli1 were down-regulated only in the lower but not the upper molars of Runx2 and Runx2/Runx3 knockouts. Interestingly, in front of the mutant upper molar, a prominent epithelial bud protruded lingually with active Shh signaling. Similar buds were also present in Runx2 heterozygotes, and they may represent the extension of dental lamina for successional teeth. The results suggest that Runx2 prevents the formation of Shh-expressing buds for successional teeth.