The histology of tooth formation in the Australian lungfish, Neoceratodus forsteri Krefft

The histology of developing toothplates of Neoceratodusforsteri from the time of first appearance of the tooth primordia to the adult condition has been investigated. The dentition develops by the formation of a shell of primary epithelial and mesenchymal matrices. Within the shell, secondary mesenchymal matrix and central material, both containing columns of tertiary matrix, are laid down. Primary epidielial matrix appears to contain collagen and is closely associated with the epithelium of the mouth. All other tooth tissues as well as the supporting bone develop in association with mesenchyme. Primary, secondary and tertiary mesenchymal matrices appear to contain collagen. Central dentine contains some fibres, possibly of reticulin or collagen, within a matrix of unknown composition.
The tooth is attached to the underlying bone by a pedestal of bone and this grows with the tooth material.
New tooth tissues are formed in the pulp cavity in layers below the older material, causing the toothplate to grow in every dimension as the animal grows.
An evolutionary pathway is suggested for lungfish with a dentition of cusps arranged in radiating ridges.     

Prismatic dentine in the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi)

The Australian lungfish, Neoceratodus forsteri, has a dentition consisting of enamel, mantle dentine and bone, enclosing circumdenteonal, core and interdenteonal dentines. Branching processes from cells that produce interdenteonal dentine leave the cell surface at different angles, with collagen fibrils aligned parallel to the long axis of each process. In the interdenteonal dentine, crystals of calcium hydroxyapatite form within fibrils of collagen, and grow within a matrix of non-collagenous protein. Crystals are aligned parallel to the cell process, as are the original collagen fibrils. Because the processes are angled to the cell surface, the crystals within the core or interdenteonal dentine are arranged in bundles set at angles to each other. Apatite crystals in circumdenteonal dentine are finer and denser than those of the interdenteonal dentine, and form outside the fibrils of collagen. In mature circumdenteonal dentine the crystals of circumdenteonal dentine form a dense tangled mass, linked to interdenteonal dentine by isolated crystals. The functional lungfish tooth plate contains prisms of large apatite crystals in the interdenteonal dentine and masses of fine tangled crystals around each denteon. This confers mechanical strength on a structure with little enamel that is subjected to heavy wear.     

Tooth development in a scincid lizard, Chalcides viridanus (Squamata), with particular attention to enamel formation

Comparative analysis of tooth development in the main vertebrate lineages is needed to determine the various evolutionary routes leading to current dentition in living vertebrates. We have used light, scanning and transmission electron microscopy to study tooth morphology and the main stages of tooth development in the scincid lizard, Chalcides viridanus, viz., from late embryos to 6-year-old specimens of a laboratory-bred colony, and from early initiation stages to complete differentiation and attachment, including resorption and enamel formation. In C. viridanus, all teeth of a jaw have a similar morphology but tooth shape, size and orientation change during ontogeny, with a constant number of tooth positions. Tooth morphology changes from a simple smooth cone in the late embryo to the typical adult aspect of two cusps and several ridges via successive tooth replacement at every position. First-generation teeth are initiated by interaction between the oral epithelium and subjacent mesenchyme. The dental lamina of these teeth directly branches from the basal layer of the oral epithelium. On replacement-tooth initiation, the dental lamina spreads from the enamel organ of the previous tooth. The epithelial cell population, at the dental lamina extremity and near the bone support surface, proliferates and differentiates into the enamel organ, the inner (IDE) and outer dental epithelium being separated by stellate reticulum. IDE differentiates into ameloblasts, which produce enamel matrix components. In the region facing differentiating IDE, mesenchymal cells differentiate into dental papilla and give rise to odontoblasts, which first deposit a layer of predentin matrix. The first elements of the enamel matrix are then synthesised by ameloblasts. Matrix mineralisation starts in the upper region of the tooth (dentin then enamel). Enamel maturation begins once the enamel matrix layer is complete. Concomitantly, dental matrices are deposited towards the base of the dentin cone. Maturation of the enamel matrix progresses from top to base; dentin mineralisation proceeds centripetally from the dentin–enamel junction towards the pulp cavity. Tooth attachment is pleurodont and tooth replacement occurs from the lingual side from which the dentin cone of the functional teeth is resorbed. Resorption starts from a deeper region in adults than in juveniles. Our results lead us to conclude that tooth morphogenesis and differentiation in this lizard are similar to those described for mammalian teeth. However, Tomes processes and enamel prisms are absent.     

Developmental regulation and ultrastructure of glycogen deposits during murine tooth morphogenesis

The distribution and ultrastructure of glycogen deposits were investigated in the murine tooth germ by histochemical periodic acid-Schiff (PAS) staining and transmission electron microscopy. Lower and upper first molars were examined in mouse embryos at embryonic days 11.5–17 (E11.5–E17) and in 2-day-old postnatal (P2) mice. The oral and dental epithelia and the mesenchymal cells were generally PAS-positive during tooth morphogenesis. PAS-negative cells were present at E13 in the distal tip of the tooth bud epithelium and in the contacting mesenchyme, and this complete lack of PAS reactivity continued in the dental papilla mesenchyme and inner enamel epithelium during the cap and bell stages. The lack of glycogen deposits in the interacting epithelium and mesenchyme during early morphogenesis may be associated with their demonstrated high signaling activities. Mesenchymal cells in the dental follicle consistently possessed small clusters or large pools of glycogen, which disappeared by P2. Since an intense PAS reaction was seen in mesenchymal cells at future bone sites, the glycogen in the dental follicle cells may be associated with their development into hard-tissue-forming cells. Ultrastructural observation of the enamel organ cells from the cap to early bell stages (E14–E15) revealed the occurrence of glycogen pools, which were associated with the Golgi apparatus and with vesicles having amorphous contents. Glycogen particles were also occasionally present inside vesicles or in the extracellular matrix. These may be associated with the exocytosis of glycosaminoglycan components into extracellular spaces and the formation of the stellate reticulum. Received: 9 November 1998 / Accepted: 17 January 1999     

Possible involvement of maspin in tooth development

Maspin is a 42 kDa serine protease inhibitor that possesses tumor suppressive and anti-angiogenic activities. Despite of a huge amount of data concerning the expression pattern of maspin in various tissues and its relevance to the biological properties of a variety of human cancer cells, little is known on the maspin expression in skeletal and tooth tissues. Recently, we reported that maspin may play an important role in extracellular matrix formation in bone by enhancing the accumulation of latent TGF-β in the extracellular matrix. This study was performed to elucidate the possible role of maspin in tooth development. First, an immunohistochemical analysis for human tooth germs at the late bell stage showed the expression of maspin by active ameloblasts and odontoblasts that were forming enamel and dentin, respectively. During rat tooth development, maspin expression was observed for the first time in inner and outer enamel epithelial cells and dental papilla cells at early bell stage. The neutralizing anti-maspin antibody inhibited the proper dental tissue formation in organ cultures of mandibular first molars obtained from 21-day-old rat embryos. In addition, the proliferation of HAT-7 cells, a rat odontogenic epithelial cell line, and human dental papilla cells were suppressed in a dose-dependent manner with anti-maspin antibody. Moreover, RT-PCR analysis showed that the expression of mRNA for tooth-related genes including dentin matrix protein 1, dentin sialophosphoprotein and osteopontin in human dental papilla cells was inhibited when treated with anti-maspin antibody. These findings suggest that maspin expressed in ameloblasts and odontoblasts plays an important physiological role in tooth development through the regulation of matrix formation in dental tissues.     

Immunolocalization of connexin 43 in the tooth germ of the neonatal rat

Summary Rabbit polyclonal antibodies to amino acids 346–360 of connexin 43, the ‘heart’ gap junction protein, were employed to immunolocalize connexin 43 gap junctions in the neonatal rat molar tooth germ. Connexin 43 appears early in the differentiation of both ectodermally derived and ectomesenchymally derived cells of the developing tooth. Connexin 43 immunoreactivity is present in the epithelial components of the enamel organ, including the area of the proximal and distal junctional complexes of the ameloblast layer, and the stratum intermedium, stellate reticulum and outer enamel epithelium. Secretory odontoblasts and developing alveolar bone also display a pattern of connexin 43 immunostaining. Both the epithelial and ectomesenchymally-derived components of the developing tooth acquire connexin 43 channels in a manner that correlates with cell differentiation. In addition, three regions can be defined by connexin 43 immunostaining: the epithelia of the enamel organ that are derived from the oral epithelium, the odontoblast layer derived from the ectomesenchyme, and the alveolar bone. The results suggest that connexin 43 may provide the mechanism for functional compartmentalization of the tissues associated with tooth formation. Compartmentalization suggested by connexin 43 expression could play important roles in the development and functions of these tissues.     

An ultrastructural study of dentinogenesis and amelogenesis in rat molar tooth germs cultured in vitro

Summary Molar tooth germs from three-day-old rats were cultured successfully for fourteen days, permitting the study of the development in vitro of both extracellular matrix and cellular elements such as odontoblasts and ameloblasts. The ultrastructure of the cultured tooth germs was compared with the ultrastructure of tooth germs in vivo at a comparable developmental stage. Progenitor cells of odontoblasts and ameloblasts were found to differentiate in vitro. Odontoblasts seemed to contain more lysosome-like bodies and fewer secretory granules than in vivo. They formed normally mineralizing dentine or a thick layer of dense, unmineralized predentine with incidentally some amorphous, extracellular material. Enamel was exclusively present opposite well developed dentine. It was often hyperor hypomineralized and enamel rods were not as regularly shaped as in vivo. In places where no enamel formation had taken place, large amounts of amorphous extracellular material were sometimes seen. From these observations it can be concluded that cellular development in cultured tooth germs appeared more or less normal, but extracellular matrix formation and mineralization were sometimes disturbed.     

Marginal tooth-bearing bones in the lower jaw of the recent australian lungfish,Neoceratodus forsteri (Osteichthyes,Dipnoi)

Developmental studies of the Recent Australian lungfish, Neoceratodus forsteri, show that this species has two sets of functional tooth-bearing bones in the lower jaw of young hatchlings. These coincide with an early stage in the life history when the fish is strictly carnivorous. In N. forsteri, a paired tooth-bearing dentary and an unpaired symphyseal bone and tooth develop slightly later than the permanent vomerine, prearticular, and pterygopalatine tooth plates, which appear at stage 44 of development, and erupt with the permanent dentition between stages 46 and 48, when the hatchling first starts to feed on small aquatic invertebrates. At these stages of development, all of the teeth are long, sharp, and conical and help to retain prey items in the mouth. Disappearance of the transient dentition coincides with complete eruption of the permanent tooth plates and precedes the change to an omnivorous diet. Existence of a transient marginal dentition in this species of lungfish suggests that the presence of an apparently similar marginal dentition in adults of many species of Palaeozoic dipnoans should be considered in phylogenetic analyses of genera within the group, and when analysing the relationships of dipnoans with other primitive animals. © 1995 Wiley-Liss, Inc.     

Fate mapping in embryos of Neoceratodus forsteri reveals cranial neural crest participation in tooth development is conserved from lungfish to tetrapods

Experimental evidence that the neural crest participates in tooth development in any osteichthyan fish has so far been lacking. Using vital dye cell-lineage tracking, we demonstrate that trigeminal stream neural crest cells contribute to the dental papilla of developing teeth in the Australian lungfish. Trigeminal neural crest cells labeled before migration have been traced during the earliest stages of tooth development. Neural crest cells from a single midbrain locus were relocated as ectomesenchyme in all developing teeth of the lungfish regardless of their topographical position in the dentition. These cells remain at the dental papilla interface and become cells committed to dentine production. Our findings provide the first cell-lineage evidence that cranial neural crest is fated to ectomesenchyme for tooth development and dentine production in the living sister-group to tetrapods. This shows that cranial neural crest contribution to teeth is conserved from this node on the tetrapod phylogeny.     

A simple,disposable, and improved organ culture system for maintaining three-dimensional development of mouse embryonic molars

Summary Mandibular first molars from 17-d-old mouse embryos were cultured in vitro for 2 to 4 d by a simple, disposable, improved floatation method. This method consisted of using a 24-well multidish and a plastic culture chamber with a membrane filter. The improved floatation method, as well as our previous method, was capable of the three-dimensional development of tooth germs. Cytodifferentiation of odontoblasts and ameloblasts and formation of extracellular matrices were accelerated by the present culture system, in comparison with our previous method. All the molars cultivated by this method were very similar in morphology to in vivo. On Day 2 of culture the terminal cytodifferentiation of odontoblasts and the formation of predentin were ascertained in the bucco-lingual sections of the cultured molars. A thick layer of predentin was formed at the tip of the cusp and gradually decreased toward the cervical loop and the fissure between the buccal and ligual cusps. On Day 4 in vitro, secretory ameloblasts produced enamel matrix, and the mineralized enamel showed prismatic structure very similar to that in vivo. Dentin and predentin also were normal in ultrastructure. The extracellular matrices (enamel, dentine, and predentin) were formed in line with the pattern of the cusp and the formation of matrices normally started at the tip of the cusp. We conclude that the three-dimensional development of whole tooth germs in vitro may be very important for normal expression of the developmental program intrinsic to mouse embryonic molars.     

High resolution transmission electron microscopy of developing enamel in the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi)

The permanent tooth plates of the Australian lungfish, Neoceratodus forsteri, are covered by enamel that develops initially in a similar manner to that of other vertebrates. As the enamel layer matures, it acquires several unusual characteristics. It has radially oriented protoprismatic structures with the long axes of the protoprisms perpendicular to the enamel surface. Protoprisms can be defined as aggregations of hydroxyapatite crystals that lack the highly ordered arrangement of the rods of mammalian enamel but are not without a specific structure of their own. The protoprisms are arranged in layers of variable thickness that are deposited sequentially as the tooth plate grows. They may be confined to the separate layers, or may cross the boundary between each layer. Crystals within the protoprisms are long and thin with hydroxyapatite c-axis dimensions of between 30 and 350 nm, and with typical a-b axis dimensions of 5-10 nm. The hydroxyapatite crystals of lungfish enamel have no centre dark lines of octacalcium phosphate, an unusual character among vertebrates. As each crystal develops, arrays of atoms may change direction, and regions exist where dislocations and extra lattice planes are inserted into the long crystal. The resulting hydroxyapatite crystal is not straight, and has a rough surface. The crystals are arranged in tangled structures with their crystallographic c-axes closely aligned with the long axis of the protoprism. Lungfish enamel differs from the enamel of higher vertebrates in that the hydroxyapatite crystals are of different shape, and, in mature enamel, the protoprisms remain as protoprisms and do not develop into the conventional prismatic structures characteristic of mammalian enamel.     

True enamel covering in teeth of the Australian lungfish Neoceratodus forsteri

Lungfish are a unique order of sarcopterygian fish cleidographically positioned between tetrapods and fish. An uninterrupted 400-million-year-old fossil record has documented lungfish skeletal elements to remain virtually unchanged since the Early Devonian. In the current study we investigated the enamel layer of lungfish teeth in order to determine whether there was evidence for higher vertebrate "true" enamel in the Australian lungfish. Juvenile lungfish from the Brisbane River were processed for light and electron microscopy and analyzed for parameters indicative of true enamel formation. Using anti-amelogenin primary antibodies for immunodetection and Western blots, enamel protein epitopes were detected in developing lungfish teeth. Using transmission electron microscopy and electron diffraction analysis, long and parallel-oriented hydroxyapatite crystals were observed in lungfish outer tooth coverings. Our findings indicate that Australian lungfish teeth are covered by a layer of true enamel. Based on the lungfish fossil record we conclude that features of true enamel formation may be as old as 400 million years. Based on taxonomic classification we confirm that true enamel is found not only in tetrapods but also in the sarcopterygian clade of the Gnathostomata.     

Development of the dentition in Alligator mississippiensis: upper jaw dental and craniofacial development in embryos, hatchlings, and young juveniles, with a comparison to lower jaw development

Development of the upper dentition in Alligator mississippiensis was investigated using a close series of accurately staged and aged embryos, hatchlings, and young juveniles up to 11 days posthatching, as well as some young and old adult specimens. Studies from scanning electron microscopy, light microscopy, acetate and computer reconstructions, radiography and macroscopy were combined to elucidate the details of embryonic dental development, tooth initiation pattern, dentitional growth, and erupted functional dentition. The results were compared with those from the lower jaw and related to the development of other craniofacial structures. Approximately 17 early teeth in each jaw half develop as surface teeth, of which 13 project for 1 to 12 days before sinking into the mesenchyme. The first three teeth initiate directly from the oral epithelium at Ferguson stages 14-15 (days 15-19 after egg laying), before there is any local trace of dental lamina formation. All other teeth develop from a dental prolamina or lamina; and with progressive lamina development, submerged teeth initiate from the aboral end leading to the formation of replacement teeth. All teeth form dentin matrix, but 12 early teeth do not form enamel. Approximately 20 embryonic teeth are resorbed, 6 are transitional, and 42 function for longer periods after hatching. The embryonic tooth initiation pattern (illustrated by defining a tooth position formula) does not support the previous models of Odontostichi, Zahnreihen, and Tooth Families, each of which postulates perfect regularity. Up to three interstitial tooth positions develop between sites of primary tooth initiation, and families with up to five generations at hatching are at first arbitrarily defined.     

Distribution of intermediate-filament proteins in the human enamel organ: unusually complex pattern of coexpression of cytokeratin polypeptides and vimentin

We applied immunohistochemical techniques and gel electrophoresis to examine the distribution of intermediate filaments in human fetal oral epithelium and the epithelia of the human enamel organ. Both methods demonstrated that human enamel epithelia contain cytokeratins 5, 14, and 17, which are typical of the basal cells of stratified epithelia, as well as smaller quantities of cytokeratins 7, 8, 19, and in trace amounts 18, which are characteristic components of simple epithelial cells. In the external enamel epithelium and stellate-reticulum cells, most of these components appeared to be simultaneously expressed. In contrast, the parental oral epithelium was negative for cytokeratin 7, thus indicating possible "neoexpression" during the course of tooth formation. Immunohistochemical procedures using various monoclonal antibodies against vimentin revealed the transient coexpression of vimentin and cytokeratins in the external enamel epithelium and in stellate-reticulum cells during enamel development. The significance of the coexpression of cytokeratins and vimentin is discussed in relation to previous findings obtained in other normal tissues and in the light of the functional processes characteristic of these epithelia.     

The pattern of tooth plate formation in the Australian lungfish, Neoceratodus forsteri Krefft

Tooth plate formation in the Queensland lungfish, Neoceratodus forsteri, Krefft begins with simple groups of isolated cusps, three in each tooth plate. The cusps fuse in ridges radiating from a point situated posterolingually. During growth, cusps are added to the labial ends of the ridges, and more ridges are added posteriorly, giving a total of seven in each tooth plate. Each tooth grows in thickness by the addition of layers of material, in line with the new cusps, beneath the tooth plate. The tooth plate grows outwards and is resorbed from the inner angle at the same time. The crushing surface is formed by the growth of cusps between the ridges. Angles between the ridges become progressively smaller, and angles between more posterior ridges are consistently less than between more anterior ridges. Similar but less pronounced changes in angles between ridges occur in a fossil genus, Sagenodus inaequalis, examined for comparison.
Vomerine teeth grow in the same way, by fusion of isolated cusps and the addition of new cusps to one end (labial) of the tooth plate. Layers of material are also added beneath the tooth plate. The vomerine tooth plates are initially low-based with long cusps but develop into high-based low cusped incisiform tooth plates in fully grown adults.
The labial dentition of the lower jaw starts to develop like the vomerine teeth, but degenerates by stage (vi) of tooth development. The single medial tooth is resorbed even earlier.
The pattern of tooth plate formation described in this paper is consistent with illustrations published by Semon (1901) and Greil (1908, 1913) but the inferred developmental processes are different.
Implications of the results for the Zahnreihe hypothesis of Edmund and for the phylogeny of Dipnoi are discussed.     

Expression pattern of E‐cadherin during development of the first tooth in zebrafish (Danio rerio)

Although the importance of cell adhesion in morphogenesis is already known for quite some time, there are remarkably few studies on the distribution and function of adhesion molecules in tooth development. We have chosen the zebrafish to study the role of specific cell adhesion molecules in the development and renewal of teeth. Zebrafish lack an oral dentition but have pharyngeal teeth which are renewed throughout life. Here we focus on the expression of E (epithelial)‐cadherin during the development of the first tooth to develop in the dentition, ‘initiator tooth’ 4V¹. E‐cadherin is expressed exclusively in the pharyngeal epithelium and in the enamel organ throughout all stages of development of this first‐generation tooth. Further studies are needed to compare this expression pattern with protein distribution, both in this and other first‐generation teeth as well as in replacement teeth.     

The dentition and dental tissues of the agamid lizard, Uromastyx

The dentition of Uromastyx hardwicki was examined in a series of carefully prepared dry skulls and was found to be very different from that of other agamid lizards. The anatomy of the dentition undergoes great changes from the time of hatching to advanced age, but no evidence of tooth replacement could be found. Extension of the tooth rows by addition of larger teeth posteriorly, together with elongation of the premaxilla, and a characteristic pattern of wear are responsible for the condition seen in aged specimens.
The structure of the dental tissues was investigated by means of a variety of histological techniques including scanning electron microscopy and it is established that the enamel has prismatic structure like that of mammalian enamel. The mode of formation of enamel with and without prisms is described and the occurrence and significance of prismatic structure in reptilian dental enamel discussed.     

Initial enamel crystals are not spatially associated with mineralized dentine

During epithelial-mesenchymal interactions associated with mammalian tooth development, epithelially-derived and mesenchymally-derived extracellular matrix molecules form a discrete dentine-enamel junction. The developmental and molecular processes required to form this junction are not known. To address this problem we designed studies to test the hypothesis that ectodermally-derived epithelial cells synthesize and secrete enamel proteins which function to nucleate and regulate the growth of enamel calcium phosphate crystals. Initial enamel crystals were detected separate from the adiacent dentine. Electron-microprobe analyses revealed that early enamel crystals were octacalciumphosphate or tricalciumphosphate rather than hydroxyapatite. Thereafter, enamel crystals became confluent with the adjacent, albeit significantly smaller hydroxyapatite crystals associated with mineralized dentine. Therefore, we interpret our data to indicate that de novo enamel crystal nucleation and growth are independent from the mineralization processes characterized for dentine. We further argue that gene expression of enamel protein appears to have a constitutive function during early enamel formation and that supramolecular aggregates of amelogenin and enamelin provide the microenvironment for the nucleation and crystal growth of the initial enamel matrix.