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Dental replacement in mammals is one of the most important evolutionary processes in these vertebrates. First mammals had many primitive features inherited from the reptilian ancestors. Already at a very early stage the teeth diversified into the incisors, canines, premolars, and molars. A tendency to reduce the tooth count also appeared. The groundbreaking innovation was the emergence of diphyodont replacement pattern (two generations of dentition, deciduous and permanent), which is a characteristic feature of mammals, in contrast to the polyphyodont reptiles. The evolution of dental exchange went towards the decreasing number of teeth being replaced. This paper aims at providing readers with information on main stages of the process initiated over 200 million years ago, which resulted in a notable diversification of both the dentition patterns and dental replacement types, observed in living mammals. Key words: dental replacement, diphyodonty, mammalian evolution, polyphyodonty

Odontomas are variably differentiated, hamartoma-like proliferations of odontogenic epithelium, pulp ectomesenchyme (odontoblasts), and dental matrix. Frogs are polyphyodont and homodont. Their teeth also differ from mammals in that they are restricted to the upper jaw in adults and lack a periodontal ligament and cementum, attaching directly to the underlying bone. Odontomas were identified in an African clawed frog ( Xenopus laevis), a false tomato frog ( Dyscophus guineti), and a tomato frog of unknown species ( Dyscophus sp.). All of the examined odontomas were composed of numerous tooth-like structures comprising an arc of dentinal matrix lined on the convex surface by ameloblasts and on the concave surface by odontoblasts. Masson’s trichrome and immunohistochemistry with pan-cytokeratin supported these findings. The pathogenesis of these lesions may be displacement of the dental lamina, which has been shown in research studies to lead to de novo proliferation of dental elements in frogs.

Many reptiles are able to continuously replace their teeth through life, an ability attributed to the existence of epithelial stem cells. Tooth replacement occurs in a spatially and temporally regulated manner, suggesting the involvement of diffusible factors, potentially over long distances. Here, we locally disrupted tooth replacement in the leopard gecko (Eublepharis macularius) and followed the recovery of the dentition. We looked at the effects on local patterning and functionally tested whether putative epithelial stem cells can give rise to multiple cell types in the enamel organs of new teeth. Second generation teeth with enamel and dentine were removed from adult geckos. The dental lamina was either left intact or disrupted in order to interfere with local patterning cues. The dentition began to reform by 1 month and was nearly recovered by 2–3 months as shown in μCT scans and eruption of teeth labeled with fluorescent markers. Microscopic analysis showed that the dental lamina was fully healed by 1 month. The deepest parts of the dental lamina retained odontogenic identity as shown by PITX2 staining. A pulse-chase was carried out to label cells that were stimulated to enter the cell cycle and then would carry BrdU forward into subsequent tooth generations. Initially we labeled 70–78% of PCNA cells with BrdU. After a 1-month chase, the percentage of BrdU + PCNA labeled cells in the dental lamina had dropped to 10%, consistent with the dilution of the label. There was also a population of single, BrdU-labeled cells present up to 2 months post surgery. These BrdU-labeled cells were almost entirely located in the dental lamina and were the likely progenitor/stem cells because they had not entered the cell cycle. In contrast fragmented BrdU was seen in the PCNA-positive, proliferating enamel organs. Homeostasis and recovery of the gecko dentition was therefore mediated by a stable population of epithelial stem cells in the dental lamina.

Synopsis In recent years, nonclassical models have emerged as mainstays for studies of evolutionary, developmental, and regenerative biology. Genomic advances have promoted the use of alternative taxa for the study of developmental biology, and the shark is one such emerging model vertebrate. Our research utilizes the embryonic shark (Scyliorhinus canicula) to characterize key developmental and regenerative processes that have been overlooked or not possible to study with more classic developmental models. Tooth development is a major event in the construction of the vertebrate body plan, linked in part with the emergence of jaws. Early development of the teeth and morphogenesis is well known from the murine model, but the process of tooth redevelopment and regeneration is less well known. Here we explore the role of the dental lamina in the development of a highly regenerative dentition in sharks. The shark represents a polyphyodont vertebrate with continuously repeated whole tooth regeneration. This is presented as a major developmental shift from the more derived renewal process that the murine model offers, where incisors exhibit continuous renewal and growth of the same tooth. Not only does the shark offer a study system for whole unit dental regeneration, it also represents an important model for understanding the evolutionary context of vertebrate tooth regeneration.

Deep understanding of tooth regeneration is hampered by the lack of lifelong replacing oral dentition in most conventional models. Here, we show that the bearded dragon, one of the rare vertebrate species with both polyphyodont and monophyodont teeth, constitutes a key model for filling this gap, allowing direct comparison of extreme dentition types. Our developmental and high-throughput transcriptomic data of microdissected dental cells unveils the critical importance of successional dental lamina patterning, in addition to maintenance, for vertebrate tooth renewal. This patterning process happens at various levels, including directional growth but also gene expression levels, dynamics, and regionalization, and involves a large number of yet uncharacterized dental genes. Furthermore, the alternative renewal mechanism of bearded dragon dentition, with dual location of slow-cycling cells, demonstrates the importance of cell migration and functional specialization of putative epithelial stem/progenitor niches in tissue regeneration, while expanding the diversity of dental replacement strategies in vertebrates., eLife digest All multicellular organisms, from lizards to humans, must be able to repair and regrow damaged tissue. This includes not only healing after an injury, but also replacing parts of the body that suffer wear and tear. For example, many animals shed and replace worn out teeth throughout their life, but the number of times this occurs varies greatly between species. Much of the understanding about how teeth grow and develop has come from researching mice. However, mice only develop one set of teeth, making them a poor ‘model’ for studying how species such as fish and reptiles can re-grow and replace their teeth. Recent studies of these species has shown that regenerating teeth relies on a specialised structure known as the dental lamina. In mice, the dental lamina forms but then quickly disappears, preventing new sets of teeth from developing. In most animals that regrow their teeth, however, the dental lamina keeps growing beyond the most recently produced tooth to create an area where its replacement will emerge. Now, Salomies et al. have identified other strategies involved in tooth replacement from studying the bearded dragon lizard, a rare example of an animal that continuously regenerates some, but not all, of its teeth. Analysing the cells in different parts of the re-growing teeth from bearded dragon lizards revealed new features of the dental lamina. Specifically, Salomies et al. found that a previously uncharacterized set of genes within the dental lamina could determine whether or not a tooth will be replaced. Further experiments using microscope imaging revealed that bearded dragon lizards use two distinct groups of stem cells – specialised cells that have the potential to develop into various cell types in the body – to re-grow their teeth. These experiments demonstrate how the bearded dragon lizard uses a previously unknown mechanism to regenerate its teeth, combining elements used by gecko lizards and sharks. These findings are an important step towards understanding the different strategies animals can use to maintain and regenerate their teeth. The knowledge gained could one day help design better therapies for patients suffering from inherited dental disorders or tooth loss.

Sharks and rays (elasmobranchs) have the remarkable capacity to continuously regenerate their teeth. The polyphyodont system is considered the ancestral condition of the gnathostome dentition. Despite this shared regenerative ability, sharks and rays exhibit dramatic interspecific variation in their tooth morphology. Ray (batoidea) teeth typically constitute crushing pads of flattened teeth, whereas shark teeth are pointed, multi-cuspid units. Although recent research has addressed the molecular development of the shark dentition, little is known about that of the ray. Furthermore, how dental diversity within the elasmobranch lineage is achieved remains unknown. Here, we examine dental development and regeneration in two Batoid species: the thornback skate (Raja clavata) and the little skate (Leucoraja erinacea). Using in situ hybridization and immunohistochemistry, we examine the expression of a core gnathostome dental gene set during early development of the skate dentition and compare it to development in the shark. Elasmobranch tooth development is highly conserved, with sox2 likely playing an important role in the initiation and regeneration of teeth. Alterations to conserved genes expressed in an enamel knot-like signalling centre may explain the morphological diversity of elasmobranch teeth, thereby enabling sharks and rays to occupy diverse dietary and ecological niches.

Summary Mammals and reptiles have evolved divergent adaptations for processing abrasive foods. Mammals have occluding, diphyodont dentitions with taller teeth (hypsodonty), more complex occlusal surfaces, continuous tooth eruption, and forms of prismatic enamel that prolong the functional life of each tooth [ 1 , 2 ]. The evolution of prismatic enamel in particular was a key innovation that made individual teeth more resilient to abrasion in early mammals [ 2 , 3 , 4 ]. In contrast, reptiles typically have thin, non-prismatic enamel, and shearing, polyphyodont dentitions with multi-cusped or serrated tooth crowns, multiple tooth rows, rapid tooth replacement rates, or batteries made of hundreds of teeth [ 5 , 6 , 7 , 8 , 9 ]. However, there are rare cases where reptiles have evolved alternative solutions to cope with abrasive diets. Here, we show that the combined effects of herbivory and an ancestral loss of tooth replacement in a lineage of extinct herbivorous sphenodontians, distant relatives of the modern tuatara (Sphenodon punctatus) [ 10 ], are associated with the evolution of wear-resistant and highly complex teeth. Priosphenodon avelasi, an extinct sphenodontian from the Cretaceous of Argentina, possesses a unique cone-in-cone dentition with overlapping generations of teeth forming a densely packed tooth file. Each tooth is anchored to its predecessor via a rearrangement of dental tissues that results in a novel enamel-to-bone tooth attachment. Furthermore, the compound occlusal surfaces, thickened enamel, and the first report of prismatic enamel in a sphenodontian are convergent strategies with those in some mammals, challenging the perceived simplicity of acrodont dentitions [ 11 , 12 , 13 , 14 , 15 ] and showcasing the reptilian capacity to produce complex and unusual dentitions.

The three major enamel matrix proteins (EMPs): amelogenin (AMEL), ameloblastin (AMBN), and enamelin (ENAM), are intrinsically linked to tooth development in tetrapods. However, reptiles and mammals exhibit significant differences in dental patterning and development, potentially affecting how EMPs evolve in each group. In most reptiles, teeth are replaced continuously throughout life, while mammals have reduced replacement to only one or two generations. Reptiles also form structurally simple, aprismatic enamel while mammalian enamel is composed of highly organized hydroxyapatite prisms. These differences, combined with reported low sequence homology in reptiles, led us to predict that reptiles may experience lower selection pressure on their EMPs as compared with mammals. However, we found that like mammals, reptile EMPs are under moderate purifying selection, with some differences evident between AMEL, AMBN, and ENAM. We also demonstrate that sequence homology in reptile EMPs is closely associated with divergence times, with more recently diverged lineages exhibiting high homology, along with strong phylogenetic signal. Lastly, despite sequence divergence, none of the reptile species in our study exhibited mutations consistent with diseases that cause degeneration of enamel (e.g. amelogenesis imperfecta). Despite short tooth retention time and simplicity in enamel structure, reptile EMPs still exhibit purifying selection required to form durable enamel.

The appearance, cellular distribution, and changes of sugar residues during tooth development in adults of the polyphyodont, Liolaemus gravenhorsti, were investigated by using horseradish-peroxidase-conjugate lectins (lectin-HRP). With Con A (Canavalia ensiformis), the ameloblasts (late bell stage) show granular supranuclear positivity and also at the Golgi zone and on their tomes process. Reactivity also appears at the apical surface of the odontoblasts and odontoblastic process. With WGA (Triticum vulgaris), the tooth germs (late bell stage) show cytoplasmatic granular positivity in the ameloblast cells, Golgi regions, and in a lesser extent of the cytoplasm. Also, the apical surface and the odontoblastic process react. WGA reaction is depressed following sialidase treatment. The significance in tooth germs of α-D-mannose, α-D-glucose as well as β-D-N-acetylglucosamine and sialic acid is difficult to ascertain. These oligosaccharides may have some significance in odontogenesis. In fact, Con A-HRP- and WGA-HRP-binding components in ameloblasts and odontoblasts may be functionally related to molecules that are thought to contribute to odontogenesis in lizards. © 1994 Wiley-Liss, Inc.

Tooth replacement in polyphyodont is a well-organized system for maintenance of homeostasis of teeth, containing the dynamic structural change in skeletal tissues such as the attachment bone, which is the supporting element of teeth. Histological analyses have revealed the character of tooth replacement, however, the cellular mechanism of how skeletal tissues are modified during tooth replacement is largely unknown. Here, we showed the important role of osteoblasts for controlling osteoclasts to modify the attachment bone during tooth replacement in medaka pharyngeal teeth, coupled with an osterix-DsRed/TRAP-GFP transgenic line to visualize osteoblasts and osteoclasts. In the turnover of the row of attachment bones, these bones were resorbed at the posterior side where most developed functional teeth were located, and generated at the anterior side where teeth were newly erupted, which caused continuous tooth replacement. In the cellular analysis, osteoclasts and osteoblasts were located at attachment bones separately, since mature osteoclasts were localized at the resorbing side and osteoblasts gathered at the generating side. To demonstrate the role of osteoclasts in tooth replacement, we established medaka made deficient in c-fms-a by TALEN. c-fms-a deficient medaka showed hyperplasia of attachment bones along with reduced bone resorption accompanied by a low number of TRAP-positive osteoclasts, indicating an important role of osteoclasts in the turnover of attachment bones. Furthermore, nitroreductase-mediated osteoblast-specific ablation induced disappearance of osteoclasts, indicating that osteoblasts were essential for maintenance of osteoclasts for the proper turnover. Taken together, our results suggested that the medaka attachment bone provides the model to understand the cellular mechanism for tooth replacement, and that osteoblasts act in the coordination of bone morphology by supporting osteoclasts.

We performed a test of how function impacts a genetically programmed process that continues into postnatal life. Using the dentition of the polyphyodont gecko as our model, tooth shedding was recorded longitudinally across the jaw. We compared two time periods: one in which teeth were patterned symmetrically in ovo and a later period when teeth were initiated post-hatching. By pairing shedding events on the right and left sides, we found the patterns of tooth loss are symmetrical and stable between periods, with only subtle deviations. Contralateral tooth positions shed within three to four days of each other in most animals (7/10). A minority of animals (3/10) had systematic tooth position shifts between right and left sides, likely due to changes in functional tooth number. Our results suggest that in addition to reproducible organogenesis of individual teeth, there is also a neotenic retention of jaw-wide dental patterning in reptiles. Finer analysis of regional asymmetries revealed changes to which contralateral position shed first, affecting up to one quarter of the jaw (10 tooth positions). Once established, these patterns were retained longitudinally. Taken together, the data support regional and global mechanisms of coordinating tooth cycling post-hatching.

Nearly all non-mammalian vertebrates are polyphyodont: that is, the teeth are replaced continually throughout life. Polyphyodonty has several functions. First, increases in number, size, or both of successive generations of teeth is an essential component of the growth of the dentition. Second, morphological changes in successive generations can allow exploitation of different food at different stages of the life cycle. Third, continual replacement compensates for wear. The teeth of elasmobranchs are connected to a common fibrous band which moves forward continuously, carrying all the teeth with it. Among amphibians and reptiles, a common pattern of replacement involves “waves” of replacement passing along the tooth row, and affecting alternate tooth loci. The number and pattern of functional teeth depends on the rapidity with which the waves propagate along the dentition. In bony fish, replacement sometimes occurs in waves, but other patterns, ranging from apparently random to near-simultaneous replacement of all teeth in a jaw quadrant, also occur. The number of replacing teeth at a tooth locus is directly related to the rate of tooth replacement. The development of the dentition requires initiation of teeth in a specific sequence and pattern and this is related to the pattern of tooth replacement. In mammals, it appears that these are controlled by differential expression of homeobox genes in different parts of the mouth. Less is known about non-mammalian vertebrates but research on reptiles and amphibians indicate that WNT is important in regulating the activities of stem cells in the dental lamina, and hence the rate of replacement.

The numerous cushion-shaped tooth-bearing plates attributed to the stem group osteichthyan Lophosteus superbus , which are argued here to represent an early form of the osteichthyan inner dental arcade, display a previously unknown and presumably primitive mode of tooth shedding by basal hard tissue resorption. They carry regularly spaced, recumbent, gently recurved teeth arranged in transverse tooth files that diverge towards the lingual margin of the cushion. Three-dimensional reconstruction from propagation phase-contrast synchrotron microtomography (PPC-SRµCT) reveals remnants of the first-generation teeth embedded in the basal plate, a feature never previously observed in any taxon. These teeth were shed by semi-basal resorption with the periphery of their bases retained as dentine rings. The rings are highly overlapped, which evidences tooth shedding prior to adding the next first-generation tooth at the growing edge of the plate. The first generation of teeth is thus diachronous. Successor teeth at the same sites underwent cyclical replacing and shedding through basal resorption, producing stacks of buried resorption surfaces separated by bone of attachment. The number and spatial arrangement of resorption surfaces elucidates that basal resorption of replacement teeth had taken place at the older tooth sites before the addition of the youngest first-generation teeth at the lingual margin. Thus, the replacement tooth buds cannot have been generated by a single permanent dental lamina at the lingual edge of the tooth cushion, but must have arisen either from successional dental laminae associated with the individual predecessor teeth, or directly from the dental epithelium of these teeth. The virtual histological dissection of these Late Silurian microfossils broadens our understanding of the development of the gnathostome dental systems and the acquisition of the osteichthyan-type of tooth replacement.

To study tooth cycling in polyphyodont animals, we chose to work on alligators. Alligators have teeth in three phases of development at each tooth location. This assembly of three teeth is called a tooth family unit. As part of the study, in order to study tooth cycling in alligators, we wanted to know the configuration of the tooth family unit in every tooth position. From the surface of the mouth, this is difficult to assess. Therefore, we decided to use MicroCT which can image X-ray dense materials providing a three-dimensional view. MicroCT provided us with valuable information for this study. The method described below can be applied to study tooth cycling in other vertebrate species.

Alligators have robust regenerative potential for tooth renewal. In contrast, extant mammals can either renew their teeth once (diphyodont dentition, as found in humans) or not at all (monophyodont dentition, present in mice). Previously, the authors used multiple mitotic labeling to map putative stem cells in alligator dental laminae, which contain quiescent odontogenic progenitors. The authors demonstrated that alligator tooth cycle initiation is related to β-catenin/Wnt pathway activity in the dental lamina bulge. However, the molecular circuitry underlying the developmental progression of polyphyodont teeth remains elusive. Here, the authors used transcriptomic analyses to examine the additional molecular pathways related to the process of alligator tooth development. The authors collected juvenile alligator dental laminae at different developmental stages and performed RNA-seq. This data shows that Wnt, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) pathways are activated at the transition from pre-initiation stage (bud) to initiation stage (cap). Intriguingly, the activation of Wnt ligands, receptors and co-activators accompanies the inactivation of Wnt antagonists. In addition, the authors identified the molecular circuitry at different stages of tooth development. The authors conclude that multiple pathways are associated with specific stages of tooth development in the alligator. This data shows that Wnt pathway activation may play the most important role in the initiation of tooth development. This result may offer insight into ways to modulate the genetic controls involved in mammalian tooth renewal.

The squaliform sharks represent one of the most speciose shark clades. Many adult squaliforms have blade-like teeth, either on both jaws or restricted to the lower jaw, forming a continuous, serrated blade along the jaw margin. These teeth are replaced as a single unit and successor teeth lack the alternate arrangement present in other elasmobranchs. Micro-CT scans of embryos of squaliforms and a related outgroup (Pristiophoridae) revealed that the squaliform dentition pattern represents a highly modified version of tooth replacement seen in other clades. Teeth ofSqualusembryos are arranged in an alternate pattern, with successive tooth rows containing additional teeth added proximally. Asynchronous timing of tooth production along the jaw and tooth loss prior to birth cause teeth to align in oblique sets containing teeth from subsequent rows; these become parallel to the jaw margin during ontogeny, so that adultSqualushas functional tooth rows comprising obliquely stacked teeth of consecutive developmental rows. In more strongly heterodont squaliforms, initial embryonic lower teeth develop into the oblique functional sets seen in adultSqualus, with no requirement to form, and subsequently lose, teeth arranged in an initial alternate pattern.

This paper describes tooth development in a basal squamate, Paroedura picta. Due to its reproductive strategy, mode of development and position within the reptiles, this gecko represents an excellent model organism for the study of reptile development. Here we document the dental pattern and development of non-functional (null generation) and functional generations of teeth during embryonic development. Tooth development is followed from initiation to cytodifferentiation and ankylosis, as the tooth germs develop from bud, through cap to bell stages. The fate of the single generation of non-functional (null generation) teeth is shown to be variable, with some teeth being expelled from the oral cavity, while others are incorporated into the functional bone and teeth, or are absorbed. Fate appears to depend on the initiation site within the oral cavity, with the first null generation teeth forming before formation of the dental lamina. We show evidence for a stratum intermedium layer in the enamel epithelium of functional teeth and show that the bicuspid shape of the teeth is created by asymmetrical deposition of enamel, and not by folding of the inner dental epithelium as observed in mammals.

Functional tooth germs in mammals, reptiles, and chondrichthyans are initiated from a dental lamina. The longevity of the lamina plays a role in governing the number of tooth generations. Monophyodont species have no replacement dental lamina, while polyphyodont species have a permanent continuous lamina. In diphyodont species, the dental lamina fragments and regresses after initiation of the second tooth generation. Regression of the lamina seems to be an important mechanism in preventing the further development of replacement teeth. Defects in the complete removal of the lamina lead to cyst formation and has been linked to ameloblastomas. Here, we show the previously unknown mechanisms behind the disappearance of the dental lamina, involving a combination of cell migration, cell-fate transformation, and apoptosis. Lamina regression starts with the loss of the basement membrane, allowing the epithelial cells to break away from the lamina and migrate into the surrounding mesenchyme. Cells deactivate epithelial markers (E-cadherin, cytokeratin), up-regulate Slug and MMP2, and activate mesenchymal markers (vimentin), while residual lamina cells are removed by apoptosis. The uncovering of the processes behind lamina degradation allows us to clarify the evolution of diphyodonty, and provides a mechanism for future manipulation of the number of tooth generations.

Most dentate vertebrates, including humans, replace their teeth and yet the process is poorly understood. Here, we investigate whether dental epithelial stem cells exist in a polyphyodont species, the leopard gecko (Eublepharis macularius). Since the gecko dental epithelium lacks a histologically distinct site for stem cells analogous to the mammalian hair follicle bulge, we performed a pulse-chase experiment on juvenile geckos to identify label-retaining cells (LRCs). We detected LRCs exclusively on the lingual side of the dental lamina, which exhibits low proliferation rates and is not involved in tooth morphogenesis. Lingual LRCs were organized into pockets of high density close to the successional lamina. A subset of the LRCs expresses Lgr5 and other genes that are markers of adult stem cells in mammals. Also similar to mammalian stem cells, the LRCs appear to proliferate in response to gain of function of the canonical Wnt pathway. We suggest that the LRCs in the lingual dental lamina represent a population of stem cells, the immediate descendents of which form the successional lamina and, ultimately, the replacement teeth in the gecko. Furthermore, their location on the non-tooth-forming side of the dental lamina implies that dental stem cells are sequestered from signals that might otherwise induce them to differentiate.

Tooth enameloid of medaka as a representative of human tooth enamel was analyzed by high-resolution transmission electron microscope (HR-TEM), electron probe microanalyzer (EPMA) and micro-X-ray diffraction to deepen the knowledge on the crystal formation of tooth enamel. HR-TEM observation showed that enameloid crystals, with incomplete slender hexagonal shape, sometimes became fused with each other when the density of crystals increased. EPMA analysis showed clear image maps of calcium, phosphate and iron. The element analysis indicated high concentration of calcium and phosphate. Micro-X-ray diffraction showed that enameloid crystals are composed of relatively well-crystallized hydroxyapatite. Each (211), (112), (210) and (002) reflection peaks was clearly detected. It is suggested that the medaka is useful for research of developing tooth because it is polyphyodont and has a short lifespan.

The single replacement from milk teeth to permanent teeth makes mammalian teeth different from teeth of most nonmammalian vertebrates and other epithelial organs such as hair and feathers, whose continuous replacement has been linked to Wnt signaling. Here we show that mouse tooth buds expressing stabilized β-catenin in epithelium give rise to dozens of teeth. The molar crowns, however, are typically simplified unicusped cones. We demonstrate that the supernumerary teeth develop by a renewal process where new signaling centers, the enamel knots, bud off from the existing dental epithelium. The basic aspects of the unlocked tooth renewal can be reproduced with a computer model on tooth development by increasing the intrinsic level of activator production, supporting the role of β-catenin pathway as an upstream activator of enamel knot formation. These results may implicate Wnt signaling in tooth renewal, a capacity that was all but lost when mammals evolved progressively more complicated tooth shapes.

Tooth replacement poses many questions about development, pattern formation, tooth attachment mechanisms, functional morphology and the evolution of vertebrate dentitions. Although most vertebrate species have polyphyodont dentitions, detailed knowledge of tooth structure and replacement is poor for most groups, particularly actinopterygians. We examined the oral dentition of the bluefish, Pomatomus saltatrix, a pelagic and coastal marine predator, using a sample of 50 individuals. The oral teeth are located on the dentary and premaxillary bones, and we scored each tooth locus in the dentary and premaxillary bones using a four-part functional classification: absent (A), incoming (I), functional (F=fully ankylosed) or eroding (E). The homodont oral teeth of Pomatomus are sharp, deeply socketed and firmly ankylosed to the bone of attachment. Replacement is intraosseus and occurs in alternate tooth loci with long waves of replacement passing from rear to front. The much higher percentage of functional as opposed to eroding teeth suggests that replacement rates are low but that individual teeth are quickly lost once erosion begins. Tooth number increases ontogenetically, ranging from 15-31 dentary teeth and 15-39 premaxillary teeth in the sample studied. Teeth increase in size with every replacement cycle. Remodeling of the attachment bone occurs continuously to accommodate growth. New tooth germs originate from a discontinuous dental lamina and migrate from the lingual (dentary) or labial (premaxillary) epithelium through pores in the bone of attachment into the resorption spaces beneath the existing teeth. Pomatomus shares unique aspects of tooth replacement with barracudas and other scombroids and this supports the interpretation that Pomatomus is more closely related to scombroids than to carangoids.

A technique for organotypic in vitro culture with serum-free medium was tested for its appropriateness to mimic normal odontogenesis in the cichlid fish Hemichromis bimaculatus and the zebrafish Danio rerio. Serial semithin sections were observed by light microscopy to collect data on tooth patterning and transmission electron microscopy was used to compare cellular and extracellular features of tooth germs developing in vitro with the situation in vivo. Head explants of H. bimaculatus from 120 h post-fertilization (hPF) to 8.5 days post-fertilization (dPF) and of zebrafish from 45 hPF to 79 hPF and adults kept in culture for 3, 4 or 7 days revealed that tooth germs developed in vitro from explants in which the buccal or pharyngeal epithelium was apparently undifferentiated and, when present at the time of explantation, they continued their development up to a stage of attachment. In addition, the medium allowed the morphogenesis and cytodifferentiation of the tooth germs similar to that observed in vivo and the establishment of a dental pattern (place and order of tooth appearance and of attachment) that mimicked that in vivo. Organotypic culture in serum-free conditions thus provides us with the means of studying epithelial-mesenchymal interactions during tooth development in teleost fish and of analysing the genetic control of either mandibular or pharyngeal tooth development and replacement in these polyphyodont species. Importantly, it allows heads from embryonically lethal (zebrafish) mutants or from early lethal knockdown experiments to develop beyond the point at which the embryos normally die. Such organotypic culture in serum-free conditions could therefore become a powerful tool in developmental studies and open new perspectives for craniofacial research.

The canonical (13-catenin dependent) Wnt signaling pathway has emerged as a likely candidate for regulating tooth replacement in continuously renewing dentitions. So far, the involvement of canonical Wnt signaling has been experimentally demonstrated predominantly in amniotes. These studies tend to show stimulation of tooth formation by activation of the Wnt pathway, and inhibition of tooth formation when blocking the pathway. Here, we report a strong and dynamic expression of the soluble V\int inhibitor dickkopfl (dkkl) in developing zebrafish (Danio rerio) tooth germs, suggesting an active repression of V\int signaling during morphogenesis and cytodifferentiation of a tooth, and derepression of Wnt signaling during start of replacement tooth formation. To further analyse the role of Wnt signaling, we used different gain-of-function approaches. These yielded disjunct results, yet none of them indicating enhanced tooth replacement. Thus, masterblind (mbl) mutants, defective in axinl, mimic overexpression of Mt, but display a normally patterned dentition in which teeth are replaced at the appropriate times and positions. Activating the pathway with LICI had variable outcomes, either resulting in the absence, or the delayed formation, of first-generation teeth, or yielding a regular dentition with normal replacement, but no supernumerary teeth or accelerated tooth replacement. The failure so far to influence tooth replacement in the zebrafish by perturbing Wnt signaling is discussed in the light of (i) potential technical pitfalls related to dose- or time-dependency, (ii) the complexity of the canonical V\int pathway, and (iii) species-specific differences in the nature and activity of pathway components. Finally, we emphasize the importance of in-depth knowledge of the wild-type pattern for reliable interpretations. It is hoped that our analysis can be inspiring to critically assess and elucidate the role of V\int signaling in tooth development in polyphyodonts.

To test whether the formation of replacement teeth in arbitrarily chosen zebrafish follows the same pattern as described for larval and young zebrafish of known age, dentitions of more than 90 animals of different ages and standard lengths were observed under the stereomicroscope. Only the teeth of the ventral tooth row (1V-5V) were considered. Statistical results (G-tests) suggested that tooth replacement does not occur randomly. The most common order of replacement can be represented as the formula 5V-2V-3V-1V-4V and counts for approximately 70% of the observed patterns. Initiation of replacement teeth at positions 4V and 5V is separated by a larger time interval than between any other combination. It is hypothesized that in older juveniles and adults, replacement teeth may be formed during three odontogenic waves.

Using tartrate-resistant acid phosphatase (TRAP), we examined the cytodifferentiation of odontoclast cells in resorbing areas of dental tissues during the replacement of teeth in a polyphyodont lizard, Liolaemus gravenhorsti. We also report, by means of Lectin-HRP histochemistry, the distribution pattern of some specific sugar residues of TRAPase-positive cells. For detection of TRAPase activity, the azo dye-coupling technique was used. Lectin binding sites were demonstrated by means of specific HRP-lectins. The process of tooth resorption was divided into four stages: 1) preresorption—the wall of the dental pulp is covered with an odontoblast layer, and no TRAP-positive cells are in the dental pulp; 2) early resorption—TRAP-positive multinucleate odontoclasts are present on the dental wall, but the rest of the pulp surface is still covered with an odontoblast layer; 3) later resorption—the entire surface of the pulp chamber is lined with multinucleate odontoclasts; and 4) final resorption—the tooth has been totally resorbed. Odontoclasts are usually detached from the resorbed surface, and show signs of degeneration. Of the six lectins used, PNA, ECA, and UEA-1 bind to multinucleated but not mononuclear cells. All the remaining lectins, BS-1, RCA120, and LTA showed no binding to any cells of the teeth. The significance of saccharidic moieties such as acetyl-galactosamine, acetyl-glucosamine, and fucose sugar residues is difficult to ascertain. Perhaps these oligosaccharides might be borne on molecules associated with odontoclastic resorption or associated with multinucleation of odontoclasts after attachment to the dentine surface. J. Morphol. 242:295–309, 1999. © 1999 Wiley-Liss, Inc.

Reptiles and fish have robust regenerative powers for tooth renewal. However, extant mammals can either renew their teeth one time (diphyodont dentition) or not at all (monophyodont dentition). Humans replace their milk teeth with permanent teeth and then lose their ability for tooth renewal. Here, we study tooth renewal in a crocodilian model, the American alligator, which has well-organized teeth similar to mammals but can still undergo life-long renewal. Each alligator tooth is a complex family unit composed of the functional tooth, successional tooth, and dental lamina. Using multiple mitotic labeling, we map putative stem cells to the distal enlarged bulge of the dental lamina that contains quiescent odontogenic progenitors that can be activated during physiological exfoliation or artificial extraction. Tooth cycle initiation correlates with β-catenin activation and soluble frizzled-related protein 1 disappearance in the bulge. The dermal niche adjacent to the dermal lamina dynamically expresses neural cell adhesion molecule, tenascin-C, and other molecules. Furthermore, in development, asymmetric β-catenin localization leads to the formation of a heterochronous and complex tooth family unit configuration. Understanding how these signaling molecules interact in tooth development in this model may help us to learn how to stimulate growth of adult teeth in mammals.

The study of fossil and extant dipnoan dentitions shows that the shagreen of denticles found in such Devonian genera as Uranolophus and Griphognathus is not primitive for the group, that the ontogeny of the extant Neoceratodus is not a guide to the primitive adult tooth plate, that homology between teeth on dipnoan tooth plates and polyphyodont marginal teeth of other osteichthyans has not been established, and that the Chinese Devonian genus Diabolepis should not be regarded as a dipnoan. We conclude that no convincing argument has been advanced to destroy our contention that the primitive dipnoan plate was a relatively featureless sheet that was added to at the margins by the incorporation of small enamel-covered dentine blisters, and was thickened by resorption of the basal bone and subsequent ingrowth by pleromic dentine.

Most dentate vertebrates, from fish to humans, replace their teeth and yet the molecular basis of tooth replacement is poorly understood. Canonical Wnt signaling regulates tooth number in mice and humans, but it is unclear what role it plays in tooth replacement as it naturally occurs. To clarify this, we characterized Wnt signaling activity in the dental tissues of the ball python Python regius. This species replaces teeth throughout life (polyphyodonty) and in the same manner as in humans, i.e., sequential budding of teeth from the tip of the dental lamina. From initiation stage onwards, canonical Wnt read-out genes (Lef1 and Axin2) are persistently expressed by cells in the dental lamina tip and surrounding mesenchyme. This implies that molecular signaling at work during dental initiation carries over to tooth replacement. We show that canonical Wnt signaling promotes cell proliferation in python dental tissues and that by confining Wnt activity in the dental lamina the structure extends instead of thickens. Presumably, lamina extension creates space between successive tooth buds, ensuring that tooth replacement occurs in an ordered manner. We suggest that hedgehog signaling confines Wnt activity in the dental epithelium by direct planar repression and, during tooth replacement stages, by negatively regulating BMP levels in the dental mesenchyme. Finally, we propose that Wnt-active cells at the extending tip of the python dental lamina represent the immediate descendents of putative stem cells housed in the lingual face of the lamina, similar to what we have recently described for another polyphyodont squamate species.

The successional dental lamina (SDL) plays an essential role in the development of replacement teeth in diphyodont and polyphyodont animals. A morphologically similar structure, the rudimental successional dental lamina (RSDL), has been described in monophyodont (only one tooth generation) lizards on the lingual side of the developing functional tooth. This rudimentary lamina regresses, which has been proposed to play a role in preventing the formation of future generations of teeth. A similar rudimentary lingual structure has been reported associated with the first molar in the monophyodont mouse, and we show that this structure is common to all murine molars. Intriguingly, a lingual lamina is also observed on the non-replacing molars of other diphyodont mammals (pig and hedgehog), initially appearing very similar to the successional dental lamina on the replacing teeth. We have analyzed the morphological as well as ultrastructural changes that occur during the development and loss of this molar lamina in the mouse, from its initiation at late embryonic stages to its disappearance at postnatal stages. We show that loss appears to be driven by a reduction in cell proliferation, down-regulation of the progenitor marker Sox2, with only a small number of cells undergoing programmed cell death. The lingual lamina was associated with the dental stalk, a short epithelial connection between the tooth germ and the oral epithelium. The dental stalk remained in contact with the oral epithelium throughout tooth development up to eruption when connective tissue and numerous capillaries progressively invaded the dental stalk. The buccal side of the dental stalk underwent keratinisation and became part of the gingival epithelium, while most of the lingual cells underwent programmed cell death and the tissue directly above the erupting tooth was shed into the oral cavity.

The rainbow trout (Oncorhynchus mykiss) as a developmental model surpasses both zebrafish and mouse for a more widespread distribution of teeth in the oro-pharynx as the basis for general vertebrate odontogenesis, one in which replacement is an essential requirement. Studies on the rainbow trout have led to the identification of the initial sequential appearance of teeth, through differential gene expression as a changing spatio-temporal pattern, to set in place the primary teeth of the first generation, and also to regulate the continuous production of replacement tooth families. Here we reveal gene expression data that address both the field and clone theories for patterning a polyphyodont osteichthyan dentition. These data inform how the initial pattern may be established through up-regulation at tooth loci from a broad odontogenic band. It appears that control and regulation of replacement pattern resides in the already primed dental epithelium at the sides of the predecessor tooth. A case is presented for the developmental changes that might have occurred during vertebrate evolution, for the origin of a separate successional dental lamina, by comparison with an osteichthyan tetrapod dentition (Ambystoma mexicanum). The evolutionary origins of such a permanent dental lamina are proposed to have occurred from the transient one demonstrated here in the trout. This has implications for phylogenies based on the homology of teeth as only those developed from a dental lamina. Utilising the data generated from the rainbow trout model, we propose this as a standard for comparative development and evolutionary theories of the vertebrate dentition.

Dental polymorphism in teleost fishes often involves production of a robust dentition, or “molarization”, in one morph. The lower pharyngeals of a sample of wild-caught individuals of the polymorphic Cuatro Cienegas cichlid, Cichlasoma minckleyi (Kornfield and Taylor) (Proc. Biol. Soc. Wash. 96 (1983) 253), were measured to investigate morphological changes associated with molarization. Principal components analysis demonstrates that dental variability in this species increases in larger fish, and that only the molariform morph contributes to this increase. Reduced major axis regression analyses between pairs of variables indicate that the papilliform morph increases both tooth measures and numbers, whereas the molariform morph maintains a relatively constant number of teeth as it produces teeth of progressively larger size. In the papilliform morph, negative allometric scaling between tooth size and dentigerous area is compensated for by addition of teeth. Tooth size variables are isometric in the molariform morph, and tooth numbers are nearly static. These results are consistent with those reported for other polymorphic cichlid species. Further study is required to elucidate the mechanisms whereby tooth form in polyphyodont species may respond to environmental factors (like food hardness), but possibilities include direct mechanical influences or transmission of signals via nerves to developing replacement teeth.

Summary The shape of the teeth and their sex-dependent dimorphic expression in three species of Costa Rican plethodontids ( Bolitoglossa subpalmata, Oedipina uniformis and Nototriton abscondens ) were studied using light microscopy and scanning electron microscopy. The teeth of the vomerine tooth patches are about one third larger than the teeth of the jaws in B. subpalmata and O. uniformis , whereas all teeth of N. abscondens are of about uniform size. The occurrence of bicuspid tooth germs in the fetus proves that primary teeth are bicuspid in these directly developing plethodontids. Females possess only bicuspid teeth consisting of a pedicel and a crown, as is considered characteristic for urodeles after metamorphosis. Adult males possess conical monocuspid teeth on the premaxillary. These teeth — which are similar to the typical late larval tooth of salamanders presenting a larval stage — are about twice as big as the neighbouring bicuspid maxillary teeth. N. abscondens males possess some monocuspid teeth and teeth of aberrant shapes on the premaxillary and the maxillaries. A tendency to build more monocuspid teeth in the premaxillary region than in the maxillary region can be observed in this species. We suppose that different degrees of sensitivity to androgens in each section of the dental lamina of the upper jaw cause the secondary occurrence of conical monocuspid teeth predominantly on the premaxillary section.

It has been suggested that nerve endings emanating from the dental nerve plexus of the jaw might be involved in the formation of tooth germs. In the present study we examine the effect of unilateral denervation on the formation of tooth germs in the lower jaw of a polyphyodont teleost--the cichild Tilapia mariae. Repeated inspection of the lower jaw dentition in normal animals over a period of about 300 days showed that the functional time of an average individual tooth is 101 days. In operated animals, the functional time was normal on the unoperated side, but on the denervated side tooth turnover ceased about 100 days after surgery. Radiographic plates from lower jaw specimens revealed that mineralized replacement teeth were present on the unoperated side, but not on the denervated side, 300 days after denervation. Light microscopic examination of semi-thin transverse sections from decalcified plastic-embedded lower jaws showed that soft-tissue tooth primordia and nerves were lacking on the denervated side, while present within the undisturbed half-jaw. It is concluded that the local presence of mandibular nerve branches is necessary for the formation of tooth germs in the lower jaw of the cichlid T. mariae.

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.

In Hemiphractus fang–like teeth are ankylosed to the premaxilla, maxilla and prevomer, and bony odontoids are found on the dentary, angular and palatine bones. The odontoids are small, but a larger pair at the front of the lower jaw project upwards and backwards into the mouth and fit into a diastema between the anterior premaxillary teeth when the mouth is closed. The teeth are unipartite and monocuspid, and each consists of a strongly recurved and elongated cone of orthodentine, capped at the tip by a thin layer of enamel. The inner circumpulpal layer of the dentine is tubular, but no tubules are present in the outer pallial layer. During tooth development, dentine is formed before the enamel matrix is produced, and the tooth germs lie horizontally beneath the ventral surface of each dentigerous bone. On eruption, the tooth germs migrate horizontally and become ankylosed to the outer edge of the jaw bone by a layer of cellular cementum. During tooth replacement, the vast majority of the dentine of each tooth, and the cementum at the tooth base, are resorbed by osteoclasts. It is not clear whether the tips of the teeth are shed or not.

Serial coronal sections of the teeth and their surrounding tissues in the agamid lizard, Uromastix aegyptius, were examined with the light microscope in order to determine how these structures change as the teeth wear. Because new teeth are added only at the posterior end of the tooth row and older teeth are not replaced, the series of sections included the youngest as well as the oldest teeth. Two types of changes occur as the teeth become older: bone under the teeth changes from cancellous to compact, and the pulp chamber of the tooth is obliterated. Although the labial surface of the dentary lacks a periosteal covering and some of the bone lacks any covering at all, it remains functional throughout the life of the animal.

Two characteristics clearly distinguish between the dentitions of typical mammals and reptiles: generally the former are heterodont and diphyodont while the latter are homodont and polyphyodont. Superficially it might seem that by progressively reducing the number of replacement teeth in an evolutionary sequence of animals, it was a simple matter to arrive at a diphyodont mammalian descendant from a polyphyodont reptilian ancestor. But data on the sequences in which teeth are initiated in mammalian and reptilian embryos suggest that the process was more complex. In most reptilian embryos teeth are probably developed in sequences which pass through alternate positions from the back to the front of the jaw (Osborn, 1971). It is not easy to relate this sequence to that observed in mammalian embryos; for instance for the upper jaw of Eremnitalpa, Osborn (1970) gives the sequence it, i2, C, m4/M1/m3/i3/M2/M3/I1, 12, C, P3, P4/ 13 (in which the lower case represents deciduous teeth and the sequence is known only where bars separate teeth). Both heterodonty and a very limited number of replacement teeth also evolved in the postcanine dentitions of the gomphodont cynodonts and their descendants, the tritylodonts of the Triassic and Jurassic (Crompton, 1973). Although these animals finally become extinct in the middle Jurassic, it seems possible that the manner in which they evolved their limited numbers of replacement teeth from the ancestral polyphyodont condition might shed light on the evolution of diphyodonty in mammals. The earliest of the well-known gomphodont cynodonts with a markedly heterodont postcanine denition and a linited number of replacement teeth was Diademodon. The purpose of the present paper is to plot the sequence in which postcanine teeth were replaced in Diademodon and to compare this with tooth replacement in a polyphyodont reptilian dentition. If both have been correctly understood it should indirectly be possible to obtain some idea of the biological controls which regulate tooth replacement. And it can be safely assumed that by varying similar biological controls, mammalian diphyodonty could also have been evolved from polyphyodont dentitions. The pattern by which Diademodon, a lower Triassic gomphodont cynodont, replaced its postcanine teeth has recently been studied by Crompton (1963), Ziegler (1969) and Hopson (1971), and is also referred to by Crompton (1973). In view of the fact that no new data is provided in the present paper, it might seem unnecessarily contentious to propose a fourth solution to this problem within eight years. However, during the last decade almost all workers have, with varying degrees of success (e.g., DeMar, 1972) attempted to fit patterns of tooth replacement into the framework of a general model known as the Zahnreike theory. This theory, which was proposed by Edmund (1960, 1962), apparently links together tooth replacement patterns encountered throughout the vertebrate kingdom. My own studies, referred to above and later, have convinced me that the Zaknreike theory as a model describing the initiation of patterns of tooth replacement is untenable in the face of the phylogenetic and embryological evidence. I have concluded that the rate of tooth replacement is partly controlled at each tooth position.

A study of the tissues of the teeth and jaws in piranhas, using the scanning electron microscope and various techniques of light microscopy, revealed many dental adaptations related to the specialized feeding habits of these carnivorous fishes. The dentition is primarily sectorial, although some anterior teeth may be used in grasping. The scissor-like rows of teeth are maintained by the specialized pattern of tooth replacement. The bones of the jaws and the tooth attachment support the teeth very firmly. In its structural organization, the enameloid covering the teeth closely resembles that on the sectorial teeth of sharks and is adapted to the probable stress patterns set up in biting.

In this study we examined the possible inductive role of the dental papilla from polyphyodont lizard tooth germs. Flank skin sheets of quail ectoderm enzymatically separated from dermal tissue were recombined with lizard tooth papillae and placed on semisolid medium and cultured for 2 days. Subsequently, the recombinants were removed and placed on the chorioallantoic membrane of chick hosts and incubated for 6 days. After this period of 8 days in explant, control tissues differentiated according to their own phenotypes. Lizard dental papilla alone differentiated as fibroblasts. Quail flank skin ectoderm differentiated into epithelial sheets. Intact lizard tooth buds developed into teeth with dentine and incipient enamel. In the best experimental recombinants, advanced and relatively well-constructed teeth were observed, with clear indications of hard tissue deposition in association with quail epithelium. The results show that mesenchyme of the adult lizard dental papilla and embryonic quail ectoderm of heterotopic origin are capable of carrying out the complex sequence of morphogenetic interactions involved in normal odontogenesis.

A well-known fact that sexual glands move to their final location accompanied with their blood vessels. The from of blood vessels supplying teeth, in tooth germ, in attached tooth, and ln shedding tooth, was also one of the most interest things to the authors. For resolution of this problem, seventeen species of lower vertebrate were prepared and infused with 10% gelatine solution added indian ink and thick sections of infused cranii were made to be observed under a binocular microscope. Lower vertebrates showed polyphyodont, and had toothgerms and attached teeth in varios stages. Form of tooth and tooth attachment was of caurseobserved., and their relationships to vascular supply were also observed with special care. Vascular supply of teeth in various stages could be easily observed even in one section. Origin, branching and course of blood vessels which supply tooth germ were changed in the progress of tooth development. These changes in each vertebrate were compared one with another and vascular supply of tooth in lower vertebrates was classified into 9 types. Each vascular type and its developmental change could he favorably explained according to the progress of tooth succession.

The developmental processes related to odontogenesis are similar in all vertebrates and they occur during embryogenesis. The dental papilla can exercises directive morphogenetic role on epithelia of different phylogenetic origin. In our laboratory we have previously shown that cultured heterologous tissue recombinations between adult lizards dental papillae and quail epithelia were capable of producing odontogenesis and amelogenesis. Employing sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli ('70, Nature, 227:680–685.) and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) (O'Farrell, '75, J. Biol. Chem., 250:4007–4021.), we have examined the distribution of soluble proteins with respect to isoelectric point and molecular weight of dental papillae isolated from tooth germs at bell stage of adult lizard Liolaemus tenuis tenuis (polyphyodont species) and dog fetuses Canis familiaris (diphyodont species). A comparison was also made with dental pulps from adult dogs. Some dental papillae from lizard tooth at bell stage were recombined with quail ectoderm and cultured in explant for 8 days. Furthermore, the histological analysis of dental papillae from lizard and dog fetus showed that dental papillae from both species contain preodontoblast cells, scarce extracellular matrix, and some blood vessels. Dental pulp from adult dogs is composed of odontoblast and fibroblast cells, extracellular matrix, collagen, blood vessels, etc. The control heterologous recombinants after the culture period differentiated chimeric tooth structures, showing that the dental papillae were capable of producing developmental instructions. The electrophoretic study of the supernatants showed differences and similarities. Under SDS-PAGE the presence of one band of an approximate molecular weight of 49.000 daltons was disclosed, which seems to be unique to dental papilla from adult lizards and dog fetuses. Under 2-D PAGE protein patterns from adult lizard and dog fetuses disclosed also two protein spots with an approximate molecular weight of 49.000 daltons and isoelectric point of pH ranges between 6 and 7.5. Apparently these spots are unique to both patterns and would have some significance in odontogenesis.