A revised hypothesis on the evolutionary origin of the vertebrate dentition

Despite claims to the contrary, the evolutionary origin of teeth has not been definitely established. The classical ‘outside in’ theory stating that teeth derive from odontodes that invaded the oral cavity in conjunction with the origin of jaws has been challenged by an alternative, ‘inside out’, hypothesis suggesting that teeth evolved from pharyngeal denticles, as endodermal derivatives, prior to the origin of jaws. We propose a third scenario, a revised ‘outside in’ hypothesis ( Huysseune et al., 2009 ). Our hypothesis is consistent with the current data and avoids speculations about convergent tooth evolution. We suggest that teeth may indeed have arisen before the origin of jaws, a pillar of the ‘inside out’ hypothesis, but not from the endodermally lined posterior pharynx. Rather, teeth would have been the result of competent, odontode‐forming ectoderm invading the oropharyngeal cavity through the mouth as well as through the gill slits, interacting with neural‐crest derived mesenchyme. Arguments in support of this hypothesis are: (i) the observation that pharyngeal teeth are present only in species known to possess gill slits, and disappear from the pharyngeal region in early tetrapods concomitant with the closure of gill slits; (ii) the assumption that endoderm alone, together with neural crest, cannot form teeth; (iii) observations on pharyngeal tooth and gill slit formation in extant species; (iv) the observation that the dental lamina (sensu Reif, 1982 ) is not a prerequisite for tooth formation; (v) evidence that patterning does not distinguish pharyngeal from skin denticles, and (vi) the observation on zebrafish mutants affected in the dermal skeleton. This ‘modified outside in’ hypothesis can be tested both on paleontological data (it predicts a correlation of the presence of pharyngeal teeth and of gill slits), and on developmental data in extant species (it predicts the necessity of an ectodermal signal to make [pharyngeal] teeth).     

Ectoderm,endoderm, and the evolution of heterodont dentitions

Mammalian dentitions consist of different shapes/types of teeth that are positioned in different regions of the jaw (heterodont) whereas in many fish and reptiles all teeth are of similar type (homodont). The process by which heterodont dentitions have evolved in mammals is not understood. In many teleosts teeth develop in the pharynx from endoderm (endodermal teeth), whereas mammalian teeth develop from the oral ectoderm indicating that teeth can develop (and thus possibly evolve) via different mechanisms. In this article, we compare the molecular characteristics of pharyngeal/foregut endoderm with the molecular characteristics of oral ectoderm during mouse development. The expression domains of Claudin6, Hnf3β, α‐fetoprotein, Rbm35a, and Sox2 in the embryonic endoderm have boundaries overlapping the molar tooth‐forming region, but not the incisor region in the oral ectoderm. These results suggest that molar teeth (but not incisors) develop from epithelium that shares molecular characteristics with pharyngeal endoderm. This opens the possibility that the two different theories proposed for the evolution of teeth may both be correct. Multicuspid (eg. molars) having evolved from the externalization of endodermal teeth into the oral cavity and monocuspid (eg. incisors) having evolved from internalization of ectodermal armour odontodes of ancient fishes. The two different mechanisms of tooth development may have provided the developmental and genetic diversity on which evolution has acted to produce heterodont dentitions in mammals. genesis 48:382–389, 2010. © 2010 Wiley‐Liss, Inc.     

Teeth before jaws? Comparative analysis of the structure and development of the external and internal scales in the extinct jawless vertebrate Loganellia scotica

Traditional hypotheses posit that teeth evolved from dermal scales, through the expansion of odontogenetically competent ectoderm into the mouth of jawless vertebrates. The discovery of tooth‐like scales inside thelodonts, an extinct group of jawless vertebrates, led to the alternative hypothesis that teeth evolved from endodermal derivatives and that there exists a fundamental developmental and phylogenetic distinction between oral/pharyngeal and external odontodes. We set out a test of this latter hypothesis, examining the development of scales of the thelodont Loganellia scotica using synchrotron radiation X‐ray tomographic microscopy (SRXTM). We reveal that the internal scales are organized into fused patches and rows, a key distinction from the discrete dermal scales. The pattern of growth of oral scale patches is polarized, but not along a particular vector, whereas pharyngeal scale rows grew along a vector. Our test of the phylogenetic distribution of oral and pharyngeal scales and teeth in vertebrates indicates that odontodes are first expressed in an external position. Internal scales, where present, are always located near to external orifices; the sequential development of pharyngeal scales in Loganellia is peculiar among thelodonts and other stem gnathostomes. It represents a convergence on, rather than the establishment of, the developmental pattern underpinning tooth replacement in jawed vertebrates. The available evidence suggests that internal odontodes evolved through the expansion of odontogenic competence from external to internal epithelia.     

Regionalisation of early head ectoderm is regulated by endoderm and prepatterns the orofacial epithelium

The oral epithelium becomes regionalised proximodistally early in development, and this is reflected by the spatial expression of signalling molecules such as Fgf8 and Bmp4. This regionalisation is responsible for regulating the spatial expression of genes in the underlying mesenchyme. These genes are required for the spatial patterning of bone, cartilage orofacial development and, in mammals, teeth. The mechanism and timing of this important regionalisation during head epithelium development are not known. Using lipophilic dyes to fate map the oral epithelium in chick embryos, we show that the cells that will occupy the epithelium of the distal and the proximal mandible primordium already occupy different spatial locations in the developing head ectoderm prior to the formation of the first pharyngeal arch and neural crest migration. Moreover, the ectoderm cells fated to become proximal oral epithelium express Fgf8 and this expression requires the presence of endoderm. Thus, the first fundamental patterning process in jaw morphogenesis is controlled by the early separation of specific areas of ectoderm that are regulated by ectoderm-endoderm interactions, and does not involve neural crest cells.     

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.     

Sonic hedgehog in the pharyngeal endoderm controls arch pattern via regulation of Fgf8 in head ectoderm

Fgf8 signalling is known to play an important role during patterning of the first pharyngeal arch, setting up the oral region of the head and then defining the rostral and proximal domains of the arch. The mechanisms that regulate the restricted expression of Fgf8 in the ectoderm of the developing first arch, however, are not well understood. It has become apparent that pharyngeal endoderm plays an important role in regulating craniofacial morphogenesis. Endoderm ablation in the developing chick embryo results in a loss of Fgf8 expression in presumptive first pharyngeal arch ectoderm. Shh is locally expressed in pharyngeal endoderm, adjacent to the Fgf8-expressing ectoderm, and is thus a candidate signal regulating ectodermal Fgf8 expression. We show that in cultured explants of presumptive first pharyngeal arch, loss of Shh signalling results in loss of Fgf8 expression, both at early stages before formation of the first arch, and during arch formation. Moreover, following removal of the endoderm, Shh protein can replace this tissue and restore Fgf8 expression. Overexpression of Shh in the non-oral ectoderm leads to an expansion of Fgf8, affecting the rostral-caudal axis of the developing first arch, and resulting in the formation of ectopic cartilage. Shh from the pharyngeal endoderm thus regulates Fgf8 in the ectoderm and the role of the endoderm in pharyngeal arch patterning may thus be indirectly mediated by the ectoderm.     

Vertebrate dentitions at the origin of jaws: when and how pattern evolved

New evidence shows that teeth evolved with a greater degree of independence from jaws than previously considered. Pharyngeal denticles occur in jawless fish and also in early gnathostomes and precede jaw teeth in phylogeny. Many of these denticles form joined polarized sets on each branchial arch; these resemble whorl-shaped tooth sets on the jaws of stem and crown gnathostomes and are proposed as homologous units. Therefore, the source of patterning of these pharyngeal denticle and tooth sets is conserved from jawless conditions. It is proposed that developmental regulatory systems, responsible for all such tooth patterns on the jaws, are co-opted from the pharyngeal region and not from the skin as classically understood. This strongly implicates embryonic endoderm as opposed to ectoderm in the genetic control of dentition patterning. New interpretations of ontogenetic data on patterning dentitions of extant sharks are proposed, together with those of osteichthyan fish. Two entirely fossil groups, placoderms and acanthodians, at the base of gnathostome phylogeny are reassessed on the basis of a new model. It is concluded that within stem group and crown group gnathostomes several different strategies, unique to each taxon, were adopted to produce different developmental models of dentition patterning from pharyngeal denticles. One shared developmental pattern is that of initiation from primordial tooth sites, independently in each dentate zone of the jaws. The new model is proposed as a framework for data on evolutionary developmental genetics.     

Teeth outside the mouth in teleost fishes: how to benefit from a developmental accident

SUMMARY Evolution proceeds by the selection of characters that enhance survival rates so that the long-term outcome for a species is better adaptation to its environment. These new characters are "accidentally" acquired, mostly through mutations leading to modifications of developmental events. However, changes that lead to the ectopic expression of an organ are rare and, whereas their subsequent selection for a new role is even more rare, such a scenario has apparently occurred for denticles in some teleost fish. Small, conical denticles are present, mainly on the dermal bones of the head, in a few, unrelated lineages of living teleosts. Here, I show that the morphology and structure of the denticles in Atherion elymus , an atheriniform, is similar to those of teeth inside the oral cavity. These denticles are not derived evolutionarily from odontodes of early vertebrates, nor do they represent a re-expression as such (i.e., a long-lasting ability to make odontodes outside the oral cavity). Teeth and odontodes are homologous organs but the origin of the denticles is to be found in teeth, not in odontodes. The denticles are simply teeth that form outside the mouth, probably derived from a sub-population of odontogenically pre-specified neural crest cells. These "accidental" extra-oral teeth have arisen independently in these lineages and were selectively advantageous in a hydrodynamic context.     

Development of the primary mouth in Xenopus laevis

The initial opening between the gut and the outside of the deuterostome embryo breaks through at the extreme anterior. This region is unique in that ectoderm and endoderm are directly juxtaposed, without intervening mesoderm. This opening has been called the stomodeum, buccopharyngeal membrane or oral cavity at various stages of its formation, however, in order to clarify its function, we have termed this the "primary mouth". In vertebrates, the neural crest grows around the primary mouth to form the face and a "secondary mouth" forms. The primary mouth then becomes the pharyngeal opening. In order to establish a molecular understanding of primary mouth formation, we have begun to examine this process during Xenopus laevis development. An early step during this process occurs at tailbud and involves dissolution of the basement membrane between the ectoderm and endoderm. This is followed by ectodermal invagination to create the stomodeum. A subsequent step involves localized cell death in the ectoderm, which may lead to ectodermal thinning. Subsequently, ectoderm and endoderm apparently intercalate to generate one to two cell layers. The final step is perforation, where (after hatching) the primary mouth opens. Fate mapping has defined the ectodermal and endodermal regions that will form the primary mouth. Extirpations and transplants of these and adjacent regions indicate that, at tailbud, the oral ectoderm is not specifically required for primary mouth formation. In contrast, underlying endoderm and surrounding regions are crucial, presumably sources of necessary signals. This study indicates the complexity of primary mouth formation, and lays the groundwork for future molecular analyses of this important structure.     

Origin and evolution of gnathostome dentitions: a question of teeth and pharyngeal denticles in placoderms

The fossil group Placodermi is the most phylogenetically basal of the clade of jawed vertebrates but lacks a marginal dentition comparable to that of the dentate Chondrichthyes, Acanthodii and Osteichthyes (crown-group Gnathostomata). The teeth of crown-group gnathostomes are part of an ordered dentition replaced from, and patterned by, a dental lamina, exemplified by the elasmobranch model. A dentition recognised by these criteria has been previously judged absent in placoderms, based on structural evidence such as absence of tooth whorls and typical vertebrate dentine. However, evidence for regulated tooth addition in a precise spatiotemporal order can be observed in placoderms, but significantly, only within the group Arthrodira. In these fossils, as in other jawed vertebrates with statodont, non-replacing dentitions, new teeth are added at the ends of rows below the bite, but in line with biting edges of the dentition. The pattern is different on each gnathal bone and probably arises from single odontogenic primordia on each, but tooth rows are arranged in a distinctive placoderm pattern. New teeth are made of regular dentine comparable to that of crown-gnathostomes, formed from a pulp cavity. This differs from semidentine previously described for placoderm gnathalia, a type present in the external dermal tubercles. The Arthrodira is a derived taxon within the Placodermi, hence origin of teeth in placoderms occurs late in the phylogeny and teeth are convergently derived, relative to those of other jawed vertebrates. More basal placoderm taxa adopted other strategies for providing biting surfaces and these vary substantially, but include addition of denticles to the growing gnathal plates, at the margins of pre-existing denticle patches. These alternative strategies and apparent absence of regular dentine have led to previous interpretations that teeth were entirely absent from the placoderm dentition. A consensus view emerged that a dentition, as developed within a dental lamina, is a synapomorphy characterising the clade of crown-group gnathostomes. Recent comparisons between sets of denticle whorls in the pharyngeal region of the jawless fish Loganellia scotica (Thelodonti) and those in sharks suggest homology of these denticle sets on gill arches. Although the placoderm pharyngeal region appears to lack denticles (placoderm gill arches are poorly known), the posterior wall of the pharyngeal cavity, formed by a bony flange termed the postbranchial lamina, is covered in rows of patterned denticle arrays. These arrays differ significantly, both in morphology and arrangement, from those of the denticles located externally on the head and trunkshield plates. Denticles in these arrays are homologous to denticles associated with the gill arches in other crown-gnathostomes, with pattern similarities for order and position of pharyngeal denticles. From their location in the pharynx these are inferred to be under the influence of a cell lineage from endoderm, rather than ectoderm. Tooth sets and tooth whorls in crown-group gnathostomes are suggested to derive from the pharyngeal denticle whorls, at least in sharks, with the patterning mechanisms co-opted to the oral cavity. A comparable co-option is suggested for the Placodermi.     

Ablation of specific expression domains reveals discrete functions of ectoderm- and endoderm-derived FGF8 during cardiovascular and pharyngeal development

Fibroblast growth factor 8 (Fgf8) is expressed in many domains of the developing embryo. Globally decreased FGF8 signaling during murine embryogenesis results in a hypomorphic phenotype with a constellation of heart, outflow tract, great vessel and pharyngeal gland defects that phenocopies human deletion 22q11 syndromes, such as DiGeorge. We postulate that these Fgf8 hypomorphic phenotypes result from disruption of local FGF8 signaling from pharyngeal arch epithelia to mesenchymal cells populating and migrating through the third and fourth pharyngeal arches. To test our hypothesis, and to determine whether the pharyngeal ectoderm and endoderm Fgf8 expression domains have discrete functional roles, we performed conditional mutagenesis of Fgf8 using novel Crerecombinase drivers to achieve domain-specific ablation of Fgf8 gene function in the pharyngeal arch ectoderm and endoderm. Remarkably, ablating FGF8 protein in the pharyngeal arch ectoderm causes failure of formation of the fourth pharyngeal arch artery that results in aortic arch and subclavian artery anomalies in 95% of mutants; these defects recapitulate the spectrum and frequency of vascular defects reported in Fgf8 hypomorphs. Surprisingly, no cardiac, outflow tract or glandular defects were found in ectodermal-domain mutants, indicating that ectodermally derived FGF8 has essential roles during pharyngeal arch vascular development distinct from those in cardiac, outflow tract and pharyngeal gland morphogenesis. By contrast, ablation of FGF8 in the third and fourth pharyngeal endoderm and ectoderm caused glandular defects and bicuspid aortic valve, which indicates that the FGF8 endodermal domain has discrete roles in pharyngeal and valvar development. These results support our hypotheses that local FGF8 signaling from the pharyngeal epithelia is required for pharyngeal vascular and glandular development, and that the pharyngeal ectodermal and endodermal domains of FGF8 have separate functions.     

Comparison of teeth and dermal denticles (odontodes) in the teleost Denticeps clupeoides (Clupeomorpha)

The present work is a contribution to an extensive comparative structural and developmental study we have undertaken to understand the evolution of the dermal skeleton in osteichthyans. We have investigated the structure of developing and functional tooth-like dermal denticles located on the head of Denticeps clupeoides, a clupeomorph, and compared their features to those of oral teeth. Morphological (scanning electron microscopy) and structural (light microscopy and transmission electron microscopy) observations clearly demonstrate that these small, sharp, conical and slightly backward-oriented denticles are true odontodes, i.e., homologous to oral teeth. They are composed of a dentine cone surrounding a pulp cavity, the top being covered by a hypermineralized cap. These odontodes are attached to a circular pedicel of attachment bone by a ligament that mineralizes, and the attachment bone matrix merges with that of the bony support. The pedicel of attachment bone surrounds a vascular cavity that is connected to the pulp cavity which is devoid of blood vessels and of nerve endings. Once the odontode is functional, the deposition of collagen matrix (called circumpulpar dentine) continues against the dentine, ligament, and attachment bone surfaces, thereby provoking a narrowing of the pulp cavity. Odontodes are shed by resorption occurring at the base, but their pedicels of attachment bone persist at the bone surface and become embedded in the bone matrix, within which they are clearly visible. The oral teeth are similar in shape, size, and structure to the odontodes, and they show only small differences probably related to the different function of these elements: They are more firmly anchored to the attachment bone, and the amount of dentine is relatively smaller than in odontodes. Despite their different functions, this close structural agreement between teeth and odontodes in Denticeps suggests that 1) competent cells from the same (ecto)mesenchymal population might be involved and 2) the genetic control of the developmental processes could be identical. It is suggested that the odontode expression in extra-oral positions is a relatively late novelty in this lineage. J. Morphol. 237:237–255, 1998. © 1998 Wiley-Liss, Inc.     

NK-2 class homeobox genes and pharyngeal/oral patterning: Nkx2-3 is required for salivary gland and tooth morphogenesis

Head development in vertebrates requires reciprocal patterning interactions between cranial neural crest and the ectodermal, mesodermal and endodermal components of the branchial arches. Patterning elements within the pharyngeal endoderm and oral ectoderm appear to play defining roles in this process. Several homeobox genes of the NK-2 class (Nkx2-1, Nkx2-3, Nkx2-5 and Nkx2-6) are expressed regionally in the developing pharynx, and Nkx2-1 mutants and Nkx2-5/Nkx2-6 double mutants show loss of thyroid and distal lung progenitors, and pharyngeal cell viability, respectively. Here we examined the expression and genetic role of Nkx2-3 in pharyngeal development. Nkx2-3 was expressed in the pharyngeal floor and pouches, as well as in oral and branchial arch ectoderm. Expression persisted in the developing thyroid until birth, in mucous-forming cells of the lingual and sublingual salivary glands, and in odontogenic epithelium of the mandible. Examination of Nkx2-3 null mice revealed defects in maturation and cellular organisation of the sublingual glands. Furthermore, cusps were absent from mandibular molars and the third molar was occasionally missing. These data suggest roles for Nkx2-3 during pharyngeal organogenesis, although the considerable potential for genetic redundancy within and outside of this gene family may mask earlier functions in organ specification.     

Structure and Development of Teeth in Three Armoured Catfish, Corydoras aeneus, C. arcuatus and Hoplosternum littorale (Siluriformes, Callichthyidae)

We have studied the premaxillary teeth in three armoured catfish, Corydoras aeneus, C. arcuatus and Hoplosternum littorale , by means of light and electron microscopy, in order to compare their development, fine structure and mode of attachment with that of odontodes and other teleost teeth. A premaxillary dentition consisting of small (50–100 μm long) slender pointed teeth showing no true replacement is only present in larval and juvenile stages and is subsequently lost, possibly in relation to a change in feeding mode from predatory to bottom feeder. Like odontodes, teeth are composed of dentine surrounding a pulp cavity and are covered by a hypermineralized cap. Particular features, also found in odontodes, are the absence of dentinal tubules and of nerves and capillaries in the pulp cavity, both possibly related to the small size of the teeth. The irregular pattern of implantation and the variability in attachment mode (primary and/or secondary attachment bone, fusion, mere apposition or ligamentous connection) distinguish the teeth from most other teleost teeth and from odontodes and are interpreted as reflecting considerable differences in dynamics of remodeling of the supporting element (premaxillary bone vs scute). This comparison of teeth and odontodes strongly supports current views according to which teeth and odontodes are two very closely related phenotypic expressions of a single, modifiable, morphogenetic system probably rooted in the earliest stages of vertebrate evolution.     

Morphological changes in the oral (buccopharyngeal) membrane in urodelan embryos: development of the mouth opening

The ultrastructure of the oral (buccopharyngeal) membrane in the embryo of the urodelan, Hynobius tokyoensis, was examined by transmission (TEM) and scanning electron microscopy (SEM). The oral membrane consists of the stomodeal ectoderm and foregut endoderm, and is three to five cell layers thick at stage 24. The oral membrane gradually thickens as development proceeds. The stomodeal collar, derived from the ectoderm, is folded inward along the foregut endoderm. Tooth germs are formed partly by cells of the stomodeal collar and partly by mesenchymal cells and calcification takes place before hatching. Secretory granules, which are markers of epithelial differentiation, appear in some cells of the foregut endoderm. Within the oral membrane, the cells of the stomodeal collar become the basal cells, and the endodermal cells of the foregut become the apical cells of the future oral epithelium. Gaps are formed by the epithelial differentiation of the endodermal cells of the foregut in the oral membrane. The gaps connect with each other, with the stomodeum, and with the foregut. As a result of these events, the mouth opens at stage 43, just after hatching.     

Low divergence in Dlx gene expression between dentitions of the medaka (Oryzias latipes) versus high level of expression shuffling in osteichtyans

SUMMARY Serially homologous structures are believed to originate from the redeployment of a genetic cascade in different locations of the body. Serial homologs may diverge at the genetic and morphological level and acquire developmental independency (individualization). Teeth are repeated units that form dentitions found on different bones of the oral–pharyngeal cavity in gnathostomes and provide a good model to study such processes. Previous comparisons of dlx gene expression patterns between mouse oral teeth and zebrafish pharyngeal teeth showed a high level of divergence. Furthermore, these genes are differentially expressed in different teeth of the zebrafish, and in the mouse they are responsible for tooth identity (incisors vs. molars). We examined the potential divergence of dlx gene expression between oral and pharyngeal teeth by examining the expression pattern in the development of the first generation teeth of the medaka and comparing it with data from the zebrafish and the mouse. Out of the seven medaka dlx genes, five are expressed during odontogenesis compared with six in both the zebrafish and the mouse. The only difference observed between oral and pharyngeal teeth in the medaka is an earlier expression of dlx5a in the oral dental epithelium. The subset of dlx genes expressed in the medaka, zebrafish, and mouse is slightly different but their detailed expression patterns are highly divergent. Our results demonstrate a low constraint on dlx gene expression shuffling in the odontogenic cascade within osteichtyans but the non-individualization of oral and pharyngeal dentitions in the medaka.