Multiple epithelia are required to develop teeth deep inside the pharynx.

To explain the evolutionary origin of vertebrate teeth from odontodes, it has been proposed that competent epithelium spread into the oropharyngeal cavity via the mouth and other possible channels such as the gill slits [Huysseune et al., 2009, J. Anat. 214, 465-476]. Whether tooth formation deep inside the pharynx in extant vertebrates continues to require external epithelia has not been addressed so far. Using zebrafish we have previously demonstrated that cells derived from the periderm penetrate the oropharyngeal cavity via the mouth and via the endodermal pouches and connect to periderm-like cells that subsequently cover the entire endoderm-derived pharyngeal epithelium [Rosa et al., 2019, Sci. Rep. 9, 10082]. We now provide conclusive evidence that the epithelial component of pharyngeal teeth in zebrafish (the enamel organ) is derived from medial endoderm, as hitherto assumed based on position deep in the pharynx. Yet, dental morphogenesis starts only after the corresponding endodermal pouch (pouch 6) has made contact with the skin ectoderm, and only after periderm-like cells have covered the prospective tooth-forming endodermal epithelium. Manipulation of signaling pathways shown to adversely affect tooth development indicates they act downstream of these events. We demonstrate that pouch-ectoderm contact and the presence of a periderm-like layer are both required, but not sufficient, for tooth initiation in the pharynx. We conclude that the earliest interactions to generate pharyngeal teeth encompass those between different epithelial populations (skin ectoderm, endoderm, and periderm-like cells in zebrafish), in addition to the epithelial-mesenchymal interactions that govern the formation of all vertebrate teeth.

Miniaturization: How many cells are needed to build a tooth?

BACKGROUND: Organs that develop early in life, and are replaced by a larger version as the animal grows, often represent a miniature version of the adult organ. Teeth constituting the first functional dentition in small-sized teleost fish, such as medaka (Oryzias latipes), are examples of such miniature organs. With a dentin cone as small as the size of one human cell, or even smaller, these teeth raise the question how many dentin-producing cells (odontoblasts) are required to build such a tooth, and whether this number can be as little as one. RESULTS: Based on detailed observations with transmission electron microscopy (TEM) and TEM-based 3D-reconstructions, we show that only one mesenchymal cell qualifies as a true odontoblast. A second mesenchymal cell potentially participates in dentin formation, but only at a late stage of tooth development. Moreover, the fate of these cells appears to be specified very early during tooth development. CONCLUSIONS: Our observations indicate that in this system, one single odontoblast fulfills roles normally exerted by a large and communicating cell population. First-generation teeth in medaka thus provide an exciting model to study integration of multiple functions into a single cell.

Fox proteins are modular competency factors for facial cartilage and tooth specification.

Facial form depends on the precise positioning of cartilage, bone, and tooth fields in the embryonic pharyngeal arches. How complex signaling information is integrated to specify these cell types remains a mystery. We find that modular expression of Forkhead domain transcription factors (Fox proteins) in the zebrafish face arises through integration of Hh, Fgf, Bmp, Edn1 and Jagged-Notch pathways. Whereas loss of C-class Fox proteins results in reduced upper facial cartilages, loss of F-class Fox proteins results in distal jaw truncations and absent midline cartilages and teeth. We show that Fox proteins are required for Sox9a to promote chondrogenic gene expression. Fox proteins are sufficient in neural crest-derived cells for cartilage development, and neural crest-specific misexpression of Fox proteins expands the cartilage domain but inhibits bone. These results support a modular role for Fox proteins in establishing the competency of progenitors to form cartilage and teeth in the face.

Slow cycling cells in the continuous dental lamina of Scyliorhinus canicula: new evidence for stem cells in sharks.

In the lesser spotted catshark (Scyliorhinus canicula), as in most non-mammalian vertebrates, the dentition renews throughout life. To contribute to our understanding of how continuous tooth replacement is achieved, we searched for evidence for the presence of stem cells in this species. Three-dimensional reconstructions of juvenile (2-3 weeks post-hatch) specimens showed that tooth families merge imperceptibly with so-called interdental zones within a continuous and permanent dental lamina. Interdental regions are composed of three layers, continuous with cervical loop, middle, and outer dental epithelium of the tooth families, respectively. A BrdU pulse-chase experiment revealed that cell proliferation is initiated in the lingual part of the dental lamina and the resulting population shifts one tooth position towards the oral epithelium in around four to five weeks. In the longest chase time (114 days) label-retaining and arguably non-differentiated cells were present at the lingual border of the dental lamina. These were found in the outer and middle dental epithelium, both within and between tooth families. This area of the dental lamina did not show expression or distribution of Sox2. Our data support the hypothesis that stem cells reside at the lingual border of the continuous dental lamina, more specifically in the middle dental epithelium at the level of the tooth families, and in its extension between the tooth families. To demonstrate their true stemness and their role in continuous tooth replacement, it remains to be shown that these cells have the potential to give rise to a complete new successor.

The innervation of the zebrafish pharyngeal jaws and teeth.

Zebrafish (Danio rerio) teeth are increasingly used as a model to study odontogenesis in non-mammalians. Using serial semi-thin section histology and immunohistochemistry, the nerves innervating the pharyngeal jaws and teeth have been identified. The last pair of branchial arches, which are non-gill bearing but which carry the teeth, are innervated by an internal branch of a post-trematic ramus of the vagal nerve. Another, external, branch is probably responsible for the motor innervation of the branchiomeric musculature. Nerve fibres appear in the pulp cavity of the teeth only late during cytodifferentiation, and are therefore likely not involved in early steps of tooth formation. The precise role of the nervous system during continuous tooth replacement remains to be determined. Nonetheless, this study provides the necessary morphological background information to address this question.

Tooth replacement without a dental lamina: the search for epithelial stem cells in Polypterus senegalus.

Most actinopterygians replace their teeth continuously throughout life. To address the question of where and how replacement teeth form in actinopterygians, it is advisable to investigate well-chosen representatives within the lineage. The African bichir, Polypterus senegalus, belongs to the earliest diverged group of the actinopterygian lineage with currently living representatives. Its well characterized dentition, together with its phylogenetic position, make this species an attractive model to answer following questions: (1) when and where does the replacement tooth form and how is it connected with the dental organ of the predecessor, and (2) is there any evidence for the presence of epithelial stem cells, hypothesized to play a role in replacement? Serial sections show that one tooth family can contain up to three members, which are all interconnected by dental epithelium. Replacement teeth develop without the presence of a successional dental lamina. We propose that this is the plesiomorphic condition for tooth replacement in actinopterygians. BrdU pulse-chase experiments reveal cells in the outer and middle dental epithelium, proliferating at the time of initiation of a new replacement tooth. It is tempting to assume that these cell layers provide a stem cell niche. The observed absence of label-retaining cells after long chase times (up to 8 weeks) is held against the light of divergent views on cell cycling properties of stem cells. At present, our data do not support, neither reject, the hypothesis on involvement of epithelial stem cells within the process of continuous tooth replacement.

Continuous tooth replacement: what can teleost fish teach us?

Most tooth-bearing non-mammalian vertebrates have the capacity to replace their teeth throughout life. This capacity was lost in mammals, which replace their teeth only once at most. Not surprisingly, continuous tooth replacement has attracted much attention. Classical morphological studies (e.g. to analyse patterns of replacement) are now being complemented by molecular studies that investigate the expression of genes involved in tooth formation. This review focuses on ray-finned fish (actinopterygians), which have teeth often distributed throughout the mouth and pharynx, and more specifically on teleost fish, the largest group of extant vertebrates. First we highlight the diversity in tooth distribution and in tooth replacement patterns. Replacement tooth formation can start from a distinct (usually discontinuous and transient) dental lamina, but also in the absence of a successional lamina, e.g. from the surface epithelium of the oropharynx or from the outer dental epithelium of a predecessor tooth. The relationship of a replacement tooth to its predecessor is closely related to whether replacement is the result of a prepattern or occurs on demand. As replacement teeth do not necessarily have the same molecular signature as first-generation teeth, the question of the actual trigger for tooth replacement is discussed. Much emphasis has been laid in the past on the potential role of epithelial stem cells in initiating tooth replacement. The outcome of such studies has been equivocal, possibly related to the taxa investigated, and the permanent or transient nature of the dental lamina. Alternatively, replacement may result from local proliferation of undifferentiated progenitors, stimulated by hitherto unknown, perhaps mesenchymal, factors. So far, the role of the neurovascular link in continuous tooth replacement has been poorly investigated, despite the presence of a rich vascularisation surrounding actinopterygian (as well as chondrichthyan) teeth and despite a complete arrest of tooth replacement after nerve resection. Lastly, tooth replacement is possibly co-opted as a process to expand the number of teeth in a dentition ontogenetically whilst conserving features of the primary dentition. That neither a dental lamina, nor stem cells appear to be required for tooth replacement places teleosts in an advantageous position as models for tooth regeneration in humans, where the dental lamina regresses and epithelial stem cells are considered lost.

Developmental disorders of the dentition: an update.

Dental anomalies are common congenital malformations that can occur either as isolated findings or as part of a syndrome. This review focuses on genetic causes of abnormal tooth development and the implications of these abnormalities for clinical care. As an introduction, we describe general insights into the genetics of tooth development obtained from mouse and zebrafish models. This is followed by a discussion of isolated as well as syndromic tooth agenesis, including Van der Woude syndrome (VWS), ectodermal dysplasias (EDs), oral-facial-digital (OFD) syndrome type I, Rieger syndrome, holoprosencephaly, and tooth anomalies associated with cleft lip and palate. Next, we review delayed formation and eruption of teeth, as well as abnormalities in tooth size, shape, and form. Finally, isolated and syndromic causes of supernumerary teeth are considered, including cleidocranial dysplasia and Gardner syndrome.

Unravelling the blood supply to the zebrafish pharyngeal jaws and teeth.

We describe the vascular supply to the pharyngeal jaws and teeth in zebrafish, from larval stages to juveniles, using serial high quality semithin sections and 3D reconstructions. We have identified that the arterial blood supply to the last pair of branchial arches, which carries the teeth, issues from the hypobranchial artery. Surprisingly, the arteries supplying the pharyngeal jaws show an asymmetric branching pattern that is modified over ontogeny. Moreover, the blood vessel pattern that serves each jaw can best be described as a sinusoidal cavity encircling the bases of both the functional and replacement teeth. Capillaries branching from this sinusoidal cavity enter the pulp and constitute the intrinsic blood supply to the attached teeth. The role of these blood vessels during tooth development (whether instructive or nutritive) remains to be determined and requires further study. However, we have provided a firm morphological basis that will aid in the interpretation of experiments addressing this question.

Bone Formation in Zebrafish: The Significance of DAF-FM DA Staining for Nitric Oxide Detection.

DAF-FM DA is widely used as a live staining compound to show the presence of nitric oxide (NO) in cells. Applying this stain to live zebrafish embryos is known to indicate early centers of bone formation, but the precise (cellular) location of the signal has hitherto not been revealed. Using sections of zebrafish embryos live-stained with DAF-FM DA, we could confirm that the fluorescent signals were predominantly located in areas of ongoing bone formation. Signals were observed in the bone and tooth matrix, in the notochord sheath, as well as in the bulbus arteriosus. Surprisingly, however, they were exclusively extracellular, even after very short staining times. Von Kossa and Alizarin red S staining to reveal mineral deposits showed that DAF-FM DA stains both the mineralized and non-mineralized bone matrix (osteoid), excluding that DAF-FM DA binds non-specifically to calcified structures. The importance of NO in bone formation by osteoblasts is nevertheless undisputed, as shown by the absence of bone structures after the inhibition of NOS enzymes that catalyze the formation of NO. In conclusion, in zebrafish skeletal biology, DAF-FM DA is appropriate to reveal bone formation in vivo, independent of mineralization of the bone matrix, but it does not demonstrate intracellular NO.

The conundrum of pharyngeal teeth origin: the role of germ layers, pouches, and gill slits.

There are several competing hypotheses on tooth origins, with discussions eventually settling in favour of an 'outside-in' scenario, in which internal odontodes (teeth) derived from external odontodes (skin denticles) in jawless vertebrates. The evolution of oral teeth from skin denticles can be intuitively understood from their location at the mouth entrance. However, the basal condition for jawed vertebrates is arguably to possess teeth distributed throughout the oropharynx (i.e. oral and pharyngeal teeth). As skin denticle development requires the presence of ectoderm-derived epithelium and of mesenchyme, it remains to be answered how odontode-forming skin epithelium, or its competence, were 'transferred' deep into the endoderm-covered oropharynx. The 'modified outside-in' hypothesis for tooth origins proposed that this transfer was accomplished through displacement of odontogenic epithelium, that is ectoderm, not only through the mouth, but also via any opening (e.g. gill slits) that connects the ectoderm to the epithelial lining of the pharynx (endoderm). This review explores from an evolutionary and from a developmental perspective whether ectoderm plays a role in (pharyngeal) tooth and denticle formation. Historic and recent studies on tooth development show that the odontogenic epithelium (enamel organ) of oral or pharyngeal teeth can be of ectodermal, endodermal, or of mixed ecto-endodermal origin. Comprehensive data are, however, only available for a few taxa. Interestingly, in these taxa, the enamel organ always develops from the basal layer of a stratified epithelium that is at least bilayered. In zebrafish, a miniaturised teleost that only retains pharyngeal teeth, an epithelial surface layer with ectoderm-like characters is required to initiate the formation of an enamel organ from the basal, endodermal epithelium. In urodele amphibians, the bilayered epithelium is endodermal, but the surface layer acquires ectodermal characters, here termed 'epidermalised endoderm'. Furthermore, ectoderm-endoderm contacts at pouch-cleft boundaries (i.e. the prospective gill slits) are important for pharyngeal tooth initiation, even if the influx of ectoderm via these routes is limited. A balance between sonic hedgehog and retinoic acid signalling could operate to assign tooth-initiating competence to the endoderm at the level of any particular pouch. In summary, three characters are identified as being required for pharyngeal tooth formation: (i) pouch-cleft contact, (ii) a stratified epithelium, of which (iii) the apical layer adopts ectodermal features. These characters delimit the area in which teeth can form, yet cannot alone explain the distribution of teeth over the different pharyngeal arches. The review concludes with a hypothetical evolutionary scenario regarding the persisting influence of ectoderm on pharyngeal tooth formation. Studies on basal osteichthyans with less-specialised types of early embryonic development will provide a crucial test for the potential role of ectoderm in pharyngeal tooth formation and for the 'modified outside-in' hypothesis of tooth origins.

Cells at the Edge: The Dentin-Bone Interface in Zebrafish Teeth.

Bone-producing osteoblasts and dentin-producing odontoblasts are closely related cell types, a result from their shared evolutionary history in the ancient dermal skeleton. In mammals, the two cell types can be distinguished based on histological characters and the cells' position in the pulp cavity or in the tripartite periodontal complex. Different from mammals, teleost fish feature a broad diversity in tooth attachment modes, ranging from fibrous attachment to firm ankylosis to the underlying bone. The connection between dentin and jaw bone is often mediated by a collar of mineralized tissue, a part of the dental unit that has been termed "bone of attachment". Its nature (bone, dentin, or an intermediate tissue type) is still debated. Likewise, there is a debate about the nature of the cells secreting this tissue: osteoblasts, odontoblasts, or yet another (intermediate) type of scleroblast. Here, we use expression of the P/Q rich secretory calcium-binding phosphoprotein 5 (scpp5) to characterize the cells lining the so-called bone of attachment in the zebrafish dentition. scpp5 is expressed in late cytodifferentiation stage odontoblasts but not in the cells depositing the "bone of attachment". nor in bona fide osteoblasts lining the supporting pharyngeal jaw bone. Together with the presence of the osteoblast marker Zns-5, and the absence of covering epithelium, this links the cells depositing the "bone of attachment" to osteoblasts rather than to odontoblasts. The presence of dentinal tubule-like cell extensions and the near absence of osteocytes, nevertheless distinguishes the "bone of attachment" from true bone. These results suggest that the "bone of attachment" in zebrafish has characters intermediate between bone and dentin, and, as a tissue, is better termed "dentinous bone". In other teleosts, the tissue may adopt different properties. The data furthermore support the view that these two tissues are part of a continuum of mineralized tissues. Expression of scpp5 can be a valuable tool to investigate how differentiation pathways diverge between osteoblasts and odontoblasts in teleost models and help resolving the evolutionary history of tooth attachment structures in actinopterygians.

Seadragon genome analysis provides insights into its phenotype and sex determination locus.

The iconic phenotype of seadragons includes leaf-like appendages, a toothless tubular mouth, and male pregnancy involving incubation of fertilized eggs on an open "brood patch." We de novo-sequenced male and female genomes of the common seadragon (Phyllopteryx taeniolatus) and its closely related species, the alligator pipefish (Syngnathoides biaculeatus). Transcription profiles from an evolutionary novelty, the leaf-like appendages, show that a set of genes typically involved in fin development have been co-opted as well as an enrichment of transcripts for potential tissue repair and immune defense genes. The zebrafish mutants for scpp5, which is lost in all syngnathids, were found to lack or have deformed pharyngeal teeth, supporting the hypothesis that the loss of scpp5 has contributed to the loss of teeth in syngnathids. A putative sex-determining locus encoding a male-specific amhr2y gene shared by common seadragon and alligator pipefish was identified.

Zebrafish teeth as a model for repetitive epithelial morphogenesis: dynamics of E-cadherin expression.

BACKGROUND: The development of teeth is the result of interactions between competent mesenchyme and epithelium, both of which undergo extensive morphogenesis. The importance of cell adhesion molecules in morphogenesis has long been acknowledged but remarkably few studies have focused on the distribution and function of these molecules in tooth development. RESULTS: We analyzed the expression pattern of an important epithelial cadherin, E-cadherin, during the formation of first-generation teeth as well as replacement teeth in the zebrafish, using in situ hybridization and whole mount immunostaining to reveal mRNA expression and protein distribution. E-cadherin was detected in every layer of the enamel organ during the different stages of tooth development, but there were slight differences between first-generation and replacement teeth in the strength and distribution of the signal. The dental papilla, which is derived from the mesenchyme, did not show any expression. Remarkably, the crypts surrounding the functional teeth showed an uneven distribution of E-cadherin throughout the pharyngeal region. CONCLUSIONS: The slight differences between E-cadherin expression in zebrafish teeth and developing mouse and human teeth are discussed in the light of fundamental differences in structural and developmental features of the dentition between zebrafish and mammals. Importantly, the uninterrupted expression of E-cadherin indicates that down-regulation of E-cadherin is not required for formation of an epithelial tooth bud. Further research is needed to understand the role of other cell adhesion systems during the development of teeth and the formation of replacement teeth.

Evolutionary and developmental origins of the vertebrate dentition.

According to the classical theory, teeth derive from odontodes that invaded the oral cavity in conjunction with the origin of jaws (the 'outside in' theory). A recent alternative hypothesis suggests that teeth evolved prior to the origin of jaws as endodermal derivatives (the 'inside out' hypothesis). We compare the two theories in the light of current data and propose a third scenario, a revised 'outside in' hypothesis. We suggest that teeth may have arisen before the origin of jaws, as a 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. This hypothesis revives the homology between skin denticles (odontodes) and teeth. Our hypothesis is based on (1) the assumption that endoderm alone, together with neural crest, cannot form teeth; (2) 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, and (3) the observation that the dental lamina (sensu Reif, 1982) is not a prerequisite for teeth to form. We next discuss the progress that has been made to understand the spatially restricted loss of teeth from certain arches, and the many questions that remain regarding the ontogenetic loss of teeth in specific taxa. The recent advances that have been made in our knowledge on the molecular control of tooth formation in non-mammalians (mostly in some teleost model species) will undoubtedly contribute to answering these questions in the coming years.

Periderm invasion contributes to epithelial formation in the teleost pharynx.

The gnathostome pharyngeal cavity functions in food transport and respiration. In amniotes the mouth and nares are the only channels allowing direct contact between internal and external epithelia. In teleost fish, gill slits arise through opening of endodermal pouches and connect the pharynx to the exterior. Using transgenic zebrafish lines, cell tracing, live imaging and different markers, we investigated if pharyngeal openings enable epithelial invasion and how this modifies the pharyngeal epithelium. We conclude that in zebrafish the pharyngeal endoderm becomes overlain by cells with a peridermal phenotype. In a wave starting from pouch 2, peridermal cells from the outer skin layer invade the successive pouches until halfway their depth. Here the peridermal cells connect to a population of cells inside the pharyngeal cavity that express periderm markers, yet do not invade from outside. The latter population expands along the midline from anterior to posterior until the esophagus-gut boundary. Together, our results show a novel role for the periderm as an internal epithelium becomes adapted to function as an external surface.

Formation of a successional dental lamina in the zebrafish (Danio rerio): support for a local control of replacement tooth initiation.

In order to test whether the formation of a replacement tooth bud in a continuously replacing dentition is linked to the functional state of the tooth predecessor, I examined the timing of development of replacement teeth with respect to their functional predecessors in the pharyngeal dentition of the zebrafish. Observations based on serial semithin sections of ten specimens, ranging in age from four week old juveniles to adults, indicate that (i) a replacement tooth germ develops at the distal end of an epithelial structure, called the successional dental lamina, budding off from the crypt epithelium surrounding the erupted part of a functional tooth; (ii) there appears to be a developmental link between the eruption of a tooth and the formation of a successional dental lamina and (iii) there can be a time difference between successional lamina formation and initiation of the new tooth germ, i.e., the successional dental lamina can remain quiescent for some time. The data suggest that the formation of a successional lamina and the differentiation of a replacement tooth germ from this lamina, are two distinct phases of a process and possibly under a different control. The strong spatio-temporal coincidence of eruption of a tooth and development of a successional dental lamina is seen as evidence for a local control over tooth replacement.

Beta-Catenin and Plakoglobin Expression during Zebrafish Tooth Development and Replacement.

We analyzed the protein distribution of two cadherin-associated molecules, plakoglobin and ß-catenin, during the different stages of tooth development and tooth replacement in zebrafish. Plakoglobin was detected at the plasma membrane already at the onset of tooth development in the epithelial cells of the tooth. This pattern remained unaltered during further tooth development. The mesenchymal cells only showed plakoglobin from cytodifferentiation onwards. Plakoglobin 1a morpholino-injected embryos showed normal tooth development with proper initiation and differentiation. Although plakoglobin is clearly present during normal odontogenesis, the loss of plakoglobin 1a does not influence tooth development. ß-catenin was found at the cell borders of all cells of the successional lamina but also in the nuclei of surrounding mesenchymal cells. Only membranous, not nuclear, ß-catenin, was found during morphogenesis stage. However, during cytodifferentiation stage, both nuclear and membrane-bound ß-catenin was detected in the layers of the enamel organ as well as in the differentiating odontoblasts. Nuclear ß-catenin is an indication of an activated Wnt pathway, therefore suggesting a possible role for Wnt signalling during zebrafish tooth development and replacement.

Epithelial Label-Retaining Cells Are Absent during Tooth Cycling in Salmo salar and Polypterus senegalus.

The Atlantic salmon (Salmo salar) and African bichir (Polypterus senegalus) are both actinopterygian fish species that continuously replace their teeth without the involvement of a successional dental lamina. Instead, they share the presence of a middle dental epithelium: an epithelial tier enclosed by inner and outer dental epithelium. It has been hypothesized that this tier could functionally substitute for a successional dental lamina and might be a potential niche to house epithelial stem cells involved in tooth cycling. Therefore, in this study we performed a BrdU pulse chase experiment on both species to (1) determine the localization and extent of proliferating cells in the dental epithelial layers, (2) describe cell dynamics and (3) investigate if label-retaining cells are present, suggestive for the putative presence of stem cells. Cells proliferate in the middle dental epithelium, outer dental epithelium and cervical loop at the lingual side of the dental organ to form a new tooth germ. Using long chase times, both in S. salar (eight weeks) and P. senegalus (eight weeks and twelve weeks), we could not reveal the presence of label-retaining cells in the dental organ. Immunostaining of P. senegalus dental organs for the transcription factor Sox2, often used as a stem cell marker, labelled cells in the zone of outer dental epithelium which grades into the oral epithelium (ODE transition zone) and the inner dental epithelium of a successor only. The location of Sox2 distribution does not provide evidence for epithelial stem cells in the dental organ and, more specifically, in the middle dental epithelium. Comparison of S. salar and P. senegalus reveals shared traits in tooth cycling and thus advances our understanding of the developmental mechanism that ensures lifelong replacement.

Are breeding teeth in Atlantic salmon a component of the drastic alterations of the oral facial skeleton?

Upriver spawning migration of starving Atlantic salmon (Salmo salar) involves drastic skeletal alterations, among which a toothless stage followed by the appearance of a new set of so-called breeding teeth has been described. To investigate this phenomenon, we examined the patterns of tooth replacement on the lower jaws in different life stages of wild animals before and after spawning. Prior to spawning, every position held either a functional or a replacement tooth, both in first-time (grilse) and repetitive (salmon) spawners. Teeth were in a similar developmental stage every three positions along the tooth row. A functional tooth occurred in every third position and intermediate positions were taken by developing teeth. Within the process of replacement, teeth were resorbed and not shed. Our observations on an uninterrupted tooth replacement pattern provided no evidence of an intermediate toothless stage nor of a specialized breeding-teeth generation. Only animals that survived spawning (kelts) showed a highly variable tooth pattern, but with the initial "every third position" pattern still recognizable in some animals. We hypothesise that previous accounts describing a complete tooth loss/replacement relate to proliferation of the oral mucosa that conceals the teeth prior to the breeding period and to the use of maceration techniques that could have removed all teeth with an incompletely mineralised base.