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Among extant cetaceans, mysticetes are filter feeders that do not possess teeth and use their baleen for feeding, while most odontocetes are considered suction feeders, which capture prey by suction without biting or chewing with teeth. In the present study, we address the functionality of amelogenin (AMEL) genes in cetaceans. AMEL encodes a protein that is specifically involved in dental enamel formation and is located on the sex chromosomes in eutherians. The X-copy AMELX is functional in enamel-bearing eutherians, whereas the Y-copy AMELY appears to have undergone decay and was completely lost in some species. Consistent with these premises, we detected various deleterious mutations and/or non-canonical splice junctions in AMELX of mysticetes and four suction feeding odontocetes, Delphinapterus leucas, Monodon monoceros, Kogia breviceps, and Physeter macrocephalus, and in AMELY of mysticetes and odontocetes. Regardless of the functionality, both AMELX and AMELY are equally and unusually small in cetaceans, and even their functional AMELX genes presumably encode a degenerate core region, which is thought to be essential for enamel matrix assembly and enamel crystal growth. Furthermore, our results suggest that the most recent common ancestors of extant cetaceans had functional AMELX and AMELY, both of which are similar to AMELX of Platanista minor. Similar small AMELX and AMELY in archaic cetaceans can be explained by gene conversion between AMELX and AMELY. We speculate that common ancestors of modern cetaceans employed a degenerate AMELX, transferred from a decaying AMELY by gene conversion, at an early stage of their transition to suction feeders.

Tooth plates of the chimaeroids, holocephalian fishes, are unique dental hard tissues. Unlike the teeth of other animals, the tooth plates are located on the roof of the mouth and in the lower jaw. Their tooth plates consist, to a large extent, of lightly mineralized tissue (osteodentin) and hypermineralized tissue (pleromin). Notably, the mineral phase of pleromin is whitlockite, while that of other animals is apatite. Dietary habits of chimaeroids and wearing features of their tooth plates suggest an extreme hardness of pleromin, but this has never been investigated. We examined the microhardness of the tooth plate of Chimaera phantasma and found that pleromin in the biting region was extremely hard, comparable with the hardness of mature tooth enamel, whereas the hardness of immature pleromin was lower than that of bovine dentin. The hardness of osteodentin, on the other hand, was equivalent to that of bovine dentin and almost the same throughout the tooth plate. Immature pleromin was sparsely packed with oval crystals of whitlockite and, as pleromin matures, the space between crystals was filled with small intercrystalline materials. The maturing process of pleromin could partly contribute to its remarkable hardness and have some implications for designing novel biomaterials.

In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin.

Summary We resolve debate over the evolution of vertebrate hypermineralized tissues through analyses of matrix protein-encoding secretory calcium-binding phosphoprotein (SCPP) genes and phylogenetic inference of hypermineralized tissues. Among these genes, AMBN and ENAM are found in both sarcopterygians and actinopterygians, whereas AMEL and SCPP5 are found only in sarcopterygians and actinopterygians, respectively. Actinopterygian AMBN, ENAM, and SCPP5 are expressed during the formation of hypermineralized tissues on scales and teeth: ganoin, acrodin, and collar enamel in gar, and acrodin and collar enameloid in zebrafish. Our phylogenetic analyses indicate the emergence of an ancestral enamel in stem-osteichthyans, whereas ganoin emerged in stem-actinopterygians and true enamel in stem-sarcopterygians. Thus, AMBN and ENAM originated in concert with ancestral enamel, SCPP5 evolved in association with ganoin, and AMEL evolved with true enamel. Shifts in gene expression domain and timing explain the evolution of different hypermineralized tissues. We propose that hypermineralized tissues in osteichthyans coevolved with matrix SCPP genes., Graphical Abstract, Highlights • Ganoin emerged in actinopterygians; true enamel arose in sarcopterygians • Dental enamel, acrodin, and enameloid in actinopterygians are related to ganoin • SCPP5 evolved in association with ganoin, whereas AMEL evolved with true enamel • Shifts in SCPP gene expression explain the evolution of hypermineralized tissues, Evolutionary Biology; Evolutionary Processes; Phylogenetics; Paleobiology

Enameloid is a well-mineralized tissue covering the tooth surface in fish and it corresponds to the outer-most layer of dentin. It was reported that both dental epithelial cells and odontoblasts are involved in the formation of enameloid. Nevertheless, the localization and timing of secretion of ectodermal enamel matrix proteins in enameloid are unclear. In the present study, the enameloid matrix during the stages of enameloid formation in spotted gar, Lepisosteus oculatus, an actinopterygian, was examined mainly by transmission electron microscopy-based immunohistochemistry using an anti-mammalian amelogenin antibody and antiserum. Positive immunoreactivity with the antibody and antiserum was found in enameloid from the surface to the dentin-enameloid junction just before the formation of crystallites. This immunoreactivity disappeared rapidly before the full appearance of crystallites in the enameloid during the stage of mineralization. Immunolabelling was usually found along the collagen fibrils but was not seen on the electron-dense fibrous structures, which were probably derived from matrix vesicles in the previous stage. In inner dental epithelial cells, the granules in the distal cytoplasm often showed positive immunoreactivity, suggesting that the enamel matrix protein-like proteins originated from inner dental epithelial cells. Enamel matrix protein-like proteins in the enameloid matrix might be common to the enamel matrix protein-like proteins previously reported in the collar enamel of teeth and ganoine of ganoid scales, because they exhibited marked immunoreactivity with the same anti-mammalian amelogenin antibodies. It is likely that enamel matrix protein-like proteins are involved in the formation of crystallites along collagen fibrils in enameloid.

Tooth enameloid in bony fish is a well-mineralized tissue resembling enamel in mammals. It was assumed that the dental epithelial cells are deeply involved in the formation of enameloid. However, unlike enamel matrix which fully consists of several ectodermal enamel matrix proteins (EMPs), whether enameloid matrix contains ectodermal EMPs has been debated for a long time. In the present study, transmission electron microscopy-based immunohistochemical examinations, using the protein A-gold method with antibodies and antiserum against mammalian amelogenin, were performed in order to search for EMP-like proteins in the cap enameloid of basic actinopterygians, Polypterus and gar. Positive immunoreactivity was detected in the cap enameloid matrix just before the appearance of many crystallites along collagen fibrils, indicating that the cap enameloid contains EMP-like proteins. Immunolabelling was usually found along the collagen fibrils but was not seen on the electron-dense fibrous structures. Therefore, it is conceivable that the ectodermal EMP-like proteins in cap enameloid are involved in crystallite formation along collagen fibrils.

Although most fish have no enamel layer in their teeth, those belonging to Lepisosteus (gars), an extant actinopterygian fish genus, do and so can be used to study amelogenesis. In order to examine the collar enamel matrix in gar teeth, we subjected gar teeth to light and electron microscopic immunohistochemical examinations using an antibody against bovine amelogenin (27 kDa) and antiserum against porcine amelogenin (25 kDa), as well as region-specific antibodies and antiserum against the C-terminus and middle region, and N-terminus of porcine amelogenin, respectively. The enamel matrix exhibited intense immunoreactivity to the anti-bovine amelogenin antibody and the anti-porcine amelogenin antiserum in addition to the C-terminal and middle region-specific antibodies, but not to the N-terminal-specific antiserum. These results suggest that the collar enamel matrix of gar teeth contains amelogenin-like proteins and that these proteins possess domains that closely resemble the C-terminal and middle regions of porcine amelogenin. Western blot analyses of the tooth germs of Lepisosteus were also performed. As a result, protein bands with molecular weights of 78 kDa and 65 kDa were clearly stained by the anti-bovine amelogenin antibody as well as the antiserum against porcine amelogenin and the middle-region-specific antibody. It is likely that the amelogenin-like proteins present in Lepisosteus do not correspond to the amelogenins found in mammals, although they do possess domains that are shared with mammalian amelogenins.

Recent findings regarding the structure and development of jaw teeth and ganoid scales in the basic actinopterygians Polypterus and Lepisosteus have been summarized in the present review. The relationship between cap enameloid, collar enamel in teeth, and ganoine in scales has been studied to understand the evolution of hard tissues in lower vertebrates. Enameloid is a well-mineralized tissue that is formed by both odontoblasts and dental epithelial cells and is found within the outermost layer of dentin. The inner dental epithelial cells surrounding the enameloid maintain the ability to produce amelogenin-like proteins; however, this may be a rudimentary function. The collar enamel of Polypterus and Lepisosteus exhibits marked immunoreactivity to anti-mammalian amelogenin antibodies, suggesting that it accommodates amelogenin-like proteins. It is likely that the collar enamel in basic actinopterygians corresponds to the enamel in sarcopterygians and amphibians. The recent fine structural and developmental studies have shown that the ganoine of ganoid scales in Polypterus and Lepisosteus is an enamel-like tissue. Positive immunoreactivity to the anti-mammalian amelogenin antibodies by the preganoine of both Polypteridae and Lepisosteidae supports that ganoine is a homolog of tooth enamel.

The morphological features of tooth enamel and enameloid in actinopterygian fish are reviewed to provide basic data concerning the biomineralization of teeth in lower vertebrates. Enameloid, which covers the tooth surface, is a unique well-mineralized tissue and usually has the same functions as mammalian tooth enamel. However, the development of enameloid is different from that of the enamel produced by dental epithelial cells. Enameloid is made by a combination of odontoblasts and dental epithelial cells. An organic matrix that contains collagen is provided by odontoblasts, and then dental epithelial cells dissolve the degenerate matrix and supply inorganic ions during advanced crystal growth in enameloid. It is likely that enameloid is a good model for studying the growth of well-mineralized hard tissues in vertebrates. Some actinopterygian fish possess a collar enamel layer that is situated at the surface of the tooth shaft, indicating that the origin of tooth enamel is found in fish. Collar enamel is thought to be a precursor of mammalian enamel, although it is thin and not well mineralized in comparison with enameloid. In Lepisosteus and Polypterus, both of which are living actinopterygians, both enameloid and enamel are found in the same tooth. Therefore, they are suitable materials for examining the developmental processes of enameloid and enamel and the relationship among them.

The fine structure of collar enamel and the cells constituting the enamel organ during amelogenesis in Lepisosteus oculatus was observed by light, scanning electron and transmission electron microscopy. In the enamel, slender crystals were arranged perpendicular to the surface and the stripes that were parallel to the surface were observed, suggesting that the enamel in Lepisosteus shares common morphological features with that in sarcopterygian fish and amphibians. Ameloblasts containing developed Golgi apparatus, rough endoplasmic reticulum (rER) and secretory granules were found in the secretory stage. In the maturation stage, a ruffled border was not seen at the distal end of the ameloblasts, while many mitochondria and lysosome-like granules were obvious in the distal cytoplasm. The enamel organ consisted of the outer dental epithelial cells, stratum reticulum cells and ameloblasts, but there was no stratum intermedium. It is likely that the ameloblasts have less absorptive function in comparison with the inner dental epithelial cells facing cap enameloid.

This is the first report on the stratum intermedium in vertebrates other than mammals. The aim of this study is to elucidate the fine structure and cytochemical features of the stratum intermedium during the stages of enameloid formation in Lepisosteus. Inner dental epithelium, stratum intermedium, stellate reticulum, and outer dental epithelium are consistently present in the tooth germs of Lepisosteus. The stratum intermedium cells are oval in shape, contain elliptical nuclei, and extend many small processes. It is implied that the structure of the enamel organ is different among actinopterygians, and that constitution of the enamel organ in Lepisosteus resembles that in higher vertebrates. Marked Ca-ATPase activity is observed at the cell membrane of the stratum intermedium cells, suggesting that the cells are involved in calcium transport during the stages of enameloid formation.

Previous studies have demonstrated the morphological similarity of the enamel-like layer found in the teeth of the coelacanth, lungfish and gar-pike to the enamel of tetrapods. In order to clarify the phylogenetic continuity between both structures, tooth germs of the gar-pike were immunocytochemically studied using an anti-bovine amelogenin polyclonal antibody. Intense immunoreaction was shown over the enamel-like matrix layer. Certain cell organelles associated with the secretory pathway of the ameloblasts were recognized as immunoreactive. These results indicate that the enamel-like layer of the gar-pike is a tissue homologous with the mammalian enamel because both possess a common, amelogenin-like substance.

In the snake, Elaphe quadrivirgata, the occurrence of amelogenin was immunohistochemically demonstrated in the enamel of developing tooth germs. Teeth of the snake are covered with a thin true enamel layer about 1-2 microns in thickness. Light and electron microscopic immunohistochemistry indicated an intense amelogenin immunoreactivity occurring in the enamel layer during the secretory stage of tooth development. Cloning and cDNA sequence of snake amelogenin was performed by RT-PCR. The amino acid sequence of the snake amelogenin cDNA--in its portion corresponding to the area from exon 5 to exon 7 of human X189 amelogenin gene--showed 45% homology with humans. Regions of both the N-terminus and C-terminus were well conserved. Furthermore, the positions of prolin in the amino acid alignment of the snake amelogenin corresponded well with those of human amelogenin. It is suggested that prolin is an essential constituent of amelogenin and therefore its positions in the molecule have been conserved after the evolutionary divergence of reptiles and mammals. This study using reptiles is the first detection of specific amelogenin immunoreactivity by high resolutional immunoelectron microscopy and the first cloning of amelogenin cDNA in a non-mammalian animal.

This is the first detailed report about the collar enamel of the teeth of Polypterus senegalus. We have examined the fine structure of the collar enamel and enamel organ of Polypterus during amelogenesis by light and transmission electron microscopy. An immunohistochemical analysis with an antibody against bovine amelogenin, an antiserum against porcine amelogenin and region-specific antibodies or antiserum against the C-terminus, middle region and N-terminus of porcine amelogenin has also been performed to examine the collar enamel matrix present in these teeth. Their ameloblasts contain fully developed Golgi apparatus, rough endoplasmic reticulum and secretory granules. During collar enamel formation, an amorphous fine enamel matrix containing no collagen fibrils is found between the dentin and ameloblast layers. In non-demineralized sections, the collar enamel (500 nm to 1 μm thick) is distinguishable from dentin, because of its higher density and differences in the arrangement of its crystals. The fine structural features of collar enamel in Polypterus are similar to those of tooth enamel in Lepisosteus (gars), coelacanths, lungfish and amphibians. The enamel matrix shows intense immunoreactivity to the antibody and antiserum against mammalian amelogenins and to the middleregion- and C-terminal-specific anti-amelogenin antibodies. These findings suggest that the proteins in the enamel of Polypterus contain domains that closely resemble those of bovine and porcine amelogenins. The enamel matrix, which exhibits positive immunoreactivity to mammalian amelogenins, extends to the cap enameloid surface, implying that amelogenin-like proteins are secreted by ameloblasts as a thin matrix layer that covers the cap enameloid after enameloid maturation.

Teeth have been missing from Aves for almost 100 million years. However, it is believed that the avian oral epithelium retains the molecular signaling required to induce odontogenesis, and this has been widely examined using heterospecific recombinations with mouse dental mesenchyme. It has also been argued that teeth can form from the avian oral epithelium owing to contamination of the mouse mesenchyme with mouse dental epithelial cells. To investigate the possibility of tooth formation from chick oral epithelium and the characteristics of possible chick enamel, we applied LacZ transgenic mice during heterospecific recombination and examined the further tooth formation. Transmission electron microscopy was used to identify the two tissues during development after heterospecific recombination. No mixing was detected between chick oral epithelium and mouse dental mesenchyme after 2 days, and secretory ameloblasts with Tomes' processes were observed after 1 week. Teeth were formed after 3 weeks with a single cusp pattern, possibly determined by epithelial factors, which is similar to that of the avian tooth in the late Jurassic period. These recombinant teeth were smaller than mouse molars, whereas perfect structures of both ameloblasts and enamel showed histological characteristics similar to those of mice. Together these observations consistent with previous report that odontogenesis is initially directed by species-specific mesenchymal signals interplaying with common epithelial signals.

Tooth germs during cap enameloid formation stages in Polypterus senegalus were investigated by transmission electron microscopy and enzyme histo- and cytochemistry. Enameloid formation was divided into three stages: matrix formation, mineralization, and maturation. The enamel organ consisted of the inner dental epithelial cells, stellate reticulum, and outer dental epithelial cells during cap enameloid formation stages, but no stratum intermedium was found. During the matrix formation stage, the tall inner dental epithelial cells contained well-developed Golgi apparatus, abundant cisternae of rough endoplasmic reticulum and mitochondria. Spindle-shaped vesicles containing a filamentous structure were seen in the distal cytoplasm. During mineralization and maturation stages, many ACPase positive lysosomes were present in the cytoplasm, whereas the organelles were decreased in number. The infoldings of the distal plasma membrane of the inner dental epithelial cells were visible in the mineralization stage but were not marked in the maturation stage. The activity of nonspecific ALPase, Ca-ATPase, and K-NPPase was detected at the plasma membrane of the inner dental epithelial cells during the stages of mineralization and maturation. The results of fine structure and enzyme cytochemistry suggested that the dental epithelial cells were mainly involved in the degeneration and removal of enameloid matrix and in material transportation during the enameloid mineralization and maturation stages, rather than in the enameloid matrix formation. The results also showed that the structure of the dental epithelial cells in Polypterus was different from that in teleosts and gars, but that the function of the dental epithelial cells was similar to that in teleosts possessing well-mineralized cap enameloid.

The tooth in the gar-pike, Lepisosteus oculatus, an actinopterygian fish, is characterized by the occurrence of both enamel and enameloid, the former covering the tooth shaft and the latter, the tooth cap. Our previous research demonstrated that the enamel in this species was, as in the lungfish, immunoreactive for amelogenin, indicating its homologous nature with the mammalian tooth enamel, whereas the enameloid was completely immunonegative. The present study demonstrates that, during the early maturation stage of the enameloid formation, the inner enamel epithelial cells (IEECs) synthesize through a well-developed Golgi apparatus a fine-granular substance which is intensely immunoreactive for amelogenin. This substance was accumulated in a large saccule extended in a suprabasal zone of the cell; we were unable to find any morphological sign indicating a connection of the substance with the enameloid matrix. The abortive secretion of the enamel matrix-like substance in the IEEC during an enameloid formation was considered to be an instance of rudimental enamel formation. In the gar-pike, the synthesis of amelogenin in the IEEC has been demonstrated to occur independently from that of the enameloid matrix. The present findings demonstrate a prominent difference between the tooth enamel and enameloid.

The gross structure and microstructure of holocephalian tooth plates were initially investigated by Bargmann (1,2) and Brettnacher (3). Orvig (4,5,6) subsequently reported in the detail microstructural characteristics of both extant and fossil holocephalian tooth plates. These studies indicate the holocephalian tooth plate to consist of bony tissue and hypermineralized pleromin (4), with the hardness of pleromin comparable to that of enamel and enameloid. Ishiyama et al. (7) found the crystalline material of pleromin to be not hydroxyapatite but whitlockite by a powder X-ray diffractometry, and pleromin to be the sole sound dental tissue consisting of whitlockite in vertebrates.

The sea snake, E. ijimae, has no functional teeth other than maxillary fangs and pterygoid teeth in the jaws as has been observed in the present species by Voris (1966). Histological examination by light microscopy reveals that the dentary and palatine dental laminae exist close to the dentary and palatine bones respectively and the maxillary dental laminae is separated backward of the maxilla in the adult specimen. On the other hand, the fetus, nearly ready for birth, has tooth germs which are situated in superficial portion of the oral mucosa and also near the dentary bones. Because of these histological structures, the dentition of both specimens are apparently vestigial, and by taking the comparative anatomy and/or dietary habit into consideration, the present species of degenerated dentition may have originated from ancestral toothed species.