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Gars and bichirs develop scales and teeth with ancient actinopterygian characteristics. Their scale surface and tooth collar are covered with enamel, also known as ganoin, whereas the tooth cap is equipped with an enamel-like tissue, acrodin. Here, we investigated the formation and mineralization of the ganoin and acrodin matrices in spotted gar, and the evolution of the scpp5, ameloblastin (ambn), and enamelin (enam) genes, which encode matrix proteins of ganoin. Results suggest that, in bichirs and gars, all these genes retain structural characteristics of their orthologs in stem actinopterygians, presumably reflecting the presence of ganoin on scales and teeth. During scale formation, Scpp5 and Enam were initially found in the incipient ganoin matrix and the underlying collagen matrix, whereas Ambn was detected mostly in a surface region of the well-developed ganoin matrix. Although collagen is the principal acrodin matrix protein, Scpp5 was detected within the matrix. Similarities in timings of mineralization and the secretion of Scpp5 suggest that acrodin evolved by the loss of the matrix secretory stage of ganoin formation: dentin formation is immediately followed by the maturation stage. The late onset of Ambn secretion during ganoin formation implies that Ambn is not essential for mineral ribbon formation, the hallmark of the enamel matrix. Furthermore, Scpp5 resembles amelogenin that is not important for the initial formation of mineral ribbons in mammals. It is thus likely that the evolution of ENAM was vital to the origin of the unique mineralization process of the enamel matrix.

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Various secretory calcium-binding phosphoprotein (SCPP) genes are expressed in the skin and jaw during the formation of bone, teeth, and scales in osteichthyans (bony vertebrates). Among these mineralized skeletal units is the ganoid scale, found in many fossil actinopterygians (ray-finned fish) but confirmed only in Polypteriformes (bichirs, reedfish) and Lepisosteiformes (gars) among extant clades. Here, we examined SCPP genes in the genome of seven non-teleost actinopterygian species that possess or do not possess ganoid scales. As a result, 39-43 SCPP genes were identified in Polypteriformes and Lepisosteiformes, whereas 22-24 SCPP genes were found in Acipenseriformes (sturgeons, paddlefish) and Amiiformes (bowfin). Most of these genes form two clusters in the genome of Polypteriformes, Lepisosteiformes, and Amiiformes, and these two clusters are duplicated in Acipenseriformes. Despite their distant phylogenetic relationship, Polypteriformes and Lepisosteiformes retain many orthologous SCPP genes. These results imply that common ancestors of extant actinopterygians possessed a large repertoire of SCPP genes, and that many SCPP genes were lost independently in Acipenseriformes and Amiiformes. Notably, most SCPP genes originally located in one of the two SCPP gene clusters are retained in Polypteriformes and Lepisosteiformes but were secondarily lost in Acipenseriformes and Amiiformes. In Lepisosteiformes, orthologs of these lost genes show high or detectable expression levels in the skin but not in the jaw. We thus hypothesize that many SCPP genes located in this cluster are involved in the formation of ganoid scales in Polypteriformes and Lepisosteiformes, and that their orthologs and ganoid scales were convergently lost in Acipenseriformes and Amiiformes.

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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.

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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.

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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.

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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.

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OBJECTIVE: Petrodentine, the core of the lungfish tooth plate, is a well-mineralized tissue similar to mammalian enamel and analogous to enameloid in fish teeth. Petrodentine is formed solely by petroblasts, which are specialized odontoblasts, whereas enameloid is a composite tissue produced by both odontoblasts and dental epithelial cells. To clarify the details of petrodentine formation, petroblasts were investigated using histochemical and immunohistochemical techniques. METHODS: Extant lungfish (Lepidosiren paradoxa) were used in this study. Tooth plates during the stage of petrodentine formation were observed by means of histochemistry and immunohistochemistry. Commercial kits were used to detect enzyme activity. Correlative sections were immunostained using antibodies against selected peptides. Routine staining such as periodic acid-Schiff (PAS) reaction to identify glycogen and Elastica van Gieson staining for the detection of elastic fibers in histological sections were performed. In addition, conventional transmission electron microscopy was used for observing the fine structure. RESULTS: Petroblasts showed marked acid and alkaline phosphatase activities, and positive immunoreactivities against anti-nestin, anti-V-ATPase, and anti-Ca2+-ATPase, during the maturation stage, but in the matrix formation stage, reactions were much weaker than that of the maturation stage. During the maturation stage, petroblasts showed intense PAS reactivity, and glycogen particles were observed in petroblasts by transmission electron microscopy. Glucose transporter 1-immunoreactivity was observed in petroblasts in the matrix formation stage and the initial to mid part of the maturation stage. CONCLUSIONS: The results in this study suggested that petroblasts have two functional stages, matrix formation and maturation, and glycogen plays an important role in the modulation of petroblasts.

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Gar is an actinopterygian that has bone, dentin, enameloid, and ganoin (enamel) in teeth and/or scales. Mineralization of these tissues involves genes encoding various secretory calcium-binding phosphoproteins (SCPPs) in osteichthyans, but no SCPP genes have been identified in chondrichthyans to date. In the gar genome, we identified 38 SCPP genes, seven of which encode "acidic-residue-rich" proteins and 31 encode "Pro/Gln (P/Q) rich" proteins. These gar SCPP genes constitute the largest known repertoire, including many newly identified P/Q-rich genes expressed in teeth and/or scales. Among gar SCPP genes, six acidic and three P/Q-rich genes were identified as orthologs of sarcopterygian genes. The sarcopterygian orthologs of most of these acidic genes are involved in bone and/or dentin formation, and sarcopterygian orthologs of all three P/Q-rich genes participate in enamel formation. The finding of these genes in gar suggests that an elaborate SCPP gene-based genetic system for tissue mineralization was already present in stem osteichthyans. While SCPP genes have been thought to originate from ancient SPARCL1, SPARCL1L1 appears to be more closely related to these genes, because it established a structure similar to acidic SCPP genes probably in stem gnathostomes, perhaps at about the same time with the origin of tissue mineralization. Assuming enamel evolved in stem osteichthyans, all P/Q-rich SCPP genes likely arose within the osteichthyan lineage. Furthermore, the absence of acidic SCPP genes in chondrichthyans might be explained by the secondary loss of earliest acidic genes. It appears that many SCPP genes expanded rapidly in stem osteichthyans and in basal actinopterygians.

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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.

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To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences.

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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.

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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.

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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.

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During cap enameloid formation in gars (Lepisosteus oculatus), the dental epithelial cells that constitute the enamel organ were observed by means of transmission electron microscopy and enzyme cytochemistry to detect the hydrolytic enzyme activities, alkaline phosphatase (ALPase), acid phosphatase (ACPase), calcium-dependent adenosine triphosphatase (Ca-ATPase) and potassium-dependent p-nitrophenylphosphatase (K-NPPase) (sodium, potassium-activated adenoshine triphosphatase (Na-K-ATPase)). The enameloid formation process in gars was divided into three stages: matrix formation, mineralisation and maturation. The enamel organ consisted of the outer dental epithelial (ODE) cells, stellate reticulum (SR), stratum intermedium (SI) and the inner dental epithelial (IDE) cells during the whole of the cap enameloid formation stages. During the matrix formation stage, many cisternae of rough endoplasmic reticulum and widely distributed Golgi apparatus, in which the procollagen granules containing cross-striations were often found, were remarkable elements in the IDE cells. During the stage of mineralisation, the IDE cells were tall columnar, and infoldings of distal plasma membrane of the IDE cells became marked. The most developed Golgi apparatus was visible at this stage, and large secretory granules containing fine granular or tubular materials were found in the distal cytoplasm that was close to the infoldings of the distal end. Many lysosomes that were ACPase positive were seen near the Golgi apparatus and in the distal cytoplasm of the IDE cells. ACPase positive granules often contained the cross-striation structure resembling procollagen, suggesting that the procollagen is degenerated in the IDE cells. During the maturation stage, the distal infoldings became unclear, and there were no large granules containing tubular materials, but many ACPase positive lysosomes were still present in the IDE cells. Non-specific ALPase was detected at the plasma membrane of the IDE cells at the mineralisation and maturation stages. K-NPPase was markedly detected at the plasma membrane of the IDE cells at the maturation stage. These results demonstrate that the IDE cells might be mainly involved in the removal of degenerated organic matrix from enameloid during the later formation stages. Strong Ca-ATPase activity was observed at the entire plasma membrane of the stratum intermedium cells, and there was slightly weak activity at the plasma membrane of the IDE cells during the mineralisation and maturation stages, implying that these cells are related to the active Ca transport to the maturing enameloid. It is likely that although the structure of the enamel organ is different, the function, especially at the mineralisation and maturation stages, is similar to other actinopterygians having well-mineralized cap enameloid.

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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.

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Light and electron microscopic examination demonstrated two types of non-endocrine agranular cells, cavity boundary cells and stellate cells, in the adenohypophysis of the South American lungfish, Lepidosiren paradoxa. The cavity boundary cells line the hypophyseal cleft and diverticulum and display few microvilli, occasional cilia, prominent junctional complexes, and many cytoplasmic microfilaments. The stellate cells are scattered in the glandular parenchyma and are devoid of microvilli and cilia. When adjacent, they are connected to one another by desmosomes. Pinocytotic vesicles or caveolae are frequently seen along the plasma membrane of the agranular cells adjoining the endocrine cells or abutting on the basement membrane. Possible roles of the agranular cells, physically and metabolically supportive functions, are discussed on the basis of their ultrastractural features.