Fine structural and immunohistochemical detection of collar enamel in the teeth of <Emphasis Type="Italic">Polypterus senegalus</Emphasis>, an actinopterygian fish

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 middle-region- 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.     

Development of larval and transformed teeth in Ambystoma mexicanum (Urodela, Amphibia): an ultrastructural study

Odontogenesis of early larval non-pedicellate teeth, late larval teeth with a more or less distinct dividing zone and fully transformed pedicellate teeth in Ambystoma mexicanum (Urodela) was studied to obtain insights into the development of differently structured teeth in lower vertebrates. Using transmission electron microscopy we investigated five developmental stages: (1) papilla; (2) bell stage (secretion of the matrix begins); (3) primordium (mineralization and activity of ameloblasts starts); (4) replacement tooth (young, old); and (5) established, functional tooth. Development of the differently structured teeth is largely identical in the first three stages. Mineralization takes place in apico-basal direction up to the (prospective) pedicel (early and some late larvae) or up to the zone that divides the late larval and transformed tooth in pedicel and dentine shaft (pedicellate condition). Mineralization starts directly at the collagen and by means of matrix vesicles. First odontoblasts develop small processes that extend to the basal lamina of the inner epithelial layer of the enamel organ. The processes are small and lack organelles in early larval teeth, but become larger, arborescent, and contain some organelles in late larval and transformed teeth. The processes are surrounded by unmineralized matrix (predentine). Odontoblasts at the basis of the teeth, at the pedicel, and in the zone of division do not develop significant cytoplasmic processes that extend into the matrix. Cells of the inner enamel epithelium differentiate to ameloblasts that secrete the enamel. In the early larval tooth they show an extensive basal labyrinth that becomes regressive when the enamel layer is completed. In late larval and transformed teeth, however, a large cavity arises between the basal ruffled border of ameloblasts and their basal lamina. This cavity appears to mediate amelogenesis. A small apical zone in early, but not in late larval teeth directly below the thin enamel layer consists of enameloid and is free of dentine channels.     

Enameloid/enamel transition through successive tooth replacements in <Emphasis Type="Italic">Pleurodeles waltl</Emphasis> (Lissamphibia,Caudata)

Study of the evolutionary enameloid/enamel transition suffers from discontinuous data in the fossil record, although a developmental enameloid/enamel transition exists in living caudates, salamanders and newts. The timing and manner in which the enameloid/enamel transition is achieved during caudate ontogeny is of great interest, because the caudate situation could reflect events that have occurred during evolution. Using light and transmission electron microscopy, we have monitored the formation of the upper tooth region in six successive teeth of a tooth family (position I) in Pleurodeles waltl from late embryos to young adult. Enameloid has only been identified in embryonic tooth I₁ and in larval teeth I₂ and I₃. A thin layer of enamel is deposited later by ameloblasts on the enameloid surface of these teeth. From post-metamorphic juvenile onwards, teeth are covered with enamel only. The collagen-rich enameloid matrix is deposited by odontoblasts, which subsequently form dentin. Enameloid, like enamel, mineralizes and then matures but ameloblast participation in enameloid matrix deposition has not been established. From tooth I₁ to tooth I₃, the enameloid matrix becomes ever more dense and increasingly comes to resemble the dentin matrix, although it is still subjected to maturation. Our data suggest the absence of an enameloid/enamel transition and, instead, the occurrence of an enameloid/dentin transition, which seems to result from a progressive slowing down of odontoblast activity. As a consequence, the ameloblasts in post-metamorphic teeth appear to synthesize the enamel matrix earlier than in larval teeth.     

Immunochemical homology between elasmobranch scale and tooth extracellular matrix proteins in Cephaloscyllium ventriosum

Studies were designed to test the hypothesis that homologous proteins are expressed in elasmobranch scale, tooth enameloid, and mammalian enamel. Using indirect immunohistochemistry and high-resolution two-dimensional gel electrophoresis with immunoblotting, mouse enamel proteins were compared with placoid scale and enameloid proteins from the swell shark, Cephaloscyllium ventriosum. Swiss Webster mouse molar teeth show a characteristic enamel protein pattern consisting of two anionic enamel proteins of 72 kDa (pI 5.8) and 46 kDa (pI 5.5) and several more basic and lower-molecular-weight enamel polypeptides. Both anionic and basic classes of enamel proteins cross-reacted with either antiamelogenin or antienamelin antibodies. Placoid scale and tooth enameloid contained two anionic proteins identified as 58 kDa (pI 5.7) and 46 kDa (pI 5.5), which cross-reacted with either antimouse amelogenin or antihuman enamelin IgG antibodies. A minor antigenically related protein of 43 kDa (pI 6.2) was detected. Immunochemical staining showed localization within placoid scale, swell shark inner enamel epithelia, enameloid, and mouse inner enamel epithelia and enamel. We interpret these results to suggest that both placoid scale and enameloid proteins share epitopes and that these epitopes are also shared with mammalian enamel proteins. Based on molecular weights, isoelectric pH values, and amino acid compositions, placoid scale and enameloid ECM proteins do not contain amelogenin proteins. We suggest that enamelinlike proteins are highly conserved during vertebrate evolution and that these relatively anionic macromolecules may serve a primary function in the initiation of calcium hydroxyapatite formation during enameloid biomineralization.     

Ultrastructure of the dental epithelium and odontoblasts during enameloid matrix deposition in cichlid teeth

Teleost enameloid matrix has been proposed to be an ectodermal, mesodermal, or joint ectodermal-mesodermal product. To determine its origin we examined the ultrastructure of the inner dental epithelium (IDE), odontoblasts, enameloid, and dentin matrices of cichlid tooth buds at the stage of enameloid formation. © Alan R. Liss, Inc. Columnar IDE cells had apical and basal terminal webs and contained organelles associated with protein synthesis, including elongated secretory granules containing fibrillar material having cross-striations with 60-nm periodicity. The morphology of IDE secretory granules was typical of procollagen granules observed in a large variety of ectodermal and mesodermal cells synthesizing collagen. In contrast, the paucity of secretory granules within three odontoblast types indicates that these cells probably do not synthesize enameloid matrix. These observations are consistent with the idea that the bulk of the enameloid matrix is itself an ectodermal collagen synthesized and secreted by IDE cells.     

Conservation and variation in enamel protein distribution during vertebrate tooth development

Vertebrate enamel formation is a unique synthesis of the function of highly specialized enamel proteins and their effect on the growth and organization of apatite crystals. Among tetrapods, the physical structure of enamel is highly conserved, while there is a greater variety of enameloid tooth coverings in fish. In the present study, we postulated that in enamel microstructures of similar organization, the principle components of the enamel protein matrix would have to be highly conserved. In order to identify the enamel proteins that might be most highly conserved and thus potentially most essential to the process of mammalian enamel formation, we used immunoscreening with enamel protein antibodies as a means to assay for degrees of homology to mammalian enamel proteins. Enamel preparations from mouse, gecko, frog, lungfish, and shark were screened with mammalian enamel protein antibodies, including amelogenin, enamelin, tuftelin, MMP20, and EMSP1. Our results demonstrated that amelogenin was the most highly conserved enamel protein associated with the enamel organ, enamelin featured a distinct presence in shark enameloid but was also present in the enamel organ of other species, while the other enamel proteins, tuftelin, MMP20, and EMSP1, were detected in both in the enamel organ and in other tissues of all species investigated. We thus conclude that the investigated enamel proteins, amelogenin, enamelin, tuftelin, MMP20, and EMSP1, were highly conserved in a variety of vertebrate species. We speculate that there might be a unique correlation between amelogenin-rich tetrapod and lungfish enamel with long and parallel crystals and enamelin-rich basal vertebrate enameloid with diverse patterns of crystal organization.     

Structure, composition, and mechanical properties of shark teeth

The teeth of two different shark species (Isurus oxyrinchus and Galeocerdo cuvier) and a geological fluoroapatite single crystal were structurally and chemically characterized. In contrast to dentin, enameloid showed sharp diffraction peaks which indicated a high crystallinity of the enameloid. The lattice parameters of enameloid were close to those of the geological fluoroapatite single crystal. The inorganic part of shark teeth consisted of fluoroapatite with a fluoride content in the enameloid of 3.1 wt.%, i.e., close to the fluoride content of the geological fluoroapatite single crystal (3.64 wt.%). Scanning electron micrographs showed that the crystals in enameloid were highly ordered with a special topological orientation (perpendicular towards the outside surface and parallel towards the center). By thermogravimetry, water, organic matrix, and biomineral in dentin and enameloid of both shark species were determined. Dentin had a higher content of water, organic matrix, and carbonate than enameloid but contained less fluoride. Nanoindentation and Vicker's microhardness tests showed that the enameloid of the shark teeth was approximately six times harder than the dentin. The hardness of shark teeth and human teeth was comparable, both for dentin and enamel/enameloid. In contrast, the geological fluoroapatite single crystal was much harder than both kinds of teeth due to the absence of an organic matrix. In summary, the different biological functions of the shark teeth ("tearing" for Isurus and "cutting" for Galeocerdo) are controlled by the different geometry and not by the chemical or crystallographic composition.     

The penetration of exogenous tracers through the enameloid organ of developing teleost fish teeth

In order to determine whether exogenous materials permeate to the forming tooth enameloid matrix, teleost species were injected intramuscularly with horseradish peroxidase (HRP) or myoglobin, or; intracardially with lanthanum nitrate or HRP, then killed a predetermined intervals post-injection. Tooth bearing bones were processed for transmission electron microscopy. At the enameloid matrix formation stage, capillaries associated with the enameloid organ were few in number and rarely fenestrated. Both organic tracers reached the matrix at cervical but not coronal, regions of the teeth in all species examined. Lanthanum was rarely observed extravascularly and never extended to the enameloid matrix at the secretion stage. At the enameloid mineralization stage, fenestrated capillaries were closely associated with the outer dental epithelial cells (ODE). All tracers were observed in the plasma membrane invaginations of the ODE. Only intracardially injected HRP compromised the apical intercellular junctions of the inner dental epithelial cells (IDE) to reach the mineralizing enameloid Lanthanum did not extend past the ODE-IDE cell junctions. It is concluded that the close association of mineralization stage fenestrated capillaries with the highly invaginated ODE cells result in increased tracer penetration compared to the secretory stage. The deeper penetration of the organic tracers, compared with lanthanum, between mineralization stage IDE cells may be due to longer in vivo circulation of the former material. The apical junctions of mineralization stage IDE cells, however, remained impermeable to the organic tracers. The absence of mineral in secretory stage enameloid mineral could not be due to specialized cell junctions preventing access of molecules to the matrix. It is suggested that controlling factors other than cellular permeability initiate enameloid mineralization.     

An ultrastructural study of dentinogenesis and amelogenesis in rat molar tooth germs cultured in vitro

Summary Molar tooth germs from three-day-old rats were cultured successfully for fourteen days, permitting the study of the development in vitro of both extracellular matrix and cellular elements such as odontoblasts and ameloblasts. The ultrastructure of the cultured tooth germs was compared with the ultrastructure of tooth germs in vivo at a comparable developmental stage. Progenitor cells of odontoblasts and ameloblasts were found to differentiate in vitro. Odontoblasts seemed to contain more lysosome-like bodies and fewer secretory granules than in vivo. They formed normally mineralizing dentine or a thick layer of dense, unmineralized predentine with incidentally some amorphous, extracellular material. Enamel was exclusively present opposite well developed dentine. It was often hyperor hypomineralized and enamel rods were not as regularly shaped as in vivo. In places where no enamel formation had taken place, large amounts of amorphous extracellular material were sometimes seen. From these observations it can be concluded that cellular development in cultured tooth germs appeared more or less normal, but extracellular matrix formation and mineralization were sometimes disturbed.     

Evolutionary genetics of vertebrate tissue mineralization: the origin and evolution of the secretory calcium-binding phosphoprotein family

Three principal mineralized tissues are present in teeth; a highly mineralized surface layer (enamel or enameloid), body dentin, and basal bone. Similar tissues have been identified in the dermal skeleton of Paleozoic jawless vertebrates, suggesting their ancient origin. These dental tissues form on protein matrix and their mineralization is controlled by distinctive proteins. We have shown that many secretory calcium-binding phosphoproteins (SCPPs) are involved in tetrapod tissue mineralization. These SCPPs all originated from the common ancestral gene SPARCL1 (secreted protein, acidic, cysteine-rich like 1) that initially arose from SPARC. The SCPP family also includes a bird eggshell matrix protein, mammalian milk casein, and salivary proteins. The eggshell SCPP plays crucial roles in rigid eggshell production, milk SCPPs in efficient lactation and in the evolution of complex dentition, and salivary SCPPs in maintaining tooth integrity. A comparative analysis of the mammalian, avian, and amphibian genomes revealed a tandem duplication history of the SCPP genes in tetrapods. Although these tetrapod SCPP genes are fewer in teleost genomes, independent parallel duplication has created distinct SCPP genes in this lineage. These teleost SCPPs are also used for enameloid and dentin mineralization, implying essential roles of SCPPs for dental tissue mineralization in osteichthyans. However, the SCPPs used for tetrapod enamel and teleost enameloid, as well as tetrapod dentin and teleost dentin, are all different. Thus, the evolution of vertebrate mineralized tissues seems to be explained by phenogenetic drift: while mineralized tissues are retained during vertebrate evolution, the underlying genetic basis has extensively drifted.     

Selachian tooth development: II. Immunolocalization of amelogenin polypeptides in epithelium during secretory amelogenesis in Squalus acanthias

We have determined the distribution of amelogenin polypeptides in an order of elasmobranchs using indirect immunofluorescence with rabbit polyclonal antibodies prepared to purified murine amelogenins. We find that amelogenins are definitely present within the inner enamel epithelium prior to the production of the extracellular matrix component termed "enameloid" (row II developing tooth organs). During subsequent stages of selachian tooth development (row III tooth organs), immunofluorescence staining data indicated localization of amelogenin antigens within epithelium as well as the enameloid extracellular matrix. The results from these immunohistochemical studies suggest that the 16-20 kdalton amelogenins, which are characteristic of murine inner enamel epithelial cells undergoing terminal biochemical differentiation into secretory ameloblasts, may also be regarded as molecular markers for amelogenesis in developing teeth in the spiny dogfish, Squalus acanthias.     

Structure and development of first-generation teeth in the cichlid Hemichromis bimaculatus (Teleostei, Cichlidae)

In order to build a reference system to assess results of ongoing in vitro experiments on the study of epithelial-mesenchymal interactions during odontogenesis in actinopterygians, we have chosen to study the first-generation teeth of the cichlid Hemichromis bimaculatus from initiation until attachment both at the light and transmission electron microscopical level. Although their development follows the general pattern of teleost tooth formation, first-generation teeth show peculiarities compared with later tooth generations, including their size, bare emergence from the epithelium, absence of dentinal tubules and of nerves and capillaries in the pulp cavity, and organization of the outer dental epithelium. Four developmental stages (a to d) prior to attachment (stage e) have been distinguished. The oral epithelium invaginates into the underlying mesenchyme (stage a) and is later folded to form a bell-shaped dental organ (stage b) without any primordial thickening, or any other morphological indication of imminent invagination. Then, the collagenous enameloid matrix is laid down, most probably by the odontoblasts (early stage c), soon followed by predentine deposition and the beginning of enameloid mineralization (late stage c). With ongoing dentinogenesis, the enameloid matrix matures (stage d), i.e. the organic constituents are removed and the matrix further mineralizes. Finally (stage e), an annular collar of attachment bone is deposited to fix the tooth onto the underlying bone.     

Immunocytochemical localization of cathepsin B in rat kidney. II. Electron microscopic study using the protein A-gold technique

Thin sections of Lowicryl K4M-embedded materials were labeled with protein A-gold complex. Gold particles representing the antigen sites for cathepsin B were exclusively confined to lysosomes of each segment of the nephron. The heaviest labeling was noted in the lysosomes of the S1 segment of the proximal tubules. Labeling intensity varied considerably with the individual lysosomes. Lysosomes of the other tubular segments, such as the S2 and S3 segments of the proximal tubules, distal convoluted tubules, and collecting tubules were weakly labeled by gold particles. Quantitative analysis of labeling density also confirmed that lysosomes in the S1 segment have the highest labeling density and that approximately 65% of labeling in the whole renal segments, except for the glomerulus, was found in the S1 segment. These results indicate that in rat kidney the lysosomes of the S1 segment are a main location of cathepsin B. Further precise observations on lysosomes of the S1 segment revealed that apical vesicles, tubules, and vacuoles were devoid of gold particles, but when the vacuoles contained fine fibrillar materials, gold labeling was detectable in such vacuoles. As the lysosomal matrix becomes denser, the labeling density is increased. Some small vesicles around the Golgi complex were also labeled. These results indicate that the endocytotic apparatus including the apical vesicles, tubules, and vacuoles contains no cathepsin B. When the vacuoles develop into phagosomes, they acquire this enzyme to digest the absorbed proteins.     

A structural comparison of cyst and germ tube wall inPhytophthora palmivora

Summary An electron microscopic analysis of germinating cysts ofPhytophthora palmivora involving freeze-etching, thin sectioning, and replica techniques reveals that both cyst and hyphal wall comprise a two-phase system with a fibrillar and an amorphous component. The cyst wall is fibrillar throughout with the fibrils tightly interwoven and embedded in an amorphous matrix on the internal side of the wall. The hyphal wall consists of a fibrillar inner layer with the fibrils lightly covered by some amorphous material and an amorphous outer layer devoid of any fibrillar material. Both cyst and germ tube walls are wholly or partially covered by a fluffy coat of variable thickness. In the zone of germ tube emergence cyst wall and germ tube wall overlap and are tightly apposed. Thus, the germ tube wall is not a simple extension of the cyst wall but a new structural entity separated from the cyst wall by a thin line of demarcation.     

Constitutively active PTH/PTHrP receptor in odontoblasts alters odontoblast and ameloblast function and maturation

Parathyroid hormone (PTH)-related protein (PTH-rP) is an important autocrine/paracrine attenuator of programmed cell differentiation whose expression is restricted to the epithelial layer in tooth development. The PTH/PTHrP receptor (PPR) mRNA in contrast is detected in the dental papilla, suggesting that PTHrP and the PPR may modulate epithelial-mesenchymal interactions. To explore the possible interactions, we studied the previously described transgenic mice in which a constitutively active PPR is targeted to osteoblastic cells. These transgenic mice have a vivid postnatal bone and tooth phenotype, with normal tooth eruption but abnormal, widened crowns. Transgene mRNA expression was first detected at birth in the dental papilla and, at 1 week postnatally, in odontoblasts. There was no transgene expression in ameloblasts or in other epithelial structures. Prenatally, transgenic molars and incisors revealed no remarkable change. By the age of 1 week, the dental papilla was widened, with disorganization of the odontoblastic layer and decreased dentin matrix. In addition, the number of cusps was abnormally increased, the ameloblastic layer disorganized, and enamel matrix decreased. Odontoblastic and, surprisingly, ameloblastic cytodifferentiation was impaired, as shown by in situ hybridization and electron microscopy. Interestingly, ameloblastic expression of Sonic Hedgehog, a major determinant of ameloblastic cytodifferentiation, was dramatically altered in the transgenic molars. These data suggest that odontoblastic activation of the PPR may play an important role in terminal odontoblastic and, indirectly, ameloblastic cytodifferentiation, and describe a useful model to study how this novel action of the PPR may modulate mesenchymal/epithelial interactions at later stages of tooth morphogenesis and development.     

Immunocytochemical and immunochemical study of enamelins, using antibodies against porcine 89-kDa enamelin and its N-terminal synthetic peptide, in porcine tooth germs

Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytochemically using two antibodies: a polyclonal antibody raised against the porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptide antibody against the porcine enamelin amino-terminus (EN). Immunochemical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies used. In contrast, the middle and inner enamel layer mainly contained lower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mineralized collagen fibrils of dentin, while EN stained both enamel matrix islands and stippled material. At the matrix formation stage, both antibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneously, while EN primarily stained the prism sheath. The intense immunoreaction over the surface layer of enamel matrix at the matrix formation stage, following staining with 89 E and EN, disappeared by the end of the transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the late differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin continues from late differentiation to the transition stage and the cleavage of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during enamel maturation.     

Ultrastructure of odontogenic cells during enameloid matrix synthesis in tooth buds from an elasmobranch, Raja erinacae

The ultrastructure of the inner dental epithelial cells (IDE) and odontoblasts in elasmobranch (Raja erinacae) tooth buds was investigated by transmission electron microscopy to determine what contribution each cell type makes to the forming enameloid matrix. Row II, early stage, IDE cells contained few organelles associated with protein synthesis, whereas preodontoblasts appeared competent to initiate extracellular matrix production. Row III IDE cells are also devoid of organelles related to secretory protein synthesis, although these IDE cells accumulated large pools of intracellular glycogen. The glycogen appeared to be packaged into vesicles and exocytosed into the lateral extracellular space toward the forming enameloid matrix. Row III odontoblasts had a morphology consistent with an active protein secretory cell. No procollagen granules were present within the odontoblasts, however, nor were many collagen fibers observed in the enameloid matrix. Instead, non-collagenous "giant" fibers having 17.5-nm periodic cross striations were associated with the invaginations of odontoblast cell processes. Giant fibers, which spanned a clear zone adjacent to the odontoblasts, terminated within the enameloid matrix. Smaller 25-nm-wide "unit" fibers emanated from the giant fiber tips to form the bulk of the enameloid matrix. The clear zone, which separated the odontoblasts from the enameloid matrix at early stages, diminished in size at later stages until the odontoblast processes were completely embedded in the enameloid matrix. Nascent enameloid crystallites were observed only after a layer of unmineralized predentin was deposited beneath fully formed enameloid matrix. The results suggest that the major constituent of the enameloid matrix in skates is a non-collagenous protein derived from the odontoblasts. The inner dental epithelial cells appear to contribute large quantities of carbohydrates to the forming enameloid matrix.     

Development and fine structure of pharyngeal replacement teeth in juvenile zebrafish (Danio rerio) (Teleostei, Cyprinidae)

Teeth are commonly used model systems for the study of epithelial-mesenchymal interactions during organogenesis. We describe here the ultrastructural characteristics of developing pharyngeal replacement teeth in juvenile zebrafish, an increasingly important model organism for vertebrate development. Replacement teeth develop in close association with the dental organ of a functional tooth. Morphogenesis is well advanced prior to the start of cytodifferentiation. Fibrillar enameloid matrix is formed first, followed by the deposition of predentine. Initial mineralization of the enameloid proceeds quickly; maturation involves the presence of ruffled-bordered ameloblasts. Dentine mineralization is inotropic and is mediated by matrix vesicles. Woven-fibred attachment bone matrix is deposited before completion of dentine mineralization. Eruption of fully ankylosed teeth is a fast process and may involve degenerative changes in the pharyngeal epithelium. Mononucleated osteoclasts and clastic cells located in the pulp cavity intervene in tooth resorption prior to shedding. Structural differences with larval, first-generation zebrafish teeth include the presence of dentinal tubules and the absence of an electron-dense covering membrane. Part of these differences may relate to size differences of the teeth. Others, like the site of the replacement tooth bud, suggest that initiation may take place in already committed epithelium from the first initiation event in the larval stage.     

The SCPP gene repertoire in bony vertebrates and graded differences in mineralized tissues

The vertebrate tooth is covered with enamel in most sarcopterygians or enameloid in chondrichthyans and actinopterygians. The evolutionary relationship among these two tissues, the hardest tissue in the body, and other mineralized tissues has long been controversial. We have recently reported that specific combinations of secretory calcium-binding phosphoprotein (SCPP) genes are involved in the mineralization of bone, dentin, enameloid, and enamel. Thus, the early repertoire of SCPP genes would elucidate the evolutionary relationship across these tissues. However, the diversity of SCPP genes in teleosts and tetrapods and the roles of these genes in distinct tissues have remained unclear, mainly because many SCPP genes are lineage-specific. In this study, I show that the repertoire of SCPP genes in the zebrafish, frog, and humans includes many lineage-specific genes and some widely conserved genes that originated in stem osteichthyans or earlier. Expression analysis demonstrates that some frog and zebrafish SCPP genes are used primarily in bone, but also in dentin, while the reverse is true of other genes, similar to some mammalian SCPP genes. Dentin and enameloid initially use shared genes in the matrix, but enameloid is subsequently hypermineralized. Notably, enameloid and enamel use an orthologous SCPP gene in the hypermineralization process. Thus, the hypermineralization machinery ancestral to both enameloid and enamel arose before the actinopterygian–sarcopterygian divergence. However, enamel employs specialized SCPPs as structuring proteins, not used in enameloid, reflecting the divergence of enamel from enameloid. These results show graded differences in mineralized dental tissues and reinforce the hypothesis that bone–dentin–enameloid–enamel constitutes an evolutionary continuum. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

CHONDROGENESIS, STUDIED WITH THE ELECTRON MICROSCOPE

The role of the cells in the fabrication of a connective tissue matrix, and the structural modifications which accompany cytodifferentiation have been investigated in developing epiphyseal cartilage of fetal rat by means of electron microscopy. Differentiation of the prechondral mesenchymal cells to chondroblasts is marked by the acquisition of an extensive endoplasmic reticulum, enlargement and concentration of the Golgi apparatus, the appearance of membrane-bounded cytoplasmic inclusions, and the formation of specialized foci of increased density in the cell cortex. These modifications are related to the secretion of the cartilage matrix. The matrix of young hyaline cartilage consists of groups of relatively short, straight, banded collagen fibrils of 10 to 20 mµ and a dense granular component embedded in an amorphous ground substance of moderate electron density. It is postulated that the first phase of fibrillogenesis takes place at the cell cortex in dense bands or striae within the ectoplasm subjacent to the cell membrane. These can be resolved into sheaves of "primary" fibrils of about 7 to 10 mµ. They are supposedly shed (by excortication) into the matrix space between the separating chondroblasts, where they may serve as "cores" of the definitive matrix fibrils. The diameter of the fibrils may subsequently increase up to threefold, presumably by incorporation of "soluble" or tropocollagen units from the ground substance. The chondroblast also discharges into the matrix the electrondense amorphous or granular contents of vesicles derived from the Golgi apparatus, and the mixed contents of large vacuoles or blebs bounded by distinctive double membranes. Small vesicles with amorphous homogeneous contents of moderate density are expelled in toto from the chondroblasts. In their subsequent evolution to chondrocytes, both nucleus and cytoplasm of the chondroblasts undergo striking condensation. Those moving toward the osteogenic plate accumulate increasingly large stores of glycogen. In the chondrocyte, the enlarged fused Golgi vesicles with dense contents, massed in the juxtanuclear zone, are the most prominent feature of the cytoplasm. Many of these make their way to the surface to discharge their contents. The hypertrophied chondrocytes of the epiphyseal plate ultimately yield up their entire contents to the matrix.