Enamel rod formation in the monkey observed by scanning electron microscopy

Scanning electron microscopy of permanent tooth buds of the monkey confirmed that mineralizing interrod enamel surrounds Tomes' processes on three sides, forming pits that restrict enamel rod formation. The forming face of the enamel rod, which is the floor of the pit, angled toward the tooth surface at the apical edge of the pit, the side nearest the cervical region of the tooth. Consequently, the apical edge of each pit was the only site where both rod and interrod enamel were formed at the nascent tooth surface. The ameloblasts had two secretory surfaces. One was the microvillous surface of the short Tomes' process abutting the forming face of the enamel rod. The other surface, closer to the ameloblast, was between Tomes' processes, abutting the crests of interrod enamel which formed the pits. At each site forming enamel crystallites had specific orientations. Due to the angle of the forming face of the rod and the short Tomes' process, crystallites with both rod and interrod orientation form at the same time and the same plane within the apical (cervical) margin of each rod. It is hypothesized that indistinct boundaries between rod and interrod enamel at the apical margin of each rod are due to both secretory surfaces of ameloblasts secreting at the same time and at the same site.     

Enamel rod formation in the monkey observed by scanning electron microscopy.

Scanning electron microscopy of permanent tooth buds of the monkey confirmed that mineralizing interrod enamel surrounds Tomes' processes on three sides, forming pits that restrict enamel rod formation. The forming face of the enamel rod, which is the floor of the pit, angled toward the tooth surface at the apical edge of the pit, the side nearest the cervical region of the tooth. Consequently, the apical edge of each pit was the only site where both rod and interrod enamel were formed at the nascent tooth surface. The ameloblasts had two secretory surfaces. One was the microvillous surface of the short Tomes' process abutting the forming face of the enamel rod. The other surface, closer to the ameloblast, was between Tomes' processes, abutting the crests of interrod enamel which formed the pits. At each site forming enamel crystallites had specific orientations. Due to the angle of the forming face of the rod and the short Tomes' process, crystallites with both rod and interrod orientation form at the same time and the same plane within the apical (cervical) margin of each rod. It is hypothesized that indistinct boundaries between rod and interrod enamel at the apical margin of each rod are due to both secretory surfaces of ameloblasts secreting at the same time and at the same site.     

A comparison of the protein synthetic activity of presecretory and secretory ameloblasts in rat incisors

Presecretory ameloblasts were compared morphologically and functionally to secretory ameloblasts in the rat incisor. Structurally, the cell types are similar except for the absence of a Tomes' process at the presecretory stage. The developmental changes at the apical end of the ameloblast were described and correlated sequentially with the onset of extracellular events. First, a layer a amorphous dense material appeared at the dentinal surface adjacent to the ameloblasts. Second, the initial layer of enamel began to develop. Third, inner enamel secretion began with the appearance of interrod enamel. This occurred concomitantly with the appearance of the interdigitating portions of Tomes' processes. Functionally, the protein synthetic activity of presecretory ameloblasts was compared to secretory ameloblasts. Light microscopic radioautography of 1-m?m thick Epon sections was used to localize ³H-proline and ³H-tyrosine at various times after injection. At time intervals up to 20 minutes the two presursors were localized as a band of labeled protein in the supranuclear cytoplasm of both presecretory and secretory ameloblasts. At 30 minutes an additional band of radioactivity was localized within the apices of both types of ameloblasts. In presecretory cells the apical reaction band was over the proximal portion of Tomes' processes which border on the dentin. In the secretory cells, the apical reaction band was over both proximal and interdigitating portions of Tomes' processes and over the enamel. Grain counts over the secretory ameloblasts showed that the incorporation of tyrosine increased by 7.5% as opposed to a 63% increase with proline when compared to the values in presecretory cells. The different increases with the two precursors were in keeping with the different amounts of the two amino acids reported to be present in enamel protein. It was concluded that while the secretory ameloblasts synthesize and secrete enamel protein, both presecretory and secretory cell types produce another category of protein which is involved in the apical reaction band. It was proposed that this material is structural protein being contributed to the continuously lengthening Tomes' process which is speculated to occur during formation of enamel.     

Correlation of the arrangement pattern of enamel rods and secretory ameloblasts in pig and monkey teeth: A possible role of the terminal webs in ameloblast movement during secretion

Enamel rod architecture and ameloblast arrangement were examined in pig and monkey teeth using light microscopy and scanning and transmission electron microscopy. Enamel rods in the pig teeth were arranged in longitudinal straight rows in the initial enamel layer, in longitudinal wavy rows in the inner enamel layer, and in a staggered pattern in the outer enamel layer. Rod decussation was seen only in the inner layer. Cross-sectined enamel rods in the pig were arcade-shaped in the initial and inner layers, and mostly round in shape with circular boundaries in the outer layer. Arrangement of secretory ameloblasts at the level of the distal terminal web and Tomes' processes, and shape of Tomes' processes, corresponded to those of the enamel rod in the enamel layers. Distal terminal webs were well developed between straight rows of the ameloblasts forming the initial layer and between wavy rows of the ameloblasts forming the inner layer, and less developed within a row. The filament bundles in the distal terminal webs were also oriented along the rows. However, in the ameloblasts forming the outer layer, which lost their row pattern, distal terminal web filaments were distributed uniformly at the cell periphery. A similar arrangement of wavy rows of ameloblasts at the level of distal terminal web and Tomes' processes was also seen in monkey teeth.     

A freeze-fracture study of the topographic relationship between inner enamel-secretory ameloblasts in the rat incisor

A correlated study using freeze-fracture replicas and routine thin sectioning was done on the ameloblasts which secrete inner enamel in the rat incisor. These ameloblasts are columnar cells aligned in rows parallel to the cross-sectional plane of the incisor. Each cell has a Tomes' process at its distal end which is contained within a cavity of interrod enamel. Tomes' processes of a particular row of cells are inclined towards the mesial side of the tooth, while those of the next row are inclined towards the lateral side, so that the processes cross each other at 90°. Freeze-fracture replicas were used to examine the surfaces of ameloblasts between adjacent rows and the surfaces of the cells within the row. Replicas of the surfaces between rows showed that ameloblasts are curved so that their proximal and distal ends (Tomes' processes) are both directed towards the same side of the tooth. The surface of each row bears the impression left upon it by the cells in the adjacent row which have been fractured away. This cell-impression is also curved, but in the opposite direction to the cells which it crosses. It was thus shown that not only the Tomes' processes of adjacent rows but the entire cell bodies cross one another. This crossing is slight at the proximal ends of the cells, but marked at the level of the distal junctional complex. At this level a cell from a particular row curves across several cells of the neighbouring row and forms large tight junctions with at least two cells in that row. The proximal and distal junctional complexes form zones of tight junctions around the cell. The distal complex is more extensive than the proximal and the tight junctions are better developed between adjacent rows. Gap junctions are, however, larger between cells within the rows. The study demonstrated the effectiveness of freeze-fracture replicas in obtaining 3-dimensional details about ameloblast shape, and this information was used to evaluate the supposed movement of ameloblasts during inner enamel formation.     

An ultrastructural study of the effect of a diabetogenic dose of alloxan on the secretory ameloblasts of the rat incisor

The effect of a single diabetogenic dose of alloxan on the ameloblasts of enamel secretion was investigated in rat incisor teeth prior to the onset of diabetes mellitus. This was compared with tissue from animals that were sacrificed at the onset of diabetes, and with tissue from animals that had been diabetic for 1 month. Male Sprague-Dawley rats were given a single subcutaneous injection of alloxan at a dose of 150 mg/kg body weight; and the animals were sacrificed at 45 minutes, 2 hours, 24 hours, 48 hours and 29 days after injection. At the electron microscope level the following changes were observed. There was an early accumulation of secretion granules in the vicinity of the Golgi apparatus and within the proximal portion of Tomes' process. These accumulations were present at the onset of diabetes, 24 hours later. At 2 hours after injection a space appeared between the plasma membrane around Tomes' process and the interrod material. This space widened with time, and at 24 hours after injection it became continuous with enlarged intercellular spaces between the proximal portions of Tomes' processes. The widening of the intercellular space was restricted to the area above the distal cell web. At the onset of diabetes this compartment of the cells was similar to that of the control samples. Extracellular material which appeared to be the secretory product of the ameloblast was observed at two sites. On one occasion this material was seen in the wide intercellular spaces between the proximal portions of Tomes' processes 24 hours after injection. It was also seen at the onset of diabetes in the intercellular space at the level of the Golgi apparatus. The changes in the animals that had been diabetic for 1 month were a scarcity of secretion granules within Tomes' processes and an abnormal accumulation of secretion granules within the supranuclear and infranuclear compartents. This study has shown that the toxic effect of alloxan persists up to the time of onset of diabetes mellitus. However, since different morphological changes were observed during diabetes mellitus, it is suggested that the changes caused by diabetes are a separate entity, initially superimposed on and later replacing the acute, toxic effect of the drug.     

The biosynthesis and secretion of precursor enamel protein by ameloblasts as visualized by autoradiography after tryptophan administration

The incorporation of ³H-tryptophan into the inner enamel epithelium of newborn mouse incisor tooth organs has been studied in situ by light and electron microscopic autoradiography to determine the sites and kinetics of biosynthesis, migration, and secretion of precursor enamel protein during newborn mouse incisor tooth formation. Maxillary and mandibular incisor tooth amelogenesis was studied 5, 30, 60, 120, 240 minutes and 24 hours following the intraperitoneal injection of ³H-tryptophan. By 5 minutes, 40% of the total silver grains associated with the secretory ameloblasts were localized over the rough endoplasmic reticulum and 50% of the silver grains were localized over the Golgi apparatus. By 30 minutes, silver grains were observed predominately over condensing vacuoles and secretory granules within the forming Tomes' processes, and were also localized over the extracellular “granular” pre-enamel matrix. The enamel proteins were synthesized on membrane-bound polysomes, transferred within the cisternae of the rough endoplasmic reticulum and then accumulated in the inner saccules of the Golgi apparatus. The enamel proteins were then packaged in condensing vacuoles which subsequently became secretory granules which migrated to the lateral and apical secretory regions of the forming Tomes' processes. It was concluded from these in vivo studies that enamel proteins were synthesized and subsequently secreted within 30 minutes. The initially secreted precursor enamel protein was localized over a material which demonstrated a granular or stippled ultrastructure. The labeled protein then was localized over the amorphous enamel matrix per se which contained the forming calcium hydroxyapatite crystals. We assumed, therefore, that there are two different ultrastructural forms of ³H-tryptophan containing extracellular enamel proteins and suggest that the granular or “stippled” form represents newly secreted precursor enamel protein.     

Immunocytochemical localization of amelogenins in the deciduous tooth germs of the human fetus

The immunocytochemical localization of amelogenins in the developing deciduous tooth germs of 6-month-old human fetuses was investigated by the protein A-gold method using an antiserum against porcine 25K amelogenin. The inner enamel epithelial cells and underlying matrix showed no amelogenin-like immunoreactivity. Distinct immunoreactivity was initially shown by fine fibrils found beneath the intact basal lamina of preameloblasts at the early differentiation stage. At the late differentiation stage, amelogenin-like immunoreactivity was shown by a fine granular material within the extracellular matrix as well as by the Golgi apparatus, secretory granules, lysosomal structures, coated vesicles, and coated pits of preameloblasts with a disrupted basal lamina. At the formative stage, the localization of immunoreactivity in secretory ameloblasts was similar to that in preameloblasts during the late differentiation stage. However, immunopositive coated vesicles and coated pits were only found at the early stage of matrix formation. The calcified enamel matrix and stippled material showed intense immunoreactivity. Immunocytochemical labeling of the enamel matrix appeared as a gradient, decreasing from the enamel surface to the dentinoenamel junction. No maturation stage of ameloblasts existed in the tooth germs examined. In predentin and dentin, amelogenin-like immunoreactivity was occasionally detected on odontoblasts and their processes, but odontoblasts and cells of the stratum intermedium contained no immunoreactive elements. These findings confirmed that the secretory ameloblast in the human deciduous tooth germ is responsible for the synthesis and secretion of enamel proteins.     

Fine structure of differentiating ameloblasts in the kitten

The fine structure of differentiating ameloblasts was studied in the lower second molar of 1-week-old kittens after perfusion fixation with and without subsequent decalcification. The differentiation zone was divided into three phases. In Differentiation 1, ameloblasts are about 27 μm long and face an uninterrupted basal lamina. The predentin adjacent to the basal lamina contains a few collagen fibrils oriented mainly at right angles to the ameloblast surface. This specialized predentin forms a well-defined layer, up to 1.5 μm thick, referred to as the junctional layer. In Differentiation 2, ameloblast processes extend through the basal lamina and the thickness of the junctional layer. The processes consist of cytoplasmic sheets forming a honeycomb-like network. Dentin starts to calcify after process-formation is underway. Two distinct types of odontoblast processes, having different shapes and contents, come in contact with the ameloblasts and push into the ameloblastic layer. In Differentiation 3, stippled material appears in the extracellular spaces between ameloblasts. Later, stippled material-like substances appear in the predentin close to the ameloblast apex and close to odontoblast processes within the dentin. Ameloblasts now are up to 40 μm high. Enamel secretion starts in small circumscribed areas which gradually enlarge, leading to the disappearance of the ameloblast processes. These findings are compared with results obtained in other species, including man, and their possible functional significance is discussed.     

Differential Localization of Calmodulin,the 67kda Calcimedin and Calpactin II In Secretory Ameloblasts

By using specific antibodies, we have shown by Western blots that dental tissues contain calmodulin, the 67 kDa calcimedin and calpactin II. Moreover, by immunogold electron microscopy, we were able to compare the intracellular distribution of these three calcium-binding proteins in secretory ameloblasts. They were all found in the cytosol of these cells but only calcimedin was detected in the mitochondria. Calpactin II was the only one present in secretory vesicles. Twice as much calmodulin and calpactin II were detected in cell bodies as in Tomes' processes, but calcimedin was more abundant in the latter. The presence of these calcium mediators in well defined areas of ameloblasts may indicate differential ways for these cells to regulate different calcium-dependent processes during enamel formation.     

The ultrastructure of the enamel organ related to enamel formation

The ultrastructure of the cells of the enamel organ related to enamel formation was studied using the lower incisors of adult male rats. In the region of enamel deposition, stratum intermedium cells are stabilized by a system of intercellular bridges and intracellular fibrils. The mitochondria in these cells are positioned toward the extracellular channels through which any direct intercellular exchange between the capillaries and ameloblasts must occur. Tentatively, the mitochondrial arrangement is considered to be related to the movements of electrolytes and water across the capillary-ameloblast interval. In the region of transition, enamel deposition ceases and the ergastoplasm of the ameloblasts is removed, apparently by cytosegresomes, with an accompanying reduction in the height of the ameloblasts. Here, vesicles containing stippled material are infrequent compared to their occurrence in ameloblasts concerned with enamel deposition. Other vesicles, characteristically found in ameloblasts related to maturing enamel, first appear in the transition region and seem to originate from the cell membrane abutting on the enamel. In the region of maturation, cytosomes are common in the Golgi region whereas vesicles and mitochondria predominate in the distal ends of the ameloblasts. The papillary cells contain an unusually large number of mitochondria, elaborate microvilli and vesicles, which suggests that these cells are extremely active, presumably in the movement of materials related to enamel maturation. The changes in structure of the papillary cells, which occur concomitantly with those of the ameloblasts during enamel formation, are indicative of interrelated functional changes and strongly support the concept of ameloblasts and papillary cells acting together as a functional unit.     

TEM Observations on the Ameloblast/Enamel Interface in the Rat Incisor

The structure of rat incisor enamel is established at the topographically complex interface between secretory ameloblasts and forming enamel. The aim of this study was to gain additional information on this interface by sectioning parallel with the rows and the long axis of Tomes' processes and prisms. Rats were sacrificed and fixed by glutaraldehyde/paraformaldehyde perfusion. After dissection, demineralization and embedding transverse jaw/incisor segments were cut, reembedded, and reoriented. Sections were prepared for and observed in the transmission electron microscopy (TEM). The intraenamel part of Tomes' process was about 18 &#119 long. The forming prism occupied a longitudinally grooved invagination on its apical aspect. The parts of Tomes' process forming the side walls of the groove were attenuated and showed variation in extent and outline. Prism growth occurred over the whole grooved area. An estimation of Tomes' process secretory area in rat compared with data from humans suggests that there may be a relationship between secretory area and rate of prism formation. Prism crystals were oriented obliquely or parallel to the secretory surface of Tomes' process. At interprism growth sites matrix deposition was irregular and required some redistribution to conform to the pattern of interprism sheets.     

Essential roles of ameloblastin in maintaining ameloblast differentiation and enamel formation

During tooth development, dental epithelial cells interact with extracellular matrix components, such as the basement membrane and enamel matrix. Ameloblastin, an enamel matrix protein, plays a crucial role in maintaining the ameloblast differentiation state and is essential for enamel formation. Ameloblastin-null mice developed severe enamel hypoplasia. In mutant mice, dental epithelial cells started to differentiate into ameloblasts, but ameloblasts soon lost cell polarity, proliferated, and formed multiple cell layers, indicative of some aspects of preameloblast phenotypes. In addition, the expression of amelogenin, another component of the enamel matrix, was specifically reduced in mutant ameloblasts. More than 20% of amelobastin-null mice developed odontogenic tumors. We also found that recombinant ameloblastin specifically bound to ameloblasts and inhibited proliferation of dental epithelial cells. These results suggest that ameloblastin is an important regulator to maintain the differentiation state of ameloblasts.     

Correlation of the arrangement pattern of enamel rods and secretory ameloblasts in pig and monkey teeth: a possible role of the terminal webs in ameloblast movement during secretion.

Enamel rod architecture and ameloblast arrangement were examined in pig and monkey teeth using light microscopy and scanning and transmission electron microscopy. Enamel rods in the pig teeth were arranged in longitudinal straight rows in the initial enamel layer, in longitudinal wavy rows in the inner enamel layer, and in a staggered pattern in the outer enamel layer. Rod decussation was seen only in the inner layer. Cross-sectioned enamel rods in the pig were arcade-shaped in the initial and inner layers, and mostly round in shape with circular boundaries in the outer layer. Arrangement of secretory ameloblasts at the level of the distal terminal web and Tomes' processes, and shape of Tomes' processes, corresponded to those of the enamel rod in the enamel layers. Distal terminal webs were well developed between straight rows of the ameloblasts forming the initial layer and between wavy rows of the ameloblasts forming the inner layer, and less developed within a row. The filament bundles in the distal terminal webs were also oriented along the rows. However, in the ameloblasts forming the outer layer, which lost their row pattern, distal terminal web filaments were distributed uniformly at the cell periphery. A similar arrangement of wavy rows of ameloblasts at the level of distal terminal web and Tomes' processes was also seen in monkey teeth.     

The effect of strontium,cobalt and fluoride on rat incisor enamel formation

This investigation examined ultrastructurally the entire period of development of alterations in formative ameloblasts and the enamel which they produce following injection with fluoride, strontium, and cobalt ions. Rats injected with these ions were sacrificed at intervals of 1, 2, 4, 8, 16, 24 and 48 hours to elucidate the sequence and detail of cytologic and cell product alterations which occur. Undecalcified sections of rat incisor teeth were studied using electron microscopy and microradiography. All three ions initially produced disturbances in cell morphology and enamel formation consisting of dark globules, vacuoles, and pooling of stippled material on the enamel surface. While a period of decreased crystal formation occurred after injection with all three ions, only cobalt responses included a period of apparently complete absence of crystal formation. The hypermineralized layers occurring in the altered enamel are attributed to changes in the rate of enamel matrix formation and duration of its exposure to tissue fluids. Morphologic changes in Tomes' process were observed at the time of formation of abnormal enamel following injection of all three ions. These observations are compared with previous studies of altered enamel formation and analyzed with the goal of learning more about the mechanisms of amelogenesis.     

Distribution of Amelotin in Mouse Tooth Development

Amelotin is expressed and secreted by ameloblasts in tooth development, but amelotin distribution during enamel development is not clear. In this report, we first investigated amelotin expression in developing teeth by immunohistochemistry. Amelotin was detected in the enamel matrix at the secretion and maturation stages of enamel development. Amelotin was also observed at Tomes' processes on the apical ends of secretory ameloblasts. We then compared amelotin gene expression with those of amelogenin, enamelin, and ameloblastin in the mandibles of postnatal mice by RT‐PCR. The expression of amelotin was detected as early as in postnatal day 0 mandibles and amelotin was coexpressed with amelogenin, ameloblastin, and enamelin during tooth development. These data strongly suggest that amelotin is an enamel matrix protein expressed at the secretion and maturation stages of enamel development. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

Fine structural changes and lysosomal phosphatase cytochemistry of ameloblasts associated with the transitional stage of enamel formation in the rat incisor

Trimetaphosphatase (TMPase) and cytidine-5′-monophosphatase (CMPase) were used as lysosomal markers in the transitional ameloblasts (TA) to investigate the distribution of lysosomal structures and to correlate the cytochemical findings with the ultrastructural features of these cells. Of particular interest were the cytochemical and morphological changes which occur as the ameloblasts approach the maturation stage of enamel formation. The sequence of changes observed provides a basis for designation of three regions of the transitional zone (early and late TA and modulating ameloblasts). In the early TA region, the cells decreased in height and contained phagic vacuoles as well as numerous TMPase and CMPase reactive structures. Late transitional ameloblasts had invaginations at their distal ends as well as membrane-bound structures, both filled with fine granular material. Dense bodies, phagic vacuoles, and other elements of the lysosomal system were enzyme reactive. Modulating ameloblasts lacked the phagic vacuoles but exhibited large numbers of multivesicular bodies, vesicles, and secretory granules. Their distal ends were morphologically altered indicating a change towards ruffle- or smooth-ended varieties of maturation ameloblast. In the former, increased granular material was observed within cell membrane invaginations and associated membrane-bound structures. In the latter, intercellular spaces widened and were filled with granular material. The present cytochemical findings of an extensive lysosomal system in transitional ameloblasts confirm the function of those cells in reducing the secretory ameloblast population and in the selective elimination of their protein-synthesizing organelles. Furthermore, this extensive lysosomal system and the present morphological findings are consistent with a potential role for transitional ameloblasts in contributing to the marked loss of enamel protein known to occur during maturation.     

A histochemical demonstration of calcium in the maturation stage enamel organ of rat incisors

The location of calcium in a rapid-frozen and freeze-substituted maturation stage enamel organ of the rat incisors was demonstrated by means of the glyoxal bis(2-hydroxyanil) (GBHA) staining method, which formed insoluble red precipitates of calcium-GBHA complex. In the ameloblast layer, highly GBHA-reactive tubulo-vesicular structures corresponding to mitochondria and some other membrane-bound structures were localized in both ruffle-ended and smooth-ended ameloblasts, although no significant GBHA reaction was localized in the nucleus, Golgi region, nor along the plasma membrane of these cells. In addition, numerous granular GBHA reactions appeared exclusively in association with the ruffled border of ruffle-ended ameloblasts. GBHA reactions were positive, but were considerably weaker in papillary cells than in the ameloblast. These observations provide a first published histochemical mapping of calcium in the maturation stage enamel organ, and suggest the active participation of mitochondria in maturation stage ameloblasts in calcium regulation.     

Immunocytochemical and immunochemical detection of a 32 kDa nonamelogenin and related proteins in porcine tooth germs.

Porcine tooth germ was investigated immunochemically and immunocytochemically using antibodies against a synthetic N-terminal peptide fragment from a 32 kDa nonamelogenin found in the inner (old) secretory enamel. In immunochemical preparations, these antibodies reacted to many proteins of differing molecular weights, especially to 140 kDa, 89 kDa, 56 kDa, 45 kDa, and 32 kDa proteins. Analysis of the layers of enamel suggested that the 140 kDa and/or 89 kDa proteins, both of which were found in newly formed enamel, were the parental proteins secreted by the ameloblasts, and that they were degraded to produce 32 kDa and other low molecular-weight proteins associated with progressive mineralization. In immunohistochemical preparation, immunoreactivity at the differentiation stage was detected initially over the amorphous dense material or fine fibrils around calcified globules in predentin, while the stippled material was devoid of immunoreactivity. The amorphous dense material seemed to give rise to a continuous layer of initial enamel. At the matrix formation stage, the immunoreactivity of immature enamel just beneath the putative secretory face of the Tomes' processes was intense. From the surface of the enamel matrix to a depth of about 100 microns, immunoreactivity of prism sheaths was weaker than that of enamel prisms, producing a reverse honeycomb pattern. In the enamel matrix deeper than 100 microns, immunoreactivity was weak and homogeneously distributed. The Golgi apparatus and secretory granules of the secretory ameloblasts showed immunoreactivity. These results suggest that the likely parent proteins of the 32 kDa nonamelogenin protein, i.e., the 140 kDa and/or 89 kDa proteins, play a significant role in the calcification of the enamel matrix.     

Localization of Core Planar Cell Polarity Proteins,PRICKLEs, in Ameloblasts of Rat Incisors: Possible Regulation of Enamel Rod Decussation

To confirm the possible involvement of planar cell polarity proteins in odontogenesis, one group of core proteins, PRICKLE1, PRICKLE2, PRICKLE3, and PRICKLE4, was examined in enamel epithelial cells and ameloblasts by immunofluorescence microscopy. PRICKLE1 and PRICKLE2 showed similar localization in the proliferation and secretory zones of the incisor. Immunoreactive dots and short rods in ameloblasts and stratum intermedium cells were evident in the proliferation to differentiation zone, but in the secretion zone, cytoplasmic dots decreased and the distal terminal web was positive for PRICKLE1 and PRICKLE2. PRICKLE3 and PRICKLE4 showed cytoplasmic labeling in ameloblasts and other enamel epithelial cells. Double labeling of PRICKLE2 with VANGL1, which is another planar cell polarity protein, showed partial co-localization. To examine the transport route of PRICKLE proteins, PRICKLE1 localization was examined after injection of a microtubule-disrupting reagent, colchicine, and was compared with CX43, which is a membrane protein transported as vesicles via microtubules. The results confirmed the retention of immunoreactive dots for PRICKLE1 in the cytoplasm of secretory ameloblasts of colchicine-injected animals, but fewer dots were observed in control animals. These results suggest that PRICKLE1 and PRICKLE2 are transported as vesicles to the junctional area, and are involved in pattern formation of distal junctional complexes and terminal webs of ameloblasts, further implying a role in the formed enamel rod arrangement.