Fine structure of the secretory and nonsecretory ameloblasts in the frog. I. Fine structure of the secretory ameloblasts.

Amelogenesis in the tooth germs of the frog Rana pipiens was examined by electron microscopy at different stages of tooth development. Cellular changes in secretory ameloblasts during this process showed many basic similarities to those in mammalian amelogenesis. Amelogenesis can be divided into three stages based on histological criteria such as thickness of enamel and the relative position of the tooth germ within the continuous succession of teeth. These stages are early, transitional and late. The fine structure of the enamel-secreting cells reflects the functional role of these ameloblasts as primarily secretory in the early stage, possibly transporting in the late stage and reorganizing between the two functions in the transitional stage. In early amelogenesis the cell exhibits well-developed granular endoplasmic reticulum, Golgi complex, microtubules, dense granules, smooth and coated vesicles, lysosome-like bodies in supranuclear and distal portions of the cell and mitochondria initially concentrated in the basal part of the cell. Numerous autophagic vacuoles are observed concomitant with the loss of some cell organelles at the transitional stage. During late amelogenesis the ameloblasts exhibit numerous vesicles, granules, convoluted cell membranes, junctional complexes and widely distributed mitochondria. Toward the end of amelogenesis, cells become oriented parallel to the enamel surface and the number of organelles is reduced. Amelogenesis in the frog is an extracellular process and mineralization seems to occur simultaneously with matrix formation.     

Inner enamel epithelia synthesize and secrete enamel proteins during mouse molar occlusal "enamel-free area" development

Mesenchyme-derived instructions for odontogenic epithelial differentiation into ameloblasts and the production of enamel matrix has been well established. However, it is not known how position-specific differences within the enamel organ of rodent molar tooth organs regulate the enamel-forming vs. the enamel free areas in the developing cusp. Light microscopy, transmission electron microscopy, and immunocytochemistry using a rabbit anti-mouse amelogenin antibody, were used to map the position-specific patterns within the enamel organ. In the enamel-forming area, ameloblasts were associated with stratum intermedium. In the enamel-free area, another cell type was interposed between inner enamel epithelia (IEE) and stratum intermedium. IEE in the enamel-free area did not have Tomes' processes and secreted enamel matrix not only toward dentin but also between IEE cells. IEE became confluent with stellate reticulum; at this position stratum intermedium cells were no longer detected. The thickness and orientation of dentin matrix collagen fibers in the enamel-free area were different from the fibers in the enamel-forming area. These results suggest that the patterns of epithelial cell-cell and cell-matrix associations during position-specific enamel organ epithelial differentiation may regulate ameloblast matrix synthesis and/or the matrix secretion pathway.     

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 mum 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. The specialized predentin forms a well-defined layer, up to 1.5 mum 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 mum 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.     

Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts

Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. During this process, epithelial cells differentiate into enamel-secreting ameloblasts. Ameloblastin, an enamel matrix protein, is expressed by differentiating ameloblasts. Here, we report the creation of ameloblastin-null mice, which developed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of Msx2, p27, and p75 were deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation. The mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.     

Sonic hedgehog regulates growth and morphogenesis of the tooth

During mammalian tooth development, the oral ectoderm and mesenchyme coordinate their growth and differentiation to give rise to organs with precise shapes, sizes and functions. The initial ingrowth of the dental epithelium and its associated dental mesenchyme gives rise to the tooth bud. Next, the epithelial component folds to give the tooth its shape. Coincident with this process, adjacent epithelial and mesenchymal cells differentiate into enamel-secreting ameloblasts and dentin-secreting odontoblasts, respectively. Growth, morphogenesis and differentiation of the epithelium and mesenchyme are coordinated by secreted signaling proteins. Sonic hedgehog (Shh) encodes a signaling peptide which is present in the oral epithelium prior to invagination and in the tooth epithelium throughout its development. We have addressed the role of Shh in the developing tooth in mouse by using a conditional allele to remove Shh activity shortly after ingrowth of the dental epithelium. Reduction and then loss of Shh function results in a cap stage tooth rudiment in which the morphology is severely disrupted. The overall size of the tooth is reduced and both the lingual epithelial invagination and the dental cord are absent. However, the enamel knot, a putative organizer of crown formation, is present and expresses Fgf4, Wnt10b, Bmp2 and Lef1, as in the wild type. At birth, the size and the shape of the teeth are severely affected and the polarity and organization of the ameloblast and odontoblast layers is disrupted. However, both dentin- and enamel-specific markers are expressed and a large amount of tooth-specific extracellular matrix is produced. This observation was confirmed by grafting studies in which tooth rudiments were cultured for several days under kidney capsules. Under these conditions, both enamel and dentin were deposited even though the enamel and dentin layers remained disorganized. These studies demonstrate that Shh regulates growth and determines the shape of the tooth. However, Shh signaling is not essential for differentiation of ameloblasts or odontoblasts.     

Laminin alpha2 is essential for odontoblast differentiation regulating dentin sialoprotein expression

Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wild-type and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.     

Possible involvement of maspin in tooth development

Maspin is a 42 kDa serine protease inhibitor that possesses tumor suppressive and anti-angiogenic activities. Despite of a huge amount of data concerning the expression pattern of maspin in various tissues and its relevance to the biological properties of a variety of human cancer cells, little is known on the maspin expression in skeletal and tooth tissues. Recently, we reported that maspin may play an important role in extracellular matrix formation in bone by enhancing the accumulation of latent TGF-β in the extracellular matrix. This study was performed to elucidate the possible role of maspin in tooth development. First, an immunohistochemical analysis for human tooth germs at the late bell stage showed the expression of maspin by active ameloblasts and odontoblasts that were forming enamel and dentin, respectively. During rat tooth development, maspin expression was observed for the first time in inner and outer enamel epithelial cells and dental papilla cells at early bell stage. The neutralizing anti-maspin antibody inhibited the proper dental tissue formation in organ cultures of mandibular first molars obtained from 21-day-old rat embryos. In addition, the proliferation of HAT-7 cells, a rat odontogenic epithelial cell line, and human dental papilla cells were suppressed in a dose-dependent manner with anti-maspin antibody. Moreover, RT-PCR analysis showed that the expression of mRNA for tooth-related genes including dentin matrix protein 1, dentin sialophosphoprotein and osteopontin in human dental papilla cells was inhibited when treated with anti-maspin antibody. These findings suggest that maspin expressed in ameloblasts and odontoblasts plays an important physiological role in tooth development through the regulation of matrix formation in dental tissues.     

Targeted p120-Catenin Ablation Disrupts Dental Enamel Development

Dental enamel development occurs in stages. The ameloblast cell layer is adjacent to, and is responsible for, enamel formation. When rodent pre-ameloblasts become tall columnar secretory-stage ameloblasts, they secrete enamel matrix proteins, and the ameloblasts start moving in rows that slide by one another. This movement is necessary to form the characteristic decussating enamel prism pattern. Thus, a dynamic system of intercellular interactions is required for proper enamel development. Cadherins are components of the adherens junction (AJ), and they span the cell membrane to mediate attachment to adjacent cells. p120 stabilizes cadherins by preventing their internalization and degradation. So, we asked if p120-mediated cadherin stability is important for dental enamel formation. Targeted p120 ablation in the mouse enamel organ had a striking effect. Secretory stage ameloblasts detached from surrounding tissues, lost polarity, flattened, and ameloblast E- and N-cadherin expression became undetectable by immunostaining. The enamel itself was poorly mineralized and appeared to be composed of a thin layer of merged spheres that abraded from the tooth. Significantly, p120 mosaic mouse teeth were capable of forming normal enamel demonstrating that the enamel defects were not a secondary effect of p120 ablation. Surprisingly, blood-filled sinusoids developed in random locations around the developing teeth. This has not been observed in other p120-ablated tissues and may be due to altered p120-mediated cell signaling. These data reveal a critical role for p120 in tooth and dental enamel development and are consistent with p120 directing the attachment and detachment of the secretory stage ameloblasts as they move in rows.     

GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.     

Spatial and temporal activity of the dentin sialophosphoprotein gene promoter: differential regulation in odontoblasts and ameloblasts

Dentin sialoprotein and dentin phosphoprotein are non-collagenous proteins that are cleavage products of dentin sialophosphoprotein (DSPP). Although these two protein products are believed to have a crucial role in the process of tooth mineralization, their precise biological functions and the molecular mechanisms of gene regulation are not clearly understood. To understand such functions, we have developed a transgenic mouse model expressing a reporter gene (lacZ) under the control of approximately 6 kb upstream sequences of Dspp. The transgenic fusion protein was designed to reside within the cells to facilitate the precise identification of cell type and developmental stages at which the Dspp-lacZ gene is expressed. The results presented in this report demonstrate: (a) the 6 kb upstream sequences of Dspp have the necessary regulatory elements to direct the tissue specific expression of the transgene similar to endogenous Dspp, (b) both odontoblasts and ameloblasts exhibit transgene expression in a differentiation dependent manner, and (c) a differential regulation of the transgene in odontoblasts and ameloblasts occurs during tooth development and mineralization.     

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.     

Immunocytochemical study of amelogenin deposition during the early odontogenesis of molars in alendronate-treated newborn rats.

Newborn rats were treated with sodium alendronate to study how enamel is formed and the effect of alendronate during early odontogenesis. Ultrastructural analysis combined with high-resolution immunocytochemistry for amelogenin was carried out. Twelve rats were subjected to daily SC injections of sodium alendronate (2.5 mg/kg/day) for 3 days on their dorsal region, whereas three rats were daily injected with saline solution as a control. Molar tooth germs from 3-day-old rats were fixed under microwave irradiation in 0.1% glutaraldehyde + 4% formaldehyde buffered at pH 7.2 with 0.1 M sodium cacodylate. The specimens were left undecalcified, postfixed with osmium tetroxide, dehydrated, and embedded in LR White resin. Ultrathin sections were incubated with a chicken anti-24-kDa rat amelogenin antibody, a secondary antibody, and finally with a protein A-gold complex. Large patches of amelogenin were present over the unmineralized mantle dentin and at early secretory ameloblasts. At more advanced stages, they were also detected at the enamel matrix, as well as in the mineralized dentin, at the periodontoblastic space of the dentinal tubules, and at the predentin. It is likely that the main effect of alendronate at early stages of odontogenesis is the increase of synthesis/secretion of amelogenin, promoting its deposition within the forming dentin and enamel.     

Distribution of calcium-ATPase in developing teeth of embryonic American alligators (Alligator mississippiensis)

Light microscopic and ultrastructural observations were carried out to evaluate the cell morphology and histochemistry (calcium-ATPase activity) of developing teeth in embryonic American alligators (Alligator mississippiensis). Ca-ATPase activity was observed in the distal and lateral cell membranes, rough endoplasmic reticulum (rER), mitochondria, vacuoles, and other organelles of the ameloblast, but only in the distal cell membrane and process of the odontoblast. Enzyme activity in the ameloblasts increased gradually during development. These sites of enzyme activity are related to mineralization of the enamel layer, similar to that in mammalian tooth development. Alligator teeth are heavily mineralized like mammalian teeth; however, alligator ameloblasts have undeveloped distal processes during mineralization in contrast to mammalian ameloblasts in which Tomes' processes are found near the distal portion of ameloblasts at maturation stage. The localization of intense enzyme activity in the distal and lateral ameloblast cell membrane suggests that these regions are the site of accumulation of calcium as enamel differentiates in the developing tooth. © 1993 Wiley-Liss, Inc.     

A freeze-fracture study of the papillary layer of the rat incisor enamel organ

After tooth enamel has been secreted it undergoes maturation or hardening. This process is mediated by ruffled and smooth-ended ameloblasts and associated papillary layer cells. The cells of the papillary layer are characterized by large numbers of mitochondria, coated vesicles, microvilli, and gap junctions. These features have led numerous investigators to speculate that the papillary layer is an ion-transporting epithelium. We have conducted freeze-fracture studies of the rat papillary layer in order to better characterize the surface features of these cells. The cell membranes of the papillary cells contained large numbers of intramembrane particles of various sizes ranging from 4 to 9 nm in diameter. Gap junctions were present at the cell surface and in the cytoplasm in the form of annular gap junctions. The intramembrane particles or connexons of both types of gap junctions were about 8-9 nm wide and were either packed randomly or present in the so-called 'crystallized' state. At the interface between smooth-ended ameloblasts and papillary layer cells, a well-developed zonula occludens was present along the basal surfaces of the ameloblasts and several large gap junctions were formed between the two cell types. The capillary network associated with the papillary layer was characterized by a thin endothelium containing large numbers of fenestrations.     

Odontoblast cells immortalized by telomerase produce mineralized dentin-like tissue both in vitro and in vivo

The formation of dentin provides one well accepted paradigm for studying mineralized tissue formation. For the assembly of dentin, several cellular signaling pathways cooperate to provide neural crest-derived mesenchymal cells with positional information. Further, "cross-talk" between signaling pathways from the mesenchymal derived odontoblast cells and the epithelially derived ameloblasts during development is responsible for the formation of functional odontoblasts. These intercellular signals are tightly regulated, both temporally and spatially. When isolated from the developing tooth germ, odontoblasts quickly lose their potential to maintain the odontoblast-specific phenotype. Therefore, generation of an odontoblast cell line would be a valuable reproducible tool for studying the modulatory effects involved in odontoblast differentiation as well as the molecular events involved in mineralized dentin formation. In this study an immortalized odontoblast cell line, which has the required biochemical machinery to produce mineralized tissue in vitro, has been generated. These cells were implanted into animal models to determine their in vivo effects on dentin formation. After implantation, we observed a multistep, programmed cascade of gene expression in the exogenous odontoblasts as the dentin formed de novo. Some of the genes expressed include the dentin matrix proteins 1, 2, and 3, which are extracellular matrix molecules responsible for the ultimate formation of mineralized dentin. The biological response was also examined by histology and radiography and confirmed for mineral deposition by von Kossa staining. Thus, a transformed odontoblast cell line was created with high proliferative capacity that might ultimately be used for the regeneration and repair of dentin in vivo.     

Maturation ameloblasts of the porcine tooth germ do not express amelogenin

 Amelogenins are the most abundant constituent in the enamel matrix of developing teeth. Recent investigations of rodent incisors and molar tooth germs revealed that amelogenins are expressed not only in secretory ameloblasts but also in maturation ameloblasts, although in relatively low levels. In this study, we investigated expression of amelogenin in the maturation stage of porcine tooth germs by in situ hybridization and immunocytochemistry. Amelogenin mRNA was intensely expressed in ameloblasts from the differentiation to the transition stages, but was not detected in maturation stage ameloblasts. C-terminal specific anti-amelogenin antiserum, which only reacts with nascent amelogenin molecules, stained ameloblasts from the differentiation to the transition stages. This antiserum also stained the surface layer of immature enamel at the same stages. At the maturation stage, no immunoreactivity was found within the ameloblasts or the immature enamel. These results indicate that, in porcine tooth germs, maturation ameloblasts do not express amelogenins, suggesting that newly secreted enamel matrix proteins from the maturation ameloblast are not essential to enamel maturation occurring at the maturation stage. Accepted: 14 January 1999     

A light and electron microscopic study of he heart of a crayfish, Procambarus clarkii (Giraud). II. Fine structure

The adventitia of the crayfish heart is composed of cells that are separated from each other by an intercellular space about 280 Å wide. Desmosomes are present on apposing surfaces of adjacent cells. A basal lamina underlies the adventitia and consists of a dense, amorphous substance that contains numerous fine filaments. The myocardial cells are striated and an external lamina 0.1 μ thick is present on the surface of the plasma membrane. The nuclei and most of the cytoplasm, glycogen and mitochondria are located at the cell periphery. The myofibrils are composed of thick and thin filaments and confined to the core of the cell. A T system and a well-developed SR are present. Elements of these organelles form dyads at levels that correspond to the H bands, and triads at levels that correspond to the Z bands of the peripheral myofibrils. The relationship of the T tubules to the myofibrils is discussed. Locus cells exhibit a unique pattern of intracellular myofibrillar branching. They branch from a region which has a structure similar to the Z band material. The myofibrils radiate outwardly in various directions and form numerous cellular branches which form intercalated discs with adjacent myocardial cells. These discs are more complex than those observed in poikilothermic vertebrates but are simpler than those in mammals. An endocardium is lacking in the crayfish heart but interstitial cells are present in close association with the myocardial cells and neural elements. Terminal nerve processes deeply embedded in the myocardial cells are described.     

Peritubular dentin formation: crystal organization and the macromolecular constituents in human teeth.

Peritubular dentin (PTD) is a relatively dense mineralized tissue that surrounds the tubules of coronal tooth dentin. It is composed mainly of crystals of carbonated apatite together with a small amount of collagen. Its mode of formation has been investigated by studying the relatively dense particles isolated from a powdered preparation. Electron microscopic examination of the PTD particles, including 3-dimensional image reconstruction and electron diffraction, shows that the organization of the crystals of PTD is very similar to that of the adjacent intertubular dentin (ITD). The latter contains relatively large amounts of collagen and the carbonated apatite crystals are closely associated with the collagen matrix. The proteins present in the PTD particles are soluble after decalcification and stain with Stains All. The principal protein has higher molecular weight and a quite different amino acid composition than the phosphophoryns of the intertubular dentin. The interface between the PTD and the ITD shows structural continuity. These data show how two distinct carbonated apatite-based mineralized tissues can be organized and formed contiguously within the same organ by utilizing different sets of matrix proteins.     

Ameloblast differentiation in the human developing tooth: Effects of extracellular matrices

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.