Expression of cytokeratins in enamel organ, junctional epithelium and epithelial cell rests of Malassez.

BACKGROUND AND OBJECTIVE: After tooth formation is complete, it is suggested that continuity exists between the epithelial cell rests of Malassez (ERM), reduced enamel epithelium (REE) and subsequently the junctional epithelium. However, the junctional epithelium was reported to differ from REE and ERM. The developmental relationships between and among them remain controversial. Therefore, in the present study we examined the expression of cytokeratins in the three types of epithelia to investigate the epithelial phenotypes. MATERIAL AND METHODS: The maxillae of Wistar rats, 1, 2, 3 and 7 wk of age, were used, and the expression of CK14, CK17, CK19, CK10/CK13 and AE1/AE3 was detected using immunoperoxidase techniques. RESULTS: There was negative staining for CK10/CK13 in all the epithelia. ERM stained strongly for AE1/AE3, CK14, CK17 and CK19. During the transformation of inner enamel epithelial (IEE) cells into reduced ameloblasts and subsequently into junctional epithelium, strong staining for CK14 was evident in IEE, REE and junctional epithelium, whereas the expression of AE1/AE3 and of CK19 were initially negative in IEE and then strong in REE and junctional epithelium, respectively. In particular, the expression of CK17 was strongly positive in ERM and REE, but was negative in IEE and junctional epithelium. CONCLUSION: ERM are of odontogenic origin and junctional epithelium has an epithelial phenotype different from REE and ERM. This is the first report to demonstrate that CK17 can be used as a marker to distinguish junctional epithelium from ERM.

Immunohistochemical localization of connective tissue growth factor, transforming growth factor-beta1 and phosphorylated-smad2/3 in the developing periodontium of rats.

BACKGROUND AND OBJECTIVE: Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta1 (TGF-ß1), and TGF-ß1-induced CTGF expression is regulated through the SMAD pathway. CTGF is implicated in the development of cartilage, bone and tooth. However, its expression in the developing periodontium is unclear. Therefore, we aimed to investigate the immunolocalization of CTGF, TGF-ß1 and phosphorylated SMAD2/3 (pSMAD2/3) in the developing periodontium of rats. MATERIAL AND METHODS: The maxillaries of Wistar rats, 2, 3, 7 and 12 wk of age, were used and the localization of CTGF, TGF-ß1 and pSMAD2/3 was detected using immunoperoxidase techniques. RESULTS: Hertwig' s epithelial root sheath (HERS) cells were strongly positive for CTGF and TGF-ß1, but not for pSMAD2/3. Positive staining for CTGF, TGF-ß1 and pSMAD2/3 was found in bone and periodontal ligament. In cementum, most cementoblasts associated with cellular cementum and some cementocytes stained strongly for CTGF, whereas cementoblasts associated with acellular cementum did not express CTGF. No signal for TGF-ß1 was observed in cellular and acellular cementum. In addition, most cementocytes were strongly positive for pSMAD2/3. CONCLUSION: CTGF, TGF-ß1 and pSMAD2/3 are localized in bone and periodontal ligament, but are differentially expressed in HERS and cementum. The results of our study indicate that the regulation of CTGF expression by TGF-ß1 might be cell-type specific in periodontium.

The cell adhesion molecule nectin-1 is critical for normal enamel formation in mice.

Nectin-1 is a member of a sub-family of immunoglobulin-like adhesion molecules and a component of adherens junctions. In the current study, we have shown that mice lacking nectin-1 exhibit defective enamel formation in their incisor teeth. Although the incisors of nectin-1-null mice were hypomineralized, the protein composition of the enamel matrix was unaltered. While strong immunostaining for nectin-1 was observed at the interface between the maturation-stage ameloblasts and the underlying cells of the stratum intermedium (SI), its absence in nectin-1-null mice correlated with separation of the cell layers at this interface. Numerous, large desmosomes were present at this interface in wild-type mice; however, where adhesion persisted in the mutant mice, the desmosomes were smaller and less numerous. Nectins have been shown to regulate tight junction formation; however, this is the first report showing that they may also participate in the regulation of desmosome assembly. Importantly, our results show that integrity of the SI-ameloblast interface is essential for normal enamel mineralization.

Consequences for enamel development and mineralization resulting from loss of function of ameloblastin or enamelin.

Although the nonamelogenin proteins, ameloblastin and enamelin, are both low-abundance and rapidly degrading components of forming enamel, they seem to serve essential developmental functions, as suggested by findings that an enamel layer fails to appear on teeth of mice genetically engineered to produce either a truncated form of ameloblastin (exons 5 and 6 deleted) or no enamelin at all (null). The purpose of this study was to characterize, by direct micro weighing, changes in enamel mineralization occurring on maxillary and mandibular incisors of mice bred for these alterations in nonamelogenin function (Ambn(+/+, +/-5,6, -5,6/-5,6), Enam(+/+, +/- ,-/-)). The results indicated similar changes to enamel-mineralization patterns within the altered genotypes, including significant decreases by as much as 50% in the mineral content of maturing enamel from heterozygous mice and the formation of a thin, crusty, and disorganized mineralized layer, rather than true enamel, on the labial (occlusal) surfaces of incisors and molars along with ectopic calcifications within enamel organ cells in Ambn(-5,6/-5,6) and Enam(-/-) homozygous mice. These findings confirm that both ameloblastin and enamelin are required by ameloblasts to create an enamel layer by appositional growth as well as to assist in achieving its unique high level of mineralization.

An immunohistochemical study of the expression of heat-shock protein-25 and cell proliferation in the dental pulp and enamel organ during odontogenesis in rat molars.

OBJECTIVES: The aim of this study is to clarify the functional significance of heat-shock protein (HSP)-25 during tooth development. DESIGN: We compared the expression of HSP-25 in the dental epithelial and mesenchymal cells with their proliferative activity during odontogenesis in rat molars on postnatal days 1-100 by immunohistochemistry using anti-HSP-25 and anti-5-bromo-2'-deoxyuridine (BrdU) for cell proliferation assay. RESULTS: On day 1, BrdU-immunoreactive cells were densely located in the inner enamel epithelium in the cervical loop and intercusped areas and the dental pulp adjacent to them, whereas HSP-25-immunoractivity (IR) was restricted to the cusped area where odontoblasts and ameloblasts had already differentiated. Subsequently, BrdU-IR shifted in the apical direction to be localized around Hertwig's epithelial root sheath during days 5-30, never overlapping with concomitantly apically-shifted HSP-25-IR. On days 60-100, BrdU-immunoreactive cells were hardly recognizable in the dental pulp, where HSP-25-IR was exclusively localized in the odontoblast layer. Furthermore, the odontoblast- and ameloblast-lineage cells exhibited two steps in the expression of HSP-25 throughout the postnatal stages: first, dental epithelial and pulpal mesenchymal cells showed a weak IR for HSP-25 after the cessation of their proliferative activity, and subsequently odontoblasts and ameloblasts consistently expressed an intense HSP-25-IR. CONCLUSION: Odontoblast- and ameloblast-lineage cells acquire HSP-25-IR after they complete their cell division, suggesting that this protein acts as a switch between cell proliferation and differentiation during tooth development. The consistent expression of HSP-25-IR in the formative cells may be involved in the maintenance of their functional integrity.

Endogenous epidermal growth factor regulates the timing and pattern of embryonic mouse molar tooth morphogenesis.

The tooth organ provides a model for discrete patterns of morphogenesis over short periods of developmental time. Studies were designed to test the hypothesis that endogenous epidermal growth factor (EGF) functions to regulate multiple cusp molar tooth morphogenesis during embryonic mouse development. The relative levels of endogenous EGF and EGF receptor (EGFR) transcripts were determined in both enamel organ epithelia and dental ectomesenchyme by reverse transcription-polymerase chain reaction (RT-PCR) assays. EGF and EGFR were localized by immunohistochemistry; both antigenic determinants were demonstrated on the same odontogenic cells in cultured tooth explants. To examine EGF-mediated signal transduction, cap stage mouse molar tooth organs (E16) were cultured in serumless, chemically-defined medium as either (i) controls, or supplemented with (ii) tryphostin (an EGF receptor kinase inhibitor), (iii) tyrphostin plus exogenous EGF, and (iv) exogenous EGF. Antisense oligodeoxynucleotide (ODN) strategy was used to investigate the functions of endogenous EGF employing (i) non-treated control, (ii) sense ODN control, (iii) antisense ODN, (iv) exogenous EGF, (v) sense ODN with exogenous EGF, and (vi) antisense ODN with exogenous EGF. Tyrphostin inhibited DNA synthesis and produced a significant decrease in the volume of the explants. These effects were recovered by addition of exogenous EGF. Antisense ODN inhibition resulted in abnormal cusp formations, decreased DNA synthesis, total DNA, RNA and protein content, and decreased stellate reticulum and tooth explant volumes. The decreased tooth size was not uniform, the most pronounced effect was in the stellate reticulum. This pattern of changes was not seen when antisense ODN treatment was supplemented with exogenous EGF. These results suggest that during cap stage of odontogenesis endogenous EGF acts to stimulate DNA synthesis, which increases the cell number of specific phenotypes within the enamel organ epithelia, and thereby regulates molar tooth morphogenesis.

Immunolocalization of CD44 and the ezrin-radixin-moesin (ERM) family in the stratum intermedium and papillary layer of the mouse enamel organ.

We studied the immunohistochemical localization of CD44 and the ezrin-radixin-moesin (ERM) family of actin binding proteins in mouse enamel organ, using confocal laser scanning microscopy and transmission electron microscopy to clarify their role in cytoskeletal organization. At the differentiation stage of ameloblasts, immunoreactivity to CD44 was detected on the plasma membrane of the inner enamel epithelium, the stellate reticulum, the stratum intermedium, and the external enamel epithelium. In accordance with the differentiation of preameloblasts into secretory ameloblasts, immunoreactivity increased in the stratum intermedium cells. At the maturation stage, intense immunoreactivity was observed on the papillary layer cells. For the ERM family, the stratum intermedium and the papillary layer cells were stained with anti-ezrin and -radixin monoclonal antibodies but not with the anti-moesin antibody. Electron microscopic observations revealed that CD44, ezrin, and radixin were localized in the region at which preameloblasts came into contact with the stratum intermedium at the differentiation stage. At the secretory and maturation stages, they were concentrated in the microvilli of the stratum intermedium and the papillary layer cells. These findings suggest that the CD44-ezrin-radixin-actin filament system is involved in cell-cell interaction between preameloblasts and the stratum intermedium, and in the cytoskeletal organization of the cells in the stratum intermedium and the papillary layer.

Immunolocalization of epithelial and mesenchymal matrix constituents in association with inner enamel epithelial cells.

After crown formation, the enamel organ reorganizes into Hertwig's epithelial root sheath (HERS). Although it is generally accepted that HERS plays an inductive role during root formation, it also has been suggested that it may contribute enamel-related proteins to cementum matrix. By analogy to the enamel-free area (EFA) in rat molars, in which epithelial cells express not only enamel proteins but also "typical" mesenchymal matrix constituents, it has been proposed that HERS cells may also have the potential to produce cementum proteins. To test this hypothesis, we examined the nature of the first matrix layer deposited along the cervical portion of root dentin and the characteristics of the associated cells. Rat molars were processed for postembedding colloidal gold immunolabeling with antibodies to amelogenin (AMEL), ameloblastin (AMBN), bone sialoprotein (BSP), and osteopontin (OPN). To minimize the possibility of false-negative results, several antibodies to AMEL were used. The labelings were compared with those obtained at the EFA. Initial cementum matrix was consistently observed at a time when epithelial cells from HERS covered most of the forming root surface. Cells with mesenchymal characteristics were rarely seen in proximity to the matrix. Both the EFA matrix and initial cementum exhibited collagen fibrils and were intensely immunoreactive for BSP and OPN. AMEL and AMBN were immunodetected at the EFA but not over the initial cementum proper. These two proteins were, however, present at the cervical-most portion of the root where enamel matrix extends for a short distance between dentin and cementum. These data suggest that epithelial cells along the root surface are likely responsible for the deposition of the initial cementum matrix and therefore, like the cells at the EFA, may be capable of producing mesenchymal proteins.

A2a adenosine receptor gene expression in developing rat brain.

Adenosine is a neuromodulator in the adult central nervous system. Membrane-bound receptors for adenosine have been identified and cDNAs for A1, A2a, A2b, and A3 adenosine receptor subtypes have been cloned recently. The present study documents the developmental appearance of A2a adenosine receptor gene expression in the rat brain. In situ hybridization using 35S-labeled RNA probes generated from the rat A2a adenosine receptor cDNA revealed receptor gene expression in the striatum on gestational day (GD) 14. Developmental alterations in the pattern of receptor gene expression within the striatum suggest that this receptor mRNA is expressed by striatal neurons soon after they complete neurogenesis. Transient expression of the A2a adenosine receptor mRNA was observed in cerebral cortex, subiculum, parafascicularis nucleus of the thalamus, facial nucleus, trigeminal nucleus, locus coeruleus, area postrema, anterior pituitary gland and in the fetal cerebral vasculature. The ganglia of cranial nerves V, VII, VIII, IX and X expressed A2a adenosine receptor mRNA in fetuses; adults have not been examined. A2a adenosine receptor mRNA was expressed in the carotid body and intermediate lobe of the pituitary during development and also in adult rats. Northern blot analysis revealed that the A2a adenosine receptor transcript is consistent in size (ca 2.5 kb) across the developmental period examined (GD 14 through adult). Previous studies in adult rats have reported that A2a adenosine receptor gene expression is limited to a population of striatal medium spiny neurons. This study documents early developmental expression of the A2a adenosine receptor gene in the striatum and its transient expression elsewhere in the brain and cerebral vasculature. If the A2a adenosine receptor mRNA is translated into receptor protein shortly after the mRNA is expressed, adenosine could influence neuronal differentiation, migration, synaptogenesis, and angiogenesis. Expression of A2a adenosine receptor mRNA in cranial ganglia, carotid body, and intermediate lobe of the pituitary gland similarly suggests novel sites of adenosine action during development and in the adult.

Fine structure of dental epithelial cells and the enameloid during the enameloid formation stages in an elasmobranch, Heterodontus japonicus.

The structural features of the dental epithelial cells and the enameloid in tooth germs of the Japanese Port Jackson shark, Heterodontus japonicus, in the stages of enameloid formation, were investigated by light and transmission electron microscopy. At the enameloid matrix-formation stage, tall columnar inner dental epithelial cells contained large numbers of glycogen particles. At the enameloid mineralization stage, when many sharply outlined crystals appeared throughout the enameloid, the inner dental epithelial cells exhibited well-developed Golgi apparatuses and many mitochondria in the proximal cytoplasm, and abundant vesicles and vacuoles in the distal cytoplasm. Marked interdigitations of the lateral membrane were visible in the inner dental epithelial cells. The outer dental epithelial cells contained many mitochondria, lysosomal bodies, vesicles and microtubules, and the capillaries usually approached the outer dental epithelial cells. At the enameloid maturation stage, large numbers of crystals occupied the enameloid, and most of the organic matrix had disappeared from the enameloid area after demineralization. The organelles in the inner and outer dental epithelial cells decreased in number, but there were still widely distributed Golgi apparatuses, abundant intermediate filaments and granules containing an electron-dense substance in the inner dental epithelial cells. It is probable that the dental epithelial cells are involved in the removal of organic matrix from the enameloid and in the process of mineralization at the later stages of enameloid formation, i.e., the mineralization and the maturation stages.

Immunoblotting and cytochemical characterization of human enamel proteins.

Mature enamel proteins (tuft proteins) and fetal enamel proteins were extracted by an homogenizing buffer method, subjected to SDS-PAGE and immunoblotted with a polyclonal antibody raised against the mature enamel proteins. Both fetal and tuft proteins were recognized by this immunoblotting. With the same antibody, immunolocalization of the developing enamel proteins was done on semi-thin-sections of human fetal tissue at the secretory stage, using an immunoperoxidase technique. Specific labelling of the enamel protein matrix was observed. It is concluded that a polyclonal antibody against mature enamel proteins (anti-tuft) can recognize the developing protein matrix at the secretory stage. This suggests that a common antigenic determinant is maintained throughout the course of amelogenesis in human enamel.

Enameloid formation in two tetraodontiform fish species with high and low fluoride contents in enameloid.

Forming teeth of parrotfish and pufferfish were viewed by transmission electron microscopy to correlate cytological features of the enameloid organ with the species' fluoride (F) content in mature enameloid. Secretory-stage inner dental epithelial cells (IDE) of parrotfish (high F) and pufferfish (low F) secreted procollagen granules into the enameloid collagen matrix. The odontoblasts of both species, less numerous than IDE cells, also contained procollagen granules at the enameloid matrix formation stage. After the full thickness of enameloid matrix collagen had been deposited, enameloid crystallites formed parallel to the long axis of the enameloid collagen fibres. Concurrently, the plasma membranes of the outer dental epithelial cells (ODE) became invaginated in both species, but to a much greater extent in parrotfish. Highly undulating parrotfish ODE cells surrounded numerous fenestrated capillaries. In contrast, pufferfish ODE cells remained straight with few adjacent capillaries. Extensive tight junctions formed between ODE and IDE cells of both species, sealing the extracellular space. With increased mineralization, enameloid collagen fibres were no longer discernible. A thin layer of amorphous material, which subsequently mineralized, was secreted on to the enameloid surface by IDE cells in both species. Pufferfish odontoblasts secreted a mineralizing amorphous layer on the pulpal aspect of the enameloid. The results suggest that at the mineralization stage, a triad of cytostructural features, highly invaginated ODE cells, highly vascularized ODE cells, and extensive tight junctions are strongly correlated with high fluoride content of mature enameloid mineral. Species without any one of these features have lower fluoride in the enameloid.

Localization of glycosylated matrix proteins in secretory porcine enamel and their possible functional roles in enamel mineralization.

The present study was undertaken to investigate glycosylation of porcine enamel proteins secreted in the secretory stage of amelogenesis and to gain insight into functional roles of glycosylated proteins in enamel mineralization. Enamel proteins, isolated from various zones of the secretory enamel, were separated by SDS-PAGE and then transferred on to a nitrocellulose membrane. The transblotted proteins were visualized with either antibodies against porcine amelogenins or various biotin-conjugated lectins. The lectins used were Con-A, GS-II, STA, WGA, s-WGA, GS-I, MPA, VVA, PNA, RCA-I, DBA, SJA, UEA-I, Lotus-A and LPA. The results of the immuno- and lectin blottings revealed that most of the lectins did not bind to porcine amelogenins, while a large number of non-amelogenins having various molecular masses were stained strongly with the conjugated WGA, Con A and MPA lectins. On the basis of the binding specificity with the lectins, porcine non-amelogenins were classified into two groups: WGA (and Con A)-binding moieties at 60-90 kDa (WGA-HMW); and MPA-binding moieties at 13-17 kDa (MPA-LMW). These two groups of non-amelogenins differed distinctly in terms of their localization and stability in the secretory tissue and their adsorption properties onto hydroxyapatite. The WGA-HMW were concentrated in the outer region adjacent to the ameloblasts and disappeared (due to degradation) in the underlying inner secretory enamel. In contrast, the MPA-LMW were found in all zones of the secretory enamel and their quantity remained relatively constant. Histochemical studies using FITC-conjugated WGA and MPA showed that the fluorescence-labelling of WGA was localized in the core region of prism rods, while the fluorescence-labelling of MPA was locally limited at the rim of prism rods or at the prism sheath. In separate adsorption studies, it was found that the WGA-HMW, as well as the intact amelogenins, displayed a high adsorption affinity on to apatite crystals, whereas the MPA-LMW showed only marginal adsorption on to apatitic surfaces. The overall results indicate that part of the heterogeneity found in porcine enamel proteins can be ascribed to variations of carbohydrate moieties attached to non-amelogenins.(ABSTRACT TRUNCATED AT 400 WORDS)

In situ localization of tenascin mRNA in developing mouse teeth.

Tenascin is a large extracellular-matrix glycoprotein found in developing connective tissue. A cDNA probe to mouse tenascin, mTN2, was used to determine the cellular origins of this molecule in the murine tooth germ by in situ hybridization. At embryonic day 19, a hybridization signal significantly greater than background was detected with mTN2 in the subodontoblastic layer of the dental mesenchyme and in the inner enamel epithelium of the enamel organ. At postnatal day 1, a signal was detected over pre-odontoblasts and the strata intermedium and externum. No tenascin mRNA was detected in odontoblasts or the stellate reticulum at either age, and hybridization in ameloblasts was not significantly greater than background at postnatal day 1. Thus, much of the tenascin found throughout developing teeth appears to be synthesized by pre-odontoblasts and the inner enamel epithelium, the two populations of cells destined to generate mineralized matrix.

Immunohistochemical demonstration of bcl-2 protein in human tooth germs.

This study sought to detect patterns of bcl-2 protein expression that could provide more insight into the cellular dynamics of tooth development. As bcl-2 serves to prevent cell death, its occurrence in odontogenic tissues might be helpful in identifying cell populations from which odontogenic tumours may arise. The bcl-2 protein was found only in the epithelial part of the tooth germ and was present in all parts of the enamel organ except the ameloblast. This suggests that bcl-2 protein plays a part in maintaining the viability of the enamel organ. The presence of bcl-2 in the fully matured tooth germ and adjacent dental lamina might indicate that epithelial odontogenic tumours may originate from various parts of the enamel organ.

Immunohistochemical and immunochemical detection of a similar epitope in enamel proteins and monocyte-macrophage protein recognized by mouse monoclonal antibody MOMA-2.

These studies were made as a first step in elucidating unknown functions of enamel proteins in odontogenesis. The cytoplasm of ameloblasts and the proteins in dentine matrix before mineralization were stained with this monoclonal antibody. SDS-PAGE and Coomassie blue staining of proteins extracted from enamel showed several protein bands. Immunoblotting revealed that proteins recognized by this antibody were situated between 20-30 kDa. These results indicate that enamel proteins, presumably amelogenins, have an epitope resembling monocyte-macrophage protein. The findings suggest that epithelial-mesenchymal interaction in odontogenesis and preosteoblast-preosteoclast interaction in osteogenesis may be similar.

Ultrastructural and immunocytochemical characterization of cultured cells from rat molar stellate reticulum.

By scanning electron microscopy, the cultured cells were stellate and had numerous filipodia--characteristics of stellate reticulum cells in vivo. By transmission electron microscopy, they contained bundles of intermediate filaments, numerous mitochondria, large electron-dense granules and desmosomes--all features of the stellate reticulum in vivo. Moreover, the stellate reticulum was the only region of the enamel organ in vivo that contained large, electron-dense granules. By immunocytochemistry, the cultured cells contained cytokeratins, confirming their epithelial nature, and stellate reticulum cells in vivo and in vitro did not have an EGF receptor. Thus, these combined ultrastructural and immunocytochemical findings suggest that the cell culture was of stellate reticulum.

Immunocytochemical localization of laminin in the epithelial rests of Malassez of immature rat molars.

Immediately after disruption of Hertwig's root sheath, epithelial cells were found near the root apex, singly or in groups of two or three cells. They were irregular in shape with only a partial lining of basal lamina. Immunoreactivity for laminin was intense in the basal lamina and weak between projections of the epithelial cells. In 5-week-old rats, the epithelial rests consisted of about five cells and still had an incomplete basal lamina with collagen fibrils in the intercellular spaces. Immunoreaction products were seen in the basal lamina and diffusely in the intercellular spaces. The epithelial rests of 9-week-old rats had more cells, an almost complete lining of basal lamina, and narrowed intercellular spaces. Immunoreaction products were seen in the basal lamina but not in the intercellular spaces. These findings indicate the basal lamina is involved in the formation of the epithelial rests.

Immunolocalization of transforming growth factor beta 1 and epidermal growth factor receptor epitopes in mouse incisors and molars with a demonstration of in vitro production of transforming activity.

Day-14 lower incisors and day-18 first lower molars of mouse embryos produced in vitro transforming activities for non-confluent NRK cells co-cultured in agar, and mitogenic activities for exponentially growing NRK and BHK cells. The patterns of distribution of TGF beta 1 and EGF receptor, both known to regulate cell proliferation, differentiation and transformation in vitro and suspected to play important roles in developmental processes, were studied during mouse odontogenesis by means of indirect immunofluorescence on fixed or frozen fixed sections. TGF beta 1 epitopes were detected in the stellate reticulum of day-13 to day-16 incisors and of molars from day-17 onwards. Dental mesenchyme of day-14 incisors and postnatal molars, and peridental mesenchyme of bud and cap stage molars and incisors were also stained by TGF beta 1 antibodies. EGF receptor was localized in the enamel organs of incisors and molars; the inner dental epithelium and later the outer dental epithelium rapidly became negative while the stellate reticulum remained stained. Incisor apical mesenchyme showed an intense reaction with EGF receptor antibodies after birth.

Temporal and spatial patterns of transforming growth factor-beta 1 expression in developing rat molars.

Regulatory peptides of the TGF-beta family affect various aspects of embryonic development. Recent immunolocalization and in situ hybridization studies have demonstrated a specific time- and tissue-dependent expression of TGF-beta 1 in the developing mouse embryo. The purpose of this study was to evaluate the distribution of TGF-beta 1 within rat molars at different stages of development, using a well-characterized antibody, highly specific for TGF-beta 1, and immunohistochemical methods of detection. TGF-beta 1 was immunolocalized intensely within the ectodermally derived stellate reticulum and the mesenchyme of the dental papilla at the bell stage of development. Marked immunostaining was also evident in the papillary layer and the reduced dental organ subjacent to ameloblasts in the differentiation and secretory phases of amelogenesis. During the formation of coronal tissues and in the pre-eruptive phase, immunoreactive TGF-beta 1 was localized conspicuously within the dental follicle overlying the tooth germ. This temporospatial pattern of expression of TGF-beta 1 appears to correlate with specific events in morphogenesis, histogenesis and cytodifferentiation during tooth development.