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Immunohistochemistry (IHC) is a technique based on the specificity of antibody-antigen principle used commonly to detect antigens in tissue sections. The immune labeling can be performed in paraffin sections, cryostat sections, and ultrathin sections and can be observed in light confocal and transmission electron microscopy. However, the use of immunohistochemical techniques for the study of mineralized tissues has been a challenge for decades (Berdal et al., Arch Oral Biol 36:715-725, 1991; Nanci et al., Eur J Histochem 52:201-214, 2008). Specific procedures are necessary when compared with soft tissue immunohistochemistry. This chapter describes methods for IHC on Tissue-Tek O.C.T. compound and paraffin-embedded sections to detect antigens in the dental mineralized tissues.

In situ hybridization (ISH) is one of the fundamental methods in developmental biology and neurobiology. Their first ISH protocols were reported in 1969 (Gall and Pardue, Proc Natl AcadSci USA 63:378-83, 1969). Since several decades, ISH based on the specific hybridization of 100-2000 nucleotides long probes enabled the localization of DNA/RNA sequences in tissues and cells with high cellular resolution. But sometimes a limited sensitivity notably in mineralized tissues (Obernosterer et al., Nature Protocols 2:1508-14, 2007).Here we describe a recent improvement of in situ hybridization efficiency by applying nucleotide locked nucleic acid (LNA)-incorporated oligodeoxynucleotide probes (20 LNA/DNA nucleotide probes) essentially used for noncoding miRNA and messenger RNAs.

In situ hybridization (ISH) is one of the fundamental methods in developmental biology and neurobiology. Their first ISH protocols were reported in 1969 (Gall and Pardue, Proc Natl AcadSci USA 63:378-83, 1969). Since several decades, ISH based on the specific hybridization of 100-2000 nucleotides long probes enabled the localization of DNA/RNA sequences in tissues and cells with high cellular resolution. But sometimes a limited sensitivity notably in mineralized tissues (Obernosterer et al., Nature Protocols 2:1508-14, 2007).Here we describe a recent improvement of in situ hybridization efficiency by applying nucleotide locked nucleic acid (LNA)-incorporated oligodeoxynucleotide probes (20 LNA/DNA nucleotide probes) essentially used for noncoding miRNA and messenger RNAs.

Immunohistochemistry (IHC) is a technique based on the specificity of antibody-antigen principle used commonly to detect antigens in tissue sections. The immune labeling can be performed in paraffin sections, cryostat sections, and ultrathin sections and can be observed in light confocal and transmission electron microscopy. However, the use of immunohistochemical techniques for the study of mineralized tissues has been a challenge for decades (Berdal et al., Arch Oral Biol 36:715-725, 1991; Nanci et al., Eur J Histochem 52:201-214, 2008). Specific procedures are necessary when compared with soft tissue immunohistochemistry. This chapter describes methods for IHC on Tissue-Tek O.C.T. compound and paraffin-embedded sections to detect antigens in the dental mineralized tissues.

Background Amelogenesis imperfecta (AI) is a group of genetic diseases characterised by tooth enamel defects. AI was recently described in patients with familial hypercalciuria and hypomagnesaemia with nephrocalcinosis (FHHNC) caused by CLDN16 mutations. In the kidney, claudin-16 interacts with claudin-19 to control the paracellular passage of calcium and magnesium. FHHNC can be linked to mutations in both genes. Claudin-16 was shown to be expressed during amelogenesis; however, no data are available on claudin-19. Moreover, the enamel phenotype of patients with CLDN19 mutations has never been described. In this study, we describe the clinical and genetic features of nine patients with FHHNC carrying CLDN19 mutations and the claudin-19 expression profile in rat ameloblasts. Methods Six FHHNC Brazilian patients were subjected to mutational analysis. Three additional French patients were recruited for orodental characterisation. The expression profile of claudin-19 was evaluated by RT-qPCR and immunofluorescence using enamel epithelium from rat incisors. Results All patients presented AI at different degrees of severity. Two new likely pathogenic variations in CLDN19 were found: p.Arg200Gln and p.Leu90Arg. RT-qPCR revealed low Cldn19 expression in ameloblasts. Confocal analysis indicated that claudin-19 was immunolocalised at the distal poles of secretory and maturing ameloblasts. Conclusions For the first time, it was demonstrated that AI is associated with FHHNC in patients carrying CLDN19 mutations. The data suggest claudin-19 as an additional determinant in enamel formation. Indeed, the coexistence of hypoplastic and hypomineralised AI in the patients was consistent with claudin-19 expression in both secretory and maturation stages. Additional indirect systemic effects cannot be excluded.

Msx and Dlx homeobox genes encode for transcription factors that control early morphogenesis. More specifically, Msx-1, Msx-2, and Dlx-2 homeobox genes contribute to the initial patterning of the dentition. The present study is devoted to the potential role of those homeobox genes during the late formation of mineralized tissues, using the rodent incisor as an experimental system. The continuously erupting mandibular incisor allows (1) the coinvestigation of the whole sequences of amelogenesis and dentinogenesis, aligned along the main dental axis in a single sample in situ and (2) the differential characterization of transcripts generated by epithelial and ectomesenchymal odontogenic cells. Northern blot experiments on microdissected cells showed the continuing expression of Msx-2 and Dlx-2 in the later stages of dental biomineralization, differentially in epithelial and ectomesenchymal compartments. Transgenic mice produced with LacZ reporter constructs for Dlx-2 and Msx-1 were used to detect different components of the gene expression patterns with the sensitive beta-galactosidase histoenzymology. The results show a prominent epithelial involvement of Dlx-2, with stage-specific variations in the cells involved in enamel formation. Quantitative analyses identified specific modulations of Dlx-2 expression in ameloblasts depending on the anatomical sites of the incisor, showing more specifically an inverse linear relationship between the Dlx-2 promoter activity level and enamel thickness. This investigation extends the role of homeoproteins to postmitotic stages, which would control secretory cell activity, in a site-specific manner as shown here for Dlx-2.

Calbindin-D9K immunoreactivity was localized by electron microscopy in rat calcifying epiphyseal plate cartilage. Antigen-antibody reaction sites were visualized by the presence of protein A-gold complex particles on undecalcified material embedded in Lowicryl K4M. Immunoreactive calbindin-D9K was found in the hyaloplasm of hypertrophic chondrocytes and inside and at the ends of their cell processes. It was localized outside the cells, inside matrix vesicles (MVs), often against the inner face of the delimiting membrane, and inside the trilaminar membrane. Immunoreactive calbindin-D9K appeared to be extruded from the chondrocytes into the matrix vesicles when the latter were formed during the budding of cell processes. In calcifying MVs, gold particles were detected over the needle-shaped crystallites and often over the crystallites lying against the inner leaflet of the vesicular membrane. At a later stage of matrix vesicle calcification after MV membrane disruption, the number of gold particles remained unchanged over the clusters of crystallites at the loci from which the crystallites appeared to have grown and radiated. At a yet more advanced stage of calcification, they remained in the same areas, which were limited to the lateral edges of calcified cartilage longitudinal septa. These results suggest that immunoreactive calbindin-D9K plays a role in calcium input to matrix vesicles and may be involved in matrix vesicle calcification, perhaps in the initial event of matrix vesicle crystal nucleation.

Les agenesies dentaires et les fentes palatines sont associees a des mutations de l'homeogene Msx1, soulignant ainsi le role primordial de ce gene au cours du developpement initial du squelette craniofacial. Il controle la proliferation et la differenciation terminale des cellules formant les tissus mineralises cranio-faciaux. Recemment, un ARN antisens du gene Msx1 a ete identifie ; il inhibe la synthese de la proteine Msx1 dans ces memes cellules.Le but de cet article est d'examiner le role du gene Msx1 et sa regulation pendant les etapes tardives du developpement du squelette cranio-facial. Les modalites de la croissance osseuse ainsi que les schemas d'expression des transcrits sens (qui codent pour la proteine), des transcrits antisens de Msx1 et de la proteine Msx1 ont ete etudies en post-natal chez la souris normale et chez la souris transgenique dont l'expression du gene Msx1 est supprimee.La proteine Msx1 s'exprime essentiellement dans les pre-osteoblastes localises dans certains sites squelettiques specifiques comme la base de la mandibule. Ces memes sites presentent une diminution de croissance osseuse chez la souris transgenique. La comparaison des schemas d'expression de la proteine Msx1 et des transcrits sens/antisens de Msx1 suggere que la balance des transcrits sens/antisens de Msx1 controlerait la repartition des sites d'actions du gene Msx1 pendant la morphogenese et la croissance du squelette cranio-facial.Chez l'humain, une etude clinique des patients presentant une mutation du gene MSX1 a ete entreprise pour confirmer cette hypothese, dans une perspective de diagnostic et de therapeutique genetiques des anomalies de croissance cranio-faciale.

The unique hereditary enamel defect clearly related to the disturbance of one enamel matrix protein is X-linked amelogenesis imperfecta (AI), in which several mutations of amelogenin gene have been identified. The clinical phenotype of many of these subjects shows similarities with enamel defects related to rickets. Therefore, we hypothesized that rachitic dental dysplasia is related to disturbances in the amelogenin pathway. In order to test this hypothesis, combined qualitative and quantitative studies in experimental vitamin D-deficient (-D) rat model systems were performed. First, Western blot analysis of microdissected enamel matrix (secretion and maturation stages) showed no clear evidence of dysregulation of amelogenin protein processing in -D rats as compared with the controls. Second, the ultrastructural investigation permitted identification of the internal tissular defect of rachitic enamel, the irregular absence of intraprismatic enamel observed in -D animals, suggesting a possible link between prism morphogenesis and vitamin D. In addition, the steady-state levels of amelogenin mRNAs measured in microdissected dental cells was decreased in -D rats and up-regulated by an unique injection of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). The present study shows evidences that amelogenin expression is regulated by vitamin D. This is the first study of an hormonal regulation of tooth-specific genes.

Calbindin-D9k and calbindin-D-28k genes are useful systems to investigate the tissue- and stage-specificity as well as the hormonal control of gene expression. Since they regulate cellular calcium mobilization, their study may be of interest in mineralized tissues. However, thus far, immunocytochemical labelling has been mainly realized in these systems. In order to set up methods for mRNA investigation, in situ hybridization of calbindin-D28k mRNAs was performed in the continuously erupting incisor of Sprague-Dawley rats (15-, 30-, and 56-day-old). 35S UTP labelled antisense and sense riboprobes specific for brain calbindin-D 28k were used for in situ hybridization. Specific and non-specific signals could not be discerned when studying decalcified samples. In contrast, on sections not pretreated with EDTA, calbindin-D 28k transcripts (in tooth and kidney) appeared strongly labelled with antisense probes, while sense probes provided a negligible background. In ameloblasts, the signal (i.e., calbindin-D 28k mRNA levels) increased during the presecretory stage. Different mRNA gradients and subcellular distribution patterns characterized the secretory and maturation stages. A nuclear labelling was observed, associated with the highest levels of transcripts. These data suggest a developmental control of calbindin-D28k mRNA transcription. Calbindin-D28k gene expression appears to be up-regulated during the initiation of both secretory and maturation stages of enamel mineralization.

International audience; Skeletal growth and homeostasis require the finely orchestrated secretion of mineralized tissue matrices by highly specialized cells, balanced with their degradation by osteoclasts. Time- and site-specific expression of Dlx and Msx homeobox genes in the cells secreting these matrices have been identified as important elements in the regulation of skeletal morphology. Such specific expression patterns have also been reported in osteoclasts for Msx genes. The aim of the present study was to establish the expression patterns of Dlx genes in osteoclasts and identify their function in regulating skeletal morphology. The expression patterns of all Dlx genes were examined during the whole osteoclastogenesis using different in vitro models. The results revealed that Dlx1 and Dlx2 are the only Dlx family members with a possible function in osteoclastogenesis as well as in mature osteoclasts. Dlx5 and Dlx6 were detected in the cultures but appear to be markers of monocytes and their derivatives. In vivo, Dlx2 expression in osteoclasts was examined using a Dlx2/LacZ transgenic mouse. Dlx2 is expressed in a subpopulation of osteoclasts in association with tooth, brain, nerve, and bone marrow volumetric growths. Altogether the present data suggest a role for Dlx2 in regulation of skeletal morphogenesis via functions within osteoclasts.

The Msx1 homeogene plays an important role in epithelial–mesenchymal interactions leading organogenesis. Msx1 gene is submitted to bidirectional transcription generating a long non-coding antisense (AS) RNA potentially involved in Msx1 expression regulation. RT-Q-PCR and RNA-FISH studies indicated that transient overexpression of the Msx1 AS transcript in 705IC5 mouse odontoblasts decreased the abundance of endogenous Msx1 S mRNA at the post-transcriptional level. Conversely, Msx1 overexpression increased the AS RNA level probably by activating AS transcription. In vivo mapping by RT-PCR evidenced both Msx1 RNAs in all adult mouse tissues tested raising the issue of Msx1 function during adulthood. The expression patterns of the two RNAs were similar, confirming the tight S/AS relationship. In particular, both Msx1 mRNAs and Msx1 protein were similarly distributed in eyes, and were found in regions with a common ectodermic origin and in cells potentially involved in regeneration. In conclusion, we report that Msx1 S RNA is negatively controlled by its AS RNA at a post-transcriptional level, and that the AS RNA is retrocontrolled positively by Msx1. The tight link between Msx1 S and AS RNAs constitutes a regulatory loop resulting in a fine-tuned expression of Msx1 which appears to be significant for adult homeostasis. J. Cell. Physiol. 220: 303–310, 2009. © 2009 Wiley-Liss, Inc.

Dental epithelium comprises different cell populations, including ameloblasts and stratum intermedium cells. Ameloblasts are vitamin D targets, and at least five proteins undergo specific modulation of their expression following the addition of 1alpha,25(OH)2 vitamin D3[1alpha,25(OH)2D3]. Stratum intermedium cells have not been studied in any great detail regarding vitamin D impact. Interestingly, in these cells, the tissue non-specific alkaline phosphatase (TNAP) is overexpressed. On the other hand, TNAP is a reliable bone marker of vitamin D action, similar to calbindins in kidney and intestine, previously used for studies of vitamin D activity in ameloblasts. Here, TNAP expression and activity were investigated in vivo in the microdissected epithelium and mesenchyme of mandible incisors. Physiological doses of 1alpha,25(OH)2D3 injected in control rats failed to modify TNAP activity in both dental epithelium and mesenchyme. No significant differences were observed in the steady-state levels of TNAP mRNAs of dental tissues from wild-type and vitamin D nuclear receptor (VDRnuc)-deficient mice of the same litters. These data suggest that, in contrast to ameloblasts, stratum intermedium cells are not sensitive to 1alpha,25(OH)2D3. An explanation for such a responsiveness of stratum intermedium cells to 1alpha,25(OH)2D3 is proposed based on the respective expressions of both vitamin D receptors (VDRnuc and 1,25D3-[MARRS]) and the Dlx2 homeobox gene.

The rapid, nongenomic effects of 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3 have been related to a 1,25D3-membrane associated, rapid response steroid binding protein or 1,25D3-[MARRS]bp, with a molecular weight of 65 kDa, in several tissues and species. Currently, no information is available concerning the nongenomic responses to 1alpha,25-(OH)2D3 in dental tissues. In order to investigate the expression of 1,25D3-[MARRS]bp in dental cells, in the presence or absence of 1alpha,25-(OH)2D3, we have used rabbit polyclonal antibodies directed against the N-terminus of the 1,25D3-[MARRS]bp (Ab099) that recognizes the 1alpha,25-(OH)2D3 binding protein in chick intestinal basolateral membranes and a mouse odontoblast-like cell line (MO6-G3). Western blotting and flow cytometric analyses with Ab099 specifically detected 1,25D3-[MARRS]bp in MO6-G3 cells. Moreover, 1,25D3-[MARRS]bp was up-regulated, in vivo, in differentiated dental cells. Electron microscopic analysis confirmed the plasma membrane localization of this binding protein and also showed its intracellular presence. Incubation of MO6-G3 cells with different doses of 1alpha,25-(OH)2D3 for 36 h resulted in an inhibition of 1,25D3-[MARRS]bp expression with a maximal effect at 50 nM steroid. In addition, the culture media of MO6-G3 cells contains immunoreactive 1,25D3-[MARRS]bp. Immunogold positive membrane vesicle-like structures are present in the extracellular matrix of MO6-G3 cells. Altogether, these results indicate that the 1,25D3-[MARRS]bp expression in MO6-G3 cells is modulated by 1alpha,25-(OH)2D3. In conclusion, this 1alpha,25-(OH)2D3 binding protein could play an important role in the rapid, nongenomic responses to 1alpha,25-(OH)2D3 in dental cells.

The present study was designed to compare the expression pattern of Dlx3 in four different mineralized tissues because of: 1-its role in skeleton patterning, 2-its expression in dental epithelium and mesenchyme during morphogenesis, 3-the membranous and endochondral bone and tooth phenotype of tricho-dento-osseous syndrome related to Dlx3 gene mutation and 4-recently emerging knowledge on Dlx family members in the bone field. Ameloblasts, odontoblasts, osteoblasts and chondrocytes were analyzed in vitro and in vivo. Dlx3 transcripts were detected by RT-PCR in established model systems (microdissected dental epithelium and mesenchyme; primary cultures of rat chondrocytes), as recently performed in osteoblasts in vitro. A human 414-bp Dlx3 probe was generated. A 4.5-kb human Dlx3 sense RNA was identified in maxillo-facial samples by Northern blotting. Immunolabeling and in situ hybridization were performed in mice from Theiler stage E 14.5 until birth. In teeth, although Dlx3 was still expressed in differentiated ameloblasts, it was down regulated during odontoblast polarization. During endochondral bone formation, Dlx3 protein was detected in chondrocytes and was most strongly expressed in the prehypertrophic cartilage zone and in differentiating and differentiated osteoblasts of metaphyseal periosteum. In vitro, real-time PCR studies supported this upregulation in prehypertrophic chondrocytes, closely correlated with Ihh variations. In membranous bone, Dlx3 was present in preosteoblasts, osteoblasts and osteoid-osteocytes. The present data on Dlx3 and recently published functional studies show that this transcription factor may be instrumental during growth in the control of matrix deposition and biomineralization in the entire skeleton.

Tooth agenesis and clef palate are associated to the mutation of the Msx1 homeobox genes, highlighting the pivotal role of homeobox genes during the initial development of the craniofacial skeleton. Msx1 also controls the terminal differentiation of mineralised tissues forming cells. Recently, a Msx1 antisense RNA has been identified which inhibits Msx1 protein expression in odontoblastic cells. In order to investigate the role of Msx1 gene and its antisense RNAs during the late developmental stages of the craniofacial bone formation, the expression pattern of Msx1 protein, sense and antisense transcripts and the aspects of bone growth have been studied in post-natal normal and Msx1 knock-in mutant mice. Msx1 protein was strongly expressed in preosteoblasts of specific bone sites such as the basal mandible. At the same bone sites, bone growth was impaired or markedly decreased in knock-in mice. The comparison between the various expression patterns of Msx1 protein, sense and antisense RNAs suggests that the site-specific action of Msx1 protein on bone growth and craniofacial morphogenesis and that Msx1 protein level could be controlled by the local ratio of Msx1 sense and antisense RNAs. Regarding our experimental data and hypothesis, a clinical study of patients with MSX1 mutation will be performed in order to better characterize the abnormalities of the craniofacial skeleton growth.

Msx1 homeobox gene, a member of Msx family, has been implicated in numerous organs. Its participation was established in different events, such as morphogenetic field determinism and epithelio-mesenchymal interactions. Most of Msx1 target organs are also known for their sensitivity to Vitamin D: such as bone, tooth germ, and hair follicle. Whereas, the expression of Msx2, another member of Msx family, has been shown to be controlled by Vitamin D, no information is available for Msx1. This study aims to analyze the potential relationships between Vitamin D and Msx1 through: (1) comparative analysis of Vitamin D receptor (VDR) and Msx1 protein expression, (2) investigation of Msx1 expression in VDR null mutant mice, and (3) study of Msx1 overexpression impact on osteocalcin VDR expression in immortalized MO6-G3 odontoblasts. Results show the existence of cross-talks between Vitamin D and Msx1 regulation pathways. In odontoblastic cells, Msx1 overexpression decrease VDR expression, whereas in rickets Msx1 sense transcript expression is decreased. These cross-talks may open a new window in the analysis of rickets mineralized tissues physiopathology. In Vitamin D null mutants, the study of the natural Msx1 antisense transcript which has been recently described should be informative.

Msx1 is a key factor for the development of tooth and craniofacial skeleton and has been proposed to play a pivotal role in terminal cell differentiation. In this paper, we demonstrated the presence of an endogenous Msx1 antisense RNA ( Msx1 -AS RNA) in mice, rats, and humans. In situ analysis revealed that this RNA is expressed only in differentiated dental and bone cells with an inverse correlation with Msx1 protein. These in vivo data and overexpression of Msx1 sense and AS RNA in an odontoblastic cell line (MO6-G3) showed that the balance between the levels of the two Msx1 RNAs is related to the expression of Msx1 protein. To analyze the impact of this balance in the Msx-Dlx homeoprotein pathway, we analyzed the effect of Msx1, Msx2, and Dlx5 overexpression on proteins involved in skeletal differentiation. We showed that the Msx1 -AS RNA is involved in crosstalk between the Msx-Dlx pathways because its expression was abolished by Dlx5. Msx1 was shown to down-regulate a master gene of skeletal cells differentiation, Cbfa1 . All these data strongly suggest that the ratio between Msx1 sense and antisense RNAs is a very important factor in the control of skeletal terminal differentiation. Finally, the initiation site for Msx1 -AS RNA transcription was located by primer extension in both mouse and human in an identical region, including a consensus TATA box, suggesting an evolutionary conservation of the AS RNA-mediated regulation of Msx1 gene expression.

Phenotypes associated with Msx1 mutations have established the prominent role of this divergent homeogene in skeletal patterning. Previous studies have been achieved during antenatal development in relation with the early death of null mutant mice. Therefore, the present study is devoted to Msx1 homeogene in the postnatal craniofacial, axial, and appendicular skeleton. A knock-in transgenic mouse line was studied from the first postnatal week until 15 months. Whole-mount β-galactosidase enzymology identified Msx1 protein expression pattern. Maintained expression of Msx1 was observed in growing and adult mice, specifically in the sites where Msx1 plays an early morphogenetic role during initial skeletal patterning. These included the craniofacial sutures, autopodium, mandible, and alveolar bone. Furthermore, active membranous and endochondral bone formation involved Msx1 in the entire skeleton. Histologic sections showed that progenitor as well as differentiating and differentiated cells of all the bone cell lineages could express the Msx1 protein (chondrocytes, osteoblasts, tartrate-resistant acid phosphatase positive osteoclasts and chondroclasts). Recent developments in the genetic and developmental biology of skeletal morphogenesis demonstrate that genes critical for development are jointly expressed in discrete embryonic signalling and growth centers, the enamel knot in teeth, the cranial suture in skull morphogenesis, and the progress zone in the limb buds. The present study suggests that these signalling pathways are jointly important throughout the entire lifetime with an exquisite site-specificity spatially related to early patterning. © 2001 Wiley-Liss, Inc.

The unique hereditary enamel defect clearly related to the disturbance of one enamel matrix protein is X-linked amelogenesis imperfecta (AI), in which several mutations of amelogenin gene have been identified. The clinical phenotype of many of these subjects shows similarities with enamel defects related to rickets. Therefore, we hypothesized that rachitic dental dysplasia is related to disturbances in the amelogenin pathway. In order to test this hypothesis, combined qualitative and quantitative studies in experimental vitamin D-deficient (-D) rat model systems were performed. First, Western blot analysis of microdissected enamel matrix (secretion and maturation stages) showed no clear evidence of dysregulation of amelogenin protein processing in -D rats as compared with the controls. Second, the ultrastructural investigation permitted identification of the internal tissular defect of rachitic enamel, the irregular absence of intraprismatic enamel observed in -D animals, suggesting a possible link between prism morphogenesis and vitamin D. In addition, the steady-state levels of amelogenin mRNAs measured in microdissected dental cells was decreased in -D rats and up-regulated by an unique injection of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). The present study shows evidences that amelogenin expression is regulated by vitamin D. This is the first study of an hormonal regulation of tooth-specific genes.

Msx and Dlx family transcription factors are key elements of craniofacial development and act in specific combinations with growth factors to control the position and shape of various skeletal structures in mice. In humans, the mutations of MSX and DLX genes are associated with specific syndromes, such as tooth agenesis, craniosynostosis, and tricho-dento-osseous syndrome. To establish some relationships between those reported human syndromes, previous experimental data in mice, and the expression patterns of MSX and DLX homeogenes in the human dentition, we investigated MSX-2, DLX-5, and DLX-7 expression patterns and compared them in orofacial tissues of 7.5- to 9-wk-old human embryos by using in situ hybridization. Our data showed that MSX-2 was strongly expressed in the progenitor cells of human orofacial skeletal structures, including mandible and maxilla bones, Meckel's cartilage, and tooth germs, as shown for DLX-5. DLX-7 expression was restricted to the vestibular lamina and, later on, to the vestibular part of dental epithelium. The comparison of MSX-2, DLX-5, and DLX-7 expression patterns during the early stages of development of different human tooth types showed the existence of spatially ordered sequences of homeogene expression along the vestibular/lingual axis of dental epithelium. The expression of MSX-2 in enamel knot, as well as the coincident expression of MSX-2, DLX-5, and DLX-7 in a restricted vestibular area of dental epithelium, suggests the existence of various organizing centers involved in the control of human tooth morphogenesis.

Comparative investigations of odontogenic cells in normally forming teeth and tumors may provide insights into the mechanisms of the differentiation process. The present study is devoted to late phenotypic markers of ameloblast and odontoblast cells, i.e., proteins involved in biomineralization. The in situ expression of amelogenins, keratins, collagens type III and IV, vimentin, fibronectin, osteonectin, and osteocalcin was performed on normal and tumor odontogenic human cells. The pattern of protein expression showed some similarities between ameloblasts and odontoblasts present in normally developing human teeth and cells present in neoplastic tissues of ameloblastic fibroma, ameloblastic fibro-odontomas, and complex odontomas. Amelogenins (for ameloblasts) and osteocalcin (for odontoblasts) were detected in cells with well-organized enamel and dentin, respectively. In contrast, "mixed" cells located in epithelial zones of mixed odontogenic tumors co-expressed amelogenins and osteocalcin, as shown by immunostaining. The presence of osteocalcin transcripts was also demonstrated by in situ hybridization in these cells. Keratins and vimentin were detected in the same epithelial zones. Tumor epithelial cells were associated with various amounts of polymorphic matrix (amelogenin- and osteocalcin-immunoreactive), depending on the types of mixed tumors. No osteocalcin labeling was found in epithelial tumors. This study confirms that the differentiation of normal and tumor odontogenic cells is accompanied by the expression of some common molecules. Furthermore, the gene products present in normal mesenchymal cells were also shown in odontogenic tumor epithelium. These data may be related to a tumor-specific overexpression of the corresponding genes transcribed at an undetectable level during normal development and/or to an epithelial-mesenchymal transition proposed to occur during normal root formation. A plausible explanation for the results is that the odontogenic tumor epithelial cells are recapitulating genetic programs expressed during normal odontogenesis, but the tumor cells demonstrate abnormal expression patterns for these genes.

Dentin sialoprotein (DSP), a 53 kDa glycoprotein, is believed to be present exclusively in dentin. Using rat and mouse digoxigenin labeled (DIG)-DSP and 35S-DSP riboprobes, and in situ hybridization techniques, we have studied the presence of DSP mRNA at specific developmental stages of dentinogenesis. In mouse and rat molars and incisors, DSP transcripts were localized in young odontoblasts associated with early stages of predentin formation, as well as in mature odontoblasts, cells with cytoplasmic extensions embedded in the forming dentin. No DSP transcripts were detected in dental pulp, enamel organ, ameloblasts, epithelial root sheath, Meckel's cartilage, alveolar bone or tibia. Furthermore, no DSP mRNA was observed in other soft tissues including heart, lung, kidney, intestine, eye, and muscle. In addition to the intense and prolonged expression by odontoblasts, DSP mRNA was transiently expressed by pre-ameloblasts in both developing molars and incisors. These observations are consistent with the results of previous immunohistochemical studies (1). The transient expression of DSP in pre-ameloblasts across from young odontoblasts suggests an involvement of DSP in epithelial-mesenchymal interactions that are crucial to later stages of tooth development.

The aim of this study was to investigate the expression pattern of 1, 25-dihydroxyvitamin D3 receptor (VDR) and vitamin D-responsive gene expression during the steps of hard tissue formation in oro-facial development. In situ hybridization of VDR, alkaline phosphatase, and osteocalcin transcripts was performed in the mandibles of growing rats. Osteoblasts were used as the internal positive control for in situ detection of VDR messenger RNAs. Transcripts were present throughout the stages of differentiation and in differentiated osteoblasts and osteocytes, and showed some anatomical specificities in their developmental expression pattern. In dental tissues, VDR was strongly expressed in the inner dental epithelium at the beginning of the presecretion stage and, after a transient decrease at the end of the presecretion stage, in secretion stage ameloblasts. VDR was continuously expressed in epithelial supraameloblastic cells. During dentin formation, VDR was mainly present in subodontoblastic cells and was down-regulated during the terminal differentiation of odontoblasts. In these cells, VDR expression appeared to be induced by 1, 25-dihydroxyvitamin D3 injection. These data confirm that VDR is expressed in cells directly involved in mineralized tissue formation: ameloblasts, odontoblasts, and osteoblasts. Furthermore, they extend the idea of vitamin D sensitivity to cells that are not directly involved in this process: supraameloblastic, subodontoblastic, and osteoprogenitor cells. The differential expression pattern of VDR in odontoblasts and osteoblasts together with the similarity in the expression of potential vitamin D-responsive genes (osteocalcin in odontoblasts and osteoblasts, and alkaline phosphatase in osteoprogenitor and subodontoblastic cells) suggest the existence of a tissue specificity for the genomic action of 1, 25-dihydroxyvitamin D3, which may involve co-operation with additional nuclear factors.

Following their terminal differentiation, highly specialized cells, ameloblasts, odontoblasts, and osteoblasts sequentially elaborate mineralized tissues. While the developmental expression pattern of matrix proteins has been studied extensively, less attention has been paid to the molecules involved in calcium handling, such as calcium-binding proteins. This shortcoming, as well as previous conflicting data, led us to conduct studies on calbindin-D9k and calbindin-D28k in rat mandibular bone and incisor based on several methods established on rat ameloblasts in vivo. Radioimmunoassays showed that calbindin-D28k accounts for approximately 0.1% of cytosolic proteins in the ectomesenchymal fraction and 1% in the epithelial fraction of the rat incisor and is 100-fold more concentrated than calbindin-D9k in both tissue types. Western blot analysis confirmed that the anticalbindin-D28k reactive species corresponded to the well characterized renal calbindin-D28k in the ectomesenchyme. In this tissue, calbindin-D28k was ultrastructurally immunolocalized in the odontoblasts. Quantitative immunocytochemistry showed that labeling was distributed throughout their nucleus and cytoplasm. The similar cytoplasmic distribution of both calbindin-D proteins and mRNAs suggests that their expression is regulated at the subcellular level. In particular, immunoreactive calbindin-D28k appeared to be associated with rough endoplasmic reticulum. Calbindin-D9k antisense probe showed negligible labeling in odontoblasts, in parallel with the protein quantities measured (approximately 10 ng/mg of total protein). Finally, in situ hybridization showed transcripts for both calbindins-D in ameloblasts and also in osteoblasts. In summary, the present results support the concept that an elevated expression of these vitamin D-dependent calcium-binding proteins may characterize the phenotype of cells directly involved in the elaboration of mineralized tissues, enamel, dentine, and bone.

The basic features on the vitamin D endocrine system, synthesis of the main metabolite 1,25-dihydroxyvitamin D3 (1,25) and its genomic action mediated via the vitamin D receptor (VDR), are reviewed. Calbindin-D9k, calbindin-D28k and osteocalcin are presented as the most-extensively investigated vitamin D-dependent calcium-binding proteins. The action of 1,25 on the basic process of proliferation and differentiation is introduced. Then, the basis of the systemic theory of vitamin D action on teeth (clinical and experimental data and the dissimilar distribution of VDR and of potential vitamin D-dependent proteins in dental cells) are exposed. Finally, the data obtained with calbindin-D9k, calbindin-D28k, osteocalcin and VDR, which supports the theory that ameloblasts and odontoblasts are target-cells for 1,25 is presented. As a perspective, a cross-survey of the 1,25 and tooth-related literature is proposed which may indicate potential target-genes for 1,25 in teeth as done previously for calbindins-D.

The EF-hand calciumdashbinding protein parvalbumin has been extensively studied in nerve and muscle cells. Its possible role in biomineralization during tooth development was here investigated by determining its subcellular localization by immunogold cytochemistry. The developmental sequences of amelogenesis and dentinogenesis were studied in rat molars, and in continuously growing rat incisors. The findings confirm that parvalbumin is a nuclear and a cytosolic protein, not associated with any particular intracellular organelle. Epithelial and mesenchymal undifferentiated cells contained no specific parvalbumin immunolabelling. In differentiated ameloblasts, secretory-pole (Tomes' process) formation was associated with a proximal-distal gradient of parvalbumin labelling. But after the Tomes' process had formed, parvalbumin was evenly distributed throughout the cell. The parvalbumin contents of ruffle-ended and smooth-ended ameloblasts appeared to be very different. Differentiated odontoblasts were less heavily labelled than ameloblasts, and the label was restricted to the cell body during the whole of dentinogenesis. These data suggest that parvalbumin could contribute to membrane plasticity during differentiation, as shown during dendritic growth in the nervous cells. Moreover, as may occur in excitable cells, parvalbumin could buffer calcium specifically in the cells producing mineralized enamel and dentine during the later stages of tooth development.

Le syndrome de Gorlin est une maladie rare caracterisee par la survenue de tumeurs keratokystiques odontogenes (OKTs) des l’enfance, de carcinomes baso-cellulaires et de «pits» palmo-plantaires. Dans environ 98 % des cas, ce syndrome est cause par une mutation dominante du gene suppresseur de tumeur PATCH1 implique dans le developpement embryonnaire. Les OKTs sont des tumeurs benignes des maxillaires invasives et recidivantes. Cette etude a pour but de comparer les donnees cliniques, histopathologiques et moleculaires d’une serie pediatrique de 10 enfants âges de 9 a 19 ans atteints d’OKTs, 5 dans le cadre d’un syndrome de Gorlin (11 OKTs) et 5 dans un cadre sporadique (5 OKTs). Les cas d’OKTs syndromiques sont caracterises par leur precocite de survenue, leur agressivite clinique et leur recurrence. L’histopathologie montre dans les cas syndromiques une plus grande proliferation de la couche basale et des kystes filles, elements non trouves dans les cas sporadiques. Des genes du developpement dentaire et des determinants de la voie activatrice du recepteur au NFkB impliques dans l’osteoclastogenese sont explores pour une meilleure comprehension des interactions entre evolution tumorale et osteolyse peritummorale dans la perspective d’une application therapeutique medicamenteuse.

Experimental and clinical data indicate that dentin mineralization is vitamin D-dependent. This calcium-regulating steroid controls protein synthesis, for instance that of osteocalcin in osteoblasts. This protein also elaborated by odontoblasts was used as a molecular marker for vitamin D action on odontoblasts. Since the most characteristic protein synthesized by odontoblasts is the dentin phosphoprotein which is thought to regulate hydroxyapatite growth, its cellular and extracellular distribution was also studied. Tooth formation in the molars and incisors of successive generations of vitamin D-deficient animals (-D) and in controls (+D) was compared by microadiography, toluidine blue histochemistry, and immunocytochemistry. In -D samples, the presence of dentin phosphoprotein in odontoblasts indicated that their differentiation occurred despite major morphological disturbances at the cusp tips. In contralateral teeth, osteocalcin was depleted in odontoblasts and dentin, suggesting an inhibition of protein synthesis induced by vitamin D-deficiency. In the extracellular matrix of +D animals, phosphoprotein distribution was associated with dentin, especially within actively forming calcospherites at the mineralization front. In contrast, in -D dentin, the mineralization defects corresponded to irregular absence of histochemically detectable phosphoprotein. This protein indeed appeared either absent or uniformly sparse in -D dentin by immunocytochemistry. These data suggest that vitamin D acts directly on odontogenic cells at various synthetic (osteocalcin) or secretory (phosphoprotein) levels indicating that odontoblasts are target-cells for vitamin D. Therefore, this hormone could contribute to the regulation of extracellular mineralization during dentinogenesis, via different mechanisms in the processing of matrix protein.

This electron microscope study describes the subcellular occurrence and distribution of immunoreactive calbindin-D9K in the trabecular metaphyseal and compact cortical bone of normal rats, rachitic vitamin-D-deficient rats, and rachitic rats given 1,25-(OH)2D3. Undecalcified bones were embedded in Lowicryl K4M and calbindin-D9K antigenicity was detected by the protein A-gold method. Immunoreactive calbindin-D9K was localized in the cytoplasm and cell processes of osteoblasts and osteocytes. Immunoreactive calbindin-D9K was also found within matrix vesicles and calcifying matrix vesicles, where it lay over the needle-shaped crystallites, at the apparent site of initial crystal formation, but not along the whole crystallites. In fully mineralized bone it occurred at the same site, over the crystallites. Calibindin-D9K was vitamin-D-dependent in the osteoblasts and matrix vesicles, where its presence was correlated with the reappearance of crystallites in 1,25-(OH)2D3-treated vitamin-D-deficient rats. This suggests that immunoreactive calbindin-D9K is involved in mineral deposition in bone matrix vesicles. Abnormal intracellular calcification associated with calbindin-D9K antigenicity in the osteoblasts of 1,25-(OH)2D3-treated vitamin-D-deficient rats indicates that immunoreactive calbindin-D9K may also play a part in abnormal intracellular mineral deposition.

The immunocytochemical patterns of calbindin-D 9k (CaBP 9 k) and calbindin-D 28 k (CaBP 28 k) were compared by light and electron microscopy throughout amelogenesis. Labelling on serial sections and co-localization of CaBPs confirmed that the two proteins were restricted to a single cell type, the ameloblasts. Their quantity increased during presecretion, was stable during secretion and alternately high and low during the cyclic modulation of ameloblasts which occurs during maturation. Ruffle-ended ameloblasts contained the highest apparent concentration. Investigations with several fixatives indicated that the CaBPs were present in the cytosol and the nucleus, although there were slight differences with various fixatives by light microscopy. Their concentrations in these compartments varied in parallel throughout amelogenesis. However, mitochondria contained only immunoreactive CaBP 9 k. While the distribution of CaBP 9 k in zones containing Golgi apparatus and rough endoplasmic reticulum was similar, CaBP 28 k concentration has, in another paper, been shown to be higher near the rough endoplasmic reticulum.

The control of tooth development by 1,25-Dihydroxyvitamin D3 is analyzed by light- and electron-microscope immunocytochemistry and Northern-blotting in vitamin D-deficient rats. The receptor for 1,25-Dihydroxyvitamin D3, immunostained at the light microscope in all stem-cells, became immunodetectable only at the ultrastructural level in the ameloblasts which elaborate enamel and odontoblasts which synthetize dentin. Moreover, 1,25-Dihydroxyvitamin D3 induces an up-regulation specifically in these cells. In parallel, the calbindins-D9k, -D28k and osteocalcin, in contrast to the phosphoprotein, appear sensitive to vitamin D-deficiency. A single injection of 1,25-Dihydroxyvitamin D3 led to the increase of steady-state levels of the corresponding calbindin mRNAs. These data show that tooth constitutes a target-organ for 1,25-Dihydroxyvitamin D3, as other components of the phospho-calcic metabolism.

The present study focuses on the ultrastructure of enamel organ cells and the immunolocalization of calbindins D-9kDa and -28kDa during enamel secretion in Vitamin D-deficient rats. Vitamin D-deficiency disturbed the deposition of the layer of inner enamel and depleted the calbindins-content of ameloblasts. These data raise the possibility of a direct action of Vitamin D on the physiology of ameloblasts through ionic calcium homeostasis.

This report presents evidence for the presence of the vitamin D-dependent calcium-binding protein, calbindin-D9K, in bone cells and matrix. In undecalcified frozen sections of growing and adult rat bone, calbindin-D9K was immunohistochemically localized in trabecular bone of the epiphysis and metaphysis and in cortical bone of the diaphysis. It was found within the cytoplasm of osteocytes, of osteoblasts lining the osteoid, and osteoblasts inside the osteoid seams. It was also found in the osteoblast processes and the anastomosed reticulum of the processes connecting the osteocytes with each other. Extracellularly, calbindin-D9K immunoreactivity was present in compact cortical bone in the areas of the mineralized matrix surrounding the osteocyte lacunae and in the pericanalicular walls containing the cell processes. Calbindin-D9K immunoreactivity was low or absent from the cytoplasm of osteocytes in trabecular bone from severely vitamin D-deficient rats and restored in vitamin D-deficient rats given a single dose of 1,25(OH)2-VitD3. Thus, the synthesis of immunoreactive calbindin-D9K by osteoblasts and osteocytes in trabecular bone is vitamin D-dependent. The presence of immunoreactive calbindin-D9K in the osteocytes and their cell processes suggests that this calcium-binding protein is involved in the calcium fluxes regulating bone calcium homeostasis. Its locatization in osteoblasts involved in bone formation and in their cell processes suggests that it has a role in the calcium transport from these cells towards the sites of active bone mineralization. The extracellular immunoreactive calbindin-D9K in the walls of osteocyte lacunae and pericanalicula margins may have a specific role in those areas. Thus, the distribution of calbindin-D9K immunoreactivity in bone indicates that it may mediate all or part of the action of vitamin D on bone cells and bone mineralization.

The present study focuses on the ultrastructure of enamel organ cells and the immunolocalization of calbindins D-9kDa and-28kDa during enamel secretion in Vitamin D-deficient rats. Vitamin D-deficiency disturbed the deposition of the layer of inner enamel and depleted the calbindins-content of ameloblasts. These data raise the possibility of a direct action of Vitamin D on the physiology of ameloblasts through ionic calcium homeostasis.

Msx1 plays a key role in early dental and cranio-facial patterning. A systematic screening of Msx1 transcripts during late postnatal stages of development evidenced not only sense mRNA but also antisense mRNA in the skeleton. Natural antisenses are able to bind their corresponding sense RNAs and block protein expression. Specific reverse-transcription polymerase chain reaction (RT-PCR) Northern-blotting using riboprobes and primer extension analysis allowed to identify and sequence a mouse 2184-base Msx1 antisense transcript. The transcription start site was located in a region including a consensus TATA box. In situ hybridization evidenced an increase in antisense mRNA expression during dental and bone cell differentiation in prenatal (Theiler stages E15.5-18.5) and newborn mice. This upregulation was related to Msx1 protein downregulation in cells expressing Msx1 sense mRNA. In vitro, transient Msx1 sense and antisense mRNA overexpression was performed in MO6-G3 cells, which pertain to the odontoblast lineage (polarization and dentin sialoprotein and phosphoprotein synthesis). The balance between antisense and sense Msx1 mRNAs appeared to control Msx1 protein levels. These data suggest that a bidirectional transcription of Msx1 homeogene may control Msx1 protein levels, and therefore may be critical in cell communication and differentiation during dental and cranio-facial development and mineralization.