Expression and function of dopamine in odontoblasts

Dopamine (DA) is produced from tyrosine by tyrosine hydroxylase (TH). A recent study has reported that DA promotes the mineralization of murine preosteoblasts. However, the role of DA in odontoblasts has not been examined. Therefore, in this investigation, we researched the expression of TH and DA in odontoblasts and the effects of DA on the differentiation of preodontoblasts (KN-3 cells). Immunostaining showed that TH and DA were intensely expressed in odontoblasts and preodontoblasts of rat incisors and molars. KN-3 cells expressed D1-like and D2-like receptors for DA. Furthermore, DA promoted odontoblastic differentiation of KN-3 cells, whereas an antagonist of D1-like receptors and a PKA signaling blocker, inhibited such differentiation. However, antagonists of D2-like receptors promoted differentiation. These results suggested that DA in preodontoblasts and odontoblasts might promote odontoblastic differentiation through D1-like receptors, but not D2-like receptors, and PKA signaling in an autocrine or paracrine manner and plays roles in dentinogenesis.     

Change of microtubular arrangement around centrioles in rat incisor odontoblasts during cell differentiation

Three stages during cell differentiation of rat incisor odontoblasts were classified, and change of microtubular arrangement around centrioles in the odontoblasts was examined with three-dimensional analyses using serial ultrathin sections. In the undifferentiated odontoblasts, microtubules were observed to radiate from the pericentriolar area, whereas, in the differentiating odontoblasts, some microtubules became poorly related to the centrioles. In the differentiated odontoblasts, arrangement of most microtubules appeared to have a poor relationship to the centrioles. Throughout the differentiation of the odontoblasts, one of the centriolar pair was ciliated, and Golgi apparatus was invariably observed near the centrioles. The present study suggests that a pericentriolar area, or a centrosome, could function as a microtubule-organizing center (MTOC) in the undifferentiated odontoblasts, but their function might be attenuated during cell differentiation.     

35S]autoradiographic study of sulfated GAG accumulation and turnover in embryonic mouse tooth germs

The accumulation of sulfated GAG in embryonic mouse molars before, during, and after terminal differentiation of odontoblasts was localized by [35S]autoradiography combined with the use of chondroitin ABC lyase. Much more sulfated GAG were accumulated in the dental papilla than in the dental epithelium. High incorporation of [35S]sulfate occurred at the epithelio-mesenchymal junction, which is the site of dental basement membrane and predentin. Before terminal differentiation of odontoblasts, the distribution of sulfated GAG was uniform at the basement membrane. After the onset of terminal differentiation of odontoblasts, much more sulfated GAG accumulated at the tip of principal cusps than at the apical (inferior) parts of cusps, and sulfated GAG were then found to be degraded more rapidly at the epithelio-mesenchymal junction than at other parts of the tooth germ. Thus regional variation in the rate of degradation of GAG exists in the tooth germs. Trypsin-isolated dental epithelia cultured in vitro synthesized a new basement membrane that could be labeled with [3H]glucosamine but not with 35SO4(-2). The epithelial-derived basal lamina contains little or no sulfatated GAG.     

Immunohistochemical localization of four and a half LIM domains 2 in the odontoblasts of mature human teeth

Four and a half LIM domains 2 (FHL2) participates in cell differentiation and cancer development of various tissues, possessing dual functions either as an activator or as a repressor depending on the protein partners involved. Recent studies show that FHL2 plays an important role in osteoblast differentiation and bone formation. The present study was to investigate the expression and localization of FHL2 in human pulp-dentin complex by immunohistochemistry. Our results showed that in sound mature human teeth, FHL2 was expressed in odontoblasts and some endothelial cells of blood vessels. Moreover, in carious teeth FHL2 immunoreactivity was detected in odontoblasts, odontoblast-like cells and endothelial cells of blood vessels. FHL2 was mainly distributed in cytosol of the odontoblast cell bodies and partly located in nuclei of odontoblasts, but not in the odontoblast processes. Our data suggest a role of FHL2 in odontoblast differentiation and dentin formation both in normal and in carious teeth.     

Expression and localization of reelin in human odontoblasts.

Reelin is a large extracellular matrix (ECM) glycoprotein strongly expressed during embryonic development in the central nervous system and involved in architectonic brain development. It could participate in axon plasticity processes or adhesion-recognition between nerve fibers in adulthood. Previously identified from a subtractive cDNA library of fully differentiated human odontoblasts, reelin might be involved in the relationship between dental nerves and odontoblasts in as so far the latter are in close association with pulpal nerve fibers. Here, we show by in situ hybridization and immunohistochemistry that reelin is specifically expressed by human odontoblasts in vivo and in vitro and that an intense expression of the reelin gene is detected in odontoblasts in comparison with pulpal cells (PC). Co-cultures of rat trigeminal ganglion (TG) and odontoblasts allow to mimic odontoblast innervation and demonstrate that neurites contact these cells with reelin molecules as observed in vivo in human dental pulp. Moreover, by RT-PCR, we show that both reelin receptors (namely apolipoprotein E receptor [ApoER-2], very low density lipoprotein receptor [VLDLR] and cadherin-related neuronal receptor [CNR]) and the cytoplasmic adapter Disabled-1 implicated in the reelin signal transduction, were expressed by trigeminal ganglion. On the basis of these data, we suggest that reelin might be an extracellular matrix molecule involved in the terminal innervation of the dentin-pulp complex, promoting adhesion between dental nerve endings and odontoblasts.     

Intracellular distribution of desmoplakin in human odontoblasts.

Coexpression of desmosomal proteins and vimentin has been reported in a specific mesenchymal phenotype. This study investigated the expression of vimentin-binding desmosomal proteins in human dental pulp fibroblasts (DPF) and odontoblasts. The dental pulp has no cells expressing desmocollin (DSC) 1-3, desmoglein (DSG) 1-3, junction plakoglobin (JUP), or desmoplakin (DPK) 1 and 2 except for odontoblasts expressing DPK. A confocal image by laser-scanning microscopy demonstrated the diffuse distribution of DPK in the cytoplasm throughout the odontoblast processes. In culture, the mRNA expression of JUP and DPK1, but not DSC1-3 and DSG1-3, was detected in all DPF clones tested and also in odontoblast-like cells (OB) expressing osteocalcin and dentin sialophosphoprotein mRNAs established in the differentiation medium. The DPF having the potential to differentiate into OB expressed vimentin, but not DPK before culturing in the differentiation medium, whereas OB expressed vimentin-binding DPK1. These results suggest that DPF usually expresses DPK1 mRNA, and that the DPK1 production and the bonding of vimentin to DPK1 occur in DPF with the differentiation into odontoblasts.     

Expression of connexin 43 and ZO-1 in differentiating ameloblasts and odontoblasts from rat molar tooth germs

We studied the distribution of connexin (Cx) 43 and ZO-1 by confocal laser scanning microscopy at early stages of dentinogenesis and amelogenesis. Labeling for Cx43 was observed at early stages of differentiation in both the epithelial cells and differentiating odontoblasts. Immunolabeling was detected at the distal and medial regions of undifferentiated ameloblasts and between cells from stratum intermedium and stellate reticulum. Differentiating odontoblasts exhibited immunoreaction for this antibody at their distal end. Immunoreactivity for ZO-1 was observed at regions that correspond to the proximal and distal junctional complexes of differentiating ameloblasts. Staining for ZO-1 was observed at apical regions of odontoblasts with a punctate appearance. In more advanced stages, expression of Cx43 was more evident on ameloblasts, especially at the junctional complexes. Punctate immunolabeling for Cx43 was observed at the lateral sides of differentiating ameloblasts and between the other cells of the enamel organ. Immunoreaction for ZO-1 in ameloblasts was more evident than at the previous stage. It was also observed at the distal end of differentiated odontoblasts. The present study showed that differentiating ameloblasts and odontoblasts express Cx43 and ZO-1 as early as the start of the differentiation process. In addition, the expression of these junctional proteins increases as differentiation of cells continues.     

The localization of Fos B, a member of transcription factor AP-1 family, in rat odontoblasts and pulpal undifferentiated ectomesenchymal cells

It has been proposed that cellular proliferation and differentiation are accomplished by AP-1 components but different components can be responsible for different functions. The aim of this study was to compare the localization of Fos B, which is a component of AP-1, in postmitotic differentiated and undifferentiated cells via Fos B immunoreactivity. For this purpose, maxillary incisor teeth from 10 Wistar rats were obtained and Fos-B was investigated immunohistochemically in formalin-fixed, paraffin-embedded tooth sections containing odontoblasts, which are postmitotic differentiated cells, and pulpal undifferentiated ectomesenchymal cells. No significant differences in percentage of Fos B-positive cells were observed between the two cell types (p>0.05). These findings suggest that Fos B, a component of AP-1 family, seems to have a negligible effect on differentiation and proliferation in odontoblasts and pulpal undifferentiated ectomesenchymal cells.     

Caspase activation in the terminal differentiation of human epidermal keratinocytes

The epidermis is a multilayered squamous epithelium in which dividing basal cells withdraw from the cell cycle and progressively differentiate as they are displaced toward the skin surface. Eventually, the cells lose their nucleus and other organelles to become flattened squames, which are finally shed from the surface as bags of cross-linked keratin filaments enclosed in a cornified envelope [1]. Although keratinocytes can undergo apoptosis when stimulated by a variety of agents [2], it is not known whether their normal differentiation programme uses any components of the apoptotic biochemical machinery to produce the cornified cell. Differentiating keratinocytes have been reported to share some features with apoptotic cells, such as DNA fragmentation, but these features have not been seen consistently [3]. Apoptosis involves an intracellular proteolytic cascade, mainly mediated by members of the caspase family of cysteine proteases, which cleave one another and various key intracellular target proteins to kill the cell neatly and quickly [4]. Here, we show for the first time that caspases are activated during normal human keratinocyte differentiation and that this activation is apparently required for the normal loss of the nucleus.     

Inducing stem cell differentiation using low intensity laser irradiation: a possible novel therapeutic intervention

Developments in adult stem cell (ASC) potentiation have contributed to excitement in the field of stem cell-based therapy. The use of ASCs not only increases therapeutic treatment possibilities but successful use of multipotent cells for gene therapy has been demonstrated in animal models [1]. Concurrent ability of stem cells (SCs) to either contribute to disease development, as identified in cancer stem cells (CSCs), or to replace diseased tissue by induced differentiation using selected growth factors, has highlighted the intricate molecular and cellular mechanisms. Adipose derived stem cells (ADSCs) are capable of self-renewal and respond well to induced differentiation [2]. Auto-immunity and transplant rejection may become minor limitations when selective induction of immunological nonresponsiveness to specific antigens or tissues become possible using autologous cell sources [3]. CSCs initiate tumorogenesis, can generate differentiated daughter cells or undergo self-renewal while thought to instigate tumour regeneration post-treatment. Therapy targeting CSCs has failed to provide feasible alternatives to conventional cancer treatment. Low intensity laser irradiation (LILI), induce a biostimulatory response in several tissue types in addition to a dose-response effect to the detriment of cellular degeneration. Potential of LILI to induce CSC differentiation and subsequent cytotoxic therapy to prevent tumour regeneration is explored in this mini-review.     

Immortalized mouse dental papilla mesenchymal cells preserve odontoblastic phenotype and respond to bone morphogenetic protein 2

Odontogenesis is the result of the reciprocal interactions between epithelial–mesenchymal cells leading to terminally differentiated odontoblasts. This process from dental papilla mesenchymal cells to odontoblasts is regulated by a complex signaling pathway. When isolated from the developing tooth germs, odontoblasts quickly lose their potential to maintain the odontoblast-specific phenotype. Therefore, generation of an odontoblast-like cell line would be a good surrogate model for studying the dental mesenchymal cell differentiation into odontoblasts and the molecular events of dentin formation. In this study, immortalized dental papilla mesenchymal cell lines were generated from the first mouse mandibular molars at postnatal day 3 using pSV40. These transformed cells were characterized by RT-PCR, immunohistochemistry, Western blot, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iMDP-3, displayed a high proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers and demonstrated the ability to differentiate and form mineralized nodules. Furthermore, iMDP-3 cells had high transfection efficiency as well as were inducible and responded to BMP2 stimulation. We conclude that the establishment of the stable murine dental papilla mesenchymal cell line might be used for studying the mechanisms of dental cell differentiation and dentin formation.     

An Immune-Related Gene Evolved into the Master Sex-Determining Gene in Rainbow Trout, Oncorhynchus mykiss

Since the discovery of Sry in mammals [1, 2], few other master sex-determining genes have been identified in vertebrates [3-7]. To date, all of these genes have been characterized as well-known factors in the sex differentiation pathway, suggesting that the same subset of genes have been repeatedly and independently selected throughout evolution as master sex determinants [8, 9]. Here, we characterized in rainbow trout an unknown gene expressed only in the testis, with a predominant expression during testicular differentiation. This gene is a male-specific genomic sequence that is colocalized along with the sex-determining locus. This gene, named sdY for sexually dimorphic on the Y?chromosome, encodes a protein that displays similarity to the C-terminal domain of interferon regulatory factor 9. The targeted inactivation of sdY in males using zinc-finger nuclease induces ovarian differentiation, and the overexpression of sdY in females using additive transgenesis induces testicular differentiation. Together, these results demonstrate that sdY is a novel vertebrate master sex-determining gene not related to any known sex-differentiating gene. These findings highlight an unexpected evolutionary plasticity in vertebrate sex determination through the demonstration that master sex determinants can arise from the de novo evolution of genes that have not been previously implicated in sex differentiation.     

The effect of epidermal growth factor on neonatal incisor differentiation in the mouse

The effect of epidermal growth factor (EGF) on cellular differentiation of the neonatal mouse mandibular incisor was examined autoradiographically using tritiated thymidine ([3H]TDR) and tritiated proline ([3H]PRO). On days 0 (day of birth), 1, and 2, EGF was administered (3 micrograms/g body wt) sc to neonates. Mice were killed on Days 1, 4, 7, 10, and 13 after birth and were injected with either [3H]TDR or [3H]PRO 1 hr before death. [3H]TDR was used to analyze cell proliferation in eight cell types in the developing mouse incisor including upper (lingual) and lower (buccal) pulpal fibroblasts, preodontoblasts, inner and outer enamel epithelial cells (IEE and OEE), stratum intermedium (SI), stellate reticulum (SR), and periodontal ligament (PDL) fibroblasts. [3H]PRO was used to analyze protein synthesis in ameloblasts, and their secretion products (enamel and dentin), as well as PDL fibroblasts. The selected EGF injection scheme elicited acceleration of incisor eruption with minimal growth retardation. At Day 1, the upper and lower pulp, preodontoblasts, SI, and SR showed a significant decrease in labeling index (LI) 24 hr after a single EGF injection. After multiple injections (Days 0, 1, 2), two LI patterns were observed. In lower pulp, preodontoblasts, IEE, SI, SR, and OEE, a posteruptive change in LI was observed. In contrast, the upper pulp and PDL regions demonstrated a direct temporal relationship with eruption. Autoradiographic analysis with [3H]PRO indicated that EGF treatment caused significant increases in grain counts per unit area in ameloblast, odontoblast, and PDL regions studied. Significant differences were found in all four regions studied (ameloblasts, enamel, odontoblasts, dentin) at the 45-microns-tall ameloblast level as well as ameloblasts and odontoblasts at the 30-microns level at 13 days of age. The PDL demonstrated significant differences at all locations studied (base, 30 microns, 45 microns,) in 4-, 7-, and 13-day-old mice. Morphologically, EGF-treated groups demonstrated premature differentiation of ameloblasts and odontoblasts at the light microscopic level. The data indicate that EGF alters DNA and protein synthesis as well as differentiation patterns during the eruption process. While EGF affects both DNA and protein synthesis, the alteration of differentiation may be secondary to mitogenic effects on proliferative compartments. In order to determine the cellular target for EGF within the newborn mouse incisor, in vivo 125I-EGF binding was analyzed autoradiographically.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ancient DNA analysis reveals divergence of the cave bear, Ursus spelaeus, and brown bear, Ursus arctos, lineages

The cave bear, Ursus spelaeus, represents one of the most frequently found paleontological remains from the Pleistocene in Europe. The species has always been confined to Europe and was contemporary with the brown bear, Ursus arctos. Relationships between the cave bear and the two lineages of brown bears defined in Europe, as well as the origins of the two species, remain controversial, mainly due to the wide morphological diversity of the fossil remains, which makes interpretation difficult [1, 2]. Sequence analysis of ancient DNA is a useful tool for resolving such problems because it provides an independent source of data [3]. We previously amplified a short DNA fragment of the mitochondrial DNA control region (mt control region) of a 40,000-year-old Ursus spelaeus sample [4]. In this paper, we describe the DNA analysis of two mtDNA regions, the control region and the cytochrome b gene. Control region sequences were obtained from ten samples of cave bears ranging from 130,000 to 20,000 years BP, and one particularly well-conserved sample gave a complete cyt b sequence. Our data demonstrate that cave bears split largely before the lineages of brown bears around 1.2 million years ago. Given its abundance, its wide distribution in space and time, and its large morphological diversity, the cave bear is a promising model for direct observation of the evolution of sequences throughout time, extinction periods, and the differentiation of populations shaped by climatic fluctuations during the Pleistocene.     

Cell-matrix interactions and odontoblast differentiation]

The terminal differentiation of odontoblasts requires the integrity of the cytoskeleton and is controlled by cell-matrix interactions. These interactions implicate both matrix molecules and matrix-associated growth factors. On the one hand, predentin-dentin constituents were found to initiate odontoblast differentiation and to allow the maintenance of this state; TGF-beta or related molecules are implicated. Fibronectin on the other hand can induce the differentiation of second generation odontoblasts and interacts with three high molecular weight proteins present in membrane prepared from dental mesenchymal cells. One of these proteins (165 kDa) was localized on the surface of odontoblasts and is involved in the organization of microfilaments. Two main axes of research will have to be developed in the future in order to understand how matrix molecules and growth factors interactions can be modulated in time and space by epithelial and mesenchymal cells, and how such modulations can affect the phenotype of these cells.     

Caspase‐7 participates in differentiation of cells forming dental hard tissues

Apoptosis during tooth development appears dependent on the apoptotic executioner caspase‐3, but not caspase‐7. Instead, activated caspase‐7 has been found in differentiated odontoblasts and ameloblasts, where it does not correlate with apoptosis. To further investigate these findings, the mouse incisor was used as a model. Analysis of caspase‐7‐deficient mice revealed a significant thinner layer of hard tissue in the adult incisor. Micro computed tomography scan confirmed this decrease in mineralized tissues. These data strongly suggest that caspase‐7 might be directly involved in functional cell differentiation and regulation of the mineralization of dental matrices.     

Comparative analysis of genetic variability of white sea cod (Gadus morhua marisalbi) at allozyme and microsatellite markers

Variability of cod spawning and feeding schools from Kandalaksha Bay of the White Sea, was examined at six allozyme and eight microsatellite loci. The degree of genetic differentiation at allozyme loci constituted θ = 0.36% [95% bootstrap interval 0.0458; 0.6743]. The differentiation estimates obtained using microsatellite markers were higher, θ = 1.33% [0.057; 3.11]. It was demonstrated that the level of genetic diversity in the White Sea cod was lower than that established for the Atlantic cod from Barents Sea using the same set of allozyme and microsatellite markers. The genetic data obtained support the opinion that the White Sea cod is a reproductively independent group formed as a result of the Holocene dispersal of Atlantic cod.