Expression of Fas, FasL, caspase-8 and other factors of the extrinsic apoptotic pathway during the onset of interdigital tissue elimination.

Elimination of the interdigital web is considered to be the classical model for assessing apoptosis. So far, most of the molecules described in the process have been connected to the intrinsic (mitochondrial) pathway. The extrinsic (receptor mediated) apoptotic pathway has been rather neglected, although it is important in development, immunomodulation and cancer therapy. This work aimed to investigate factors of the extrinsic apoptotic machinery during interdigital regression with a focus on three crucial initiators: Fas, Fas ligand and caspase-8. Immunofluorescent analysis of mouse forelimb histological sections revealed abundant expression of these molecules prior to digit separation. Subsequent PCR Array analyses indicated the expression of several markers engaged in the extrinsic pathway. Between embryonic days 11 and 13, statistically significant increases in the expression of Fas and caspase-8 were observed, along with other molecules involved in the extrinsic apoptotic pathway such as Dapk1, Traf3, Tnsf12, Tnfrsf1A and Ripk1. These results demonstrate for the first time the presence of extrinsic apoptotic components in mouse limb development and indicate novel candidates in the molecular network accompanying the regression of interdigital tissue during digitalisation.

Concomitant multipotent and unipotent dental pulp progenitors and their respective contribution to mineralised tissue formation.

Upon in vitro induction or in vivo implantation, the stem cells of the dental pulp display hallmarks of odontoblastic, osteogenic, adipogenic or neuronal cells. However, whether these phenotypes result from genuine multipotent cells or from coexistence of distinct progenitors is still an open question. Furthermore, determining whether a single cell-derived progenitor is capable of undergoing a differentiation cascade leading to tissue repair in situ is important for the development of cell therapy strategies. Three clonal pulp precursor cell lines (A4, C5, H8), established from embryonic ED18 first molars of mouse transgenic for a recombinant plasmid adeno-SV40, were induced to differentiate towards the odonto/osteogenic, chondrogenic or adipogenic programme. Expression of phenotypic markers of each lineage was evaluated by RT-PCR, histochemistry or immunocytochemistry. The clones were implanted into mandibular incisors or calvaria of adult mice. The A4 clone was capable of being recruited towards at least 3 mesodermal lineages in vitro and of contributing to dentin-like or bone formation, in vivo, thus behaving as a multipotent cell. In contrast, the C5 and H8 clones displayed a more restricted potential. Flow cytometric analysis revealed that isolated monopotent and multipotent clones could be distinguished by a differential expression of CD90. Altogether, isolation of these clonal lines allowed demonstrating the coexistence of multipotential and restricted-lineage progenitors in the mouse pulp. These cells may further permit unravelling specificities of the different types of pulp progenitors, hence facilitating the development of cell-based therapies of the dental pulp or other cranio-facial tissues.

Pulpal progenitors and dentin repair.

Mesenchymal stem cells are present in the dental pulp. They have been shown to contribute to dentin-like tissue formation in vitro and to participate in bone repair after a mandibular lesion. However, their capacity to contribute efficiently to reparative dentin formation after pulp lesion has never been explored. After pulp exposure, we have identified proliferative cells within 3 zones. In the crown, zone I is near the cavity, and zone II corresponds to the isthmus between the mesial and central pulp. In the root, zone III, near the apex, at a distance from the inflammatory site, contains mitotic stromal cells which may represent a source of progenitor cells. Stem-cell-based strategies are promising treatments for tissue injury in dentistry. Our experiments focused on (1) location of stem cells induced to leave their quiescent state early after pulp injury and (2) implantation of pulp progenitors, a substitute for classic endodontic treatments, paving the way for pulp stem-cell-based therapies.

Controlled conversion of an immortalized mesodermal progenitor cell towards osteogenic, chondrogenic, or adipogenic pathways.

The teratocarcinoma-derived C1 clone behaves as a mesodermal tripotential progenitor cell whose choice of fate, either osteoblast, chondroblast, or adipoblast, is strictly dependent on the spatial organization of the cells and the nature of the induction. In the absence of cell contact before the addition of inducers, the C1 cells maintain a stable undifferentiated phenotype while expressing potential regulators of embryonic mesodermal stem cell fate such a M-twist and Id1. Upon establishment of cell contacts before the induction of differentiation, the early genes characteristic of the three fates become expressed. In the presence of beta glycerophosphate and ascorbate, provided the cells have formed aggregates, 95% of the C1 cells mineralize with a kinetics of gene expression close to that of osteoblasts (Poliard, A., D. Lamblin, P. J. Marie, M. H. Buc, and O. Kellerman. 1993. J. Cell Sci. 106:503-512). With 10(-6)M dexamethasone, 80% of the same aggregates differentiate into foci of chondroblast-like cells. The kinetics of expression of the genes encoding type II, IX, X, and XI collagens, aggrecan and link protein during the conversion toward cartilage hypertrophy resembles that accompanying in vivo chondrogenesis. The synergistic action of dexamethasone and insulin convert most confluent C1 cells into functional adipocytes and induce a pattern of gene expression close to that reported for adipoblast cell lines. The C1 clone with its capacity to differentiate along three alternative pathways with high frequency, therefore appears as a valid in vitro model for deciphering the molecular basis of mesoblast ontogeny.

Cellular analysis by in situ hybridization and immunoperoxidase of alpha-fetoprotein and albumin gene expression in rat liver during the perinatal period.

To analyze at the cellular level the decrease in alpha-fetoprotein (AFP) gene expression during the early postnatal growth, we searched for AFP gene transcripts by in situ hybridization using a specific cDNA probe, and for the corresponding protein by immunocytochemistry, on rat liver sections at various times of the perinatal period. The relative number of mRNA sequences was evaluated by Northern blot analysis. Albumin (ALB) gene expression was studied simultaneously with the same techniques. In 17-19-d-old fetuses all hepatocytes express simultaneously, for both genes, the mRNAs and the corresponding proteins. During the first postnatal weeks, at a time when the global number of AFP mRNA molecules decreases, all hepatocytes still contain cytoplasmic transcripts and protein. A zonal heterogeneity in the level of AFP gene expression develops around the first week, a higher number of gene products being detected in perivenous than in periportal hepatocytes. This heterogeneity persists until the fourth week when AFP mRNA sequences and protein are barely detectable. All hepatocytes express the ALB gene after birth, but at around the second week, a periportal intensification of the in situ hybridization signal and immunostaining becomes apparent. Our data indicate that co-expression of the AFP and ALB genes by all hepatocytes is a normal step in liver ontogeny; the diminution of AFP gene expression after birth is not the result of the disappearance of specialized cell clones; and zonal quantitative differences in the level of AFP and ALB gene expression are observed within the maturing liver lobule.

Coupling activation of pro-apoptotic caspases with autophagy in the Meckel´s cartilage.

Mammalian Meckel´s cartilage is a temporary structure associated with mandible development. Notably, its elimination is not executed by apoptosis, and autophagy was suggested as the major mechanism. Simultaneous reports point to pro-apoptotic caspases as novel participants in autophagic pathways in general. The aim of this research was to find out whether activation of pro-apoptotic caspases (-2, -3, -6, -7, -8 and -9) was associated with autophagy of the Meckel´s cartilage chondrocytes. Active caspases were examined in serial histological sections of mouse mandible using immunodetection and were correlated with incidence of autophagy based on Beclin-1 expression. Caspase-2 and caspase-8 were found in Beclin-1 positive regions, whereas caspase-3, -6, -7 and -9 were not present. Caspase-8 was further correlated with Fas/FasL and HIF-1alpha, potential triggers for its activation. Some Fas and FasL positivity was observed in the chondrocytes but caspase-8 activation was found also in FasL deficient cartilage. HIF-1alpha was abundantly present in the hypertrophic chondrocytes. Taken together, caspase-8 activation in the Meckel´s cartilage was demonstrated for the first time. Caspase-8 and caspase-2 were the only pro-apoptotic caspases detected in the Beclin-1 positive segment of the cartilage. Activation of caspase-8 appears FasL/Fas independent but may be switched on by HIF-1alpha.

Stepwise control of osteogenic differentiation by 5-HT(2B) receptor signaling: nitric oxide production and phospholipase A2 activation.

During development, antagonists of 5-HT(2) receptor subtypes cause morphological defects of mesodermal and neural crest derivatives including the craniofacial skeleton. We used an inducible mesoblastic cell line, C1, able to fully convert into osteocytes within 12 days, to assess the involvement of 5-HT(2) receptors during osteogenic differentiation. On day 5 of the osteogenic program, immediately before matrix mineralization, the cells selectively implement 5-HT(2B) receptors (5-HT(2B)R) which remain functional until terminal differentiation. In 5-HT-depleted medium, the receptor exhibits a constitutive activity leading to basal nitric oxide (NO) release and phospholipase A2 (PLA2)-dependent arachidonic acid (AA) production. Blockade of this intrinsic activity affects the efficiency of mineralization by decreasing calcium incorporation within the matrix by 40%. Optimal bone matrix mineralization involves both NO and PLA2 signaling pathways. Moreover, between day 5 and day 10, at the beginning of mineral deposition, the 5-HT(2B)R promotes prostaglandin E2 production through AA-dependent cyclooxygenase (COX) activation. From day 10 onwards, when C1 osteoblasts undergo conversion into osteocyte-like cells, COX activity is quenched. Altogether these observations indicate that the 5-HT(2B)R contributes in an autocrine manner to osteogenic differentiation and highlight a switch in the downstream targets of the receptor at the terminal stage of the program. Finally, in addition to its autocrine function, the 5-HT(2B)R responds to 5-HT by increasing NO production and AA release. These findings raise concern regarding the use of 5-HT(2B)R-related drugs that may interfere with bone metabolism in physiological or pathological situations.

Overexpression of murine small heat shock protein HSP25 interferes with chondrocyte differentiation and decreases cell adhesion.

Although multiple functions for the small heat shock protein HSP25 have been proposed, its specific role during developmental and differentiation processes is not known. Cartilage is one of the tissues in which HSP25 is specifically and highly expressed during development. C1 cells, able to form aggregates in vitro, can be induced to differentiate into chondrocytes. In this study, we generated two stable transfected clones overexpressing HSP25 at two different levels. Cell morphology and growth rate were modified in both clones, although the actin content and distribution did not seem to be altered. Overexpressing clones had more difficulties in coalescing, leading to smaller aggregates and they did not differentiate into chondrocytes. Subsequently, these aggregates tended to dissociate into loose masses of dying cells. The strength of all these effects was directly correlated to the level of HSP25 overexpression. These data suggest that overexpressing HSP25 decreases cellular adhesion and interferes with chondrocyte differentiation.

New cellular models for tracking the odontoblast phenotype.

Odontoblasts and osteoblasts differ functionally and histologically. Because of their close relationship, mesenchymal cells derived from teeth and bone are difficult to distinguish ex vivo. Indeed, the main non-collagenous components of the odontoblastic extracellular matrix, dentin sialoprotein (DSP) or dentin matrix protein 1 (DMP1), have also been detected in osteoblasts. The need to develop cellular models of odontoblast differentiation and to identify markers specific for the odontoblast lineage, has led us to establish clonal cell lines from tooth germs of day 18 mouse embryos transgenic for an adenovirus-SV40 recombinant plasmid. In this study, we analyzed the phenotypes of three independent clones by RT-PCR and Western blot. These clones synthesised DSP, DMP1 and other extracellular matrix proteins typical of the odontoblast and are therefore likely to be derived from the pulp. Transcripts encoding a set of homeobox proteins involved in craniofacial development, such as Pax9, Msx1, Cbfa1, Dlx2 and 5 were also expressed albeit at a different level. These features of the pulpal clones are shared by the C1 mesodermal cells that are capable of differentiating along osteogenic, chondrogenic or adipogenic lineages In contrast, transcripts for two LIM-domain homeobox family genes (Lhx6 and Lhx7) were only detected in the dental clones. Since these genes are preferentially expressed in the mesenchyme of the developing tooth, this suggests that our transgenic-derived cell lines retain intrinsic properties of odontoblastic cells. They may help to characterise genes specifying the odontoblast phenotype and the signalling pathways underlying odontoblast differentiation.

Detection of albumin mRNAs in rat liver by in situ hybridization: usefulness of paraffin embedding and comparison of various fixation procedures.

Our aim was to define optimal conditions for efficient and reproducible albumin mRNA detection in rat liver by in situ hybridization. We used an albumin-specific [3H]-labeled cDNA probe with a specific activity of 6-8.10(6) cpm/microgram DNA. In situ hybridization is as efficient on paraffin sections as on cryostat sections for detecting albumin mRNAs. Perfusion fixation with a 4% paraformaldehyde solution results in homogeneous RNA retention within tissue blocks, in contrast with immersion fixation, which yields heterogeneous RNA preservation. Comparison of immersion fixation with three different fixatives (paraformaldehyde, ethanol-acetic acid, and Bouin's fixative) shows that the highest level of hybridization signal is obtained with paraformaldehyde. Ethanol-acetic acid and Bouin's fixative appear less efficient for albumin mRNA detection. Loss of mRNAs within liver tissue blocks over time is largely although not completely prevented by paraffin embedding.

Nucleoside uptake in normal and cystic fibrosis fibroblasts in vitro.

Cultured skin fibroblasts derived from unrelated patients with diagnosed cystic fibrosis were compared with those from one normal individual in respect to thymidine and uridine uptake. The results obtained show that the two nucleosides are incorporated at the same rate by the three cell strains. Therefore, it appears that the reported abnormality [6] in nucleoside uptake in cystic fibrosis cells is not a general characteristic of these cells.

Minimal intervention dentistry: part 8. Biotherapies for the dental pulp.

The development of new therapies, the biotherapies, can now be envisioned in dentistry thanks to the important development of research in the stem cell and biomaterial fields. Indeed, the recent discovery of stem cells in different tissues of the body, and in particular in the dental pulp, should allow the development of new therapeutic approaches for pulpo-dentinal lesions. In case of moderate pulp injury, the stimulation by biomolecules of pulp stem cells located in the remaining pulp is the approach to be considered. In case of more severe lesions requiring a total removal of pulp tissues, a cell therapy could be considered. The objective of this article is to present these innovative therapeutic approaches for the future of dentistry.

ES cells-derived ectomesenchymal cells for tooth engineering.

Recent progresses in stem cell biology and tissue engineering allow considering the possible development of new therapies for compensating the dental tissue losses associated with traumas, pathologies or ageing. The possibility of generating a tooth by mimicking development through reassociations between dental epithelial cells and ectomesenchymal cells derived from the neural crest (NC) has been demonstrated in the mouse. In the search of cell sources to be used for a human transfer, pluripotent stem cells could represent a good alternative. Our study thus focuses on obtaining, ectomesenchymal cells from pluripotent ES cells, capable of promoting tooth histomorphogenesis, when reassociated with a competent dental epithelium. To this end, two ES differentiation protocols, using cyclopamine or a combination of FGF2 and BMP4, have been developed and tested for their capacity to generate such cells. The differentiated ES cells were characterized by quantitative RT-PCR. Both protocols led the cells to acquire in 10 days a mesenchymal-like cell morphology. Rapidly after induction, the cells loose their expression of pluripotent genes while sequentially activating typical NC specifiers. However, the kinetics of gene activation differed between the 2 protocols. Interestingly, Twist, a gene whose expression in the NC is associated with a commitment towards an ectomesenchymal fate, is only activated under the influence of FGF2 and BMP4. Reassociation experiments with a competent epithelium will allow testing the odontogenic potential of the differentiated ES cells. These experiments performed in the mouse system should allow defining a strategy for obtaining odontogenic competent human cells.

Matricellular molecules and odontoblast progenitors as tools for dentin repair and regeneration.

This review summarizes the in vivo experiments carried out by our group after implantation of bioactive molecules (matricellular molecules) into the exposed pulp of the first maxillary molar of the rat or the mandibular incisor of rats and mice. We describe the cascade of recruitment, proliferation and terminal differentiation of cells involved in the formation of reparative dentin. Cloned immortalized odontoblast progenitors were also implanted in the incisors and in vitro studies aimed at revealing the signaling pathways leading from undifferentiated progenitors to fully differentiated polarized cells. Together, these experimental approaches pave the way for controlled dentin regenerative processes and repair.

Noxp20 and Noxp70, two new markers of early neuronal differentiation, detected in teratocarcinoma-derived neuroectodermic precursor cells.

The murine 1C11 cell line, derived from F9 pluripotent teratocarcinoma cells, exhibits features of a bipotential neuronal precursor as it converts into serotonergic or catecholaminergic neurons under appropriate induction. In order to point out molecular markers expressed in this early neuroectodermic commitment, we used a cDNA subtractive hybridization method. The 105 different isolated cDNAs represented 75 known genes, expressed sequence tags (EST) or genomic fragments. A majority of known proteins encoded by these sequences are involved in cellular mobility or migration. We characterized two sequences showing identities with ESTs and we called them Noxp20 and Noxp70. The Noxp20 transcript encodes a putative protein with a predicted caspase recruitment domain and the Noxp70 transcript encodes a putative protein displaying a Zn-finger domain. Consistent with their roles in neuronal cell development, in situ hybridization showed that Noxp20 and Noxp70 are over-expressed in brain. At embryonic days 12 and 15, Noxp20 is strongly expressed in the ventricular and intermediate zones of the brain and of the spinal cord. At embryonic day 15, Noxp70 was found to be strongly expressed in the ventricular zone around the telencephalic ventricle, and to a lower extent in the thalamus and hypothalamus. At post-natal day 10, Noxp20 mRNA was detected in the dentate gyrus, the hippocampus, the cerebellum and the olfactory bulb.

Alpha fetoprotein and albumin gene transcripts are detected in distinct cell populations of the brain and kidney of the developing rat.

We report the cellular localization of alpha-fetoprotein (AFP) and albumin (ALB) gene transcripts in rat kidney and brain as detected by in situ hybridization on tissue sections with [35S]-labelled alpha-fetoprotein and albumin cDNA probes. Both types of mRNA were present in distinct cell populations of the developing kidney and brain. In the kidney, both gene transcripts were distributed over all developing tubular cells in the 20-day-old fetus. During the first 3 weeks of life, a gradual decrease in the expression of AFP and ALB mRNA was apparent, the rate of decrease being greater on proximal tubules than on the other tubular cells. From the 4th week onwards, a weak signal for both mRNAs persisted in the majority of the tubular cells. In the brain, all neuronal cells expressed both genes. Transcript cellular distribution was mainly cytoplasmic during fetal and early postnatal life and became predominantly nuclear at 3, 4 and 5 weeks, suggesting that posttranscriptional mechanisms are involved in the control of AFP and ALB gene expression at these stages. In the adult brain no significant signal was recorded thereafter. Coexpression of AFP and ALB transcripts by specific cell types, together with their gradual disappearance concomitant with postnatal organ maturation, suggests a possible role for these proteins in terminal differentiation processes of tubular and neuronal cells.

In situ cellular analysis of alpha-fetoprotein gene expression in regenerating rat liver after partial hepatectomy.

Cellular analysis of hepatic alpha-fetoprotein gene expression in normal adult rat and during regeneration induced by partial hepatectomy was performed at the cellular level by in situ hybridization using 35S-labeled complementary DNA probes and immunoperoxidase techniques. In normal adult rat liver sections, a few alpha-fetoprotein mRNA-cDNA hybrids are detected over all hepatocytes. No protein is detected with routine immunoperoxidase methods. However, after in vivo colchicine blockade of alpha-fetoprotein secretion, 10 to 20% alpha-fetoprotein-positive hepatocytes are observed. In regenerating livers, at 2,6 and 24 hr (before and at the time of the peak of DNA synthesis in the periportal zones), a rise of the nuclear signal level is observed selectively in periportal hepatocytes, without modification of the cytoplasmic signal. At 48 hr (when most hepatocytes have completed at least one replicative cycle), almost all hepatocytes throughout the liver lobule display a rise of the nuclear (2- to 3-fold) and cytoplasmic (1.5- to 2-fold) signal level compared to nonoperated rats. These data show that all hepatocytes in the adult liver express a small number of alpha-fetoprotein mRNA sequences; they appear to be translated in protein whose secretion can be blocked by colchicine. The moderate increase in alpha-fetoprotein gene expression induced by liver regeneration takes place in all hepatocytes, in apparently two distinct steps: a very early nuclear accumulation of alpha-fetoprotein mRNA sequences and a late cytoplasmic accumulation of alpha-fetoprotein mRNA molecules.

Coexistence of expressed and non-expressed alpha-fetoprotein genes in somatic cell hybrids.

Hybrids have been generated between mouse hepatoma cells, which actively synthesize alpha-fetoprotein (AFP), and adult hepatocytes, where AFP production is shut off. These hybrids maintain an active synthesis of mouse AFP. Using a specific radioimmunoassay, we found that rat AFP production is not activated. Southern blot analysis showed that mouse and rat AFP DNA sequences can be distinguished and that hybrid clones possessing something close to the complete chromosome sets of both parents have retained both parental AFP DNA sequences. Thus expressed and non-expressed AFP genes coexist in these hybrid cells as if their expression were dependent on a cis-acting event.

Commitment of the teratocarcinoma-derived mesodermal clone C1 towards terminal osteogenic differentiation.

The mesodermal clone C1 was derived from the multipotent embryonal carcinoma 1003 cell line transformed with the plasmid pK4 carrying SV40 oncogenes under the control of the adenovirus E1A promoter. We have shown that the C1 clone becomes committed to the osteogenic pathway when cultured in aggregates in the presence of mediators of the osteogenic differentiation. To further validate C1 as a model with which to study osteogenesis in vitro the kinetics of its differentiation was studied, focusing on the histology of the aggregates and on the expression of a set of genes corresponding to representative bone matrix proteins. The presence of ascorbic acid and beta- glycerophosphate specifically leads to mineralization in almost 100% of the aggregates. Transcription of the above genes, silent in exponentially growing cells, specifically occurred with the establishment of cell-cell contacts independently of the presence of ascorbic acid and inorganic phosphate. The latter, however, were absolutely required for matrix deposition and mineralization. In their presence, one observed an overall decline in type I collagen and alkaline phosphatase transcripts while osteocalcin and osteopontin transcripts preferentially accumulated in cells lining the mineralizing foci. Concomitantly, type I collagen and osteocalcin became extracellularly deposited. The osteogenic differentiation of C1 occurred while cells were still proliferating. The C1 clone thus behaves as a mesodermal stem cell, becoming committed to the osteogenic pathway upon: firstly, establishment of cellular contacts; and secondly, addition of ascorbate and beta-glycerophosphate. It therefore appears to be a promising in vitro system for deciphering the molecular basis of osteoblast ontogeny.(ABSTRACT TRUNCATED AT 250 WORDS)