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R N D'Souza

Publications and source records attributed to R N D'Souza.

At least 19 recordsLinked to original sources
PubMed2003

Tooth eruption and cementum formation in the Runx2/Cbfa1 heterozygous mouse.

Cleidocranial dysplasia (CCD) is an autosomal dominant human disorder that affects development of bones and teeth. The dental disorders in CCD patients include formation of supernumerary teeth, delayed tooth eruption, and lack of formation of cellular cementum in permanent teeth. This disorder involves a mutation in the osteoblast-specific transcription factor Runx2/Cbfa1, leading to haploinsufficiency of the Runx2/Cbfa1 protein. Here, we examined if Runx2/Cbfa1 heterozygous mice (with one functional allele for Runx2/Cbfa1) exhibit similar changes in tooth eruption, and dental cementum formation as in CCD patients. Heads of Runx2/Cbfa1 heterogeneous and wildtype mice aged days 16-35 postnatally were serially sectioned and stained with hematoxylin-eosin or for tartrate resistant acid phosphatase (TRAP) to identify osteoclasts. The results showed that the eruption pattern of the first and second molars in maxilla and mandible in Runx2/Cbfa1 +/- mice was the same as in wildtype animals. No clear difference in distribution or in the (estimated) number of osteoclasts was found. Cellular cement at the apical portions of the molar roots was present in both groups. The data suggests that in the mouse one allele for Runx2/Cbfa1 is sufficient for an undisturbed tooth eruption and an apparently normal formation of the periodontium.

Animals↗
PubMed2002

Cellular, molecular, and genetic determinants of tooth eruption.

Tooth eruption is a complex and tightly regulated process that involves cells of the tooth organ and the surrounding alveolus. Mononuclear cells (osteoclast precursors) must be recruited into the dental follicle prior to the onset of eruption. These cells, in turn, fuse to form osteoclasts that resorb alveolar bone, forming an eruption pathway for the tooth to exit its bony crypt. Some of the molecules possibly involved in the signaling cascades of eruption have been proposed in studies from null mice, osteopetrotic rodents, injections of putative eruption molecules, and cultured dental follicle cells. In particular, recruitment of the mononuclear cells to the follicle may require colony-stimulating factor-one (CSF-1) and/or monocyte chemotactic protein-1 (MCP-1). Osteoclastogenesis is needed for the bone resorption and may involve inhibition of osteoprotegerin transcription and synthesis in the follicle, as well as enhancement of receptor activator of NF kappa B ligand (RANKL), in the adjacent alveolar bone and/or in the follicle. Paracrine signaling by parathyroid-hormone-related protein and interleukin -1 alpha, produced in the stellate reticulum adjacent to the follicle, may also play a role in regulating eruption. Osteoblasts might also influence the process of eruption, the most important physiologic role likely being at the eruptive site, in the formation of osteoclasts through signaling via the RANKL/OPG pathway. Evidence thus far supports a role for an osteoblast-specific transcription factor, Cbfa1 (Runx2), in molecular events that regulate tooth eruption. Cbfa1 is also expressed at high levels by the dental follicle cells. This review concludes with a discussion of the several human conditions that result in a failure of or delay in tooth eruption.

Animals↗
PubMed2002

A novel mutation in human PAX9 causes molar oligodontia.

Experimental and animal studies, as well as genetic mutations in man, have indicated that the development of dentition is under the control of several genes. So far, mutations in MSX1 and PAX9 have been associated with dominantly inherited forms of human tooth agenesis that mainly involve posterior teeth. We identified a large kindred with several individuals affected with molar oligodontia that was transmitted as an isolated autosomal-dominant trait. Two-point linkage analysis using DNA from the family and polymorphic marker D14S288 in chromosome 14q12 produced a maximum lod score of 2.29 at theta = 0.1. Direct sequencing of exons 2 to 4 of PAX9 revealed a cytosine insertion mutation at nucleotide 793, leading to a premature termination of translation at aa 315. Our results support the conclusion that molar oligodontia is due to allelic heterogeneity in PAX9, and these data further corroborate the role of PAX9 as an important regulator of molar development.

Adult↗
PubMed2001

Identification of tooth-specific downstream targets of Runx2.

Odontogenesis or tooth development is a highly regulated process that involves complex epithelial-mesenchymal signaling interactions that lead to cuspal morphogenesis, cell differentiation and the subsequent formation of the specialized matrices of enamel, dentin, cementum and bone. Although studies on tooth epithelial-mesenchymal signaling interactions have greatly increased our understanding of molecules that regulate tooth initiation and early morphogenesis (review: Jernvall and Thesleff, Mech. Dev. 92 (2000) 19), the precise nature of the molecular events controlling late morphogenesis and terminal cytodifferentiation is not known. We have recently reported a unique phenotype involving dentition in mice lacking a functional Runx2 gene (D'Souza et al., Development 126 (1999) 2911). The markedly hypoplastic tooth organs as well as defects in the maturation of ameloblasts and odontoblasts point to an important and non-redundant role for Runx2 in tooth morphogenesis and cytodifferentiation. In order to identify genes that are affected by the absence of Runx2, a cDNA library was generated from Runx2(-/-) and Runx2(+/+) first molar organs. Thus far, our analysis has revealed several tooth-specific downstream target genes of Runx2 that include extracellular matrix proteins, kinases, receptors, growth factors, mitochondrial proteins and transcription molecules. Sequence analysis of 61 differentially expressed genes revealed that 96.03% of the clones matched previously described genes in the GenBank/EBML database and 3.96% did not match any entries in the database. Our preliminary expression analysis of one of the differentially expressed clones which encodes for a zinc finger transcription factor termed Zfp reveals that the gene is temporally regulated during tooth development. In conclusion, we have successfully generated a library enriched in genes expressed in Runx2(+/+) molar tooth organs and performed preliminary studies to assess the role of Zfp in tooth development.

Animals↗
PubMed2001

Cell-specific patterns of Cbfa1 mRNA and protein expression in postnatal murine dental tissues.

Cbfa1 (core binding factor alpha 1) is a transcription factor that is a key determinant of the osteoblastic lineage. Recent data showed that Cbfa1 is also highly expressed in early stages of tooth development and is involved in crown morphogenesis and cytodifferentiation of odontoblasts. Here we report the mRNA expression and protein localization of Cbfa1 in the mouse dentition in (later) stages of crown and root development. In addition to osteoblasts, osteocytes, chondrocytes, odontoblasts, dental follicle cells, cementoblasts and periodontal ligament cells, we report also Cbfa1 expression in dental epithelial cells (secretory and maturation ameloblasts) and several non-mineralizing cell types (hair follicles, ducts of salivary glands, and junctional epithelium of the gingiva).

Age Factors↗
PubMed2001

Gene expression in a pure population of odontoblasts isolated by laser-capture microdissection.

Studies of odontoblast differentiation and function have been limited due to difficulties in obtaining sufficient numbers of intact cells. We describe a novel approach of laser-capture microdissection to obtain homogenous populations of pre-odontoblasts and odontoblasts from tissue sections of mouse molar cusp tips. Fixation, processing, and staining conditions were assessed for the optimal retrieval of total RNA from microdissected odontoblasts. Fluorometric assays and RT-PCR analysis of alpha1(I) collagen, dentin sialophosphoprotein (Dspp), and osteocalcin (OC) confirmed that the total RNA from three-day-old captured odontoblasts was sufficient in quantity and quality. Odontoblast-specific gene expression was studied by RT-PCR analysis performed in a single streptavidin-coated tube. At E15.5, Days 0 and 3, gene expression in laser-captured odontoblasts resembled that seen in vivo by in situ hybridization. The use of LCM is thus a valuable means of retrieving quality RNA from discrete populations of odontoblasts at different stages of dentinogenesis.

Animals↗
PubMed2001

Molecular insights into the lineage-specific determination of odontoblasts: the role of Cbfa1.

The role of stable transcription complexes in initiating and consolidating programs of gene expression during lineage specification has been extensively studied. Despite the progress made in the identification of key molecules of tooth initiation and patterning, the mechanisms leading to cell differentiation during odontogenesis are unknown. Odontoblasts are exclusive dentin-producing cells that are phenotypically and functionally distinct from osteoblasts. However, not much is known about the precise determinants of odontoblast terminal differentiation--in particular, how the fate of these cells becomes delineated from that of osteogenic mesenchyme. Cbfa1(-/-) mice completely lack osteoblasts and bone, while tooth development arrests at the time of odontoblast differentiation. The purpose of this paper is to overview our studies on the role of Cbfa1 in odontoblast determination and differentiation using the Cbfa1(-/-) mouse model and various experimental approaches. Our expression analyses confirm the down-regulation of Cbfa1 expression in newly differentiated and functional odontoblasts. Second, we demonstrate that Cbfa1(-/-) incisor organs arrest at a later stage than molars, and that alpha 1 (I) collagen, a marker of odontoblast differentiation shared in common with osteoblasts, is not significantly affected by the absence of the transcription factor. Interestingly, Dspp expression in Cbfa1(-/-) appeared markedly down-regulated in putative odontoblasts. The overexpression of Cbfa1 in an odontoblast cell line (MDPC-23) results in the selective down-regulation of Dspp and not type I collagen. It is likely that, in addition to its influence on tooth epithelial morphogenesis, Cbfa1 plays a non-redundant and stage-specific role in the lineage determination and terminal differentiation of odontoblasts from dental papilla mesenchyme.

Animals↗
PubMed2000

Clinical, radiographic, and genetic evaluation of a novel form of autosomal-dominant oligodontia.

A frameshift mutation recently identified within the paired domain of the transcription factor, PAX9, has been linked to a unique form of oligodontia in a single, multigenerational family (Stockton et al., 2000). We now describe the phenotypic and segregation analyses of this remarkable kindred, the initial approach taken to identify a candidate gene involved in this form of oligodontia, and the power of this single-family pedigree to generate significant linkage in a genome search. Of the 43 family members enrolled in this study, 21 individuals were affected with several congenitally missing permanent teeth. The pattern of inheritance of the oligodontia trait suggested the involvement of a single gene bearing a dominant mutation. To various degrees, affected members lacked permanent first, second, and third molars in all four quadrants. Several individuals with missing molars also lacked second premolars- most commonly, maxillary second premolars and mandibular central incisors. To the best of our knowledge, this pattern of non-syndromic, familial tooth agenesis has not been previously described in the literature. Since a missense mutation in the homeobox gene, MSX1, was previously linked to tooth agenesis in a single family lacking second premolars and third molars, we performed a mutational analysis of MSX1 by PCR. The absence of a mutation in exons 1 and 2 of MSX1 suggested that allelic mutations in the coding region of MSX1 are not associated with this phenotypically distinct form of oligodontia. Computer simulation of linkage analysis further proved that this pedigree alone was sufficient to generate a significant result for a total genome scan.

Adolescent↗
PubMed1999

Cbfa1 is required for epithelial-mesenchymal interactions regulating tooth development in mice.

Osteoblasts and odontoblasts, cells that are responsible for the formation of bone and dentin matrices respectively, share several molecular characteristics. Recently, Cbfa1 was shown to be a critical transcriptional regulator of osteoblast differentiation. Mutations in this gene cause cleidocranial dysplasia (CCD), an autosomal dominant disorder in humans and mice characterized by defective bone formation. CCD also results in dental defects that include supernumerary teeth and delayed eruption of permanent dentition. The dental abnormalities in CCD suggest an important role for this molecule in the formation of dentition. Here we describe results of studies aimed at understanding the functions of Cbfa1 in tooth formation. RT-PCR and in situ hybridization analyses show that Cbfa1 has a unique expression pattern in dental mesenchyme from the bud to early bell stages during active epithelial morphogenesis. Unlike that observed in osteoblast differentiation, Cbfa1 is downregulated in fully differentiated odontoblasts and is surprisingly expressed in ectodermally derived ameloblasts during the maturation phase of enamel formation. The role of Cbfa1 in tooth morphogenesis is further illustrated by the misshapen and severely hypoplastic tooth organs in Cbfa1-/- mice. These tooth organs lacked overt odontoblast and ameloblast differentiation and normal dentin and enamel matrices. Epithelial-mesenchymal recombinants demonstrate that dental epithelium regulates mesenchymal Cbfa1 expression during the bud and cap stages and that these effects are mimicked by the FGFs but not by the BMPs as shown by our bead implantation assays. We propose that Cbfa1 regulates the expression of molecules in mesenchyme that act reciprocally on dental epithelium to control its growth and differentiation. Taken together, our data indicate a non-redundant role for Cbfa1 in tooth development that may be distinct from that in bone formation. In odontogenesis, Cbfa1 is not involved in the early signaling networks regulating tooth initiation and early morphogenesis but regulates key epithelial-mesenchymal interactions that control advancing morphogenesis and histodifferentiation of the epithelial enamel organ.

Animals↗
PubMed1998

Genomic organization, chromosomal mapping, and promoter analysis of the mouse dentin sialophosphoprotein (Dspp) gene, which codes for both dentin sialoprotein and dentin phosphoprotein.

Our laboratory has reported that two major noncollagenous dentin proteins, dentin sialoprotein and dentin phosphoprotein, are specific cleavage products of a larger precursor protein termed dentin sialophosphoprotein (MacDougall, M., Simmons, D., Luan, X., Nydegger, J., Feng, J. Q., and Gu, T. T. (1997) J. Biol. Chem. 272:835-842). To confirm our single gene hypothesis and initiate in vitro promoter studies, we have characterized the structural organization of the mouse dentin sialophosphoprotein gene. This gene has a transcription unit of approximately 9.4 kilobase pairs and is organized into 5 exons and 4 introns. Exon 1 contains a noncoding 5' sequence, and exon 2 contains the transcriptional start site, signal peptide, and first two amino acids of the NH2 terminus. Exons 3 and 4 contain coding information for 29 and 314 amino acids, respectively. The remainder of the coding information and the untranslated 3' region are contained in exon 5. Chromosomal mapping localized the gene to mouse chromosome 5q21 in close proximity to other dentin/bone matrix genes. Computer analysis of the promoter proximal 1.6-kilobase pair sequence revealed a number of potentially important cis-regulatory sequences; these include the recognition elements of AP-1, AP-2, Msx-1, serum response elements, SP-1, and TCF-1. In vitro studies showed that the DSPP promoter is active in an odontoblast cell line, MO6-G3, with basal activity mapped to -95 bp. Two potential enhancer and suppresser elements were identified in the regions between -1447 and -791 bp and -791 and -95 bp, respectively. The structural organization of the dentin sialophosphoprotein gene confirms our finding that both dentin sialoprotein and dentin phosphoprotein are encoded by a single gene with a continuous open reading frame.

Amino Acid Sequence↗
PubMed1998

TGF-beta1 is essential for the homeostasis of the dentin-pulp complex.

Among the complex network of cytokines that influence odontoblast function during development and repair, TGF-beta1 is unique in its dual abilities to function as a potent immunosuppressant and as an inducer of extracellular matrix production. These properties underscore the importance of this molecule in maintaining the homeostasis of the dentin-pulp complex after injury. The purpose of this paper is to describe new findings of our phenotypic analysis of dentition in mice in which the TGF-beta1 gene has been disrupted. The major phenotype of TGF-beta1(-/-) offspring is one of diffuse immune system activation with progressive inflammation, wasting and death. Our studies of adult TGF-beta1(-/-) dentition show widespread pulpal and periapical inflammation and necroses. In addition, the coronal surfaces of occluding molars show marked attrition. To determine whether the phenotypic changes in TGF-beta1(-/-) dentition are directly linked to the loss of TGF-beta1 rather than the inflammatory process itself, we studied adult dentition in TGF-beta1(-/-) mice backcrossed into immunodeficient backgrounds. Results of our histopathologic and radiographic analyses show that teeth of TGF-beta1(-/-) immunodeficient mice retain vitality in pulpal and periapical regions but show excessive wear of occlusal surfaces.

Animals↗
PubMed1998

Bone morphogenetic protein-7 (osteogenic protein-1, OP-1) and tooth development.

Bone morphogenetic proteins (BMPs) form a family of growth factors originally isolated from extracellular bone matrix that are capable of inducing bone formation ectopically. We studied the expression, tissue localization, and function of BMP-7 (OP-1) during tooth development in rodents. Patterns of BMP-7 gene expression and peptide distribution indicated that BMP-7 was present in dental epithelium during the dental lamina, bud, and cap stages. During the bell stage, BMP-7 mRNA expression and protein distribution shifted from dental epithelium toward the dental mesenchyme. With advancing differentiation of odontoblasts, BMP-7 protein staining in the dental papilla became restricted to the layer of fully functional odontoblasts in the process of depositing (pre)dentin. Secretory-stage ameloblasts exhibited weak immunostaining for BMP-7. A restricted pattern of staining in ameloblasts became apparent in post-secretory stages of amelogenesis. Also, cells of the forming periodontal ligament were immunopositive. Histological analysis of tooth development in neonatal BMP-7-deficient mice did not reveal obvious changes compared with wild-type mice. We conclude that, in developing dental tissues, BMP-7 has distribution and expression patterns similar to those of other BMP members but is not an essential growth factor for tooth development, possibly because of functional redundancy with other BMP members or related growth factors.

Ameloblasts↗
PubMed1998

Characterization of immunocompetent cells in the diseased canine periodontium.

The beagle dog with naturally occurring periodontal disease is one of the most widely used animal models in periodontal research for histological studies on disease pathogenesis and on the effect of potential therapeutic regimens. However, previous studies were restricted to morphological assessment of immunocompetent cells because of the lack of available cell-specific markers. In this study we systematically characterized the specificity and immunoreactivity of a panel of anti-human antibodies for identification (ABC method) of immunocompetent cells in formalin-fixed, EDTA-decalcified, paraffin-embedded inflamed periodontal tissues obtained from six beagle dogs. Canine lymph nodes and a panel of different human tissues served as positive controls. Polyclonal anti-CD3 immunolabeled canine T-lymphocytes specifically. Anti-CD79alpha (clone HM57) reacted with B-lymphocytes and plasma cells, and CD79alpha (clone JCP117) showed no staining in canine tissues. Neutrophils, monocytes, small macrophages, and keratinocytes reacted with an anti-myeloid/histiocyte antibody (clone MAC387). Anti-CD68 (clones PG-M1 and EBM11) immunolabeled large macrophages and plasma cells. Clone EBM11 also stained osteoclasts and cementoclasts. With the exception of JCB117, all antibodies revealed similarly favorable immunolabeling of canine and human immunocompetent cells. Long-term EDTA decalcification appeared to weaken immunostaining of plasma cells with HM57. MAC387 and CD68 can be used to distinguish macrophages in different differentiation stages in canine periodontal tissues. (J Histochem Cytochem 46:1443-1454, 1998)

Animals↗
PubMed1998

Developmental regulation of dentin sialophosphoprotein during ameloblast differentiation: a potential enamel matrix nucleator.

The two major dentin matrix proteins, dentin sialoprotein and dentin phosphoprotein have been shown to be expressed as a single large transcript termed dentin sialophosphoprotein (DSPP). These non-collagenous matrix proteins, identified biochemically by their unique physical-chemical properties, are specific cleavage products of a large parent acidic phosphorylated protein (pI 4.0). Previous studies have shown expression of dentin sialoprotein at the protein level by ameloblasts. The purpose of this study was to determine the temporal-spatial pattern of DSPP expression during amelogenesis. In situ hybridization and immunohistochemistry were performed on sections of developing mouse molars. These data were correlated with RT-PCR analysis of in vitro enamel organ epithelium monolayer cell cultures enriched for ameloblasts. Our data indicates initial expression of the DSPP transcripts and protein during early ameloblast differentiation prior to the secretory phase when the majority of the enamel matrix is formed. Ameloblasts appear to tightly down-regulate DSPP transcription as enamel matrix formation is up-regulated. These data demonstrate DSPP expression during amelogenesis is under highly controlled developmental regulation. Therefore, DSPP may have a primary role in the initial mineralization events of both enamel and dentin, acting as a potential nucleator of hydroxyapatite crystal formation.

Ameloblasts↗
PubMed1997

OP-1 (BMP-7) affects mRNA expression of type I, II, X collagen, and matrix Gla protein in ossifying long bones in vitro.

In long bone development, a regulating role of OP-1 is suggested by the local correlated expression of both OP-1 ligand and OP-1 binding receptors in developing mouse hind limbs. OP-1 is expressed in the interdigital mesenchyme, whereas OP-1 binding receptors are found in the bordering perichondrium, and both OP-1 ligand and receptors are present in the zone of (pre)hypertrophic chondrocytes. We investigated the role of OP-1 in long bone development experimentally by treating organ cultures of embryonic mouse metatarsals with rhOP-1. The mRNA expression patterns of type I, II, X collagen, and matrix Gla protein (MGP) were studied using in situ hybridization and cell proliferation using [3H]thymidine and BrdU labeling. In the epiphyseal perichondrium, treatment with 40 ng/ml OP-1 enhanced cell proliferation after day 2, while 6-day treatment caused a shift in expression from type I collagen to type II collagen mRNA. This supports previous histochemical findings that OP-1 induced the transition of perichondrium into cartilage. In the center of the rudiment, OP-1 inhibited the expression of type X collagen mRNA, indicating inhibition of chondrocyte hypertrophy. An arrest of differentiation at the (pre)hypertrophic chondrocyte stage was also indicated by the large area of cells expressing MGP mRNA in the OP-1-treated rudiments. We conclude that OP-1 affected the expression of marker genes of chondrocyte differentiation by acting on two steps in endochondral ossification. First, cell proliferation was enhanced, particularly so in the perichondrium where cells started to express the chondrocyte phenotype. Second, the terminal differentiation of mature chondrocytes into hypertrophic chondrocytes was inhibited. These results, combined with the known pattern of OP-1 ligand and BMP receptor expression in the embryo, suggest that OP-1 plays a local role in the cascade of events during endochondral ossification.

Animals↗
PubMed1997

Gene expression patterns of murine dentin matrix protein 1 (Dmp1) and dentin sialophosphoprotein (DSPP) suggest distinct developmental functions in vivo.

Although the precise mechanisms of the conversion of predentin to dentin are not well understood, several lines of evidence implicate the noncollagenous proteins (NCPs) as important regulators of dentin biomineralization. Here we compared the in vivo temporospatial expression patterns of two dentin NCP genes, dentin matrix protein 1 (Dmp1), and dentin sialophosphoprotein (DSPP) in developing molars. Reverse transcription-polymerase chain reaction was performed on embryonic day 13 to 1-day-old first molars using Dmp1- and DSPP-specific primer sets. Dmp1 transcripts appeared at the late bud stage, while DSPP mRNA was seen at the cap stage. Expression of both genes was sustained throughout odontogenesis. In situ hybridization analysis revealed interesting differences in the expression patterns of these genes. While Dmp1 and DSPP showed coexpression in young odontoblasts before the start of mineralization, the expression of these genes was notably distinct at later stages. Dmp1 expression decreased in secretory odontoblasts after the appearance of mineral, while high levels of DSPP were sustained in odontoblasts. In early secretory ameloblasts, DSPP expression was transient and down-regulated with the appearance ofdentin matrix. Interestingly, Dmp1 expression became evident in ameloblasts during the maturative phase of amelogenesis. In contrast to Dspp expression that was tooth-specific, Dmp1 was expressed by osteoblasts throughout ossification in the skeleton. Probes directed to the "DSP" and "DPP" regions of the DSPP gene showed identical patterns of mRNA expression. These data show that the developmental expression patterns of Dmp1 and DSPP are distinct, implying that these molecules serve different biological functions in vivo.

Animals↗