Rena N. D'Souza

Mutation of PAX9 is associated with oligodontia

We identified a frameshift mutation in the paired domain of PAX9 following genome-wide analysis of a family segregating autosomal dominant oligodontia. Affected members have normal primary dentition but lacked most permanent molars.

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 of dentin 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.

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.

Dentin Matrix Protein 1 (DMP1): New and Important Roles for Biomineralization and Phosphate Homeostasis

Previously, non-collagenous matrix proteins, such as DMP1, were viewed with little biological interest. The last decade of research has increased our understanding of DMP1, as it is now widely recognized that this protein is expressed in non-mineralized tissues, as well as in cancerous lesions. Protein chemistry studies have shown that the full length of DMP1, as a precursor, is cleaved into two distinct forms: the C-terminal and N-terminal fragments. Functional studies have demonstrated that DMP1 is essential in the maturation of odontoblasts and osteoblasts, as well as in mineralization via local and systemic mechanisms. The identification of DMP1 mutations in humans has led to the discovery of a novel disease: autosomal-recessive hypophosphatemic rickets. Furthermore, the regulation of phosphate homeostasis by DMP1 through FGF23, a newly identified hormone that is released from bone and targeted in the kidneys, sets a new direction for research that associates biomineralization with phosphate regulation.

Isolation, characterization and immunolocalization of a 53-kDal dentin sialoprotein (DSP).

We isolated a sialic-rich protein from rat dentin extracts and have named it dentin sialoprotein, DSP (formerly called 95K glycoprotein). DSP is rich in aspartic acid, glutamic acid, glycine and serine, but contains no cysteine or phosphate. The 30% carbohydrate content includes about 9% sialic acid and indicates that several N-glycosides and O-glycosides are present. Sedimentation equilibrium analysis gave a M(r) of 52,570. Based on this molecular weight we calculated that DSP contains about 350-amino acids and 75 monosaccharides. With automated Edman degradation the sequence of the first 8-amino acids was shown to be: Ile-Pro-Val-Pro-Gln-Leu-Val-Pro. The initial 3 residues of this sequence are identical to the first 3 in human osteopontin (OPN) and are closely similar to the Leu-Pro-Val sequences of OPN from other species, as well as at the beginning of bone acidic glycoprotein-75 (BAG-75). On Western immunoblots, purified polyclonal antibodies reacted only with DSP in dentin extracts and with none of the proteins from bone. Similarly, immunolocalization experiments showed the presence of DSP in dentin but not in enamel or alveolar bone. Along with immunohistochemical localization data reported elsewhere, these observations suggest that DSP may be an important marker for cells in the odontoblast lineage.

Self-Assembling Peptide Amphiphile Nanofibers as a Scaffold for Dental Stem Cells

Dental caries remains one of the most prevalent infectious diseases in the world. So far, available treatment methods rely on the replacement of decayed soft and mineralized tissue with inert biomaterials alone. As an approach to develop novel regenerative strategies and engineer dental tissues, two dental stem cell lines were combined with peptide-amphiphile (PA) hydrogel scaffolds. PAs self-assemble into three-dimensional networks of nanofibers, and living cells can be encapsulated. Cell-matrix interactions were tailored by incorporation of the cell adhesion sequence RGD and an enzyme-cleavable site. SHED (stem cells from human exfoliated deciduous teeth) and DPSC (dental pulp stem cells) were cultured in PA hydrogels for 4 weeks using different osteogenic supplements. Both cell lines proliferate and differentiate within the hydrogels. Histologic analysis shows degradation of the gels and extracellular matrix production. However, distinct differences between the two cell lines can be observed. SHED show a spindle-shaped morphology, high proliferation rates, and collagen production, resulting in soft tissue formation. In contrast, DPSC reduce proliferation, but exhibit an osteoblast-like phenotype, express osteoblast marker genes, and deposit mineral. Since the hydrogels are easy to handle and can be introduced into small defects, this novel system might be suitable for engineering both soft and mineralized matrices for dental tissue regeneration.

Phenotypic changes in dentition of Runx2 homozygote-null mutant mice

Genetic and molecular studies in humans and mice indicate that Runx2 (Cbfa1) is a critical transcriptional regulator of bone and tooth formation. Heterozygous mutations in Runx2 cause cleidocranial dysplasia (CCD), an inherited disorder in humans and mice characterized by skeletal defects, supernumerary teeth, and delayed eruption. Mice lacking the Runx2 gene die at birth and lack bone and tooth development. Our extended phenotypic studies of Runx2 mutants showed that developing teeth fail to advance beyond the bud stage and that mandibular molar organs were more severely affected than maxillary molar organs. Runx2 (−/−) tooth organs, when transplanted beneath the kidney capsules of nude mice, failed to progress in development. Tooth epithelial-mesenchymal recombinations using Runx2 (+/+) and (−/−) tissues indicate that the defect in mesenchyme cannot be rescued by normal dental epithelium. Finally, our molecular analyses showed differential effects of the absence of Runx2 on tooth extracellular matrix (ECM) gene expression. These data support the hypothesis that Runx2 is one of the key mesenchymal factors that influences tooth morphogenesis and the subsequent differentiation of ameloblasts and odontoblasts.

Characterization of cellular responses involved in reparative dentinogenesis in rat molars.

During primary dentin formation, differentiating primary odontoblasts secrete an organic matrix, consisting principally of type I collagen and non-collagenous proteins, that is capable of mineralizing at its distal front. In contrast to ameloblasts that form enamel and undergo programed cell death, primary odontoblasts remain metabolically active in a functional tooth. When dentin is exposed to caries or by operative procedures, and when exposed dentinal tubules are treated with therapeutic dental materials, the original population of odontoblasts is often injured and destroyed. The characteristics of the replacement pool of cells that form reparative dentin and the biologic mechanisms that modulate the formation of this matrix are poorly understood. Based on the hypothesis that events governing primary dentinogenesis are reiterated during dentin repair, the present study was designed to test whether cells that form reparative dentin are odontoblast-like. Cervical cavities were prepared in rat first molars to generate reparative dentin, and animals were killed at various time intervals. In situ hybridization with gene-specific riboprobes for collagen types I and III was used to study de novo synthesis by cells at the injured dentin-pulp interface. Polyclonal antibodies raised against dentin sialoprotein (DSP), a dentin-specific protein that marks the odontoblast phenotype, were used in immunohistochemical experiments. Data from our temporal and spatial analyses indicated that cells forming reparative dentin synthesize type I but not type III collagen and are immunopositive for DSP. Our results suggest that cells that form reparative dentin are odontoblast-like.

Developmental expression of a 53 KD dentin sialoprotein in rat tooth organs.

Rat dentin contains a major sialic acid-rich glycoprotein, DSP, with an overall composition similar to that of bone sialoproteins but whose biological role in dentinogenesis is unknown. Using polyclonal affinity-purified antibodies to rat DSP and four immunohistochemical methods of detection, we studied the cell and tissue localization of DSP and the time course of its appearance during odontoblast differentiation. DSP first appeared within young odontoblasts concomitant with early secretion of pre-dentin matrix and before the onset of mineralization but was absent in pre-odontoblasts. DSP immunostaining also localized within secretory odontoblasts and was intense in odontoblastic processes. Early pre-dentin stained positive for DSP, in contrast to more mature pre-dentin, where immunoreactivity was less intense and more restricted to odontoblastic processes. In the zone of mineralized dentin matrix, a moderate and uniform staining pattern was evident. Intense immunostaining was also seen within the cells and matrix of dental pulp during dentinogenesis. Other cells and tissues within the tooth organ and those surrounding it were non-reactive. These findings suggest that DSP is developmentally expressed in cells of the odontoblastic lineage and may be a biochemical marker of odontoblastic activity.

Functional consequences of interactions between Pax9 and Msx1 genes in normal and abnormal tooth development

Pax9 and Msx1 encode transcription factors that are known to be essential for the switch in odontogenic potential from the epithelium to the mesenchyme. Multiple lines of evidence suggest that these molecules play an important role in the maintenance of mesenchymal Bmp4 expression, which ultimately drives morphogenesis of the dental organ. Here we demonstrate that Pax9 is able to directly regulate Msx1 expression and interact with Msx1 at the protein level to enhance its ability to transactivate Msx1 and Bmp4 expression during tooth development. In addition, we tested how a missense mutation (T62C) in the paired domain of PAX9 that is responsible for human tooth agenesis (1) affects its functions. Our data indicate that although the mutant Pax9 protein (L21P) can bind to the Msx1 protein, it fails to transactivate the Msx1 and Bmp4 promoter, presumably because of its inability to bind cognate paired domain recognition sequences. In addition, synergistic transcriptional activation of the Bmp4 promoter was lost with coexpression of mutant Pax9 and wild-type Msx1. This suggests that Pax9 is critical for the regulation of Bmp4 expression through its paired domain rather than Msx1. Our findings demonstrate the partnership of Pax9 and Msx1 in a signaling pathway that involves Bmp4. Furthermore, the regulation of Bmp4 expression by the interaction of Pax9 with Msx1 at the level of transcription and through formation of a protein complex determines the fate of the transition from bud to cap stage during tooth development.