Adriana Cavender

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.

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.

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).

Functional Analysis of a Mutation in PAX9 Associated with Familial Tooth Agenesis in Humans

Pax9 is a paired domain-containing transcription factor that plays an essential role in the patterning of murine dentition. In humans, mutations in PAX9 are associated with unique phenotypes of familial tooth agenesis that mainly involve posterior teeth. Among these, a frameshift mutation (219InsG) within the paired domain of PAX9 produces a protein product associated with a severe form of molar agenesis in a single family. The objectives of this study were to gain new insights into the molecular pathogenesis of the 219InsG mutation and its role in tooth agenesis. Here we describe functional defects in DNA binding and transactivation of mutant 219InsGPax9. Although wild type Pax9 binds to the high affinity paired domain recognition sequences, e5 and CD19-2(A-ins), the 219InsGPax9 mutant protein was unable to bind to these cognate DNA-binding sites. In co-transfection assays, wild type Pax9 activated reporter gene transcription although the mutant was transcriptionally inactive. Immunolocalization data show that Pax9 and 219InsGPax9 proteins are synthesized in mammalian cells but that the nuclear localization of the mutant Pax9 protein is altered. Furthermore, transactivation by the full-length Pax9 protein from paired domain binding sites was not impaired by the 219InsGPax9 mutant. The latter did not alter the DNA binding activities of wild type Pax9 in gel mobility shift assays. The combined defects in DNA binding activities and transactivation function of mutant 219InsGPAX9 likely alter the selective activation and/or repression of PAX9 effector genes during odontogenesis. This loss-of-function of PAX9 most likely results in its haploinsufficiency during the patterning of dentition and the subsequent loss of posterior teeth.

Differentiation of human dental stem cells reveals a role for microRNA‐218

BACKGROUND Regeneration of lost periodontium is the ultimate goal of periodontal therapy. Advances in tissue engineering have demonstrated the multilineage potential and plasticity of adult stem cells located in periodontal apparatus. However, it remains unclear how epigenetic mechanisms controlling signals determine tissue specification and cell lineage decisions. To date, no data are available on micro-RNA (miRNA) activity behind human-derived dental stem cells (DSCs). MATERIAL AND METHODS In this study, we isolated periodontal ligament stem cells, dental pulp stem cells and gingival stem cells from extracted third molars; human bone marrow stem cells were used as a positive control. The expression of OCT4A and NANOG was confirmed in these undifferentiated cells. All cells were cultured under osteogenic inductive conditions and RUNX2 expression was analyzed as a marker of mineralized tissue differentiation. The miRNA expression profile was obtained at baseline and after osteogenic induction in all cell types. RESULTS The expression of RUNX2 demonstrated successful osteogenic induction of all cell types, which was confirmed by alizarin red stain. The analysis of 765 miRNAs demonstrated a shift in miRNA expression that occurred in all four stem cell types, including a decrease in hsa-mir-218 across all differentiated cell populations. Hsa-mir-218 targets RUNX2 and decreases RUNX2 expression in undifferentiated human DSCs. DSC mineralized tissue type differentiation is associated with a decrease in hsa-mir-218 expression. CONCLUSION These data reveal a miRNA-regulated pathway for the differentiation of human DSCs and a select network of human miRNAs that control DSC osteogenic differentiation.

Novel expression and transcriptional regulation of FoxJ1 during oro-facial morphogenesis.

Axenfeld-Rieger syndrome (ARS) patients with PITX2 point mutations exhibit a wide range of clinical features including mild craniofacial dysmorphism and dental anomalies. Identifying new PITX2 targets and transcriptional mechanisms are important to understand the molecular basis of these anomalies. Chromatin immunoprecipitation assays demonstrate PITX2 binding to the FoxJ1 promoter and PITX2C transgenic mouse fibroblasts and PITX2-transfected cells have increased endogenous FoxJ1 expression. FoxJ1 is expressed at embryonic day 14.5 (E14.5) in early tooth germs, then down-regulated from E15.5-E17.5 and re-expressed in the inner enamel epithelium, oral epithelium, tongue epithelium, sub-mandibular salivary gland and hair follicles during E18.5 and neonate day 1. FoxJ1 and Pitx2 exhibit overlapping expression patterns in the dental and oral epithelium. PITX2 activates the FoxJ1 promoter and, Lef-1 and beta-catenin interact with PITX2 to synergistically regulate the FoxJ1 promoter. FoxJ1 physically interacts with the PITX2 homeodomain to synergistically regulate FoxJ1, providing a positive feedback mechanism for FoxJ1 expression. Furthermore, FoxJ1, PITX2, Lef-1 and beta-catenin act in concert to activate the FoxJ1 promoter. The PITX2 T68P ARS mutant protein physically interacts with FoxJ1; however, it cannot activate the FoxJ1 promoter. These data indicate a mechanism for the activity of the ARS mutant proteins in specific cell types and provides a basis for craniofacial/ tooth anomalies observed in these patients. These data reveal novel transcriptional mechanisms of FoxJ1 and demonstrate a new role of FoxJ1 in oro-facial morphogenesis.

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 Cbfal expression in newly differentiated and functional odontoblasts. Second, we demonstrate that Cbfa1(-/-) incisor organs arrest at a later stage than molars, and that al(I) :ollagen, 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.

Expression of Runx2/Cbfa1/Pebp2αA During Angiogenesis in Postnatal Rodent and Fetal Human Orofacial Tissues†

Transient expression of Runx2 is reported in endothelial cells and vascular smooth muscle cells during vessel formation in skin, stroma of forming bones and developing periodontal ligament, developing skeletal muscle cells, and fat tissue. The data suggest that Runx2 is expressed in a multipotential mesenchymal cell population that gives rise to various osseous and nonosseous cell lineages.

TWIST1 Promotes the Odontoblast-like Differentiation of Dental Stem Cells:

Stem cells derived from the dental pulp of extracted human third molars (DPSCs) have the potential to differentiate into odontoblasts, osteoblasts, adipocytes, and neural cells when provided with the appropriate conditions. To advance the use of DPSCs for dentin regeneration, it is important to replicate the permissive signals that drive terminal events in odontoblast differentiation during tooth development. Such a strategy is likely to restore a dentin matrix that more resembles the tubular nature of primary dentin. Due to the limitations of culture conditions, the use of ex vivo gene therapy to drive the terminal differentiation of mineralizing cells holds considerable promise. In these studies, we asked whether the forced expression of TWIST1 in DPSCs could alter the potential of these cells to differentiate into odontoblast-like cells. Since the partnership between Runx2 and Twist1 proteins is known to control the onset of osteoblast terminal differentiation, we hypothesized that these genes act to control lineage determination of DPSCs. For the first time, our results showed that Twist1 overexpression in DPSCs enhanced the expression of DSPP, a gene that marks odontoblast terminal differentiation. Furthermore, co-transfection assays showed that Twist1 stimulates Dspp promoter activity by antagonizing Runx2 function in 293FT cells. Analysis of our in vitro data, taken together, suggests that lineage specification of DPSCs can be modulated through ex vivo gene modifications.

Mutational analysis of families affected with molar oligodontia

Oligodontia, the congenital absence of six or more permanent teeth, is a common developmental anomaly of human dentition whose genetic basis is poorly understood. We recently reported a nonsyndromic form of oligodontia involving mostly all permanent molars in a large kindred, caused by a frameshift mutation in exon 2 of the human PAX9 gene [3]. To better understand the genotype/phenotype correlation in non-syndromic familial oligodontia, we identified additional families with a similar pattern of molar oligodontia. We hypothesize that molar oligodontia is due to allelic heterogeneity involving different mutations in PAX9 . To test this hypothesis, we performed pedigree analysis followed by mutational analysis. Pedigree analysis revealed that the oligodontia trait is inherited in an autosomal dominant fashion. Mutational analysis of PAX9 thus far excludes the presence of the previously identified frameshift mutation.