Mesenchymal TGF-ß signaling orchestrates dental epithelial stem cell homeostasis through Wnt signaling.

In mouse, continuous growth of the postnatal incisor is coordinated by two populations of multipotent progenitor cells, the dental papilla mesenchymal cells and dental epithelial stem cells, residing at the proximal end of the incisor, yet the molecular mechanism underlying the cooperation between mesenchymal and epithelial cells is largely unknown. Here, transforming growth factor-ß (TGF-ß) type II receptor (Tgfbr2) was specifically deleted within the postnatal dental papilla mesenchyme. The Tgfbr2-deficient mice displayed malformed incisors with wavy mineralized structures at the labial side as a result of increased differentiation of dental epithelial stem cells. We found that mesenchymal Tgfbr2 disruption led to upregulated expression of Wnt5a and downregulated expression of Fgf3/10 in the mesenchyme, both of which synergistically enhanced Lrp5/6-ß-catenin signaling in the cervical loop epithelium. In accord with these findings, mesenchyme-specific depletion of the Wnt transporter gene Wls abolished the aberrant mineralized structures caused by Tgfbr2 deletion. Thus, mesenchymal TGF-ß signaling provides a unifying mechanism for the homeostasis of dental epithelial stem cells via a Wnt signaling-mediated mesenchymal-epithelial cell interaction.

Different expression patterns of Gli1-3 in mouse embryonic maxillofacial development.

The Sonic hedgehog (SHH) signaling pathway plays many key roles in the development of Drosophila and vertebrate embryos, including regulating craniofacial development. The GLI family of transcription factors (GLI1, 2, and 3) mediates the SHH morphogenetic signal by regulating the expression of downstream target genes. Signaling aberrations seriously affect vertebrate development. To better understand the regulation of GLI transcription factors, we investigated the expression patterns of Gli1-3 during murine embryonic craniofacial development using in situ hybridization with whole-mounts and sections and quantitative real-time PCR. We found that Gli1 expression was mostly detected in the mesenchyme in embryonic dermis, and strongly detected in embryonic oral mucosa at late stages of face development. Gli2 was strong in mesenchyme, both in dermis and oral mucosa. Gli3 expression was detected fainter in the epithelium than the mesenchyme at 11 days post-coitum (dpc), but at 14.5 dpc, expression was stronger in the epithelium. Of the three genes, Gli1 expression was highest in early stages of face development, Gli3 expression was highest in the second half of facial development (and remained stable thereafter), whereas Gli2 expression remained low throughout facial development. The present findings suggest that Gli1-3 may play different roles during facial development.

Expression of Dpp6 in mouse embryonic craniofacial development.

Dipeptidyl-peptidase-like protein 6 (DPP6), a member of the dipeptidyl aminopeptidase family, plays distinct roles in brain development, but its expression in embryonic craniofacial development is unknown. The expression pattern of Dpp6 in the maxillofacial region during mouse embryonic craniofacial development was analyzed by whole-mount in situ hybridization on sections and by real-time PCR analysis. Dpp6 expression was detected during mouse embryonic craniofacial development in embryos 11-13.5 days post-coitum (dpc). Real-time PCR showed high Dpp6 expression present in 11.5-13.5dpc, and this then decreased as development of maxillofacial region progressed. The expression pattern of Dpp6 suggests that Dpp6 may be involved in embryonic craniofacial development.

Disruption of Smad4 in odontoblasts causes multiple keratocystic odontogenic tumors and tooth malformation in mice.

Keratocystic odontogenic tumors (KCOTs) are cystic epithelial neoplasias with a high recurrence rate. However, the molecular mechanisms underlying the initiation and progression of KCOTs are still largely unknown. Here, we show that specific ablation of Smad4 in odontoblasts unexpectedly resulted in spontaneous KCOTs in mice. The mutant mice exhibited malformed teeth characterized by fractured incisors and truncated molar roots. These abnormalities were mainly caused by disrupted odontoblast differentiation that led to irregular dentin formation. The cystic tumors arising from the reactivation of epithelial rests of Malassez (ERM), in which Smad4 remained intact, proliferated and formed stratified and differentiated squamous epithelia that exhibited a dramatic upregulation of Hedgehog signaling. Odontoblasts, which are responsive to transforming growth factor beta (TGF-beta)/bone morphogenetic protein (BMP) signals, may produce signal molecules to inhibit the activation of ERM. Indeed, we observed a downregulation of BMP signals from Smad4 mutant odontoblasts to the adjacent Hertwig's epithelial root sheath (HERS). Intriguingly, KCOTs frequently emerged from Smad4-deficient ERM in keratinocyte-specific Smad4 knockout mice, suggesting a novel mechanism in which reciprocal TGF-beta/BMP signaling between odontoblasts and HERS was required for tooth root development and suppression of KCOT formation. These findings provide insight into the genetic basis underlying KCOTs and have important implications for new directions in KCOT treatment.

Collagen1alpha1 promoter drives the expression of Cre recombinase in osteoblasts of transgenic mice.

Osteoblasts participate in bone formation, bone mineralization, osteoclast differentiation and many pathological processes. To study the function of genes in osteoblasts using Cre-LoxP system, we generated a mouse line expressing the Cre recombinase under the control of the rat Collagen1alpha1 (Col1alpha1) promoter (Col1alpha1-Cre). Two founders were identified by genomic PCR from 16 offsprings, and the integration efficiency is 12.5%. In order to determine the tissue distribution and the activity of Cre recombinase in the transgenic mice, the Col1alpha1-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4(Co/Co)). Multiple tissue PCR of Col1alpha1-Cre;Smad4(Co/+)mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon. LacZ staining in the Col1alpha1-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5. Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice. All these data indicated that the Col1alpha1-Cre transgenic mice could serve as a valuable tool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.