Activation of Wnt signaling in Axin2+ cells leads to osteodentin formation and cementum overgrowth.

OBJECTIVES: In this study, we used the mouse incisor model to investigate the regulatory mechanisms of Wnt/ß-catenin signaling on Axin2+ cells in tooth development. MATERIALS AND METHODS: Axin2lacZ/+ reporter mice were used to define the expression pattern of Axin2 in mouse incisors. We traced the fate of Axin2+ cells from postnatal Day 21 (P21) to P56 using Axin2CreERT2/+ and R26RtdTomato/+ reporter mice. For constitutive activation of Wnt signaling, Axin2CreERT2/+ , ß-cateninflox(Ex3)/+ , and R26RtdTomato/+ (CA-ß-cat) mice were generated to investigate the gain of function (GOF) of ß-catenin in mouse incisor growth. RESULTS: The X-gal staining of Axin2lacZ/+ reporter mice and lineage tracing showed that Axin2 was widely expressed in dental mesenchyme of mouse incisors, and Axin2+ cells were essential cell sources for odontoblasts, pulp cells, and periodontal ligament cells. The constitutive activation of Wnt signaling in Axin2+ cells resulted in the formation of osteodentin featured with increased DMP1 and dispersed DSP expression and overgrowth of cementum. CONCLUSION: Wnt signaling plays a key role in the differentiation and maturation of Axin2+ cells in mouse incisors.

The spatial distribution of focal stacks within the inner enamel layer of mandibular mouse incisors.

Focal stacks are an alternative spatial arrangement of enamel rods within the inner enamel of mandibular mouse incisors where short rows comprised of 2-45 enamel rods are nestled at the side of much longer rows, both sharing the same rod tilt directed mesially or laterally. The significance of focal stacks to enamel function is unknown, but their high frequency in transverse sections (30% of all rows) suggests that they serve some purpose beyond representing an oddity of enamel development. In this study, we characterized the spatial distribution of focal stacks in random transverse sections relative to different regions of the inner enamel and to different locations across enamel thickness. The curving dentinoenamel junction (DEJ) in transverse sections complicated spatial distribution analyses, and a technique was developed to "unbend" the curving DEJ allowing for more linear quantitative analyses to be carried out. The data indicated that on average there were 36 ± 7 focal stacks located variably within the inner enamel in any given transverse section. Consistent with area distributions, focal stacks were four times more frequent in the lateral region (53%) and twice as frequent in the mesial region (33%) compared to the central region (14%). Focal stacks were equally split by tilt (52% mesial vs. 48% lateral, not significant), but those having a mesial tilt were more frequently encountered in the lateral and central regions (2:1) and those having a lateral tilt were more numerous in the mesial region (1:3). Focal stacks having a mesial tilt were longer on average compared to those having a lateral tilt (7.5 ± 5.6 vs. 5.9 ± 4.0 rods per row, p < 0.01). There was no relationship between the length of a focal stack and its location within the inner enamel. All results were consistent with the notion that focal stacks travel from the DEJ to the outer enamel the same as the longer and decussating companion rows to which they are paired. The spatial distribution of focal stacks within the inner enamel was not spatially random but best fit a null model based on a heterogenous Poisson point process dependent on regional location within the transverse plane of the enamel layer.

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.

Loss of Autophagy Disrupts Stemness of Ameloblast-Lineage Cells in Aging.

Autophagy is one of the intracellular degradation pathways and maintains cellular homeostasis, regulating the stress response, cell proliferation, and signal transduction. To elucidate the role of autophagy in the maintenance of dental epithelial stem cells and the subsequent enamel formation, we analyzed autophagy-deficient mice in epithelial cells (Atg7f/f;KRT14-Cre mice), focusing on the influence of aging and stress environments. We also performed in vitro cell and organ culture experiments with an autophagy inhibitor. In young Atg7f/f;KRT14-Cre mice, morphological change was not obvious in maxillary incisors, except for the remarkable cell death in the stratum intermedium of the transitional stage. However, under stress conditions of hyperglycemia, the incisor color changed to white in diabetes Atg7f/f;KRT14-Cre mice. Regarding dental epithelial stem cells, the shape of the apical bud region of the incisor became irregular with age, and odontoma was formed in aged Atg7f/f;KRT14-Cre mice. In addition, the shape of apical bud culture cells of Atg7f/f;KRT14-Cre mice became irregular and enlarged atypically, with epigenetic changes during culture, suggesting that autophagy deficiency may induce tumorigenesis in dental epithelial cells. The epigenetic change and upregulation of p21 expression were induced by autophagy inhibition in vivo and in vitro. These findings suggest that autophagy is important for the regulation of stem cell maintenance, proliferation, and differentiation of ameloblast-lineage cells, and an autophagy disorder may induce tumorigenesis in odontogenic epithelial cells.

Eda controls the size of the enamel knot during incisor development.

Ectodysplasin (Eda) plays important roles in both shaping the developing tooth and establishing the number of teeth within the tooth row. Sonic hedgehog (Shh) has been shown to act downstream of Eda and is involved in the initiation of tooth development. Eda-/- mice possess hypoplastic and hypomineralized incisors and show changes in tooth number in the molar region. In the present study we used 3D reconstruction combined with expression analysis, cell lineage tracing experiments, and western blot analysis in order to investigate the formation of the incisor germs in Eda-/- mice. We show that a lack of functional Eda protein during early stages of incisor tooth germ development had minimal impact on development of the early expression of Shh in the incisor, a region proposed to mark formation of a rudimental incisor placode and act as an initiating signalling centre. In contrast, deficiency of Eda protein had a later impact on expression of Shh in the primary enamel knot of the functional tooth. Eda-/- mice had a smaller region where Shh was expressed, and a reduced contribution from Shh descendant cells. The reduction in the enamel knot led to the formation of an abnormal enamel organ creating a hypoplastic functional incisor. Eda therefore appears to influence the spatial formation of the successional signalling centres during odontogenesis.

Sox2 controls asymmetric patterning of ameloblast lineage commitment by regulation of FGF signaling in the mouse incisor.

Mouse incisors are covered by enamel only on the labial side and the lingual side is covered by dentin without enamel. This asymmetric distribution of enamel makes it possible to be abrased on the lingual side, generating the sharp cutting edge of incisor on the labial side. The abrasion of mouse incisors is compensated by the continuous growth throughout life. Epithelium stem cells responsible for its continuous growth are reported to localize within the labial cervical loop. The transcription factor Sox2 plays important roles in the maintenance of stem cell pluripotency and organ formation. We previously found that Sox2 mainly expressed in the dental epithelium. Besides, Sox2 has been reported to be a dental epithelium stem cell marker in the incisor. However, the exact mechanism of Sox2 controlling amelogenesis is still not quite clearly elucidated. Here we report that conditional deletion of Sox2 in the dental epithelium using Shhcre caused impaired ameloblast differentiation in the labial side and induced ectopic ameloblast-like cell differentiation on the lingual side. Abnormal FGF gene expression was detected by RNAscope in situ hybridization in the mutant incisor. Collectively, we speculate that asymmetric ameloblast lineage commitment of mouse incisor might be regulated by Sox2 through FGF signaling.

Odontoblast processes of the mouse incisor are plates oriented in the direction of growth.

Odontoblast processes are thin cytoplasmic projections that extend from the cell body at the periphery of the pulp toward the dentin-enamel junction. The odontoblast processes function in the secretion, assembly and mineralization of dentin during development, participate in mechanosensation, and aid in dentin repair in mature teeth. Because they are small and densely arranged, their three-dimensional organization is not well documented. To gain further insight into how odontoblast processes contribute to odontogenesis, we used serial section electron microscopy and three-dimensional reconstructions to examine these processes in the predentin region of mouse molars and incisors. In molars, the odontoblast processes are tubular with a diameter of ~1.8 µm. The odontoblast processes near the incisor tip are similarly shaped, but those midway between the tip and apex are shaped like plates. The plates are radially aligned and longitudinally oriented with respect to the growth axis of the incisor. The thickness of the plates is approximately the same as the diameter of molar odontoblast processes. The plates have an irregular edge; the average ratio of width (midway in the predentin) to thickness is 2.3 on the labial side and 3.6 on the lingual side. The plate geometry seems likely to be related to the continuous growth of the incisor and may provide a clue as to the mechanisms by which the odontoblast processes are involved in tooth development.

Runx2+ Niche Cells Maintain Incisor Mesenchymal Tissue Homeostasis through IGF Signaling.

Stem cell niches provide a microenvironment to support the self-renewal and multi-lineage differentiation of stem cells. Cell-cell interactions within the niche are essential for maintaining tissue homeostasis. However, the niche cells supporting mesenchymal stem cells (MSCs) are largely unknown. Using single-cell RNA sequencing, we show heterogeneity among Gli1+ MSCs and identify a subpopulation of Runx2+/Gli1+ cells in the adult mouse incisor. These Runx2+/Gli1+ cells are strategically located between MSCs and transit-amplifying cells (TACs). They are not stem cells but help to maintain the MSC niche via IGF signaling to regulate TAC proliferation, differentiation, and incisor growth rate. ATAC-seq and chromatin immunoprecipitation reveal that Runx2 directly binds to Igfbp3 in niche cells. This Runx2-mediated IGF signaling is crucial for regulating the MSC niche and maintaining tissue homeostasis to support continuous growth of the adult mouse incisor, providing a model for analysis of the molecular regulation of the MSC niche.

Dental Epithelial Stem Cells Express the Developmental Regulator Meis1.

MEIS1 is a key developmental regulator of several organs and participates in stem cell maintenance in different niches. However, despite the murine continuously growing incisor being a well described model for the study of adult stem cells, Meis1 has not been investigated in a dental context. Here, we uncover that Meis1 expression in the tooth is confined to the epithelial compartment. Its expression arises during morphogenesis and becomes restricted to the mouse incisor epithelial stem cell niche, the labial cervical loop. Meis1 is specifically expressed by Sox2+ stem cells, which give rise to all dental epithelial cell lineages. Also, we have found that Meis1 in the incisor is coexpressed with potential binding partner Pbx1 during both embryonic and adult stages. Interestingly, Meis2 is present in different areas of the forming tooth and it is not expressed by dental epithelial stem cells, suggesting different roles for these two largely homologous genes. Additionally, we have established the expression patterns of Meis1 and Meis2 during tongue, hair, salivary gland and palate formation. Finally, analysis of Meis1-null allele mice indicated that, similarly, to SOX2, MEIS1 is not essential for tooth initiation, but might have a role during adult incisor renewal.

Isl1 Controls Patterning and Mineralization of Enamel in the Continuously Renewing Mouse Incisor.

Rodents are characterized by continuously renewing incisors whose growth is fueled by epithelial and mesenchymal stem cells housed in the proximal compartments of the tooth. The epithelial stem cells reside in structures known as the labial (toward the lip) and lingual (toward the tongue) cervical loops (laCL and liCL, respectively). An important feature of the rodent incisor is that enamel, the outer, highly mineralized layer, is asymmetrically distributed, because it is normally generated by the laCL but not the liCL. Here, we show that epithelial-specific deletion of the transcription factor Islet1 (Isl1) is sufficient to drive formation of ectopic enamel by the liCL stem cells, and also that it leads to production of altered enamel on the labial surface. Molecular analyses of developing and adult incisors revealed that epithelial deletion of Isl1 affected multiple, major pathways: Bmp (bone morphogenetic protein), Hh (hedgehog), Fgf (fibroblast growth factor), and Notch signaling were upregulated and associated with liCL-generated ectopic enamel; on the labial side, upregulation of Bmp and Fgf signaling, and downregulation of Shh were associated with premature enamel formation. Transcriptome profiling studies identified a suite of differentially regulated genes in developing Isl1 mutant incisors. Our studies demonstrate that ISL1 plays a central role in proper patterning of stem cell-derived enamel in the incisor and indicate that this factor is an important upstream regulator of signaling pathways during tooth development and renewal. © 2017 American Society for Bone and Mineral Research.

Spatial and temporal expression of Sox9 during murine incisor development.

Mouse incisors are capable of continuously growing due to the renewal of dental epithelium stem cells and mesenchymal stem cells residing at the proximal ends. The transcription factor Sox9 plays important roles in maintaining the stem cells of hair follicles, retinal progenitor cells and neural crest stem cells. Whether Sox9 is involved during mouse incisor development is not reported yet. In this study, we examined the expression pattern of Sox9 during mouse incisor development by in situ hybridization and immunohistochemistry. Sox9 mRNA and protein showed similar expression pattern from embryonic day (E) 13.5 to postnatal (PN) day 10. At E13.5 and E14.5, Sox9 was strongly expressed in the dental epithelium. At E16.5, Sox9 started to be detected in the mesenchymal cells within the dental pulp, especially the dental pulp cells that adjacent to the labial cervical loop. Similarly with E14.5, Sox9 was strongly detected in the labial cervical loop, including the basal epithelium, the stellate reticulum and the outer enamel epithelium from E16.5 to PN10. The mesenchyme adjacent to the labial cervical loop also showed strong signal of Sox9. The spatiotemporal expression of Sox9 suggested its possible involvement during mouse incisor development.

Stem Cells in Tooth Development, Growth, Repair, and Regeneration.

Human teeth contain stem cells in all their mesenchymal-derived tissues, which include the pulp, periodontal ligament, and developing roots, in addition to the support tissues such as the alveolar bone. The precise roles of these cells remain poorly understood and most likely involve tissue repair mechanisms but their relative ease of harvesting makes teeth a valuable potential source of mesenchymal stem cells (MSCs) for therapeutic use. These dental MSC populations all appear to have the same developmental origins, being derived from cranial neural crest cells, a population of embryonic stem cells with multipotential properties. In rodents, the incisor teeth grow continuously throughout life, a feature that requires populations of continuously active mesenchymal and epithelial stem cells. The discrete locations of these stem cells in the incisor have rendered them amenable for study and much is being learnt about the general properties of these stem cells for the incisor as a model system. The incisor MSCs appear to be a heterogeneous population consisting of cells from different neural crest-derived tissues. The epithelial stem cells can be traced directly back in development to a Sox10(+) population present at the time of tooth initiation. In this review, we describe the basic biology of dental stem cells, their functions, and potential clinical uses.

In vivo administration of dental epithelial stem cells at the apical end of the mouse incisor.

Cell-based tissue regeneration is an attractive approach that complements traditional surgical techniques for replacement of injured and lost tissues. The continuously growing rodent incisor provides an excellent model system for investigating cellular and molecular mechanisms that underlie tooth renewal and regeneration. An active population of dental epithelial progenitor/stem cells located at the posterior part of the incisor, commonly called cervical loop area, ensures the continuous supply of cells that are responsible for the secretion of enamel matrix. To explore the potential of these epithelial cells in therapeutic approaches dealing with enamel defects, we have developed a new method for their in vivo administration in the posterior part of the incisor. Here, we provide the step-by-step protocol for the isolation of dental epithelial stem cells and their delivery at targeted areas of the jaw. This simple and yet powerful protocol, consisting in drilling a hole in the mandibular bone, in close proximity to the cervical loop area of the incisor, followed up by injection of stem cells, is feasible, reliable, and effective. This in vivo approach opens new horizons and possibilities for cellular therapies involving pathological and injured dental tissues.

An In vivo Model for Short-Term Evaluation of the Implantation Effects of Biomolecules or Stem Cells in the Dental Pulp.

The continuously growing rodent incisor is a widely used model to investigate odontogenesis and mineralized tissue formation. This study focused on evaluating the mouse mandibular incisor as an experimental biological tool for analyzing in vivo the capacity of odontoblast-like progenitors or bioactive molecules to contribute to reparative dentinogenesis. We describe here a surgical procedure allowing direct access to the forming part of the incisor dental pulp Amelogenin peptide A+4 adsorbed on agarose beads, or dental pulp progenitor cells were implanted in the pulp following this procedure. After 10 days A+4 induced the formation of an osteodentin occluding almost the totality of the pulp compartment. Implantation of progenitor cells leads to formation of islets of osteodentin-like structures located centrally in the pulp. These pilot studies validate the incisor as an experimental model to test the capacity of progenitor cells or bioactive molecules to induce the formation of reparative dentin.