Ror2-mediated non-canonical Wnt signaling regulates Cdc42 and cell proliferation during tooth root development.

The control of size and shape is an important part of regulatory process during organogenesis. Tooth formation is a highly complex process that fine-tunes the size and shape of the tooth, which are crucial for its physiological functions. Each tooth consists of a crown and one or more roots. Despite comprehensive knowledge of the mechanism that regulates early tooth crown development, we have limited understanding of the mechanism regulating root patterning and size during development. Here, we show that Ror2-mediated non-canonical Wnt signaling in the dental mesenchyme plays a crucial role in cell proliferation, and thereby regulates root development size in mouse molars. Furthermore, Cdc42 acts as a potential downstream mediator of Ror2 signaling in root formation. Importantly, activation of Cdc42 can restore cell proliferation and partially rescue the root development size defects in Ror2 mutant mice. Collectively, our findings provide novel insights into the function of Ror2-mediated non-canonical Wnt signaling in regulating tooth morphogenesis, and suggest potential avenues for dental tissue engineering.

Site-specific function and regulation of Osterix in tooth root formation.

Congenital diseases of tooth roots, in terms of developmental abnormalities of short and thin root phenotypes, can lead to loss of teeth. A more complete understanding of the genetic molecular pathways and biological processes controlling tooth root formation is required. Recent studies have revealed that Osterix (Osx), a key mesenchymal transcriptional factor participating in both the processes of osteogenesis and odontogenesis, plays a vital role underlying the mechanisms of developmental differences between root and crown. During tooth development, Osx expression has been identified from late embryonic to postnatal stages when the tooth root develops, particularly in odontoblasts and cementoblasts to promote their differentiation and mineralization. Furthermore, the site-specific function of Osx in tooth root formation has been confirmed, because odontoblastic Osx-conditional knockout mice demonstrate primarily short and thin root phenotypes with no apparent abnormalities in the crown (Journal of Bone and Mineral Research 30, 2014 and 742, Journal of Dental Research 94, 2015 and 430). These findings suggest that Osx functions to promote odontoblast and cementoblast differentiation and root elongation only in root, but not in crown formation. Mechanistic research shows regulatory networks of Osx expression, which can be controlled through manipulating the epithelial BMP signalling, mesenchymal Runx2 expression and cellular phosphorylation levels, indicating feasible routes of promoting Osx expression postnatally (Journal of Cellular Biochemistry 114, 2013 and 975). In this regard, a promising approach might be available to regenerate the congenitally diseased root and that regenerative therapy would be the best choice for patients with developmental tooth diseases.

Cell dynamics in cervical loop epithelium during transition from crown to root: implications for Hertwig's epithelial root sheath formation.

BACKGROUND AND OBJECTIVE: Some clinical cases of hypoplastic tooth root are congenital. Because the formation of Hertwig's epithelial root sheath (HERS) is an important event for root development and growth, we have considered that understanding the HERS developmental mechanism contributes to elucidate the causal factors of the disease. To find integrant factors and phenomenon for HERS development and growth, we studied the proliferation and mobility of the cervical loop (CL). MATERIAL AND METHODS: We observed the cell movement of CL by the DiI labeling and organ culture system. To examine cell proliferation, we carried out immunostaining of CL and HERS using anti-Ki67 antibody. Cell motility in CL was observed by tooth germ slice organ culture using green fluorescent protein mouse. We also examined the expression of paxillin associated with cell movement. RESULTS: Imaging using DiI labeling showed that, at the apex of CL, the epithelium elongated in tandem with the growth of outer enamel epithelium (OEE). Cell proliferation assay using Ki67 immunostaining showed that OEE divided more actively than inner enamel epithelium (IEE) at the onset of HERS formation. Live imaging suggested that mobility of the OEE and cells in the apex of CL were more active than in IEE. The expression of paxillin was observed strongly in OEE and the apex of CL. CONCLUSION: The more active growth and movement of OEE cells contributed to HERS formation after reduction of the growth of IEE. The expression pattern of paxillin was involved in the active movement of OEE and HERS. The results will contribute to understand the HERS formation mechanism and elucidate the cause of anomaly root.

Molecular regulatory mechanism of root development.

TGF-beta signaling is known to function during tooth formation. The authors' study investigated the role of TGF-beta signaling during tooth root development and determined how the common mediator for TGF-beta signaling, Smad4, affected root formation in mice. Smod4 was specifically inactivated in all epidermal-derived tissues by using a two-component genetic system. The authors' findings show that when Smad4 expression is eliminated in the dental epithelium, there is lack of root formation and severe crown defects.

Antagonistic interaction between Ezh2 and Arid1a coordinates root patterning and development via Cdkn2a in mouse molars.

Patterning is a critical step during organogenesis and is closely associated with the physiological function of organs. Tooth root shapes are finely tuned to provide precise occlusal support to facilitate the function of each tooth type. However, the mechanism regulating tooth root patterning and development is largely unknown. In this study, we provide the first in vivo evidence demonstrating that Ezh2 in the dental mesenchyme determines patterning and furcation formation during dental root development in mouse molars. Mechanistically, an antagonistic interaction between epigenetic regulators Ezh2 and Arid1a controls Cdkn2a expression in the dental mesenchyme to regulate dental root patterning and development. These findings indicate the importance of balanced epigenetic regulation in determining the tooth root pattern and the integration of roots with the jaw bones to achieve physiological function. Collectively, our study provides important clues about the regulation of organogenesis and has general implications for tooth regeneration in the future.

Guanine and nucleotide binding protein 3 promotes odonto/osteogenic differentiation of apical papilla stem cells via JNK and ERK signaling pathways.

Odonto/osteogenic differentiation of stem cells from the apical papilla (SCAPs) is a key process in tooth root formation and development. However, the molecular mechanisms underlying this process remain largely unknown. In the present study, it was identified that guanine and nucleotide binding protein 3 (GNAI3) was at least in part responsible for the odonto/osteogenic differentiation of SCAPs. GNAI3 was markedly induced in mouse tooth root development in vivo and in human SCAPs mineralization in vitro. Notably, knockdown of GNAI3 by lentiviral vectors expressing short­hairpin RNAs against GNAI3 significantly inhibited the proliferation, cell cycle progression and migration of SCAPs, as well as odonto/osteogenic differentiation of SCAPs in vitro, suggesting that GNAI3 may play an essential role in tooth root development. The promotive role of GNAI3 in odonto/osteogenic differentiation was further confirmed by downregulation of odonto/osteogenic makers in GNAI3­deficient SCAPs. In addition, knockdown of GNAI3 effectively suppressed activity of c­Jun N­terminal kinase (JNK) and extracellular­signal regulated kinase (ERK) signaling pathways that was induced during SCAPs differentiation, suggesting that GNAI3 promotes SCAPs mineralization at least partially via JNK/ERK signaling. Taken together, the present results implicate GNAI3 as a critical regulator of odonto/osteogenic differentiation of SCAPs in tooth root development, and suggest a possible role of GNAI3 in regeneration processes in dentin or other tissues.

Hedgehog pathway gene expression during early development of the molar tooth root in the mouse.

Sonic hedgehog is a secreted protein important for many aspects of embryonic development. In the developing tooth, Shh expression is restricted to the epithelial compartment and plays an important role during both initiation and subsequent coronal morphogenesis. We have investigated the expression of Shh and constituent members of the signalling pathway during early development of the molar tooth root in the mouse and find the presence of transcripts in Hertwig's epithelial root sheath. These epithelial cells of the root sheath and the surrounding apical mesenchyme of the dental papilla and follicle also expressed the Shh receptor Ptc1, agonist Smo and Gli downstream transcriptional effectors; however, this response occurred over short range. In contrast, the Shh antagonists Hip1 and Gas1 were both expressed at a distance from these responding cells, in more peripheral regions of the developing root. Transcripts of the Skn acyl transferase lacked specific expression in early root structures.

Regulation of root patterns in mammalian teeth.

Mammalian teeth have diverse pattern of the crown and root. The patterning mechanism of the root position and number is relatively unknown compared to that of the crown. The root number does not always match to the cusp number, which has prevented the complete understanding of root patterning. In the present study, to elucidate the mechanism of root pattern formation, we examined (1) the pattern of cervical tongues, which are tongue-like epithelial processes extending from cervical loops, (2) factors influencing the cervical tongue pattern and (3) the relationship among patterns of cusp, cervical tongue and root in multi-rooted teeth. We found a simple mechanism of cervical tongue formation in which the lateral growth of dental mesenchyme in the cuspal region pushes the cervical loop outward, and the cervical tongue appears in the intercuspal region subsequently. In contrast, when lateral growth was physically inhibited, cervical tongue formation was suppressed. Furthermore, by building simple formulas to predict the maximum number of cervical tongues and roots based on the cusp pattern, we demonstrated a positive relationship among cusp, cervical tongue and root numbers. These results suggest that the cusp pattern and the lateral growth of cusps are important in the regulation of the root pattern.

The Impact of the Eda Pathway on Tooth Root Development.

The Eda pathway ( Eda, Edar, Edaradd) plays an important role in tooth development, determining tooth number, crown shape, and enamel formation. Here we show that the Eda pathway also plays a key role in root development. Edar (the receptor) is expressed in Hertwig's epithelial root sheath (HERS) during root development, with mutant mice showing a high incidence of taurodontism: large pulp chambers lacking or showing delayed bifurcation or trifurcation of the roots. The mouse upper second molars in the Eda pathway mutants show the highest incidence of taurodontism, this enhanced susceptibility being matched in human patients with mutations in EDA-A1. These taurodont teeth form due to defects in the direction of extension of the HERS from the crown, associated with a more extensive area of proliferation of the neighboring root mesenchyme. In those teeth where the angle at which the HERS extends from the crown is very wide and therefore more vertical, the mutant HERSs fail to reach toward the center of the tooth in the normal furcation region, and taurodont teeth are created. The phenotype is variable, however, with milder changes in angle and proliferation leading to normal or delayed furcation. This is the first analysis of the role of Eda in the root, showing a direct role for this pathway during postnatal mouse development, and it suggests that changes in proliferation and angle of HERS may underlie taurodontism in a range of syndromes.

Signaling Pathways Critical for Tooth Root Formation.

Tooth is made of an enamel-covered crown and a cementum-covered root. Studies on crown dentin formation have been a major focus in tooth development for several decades. Interestingly, the population prevalence for genetic short root anomaly (SRA) with no apparent defects in crown is close to 1.3%. Furthermore, people with SRA itself are predisposed to root resorption during orthodontic treatment. The discovery of the unique role of Nfic (nuclear factor I C; a transcriptional factor) in controlling root but not crown dentin formation points to a new concept: tooth crown and root have different control mechanisms. Further genetic mechanism studies have identified more key molecules (including Osterix, ß-catenin, and sonic hedgehog) that play a critical role in root formation. Extensive studies have also revealed the critical role of Hertwig's epithelial root sheath in tooth root formation. In addition, Wnt10a has recently been found to be linked to multirooted tooth furcation formation. These exciting findings not only fill the critical gaps in our understanding about tooth root formation but will aid future research regarding the identifying factors controlling tooth root size and the generation of a whole "bio-tooth" for therapeutic purposes. This review starts with human SRA and mainly focuses on recent progress on the roles of NFIC-dependent and NFIC-independent signaling pathways in tooth root formation. Finally, this review includes a list of the various Cre transgenic mouse lines used to achieve tooth root formation-related gene deletion or overexpression, as well as strengths and limitations of each line.

Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) expression and possible function in mouse tooth germ development.

Abnormal expression of Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is involved in the pathogenesis of FSHD. FRG1 is also important for the normal muscular and vascular development. Our previous study showed that FRG1 is one of the highly expressed genes in the mandible on embryonic day 10.5 (E10.5) than on E12.0. In this study, we investigated the temporospatial expression pattern of FRG1 mRNA and protein during the development of the mouse lower first molar, and also evaluated the subcellular localization of the FRG1 protein in mouse dental epithelial (mDE6) cells. The FRG1 expression was identified in the dental epithelial and mesenchymal cells at the initiation and bud stages. It was detected in the inner enamel epithelium at the cap and early bell stages. At the late bell and root formation stages, these signals were detected in ameloblasts and odontoblasts during the formation of enamel and dentin matrices, respectively. The FRG1 protein was localized in the cytoplasm in the mouse tooth germ in vivo, while FRG1 was detected predominantly in the nucleus and faintly in the cytoplasm in mDE6 cells in vitro. In mDE6 cells treated with bone morphogenetic protein 4 (BMP4), the protein expression of FRG1 increased in cytoplasm, suggesting that FRG1 may translocate to the cytoplasm. These findings suggest that FRG1 is involved in the morphogenesis of the tooth germ, as well as in the formation of enamel and dentin matrices and that FRG1 may play a role in the odontogenesis in the mouse following BMP4 stimulation.

Altered distribution of Ghrelin protein in mice molar development.

OBJECTIVE: Ghrelin, an appetite-stimulating hormone, plays diverse regulatory functions in cell growth, proliferation, differentiation and apoptosis during mammalian development. There is limited information currently available regarding Ghrelin expression during mammalian tooth development, thus we aimed to establish the spatiotemporal expression of Ghrelin during murine molar odontogenesis. DESIGN: Immunohistochemistry was performed to detect the expression pattern of Ghrelin in mandible molar from E15.5 to PN7 during murine tooth development. RESULTS: The results showed that Ghrelin initially expressed in the inner enamel epithelium and the adjacent mesenchymal cells below, further with persistent expression in the ameloblasts and odontoblasts throughout the following developmental stages. In addition, Ghrelin was also present in Hertwig's epithelial root sheath at the beginning of tooth root formation. CONCLUSIONS: These results suggest that Ghrelin was present in tooth organs throughout the stages of tooth development, especially in ameloblasts and odontoblasts with little spatiotemporal expression differences. However, the potential regulatory roles of this hormone in tooth development still need to be validated by functional studies.

Odontoblast ß-catenin signaling regulates fenestration of mouse Hertwig's epithelial root sheath.

The interaction between Hertwig's epithelial root sheath (HERS) and the adjacent mesenchyme is vitally important in mouse tooth root development. We previously generated odontoblast-specific Ctnnb1 (encoding ß-catenin) deletion mice, and demonstrated that odontoblast ß-catenin signaling regulates odontoblast proliferation and differentiation. However, the role of odontoblast ß-catenin signaling in regulation of HERS behavior has not been fully investigated. Here, using the same odontoblast- specific Ctnnb1 deletion mice, we found that ablation of ß-catenin signaling in odontoblasts led to aberrant HERS formation. Mechanistically, odontoblast-specific Ctnnb1 deletion resulted in elevated bone morphogenetic protein 7 (Bmp7) expression and reduced expression of noggin and follistatin, both of which encode extracellular inhibitors of BMPs. Furthermore, the levels of phosphorylated Smad1/5/8 were increased in HERS cells. In vitro tissue culture confirmed that BMP7 treatment disrupted the HERS structure. Taken together, we demonstrated that odontoblast ß-catenin signaling may act through regulation of BMP signaling to maintain the integrity of HERS cells.

Development and evolution of dentition pattern and tooth order in the skates and rays (batoidea; chondrichthyes).

Shark and ray (elasmobranch) dentitions are well known for their multiple generations of teeth, with isolated teeth being common in the fossil record. However, how the diverse dentitions characteristic of elasmobranchs form is still poorly understood. Data on the development and maintenance of the dental patterning in this major vertebrate group will allow comparisons to other morphologically diverse taxa, including the bony fishes, in order to identify shared pattern characters for the vertebrate dentition as a whole. Data is especially lacking from the Batoidea (skates and rays), hence our objective is to compile data on embryonic and adult batoid tooth development contributing to ordering of the dentition, from cleared and stained specimens and micro-CT scans, with 3D rendered models. We selected species (adult and embryonic) spanning phylogenetically significant batoid clades, such that our observations may raise questions about relationships within the batoids, particularly with respect to current molecular-based analyses. We include developmental data from embryos of recent model organisms Leucoraja erinacea and Raja clavata to evaluate the earliest establishment of the dentition. Characters of the batoid dentition investigated include alternate addition of teeth as offset successional tooth rows (versus single separate files), presence of a symphyseal initiator region (symphyseal tooth present, or absent, but with two parasymphyseal teeth) and a restriction to tooth addition along each jaw reducing the number of tooth families, relative to addition of successor teeth within each family. Our ultimate aim is to understand the shared characters of the batoids, and whether or not these dental characters are shared more broadly within elasmobranchs, by comparing these to dentitions in shark outgroups. These developmental morphological analyses will provide a solid basis to better understand dental evolution in these important vertebrate groups as well as the general plesiomorphic vertebrate dental condition.

Calbindin-D28 kappa localization in rat molars during odontogenesis.

Specific antiserum raised against Calbindin-D28 kappa, a vitamin D-dependent calcium-binding protein (CaBP) isolated from chick intestine, was used for localization of the protein in developing rat molars. Previously, CaBP had been localized in specific cells associated with the continuously erupting rat incisor: late pre-secretory ameloblasts, secretory and maturation zone ameloblasts, stratum intermedium cells adjacent to ameloblasts in the late zone of enamel secretion, and papillary cells underlying maturation zone ameloblasts. In this study, the peroxidase anti-peroxidase technique was used for localization of the peroxidase anti-peroxidase technique was used for localization of CaBP in histological sections of rat mandibles from 18-day-old rat embryos through 20-day-old neonates. CaBP was not detected in any cells of the enamel organ, dental papilla, or dental sac during early odontogenesis from the dental lamina stage through the advanced bell stage. The protein first appeared in secretory ameloblasts which were situated opposite odontoblasts with newly secreted dentin. CaBP was present in the cytoplasm of more mature ameloblasts, but not in less mature ameloblasts. Some stratum intermedium cells subjacent to well-developed secretory and maturation zone ameloblasts also contained CaBP. The protein was not detected in odontoblasts, pulp cells, or other cells associated with the developing molars. It was also absent from the demineralized enamel and dentin matrix. In developing rat molars, the time-course of appearance of CaBP, a protein dependent for its synthesis on the vitamin D endocrine system in other organ systems, suggests a potential direct role of this hormonal system in enamel mineralization.

A new look at the rests of Malassez. A review of their embryological origin, anatomy, and possible role in periodontal health and disease.

The embryological formation and anatomy of the rests of Malassez are described and their possible roles of health and disease are discussed. It is emphasized that the rests are worthy of new interest and further study. They comprise a structure whose precise anatomy still is in need of accurate description and in which the presence or absence of a physiological role is unproven.

Development and cell fate in interspecific (Mus musculus/Mus caroli) intraocular transplants of mouse molar tooth-germ tissues detected by in situ hybridization.

Mandibular first molar tooth germs were dissected from Mus musculus (CDI) and Mus caroli (age range: 14-day embryo to 1-day postnatal). Most of the tooth germs were separated enzymically into epithelial and mesenchymal components. Interspecific tissue recombinations and intact M. caroli tooth germs were grown in the anterior chamber of the eye of adult CDI mice for 24 weeks. Recombinations of M. caroli enamel-organ epithelium with M. musculus, dental papilla and follicle mesenchyme developed into normal teeth with advanced root, periodontal ligament and bone formation, thereby confirming extensive epithelial-mesenchymal interactions across the species barrier. Labelling sections by in situ hybridization with a M. musculus-specific DNA probe (pMSat5) showed that almost all cells in the pulp, periodontal ligament and bone were M. musculus, including cementoblasts. Reduced enamel epithelium and epithelial cell rests derived from donor M. caroli enamel organ were unlabelled. This indicates that any cementogenic role of Hertwig's epithelial root sheath must be short-lived. The immunological privilege of the intraocular transplantation site in M. musculus CDI mice did not extend to grafts including xenogeneic M. caroli dental mesenchyme. Thus, intact M. caroli tooth germs and recombinations of M. musculus enamel organ with M. caroli dental papilla and follicle showed limited development, with no root formation, and were populated almost exclusively with labelled host M. musculus lymphocytes.

[The cellular and molecular bases of "tooth framing"]. / Le basi cellulari e molecolari della "costruzione" dei denti.

Progress in molecular biology in recent years has enormously increased interest in tooth generation. The enamel knot has been discovered, in consequence. This is a transient structure acting as molecular signaling center, responsible for controlling cusp formation, stimulating growth of surrounding epithelium, and generating new knots or their disappearance through apoptosis. Both tooth development and enamel knots are regulated by a cascade of gene activity where Fgf4, Shh, BMP4, Lef1 and p21 are the prime movers of the processes. Homeobox genes (Msx, Dlx) are the orchestrators of the framing and a series of proteins (adhesion molecules, extracellular matrix components) are the executors of "tooth framing". An important concept has emerged from developmental biology through the identification of the basic mechanisms involved in tooth development: the molecular basis of structure framing shares common rules. Thus similar genetic programs are involved in body structure generation (limb bud, tooth, branching morphogenesis). A deeper understanding of developmental rules regulating tooth formation will make it possible in the near future: a) to modify in vivo homeobox gene expression and restore tooth generation hampered by tooth agenesia due to homeobox gene deregulation; b) to induce complete tooth formation, in case of tooth loss due to trauma or diseases, through implantation in the patient's oral cavity of a synthetic ball containing morphogens and growth factors to stimulate, in the right spatio-temporal sequence, the entire tooth genetic cascade. These concepts will certainly enforce cultural and practical interaction between biology and dentistry.

Molecular regulatory mechanism of tooth root development.

The root is crucial for the physiological function of the tooth, and a healthy root allows an artificial crown to function as required clinically. Tooth crown development has been studied intensively during the last few decades, but root development remains not well understood. Here we review the root development processes, including cell fate determination, induction of odontoblast and cementoblast differentiation, interaction of root epithelium and mesenchyme, and other molecular mechanisms. This review summarizes our current understanding of the signaling cascades and mechanisms involved in root development. It also sets the stage for de novo tooth regeneration.

Morphogenetic roles of perlecan in the tooth enamel organ: an analysis of overexpression using transgenic mice.

Perlecan, a heparan sulfate proteoglycan, is enriched in the intercellular space of the enamel organ. To understand the role of perlecan in tooth morphogenesis, we used a keratin 5 promoter to generate transgenic (Tg) mice that over-express perlecan in epithelial cells, and examined their tooth germs at tissue and cellular levels. Immunohistochemistry showed that perlecan was more strongly expressed in the enamel organ cells of Tg mice than in wild-type mice. Histopathology showed wider intercellular spaces in the stellate reticulum of the Tg molars and loss of cellular polarity in the enamel organ, especially in its cervical region. Hertwig's epithelial root sheath (HERS) cells in Tg mice were irregularly aligned due to excessive deposits of perlecan along the inner, as well as on the outer sides of the HERS. Tg molars had dull-ended crowns and outward-curved tooth roots and their enamel was poorly crystallized, resulting in pronounced attrition of molar cusp areas. In Tg mice, expression of integrin ß1 mRNA was remarkably higher at E18, while expression of bFGF, TGF-ß1, DSPP and Shh was more elevated at P1. The overexpression of perlecan in the enamel organ resulted in irregular morphology of teeth, suggesting that the expression of perlecan regulates growth factor signaling in a stage-dependent manner during each step of the interaction between ameloblast-lineage cells and mesenchymal cells.