WNT10A mutation results in severe tooth agenesis in a family of three sisters.

OBJECTIVES: To identify the genetic basis of severe tooth agenesis in a family of three affected sisters. PATIENTS AND METHODS: A family of three sisters with severe tooth agenesis was recruited for whole-exome sequencing to identify potential genetic variation responsible for this penetrant phenotype. The unaffected father was tested for specific mutations using Sanger sequencing. Gene discovery was supplemented with in situ hybridization to localize gene expression during human tooth development. RESULTS: We report a nonsense heterozygous mutation in exon 2 of WNT10A c.321C>A[p.Cys107*] likely to be responsible for the severe tooth agenesis identified in this family through the creation of a premature stop codon, resulting in truncation of the amino acid sequence and therefore loss of protein function. In situ hybridization showed expression of WNT10A in odontogenic epithelium during the early and late stages of human primary tooth development. CONCLUSIONS: WNT10A has previously been associated with both syndromic and non-syndromic forms of tooth agenesis, and this report further expands our knowledge of genetic variation underlying non-syndromic forms of this condition. We also demonstrate expression of WNT10A in the epithelial compartment of human tooth germs during development.

Redefining Mucosal Inflammation with Spatial Genomics.

The human oral mucosa contains one of the most complex cellular systems that are essential for normal physiology and defense against a wide variety of local pathogens. Evolving techniques and experimental systems have helped refine our understanding of this complex cellular network. Current single-cell RNA sequencing methods can resolve subtle differences between cell types and states, thus providing a great tool for studying the molecular and cellular repertoire of the oral mucosa in health and disease. However, it requires the dissociation of tissue samples, which means that the interrelationships between cells are lost. Spatial transcriptomic methods bypass tissue dissociation and retain this spatial information, thereby allowing gene expression to be assessed across thousands of cells within the context of tissue structural organization. Here, we discuss the contribution of spatial technologies in shaping our understanding of this complex system. We consider the impact on identifying disease cellular neighborhoods and how space defines cell state. We also discuss the limitations and future directions of spatial sequencing technologies with recent advances in machine learning. Finally, we offer a perspective on open questions about mucosal homeostasis that these technologies are well placed to address.

Computational Methods for Image Analysis in Craniofacial Development and Disease.

Observation is at the center of all biological sciences. Advances in imaging technologies are therefore essential to derive novel biological insights to better understand the complex workings of living systems. Recent high-throughput sequencing and imaging techniques are allowing researchers to simultaneously address complex molecular variations spatially and temporarily in tissues and organs. The availability of increasingly large dataset sizes has allowed for the evolution of robust deep learning models, designed to interrogate biomedical imaging data. These models are emerging as transformative tools in diagnostic medicine. Combined, these advances allow for dynamic, quantitative, and predictive observations of entire organisms and tissues. Here, we address 3 main tasks of bioimage analysis, image restoration, segmentation, and tracking and discuss new computational tools allowing for 3-dimensional spatial genomics maps. Finally, we demonstrate how these advances have been applied in studies of craniofacial development and oral disease pathogenesis.

A mathematical model for mechanotransduction at the early steps of suture formation.

Growth and patterning of craniofacial sutures is subjected to the effects of mechanical stress. Mechanotransduction processes occurring at the margins of the sutures are not precisely understood. Here, we propose a simple theoretical model based on the orientation of collagen fibres within the suture in response to local stress. We demonstrate that fibre alignment generates an instability leading to the emergence of interdigitations. We confirm the appearance of this instability both analytically and numerically. To support our model, we use histology and synchrotron X-ray microtomography and reveal the fine structure of fibres within the sutural mesenchyme and their insertion into the bone. Furthermore, using a mouse model with impaired mechanotransduction, we show that the architecture of sutures is disturbed when forces are not interpreted properly. Finally, by studying the structure of sutures in the mouse, the rat, an actinopterygian (Polypterus bichir) and a placoderm (Compagopiscis croucheri), we show that bone deposition patterns during dermal bone growth are conserved within jawed vertebrates. In total, these results support the role of mechanical constraints in the growth and patterning of craniofacial sutures, a process that was probably effective at the emergence of gnathostomes, and provide new directions for the understanding of normal and pathological suture fusion.

Shape and volume of craniofacial cavities in intentional skull deformations.

Intentional cranial deformations (ICD) have been observed worldwide but are especially prevalent in preColombian cultures. The purpose of this study was to assess the consequences of ICD on three cranial cavities (intracranial cavity, orbits, and maxillary sinuses) and on cranial vault thickness, in order to screen for morphological changes due to the external constraints exerted by the deformation device. We acquired CT-scans for 39 deformed and 19 control skulls. We studied the thickness of the skull vault using qualitative and quantitative methods. We computed the volumes of the orbits, of the maxillary sinuses, and of the intracranial cavity using haptic-aided semi-automatic segmentation. We finally defined 3D distances and angles within orbits and maxillary sinuses based on 27 anatomical landmarks and measured these features on the 58 skulls. Our results show specific bone thickness patterns in some types of ICD, with localized thinning in regions subjected to increased pressure and thickening in other regions. Our findings confirm that volumes of the cranial cavities are not affected by ICDs but that the shapes of the orbits and of the maxillary sinuses are modified in circumferential deformations. We conclude that ICDs can modify the shape of the cranial cavities and the thickness of their walls but conserve their volumes. These results provide new insights into the morphological effects associated with ICDs and call for similar investigations in subjects with deformational plagiocephalies and craniosynostoses.

GSK3 Inhibitor-Induced Dentinogenesis Using a Hydrogel.

Small-molecule drugs targeting glycogen synthase kinase 3 (GSK3) as inhibitors of the protein kinase activity are able to stimulate reparative dentine formation. To develop this approach into a viable clinical treatment for exposed pulp lesions, we synthesized a novel, small-molecule noncompetitive adenosine triphosphate (ATP) drug that can be incorporated into a biodegradable hydrogel for placement by syringe into the tooth. This new drug, named NP928, belongs to the thiadiazolidinone (TDZD) family and has equivalent activity to similar drugs of this family such as tideglusib. However, NP928 is more water soluble than other TDZD drugs, making it more suitable for direct delivery into pulp lesions. We have previously reported that biodegradable marine collagen sponges can successfully deliver TDZD drugs to pulp lesions, but this involves in-theater preparation of the material, which is not ideal in a clinical context. To improve surgical handling and delivery, here we incorporated NP928 into a specifically tailored hydrogel that can be placed by syringe into a damaged tooth. This hydrogel is based on biodegradable hyaluronic acid and can be gelled in situ upon dental blue light exposure, similarly to other common dental materials. NP928 released from hyaluronic acid-based hydrogels upregulated Wnt/ß-catenin activity in pulp stem cells and fostered reparative dentine formation compared to marine collagen sponges delivering equivalent concentrations of NP928. This drug-hydrogel combination has the potential to be rapidly developed into a therapeutic procedure that is amenable to general dental practice.

Scaffold-based developmental tissue engineering strategies for ectodermal organ regeneration.

Tissue engineering (TE) is a multidisciplinary research field aiming at the regeneration, restoration, or replacement of damaged tissues and organs. Classical TE approaches combine scaffolds, cells and soluble factors to fabricate constructs mimicking the native tissue to be regenerated. However, to date, limited success in clinical translations has been achieved by classical TE approaches, because of the lack of satisfactory biomorphological and biofunctional features of the obtained constructs. Developmental TE has emerged as a novel TE paradigm to obtain tissues and organs with correct biomorphology and biofunctionality by mimicking the morphogenetic processes leading to the tissue/organ generation in the embryo. Ectodermal appendages, for instance, develop in vivo by sequential interactions between epithelium and mesenchyme, in a process known as secondary induction. A fine artificial replication of these complex interactions can potentially lead to the fabrication of the tissues/organs to be regenerated. Successful developmental TE applications have been reported, in vitro and in vivo, for ectodermal appendages such as teeth, hair follicles and glands. Developmental TE strategies require an accurate selection of cell sources, scaffolds and cell culture configurations to allow for the correct replication of the in vivo morphogenetic cues. Herein, we describe and discuss the emergence of this TE paradigm by reviewing the achievements obtained so far in developmental TE 3D scaffolds for teeth, hair follicles, and salivary and lacrimal glands, with particular focus on the selection of biomaterials and cell culture configurations.

Dlx homeobox gene family expression in osteoclasts.

Skeletal growth and homeostasis require the finely orchestrated secretion of mineralized tissue matrices by highly specialized cells, balanced with their degradation by osteoclasts. Time- and site-specific expression of Dlx and Msx homeobox genes in the cells secreting these matrices have been identified as important elements in the regulation of skeletal morphology. Such specific expression patterns have also been reported in osteoclasts for Msx genes. The aim of the present study was to establish the expression patterns of Dlx genes in osteoclasts and identify their function in regulating skeletal morphology. The expression patterns of all Dlx genes were examined during the whole osteoclastogenesis using different in vitro models. The results revealed that Dlx1 and Dlx2 are the only Dlx family members with a possible function in osteoclastogenesis as well as in mature osteoclasts. Dlx5 and Dlx6 were detected in the cultures but appear to be markers of monocytes and their derivatives. In vivo, Dlx2 expression in osteoclasts was examined using a Dlx2/LacZ transgenic mouse. Dlx2 is expressed in a subpopulation of osteoclasts in association with tooth, brain, nerve, and bone marrow volumetric growths. Altogether the present data suggest a role for Dlx2 in regulation of skeletal morphogenesis via functions within osteoclasts.

Translation Approach for Dentine Regeneration Using GSK-3 Antagonists.

The canonical Wnt/ß-catenin signaling pathway is crucial for reparative dentinogenesis following tooth damage, and the modulation of this pathway affects the rate and extent of reparative dentine formation in damaged mice molars by triggering the natural process of dentinogenesis. Pharmacological stimulation of Wnt/ß-catenin signaling activity by small-molecule GSK-3 inhibitor drugs following pulp exposure in mouse molars results in reparative dentinogenesis. The creation of similar but larger lesions in rat molars shows that the adenosine triphosphate (ATP)-competitive GSK-3 inhibitor, CHIR99021 (CHIR), and the ATP noncompetitive inhibitor, Tideglusib (TG), can equally enhance reparative dentine formation to fully repair an area of dentine damage up to 10 times larger, mimicking the size of small lesions in humans. To assess the chemical composition of this newly formed dentine and to compare its structure with surrounding native dentine and alveolar bone, Raman microspectroscopy analysis is used. We show that the newly formed dentine comprises equal carbonate to phosphate ratios and mineral to matrix ratios to that of native dentine, both being significantly different from bone. For an effective dentine repair, the activity of the drugs needs to be restricted to the region of damage. To investigate the range of drug-induced Wnt-activity within the dental pulp, RNA of short-term induced (24-h) molars is extracted from separated roots and crowns, and quantitative Axin2 expression is assayed. We show that the activation of Wnt/ß-catenin signaling is highly restricted to pulp cells in the immediate location of the damage in the coronal pulp tissue with no drug action detected in the root pulp. These results provide further evidence that this simple method of enhancement of natural reparative dentinogenesis has the potential to be translated into a clinical direct capping approach.

Transformation of tooth type induced by inhibition of BMP signaling.

Mammalian dentitions are highly patterned, with different types of teeth positioned in different regions of the jaws. BMP4 is an early oral epithelial protein signal that directs odontogenic gene expression in mesenchyme cells of the developing mandibular arch. BMP4 was shown to inhibit expression of the homeobox gene Barx-1 and to restrict expression to the proximal, presumptive molar mesenchyme of mouse embryos at embryonic day 10. The inhibition of BMP signaling early in mandible development by the action of exogenous Noggin protein resulted in ectopic Barx-1 expression in the distal, presumptive incisor mesenchyme and a transformation of tooth identity from incisor to molar.

Perivascular-Derived Mesenchymal Stem Cells.

Cells have been identified in postnatal tissues that, when isolated from multiple mesenchymal compartments, can be stimulated in vitro to give rise to cells that resemble mature mesenchymal phenotypes, such as odontoblasts, osteoblasts, adipocytes, and myoblasts. This has made these adult cells, collectively called mesenchymal stem cells (MSCs), strong candidates for fields such as tissue engineering and regenerative medicine. Based on evidence from in vivo genetic lineage-tracing studies, pericytes have been identified as a source of MSC precursors in vivo in multiple organs, in response to injury or during homeostasis. Questions of intense debate and interest in the field of tissue engineering and regenerative studies include the following: 1) Are all pericytes, irrespective of tissue of isolation, equal in their differentiation potential? 2) What are the mechanisms that regulate the differentiation of MSCs? To gain a better understanding of the latter, recent work has utilized ChIP-seq (chromatin immunoprecipitation followed by sequencing) to reconstruct histone landscapes. This indicated that for dental pulp pericytes, the odontoblast-specific gene Dspp was found in a transcriptionally permissive state, while in bone marrow pericytes, the osteoblast-specific gene Runx2 was primed for expression. RNA sequencing has also been utilized to further characterize the 2 pericyte populations, and results highlighted that dental pulp pericytes are already precommitted to an odontoblast fate based on enrichment analysis indicating overrepresentation of key odontogenic genes. Furthermore, ChIP-seq analysis of the polycomb repressive complex 1 component RING1B indicated that this complex is likely to be involved in inhibiting inappropriate differentiation, as it localized to a number of loci of key transcription factors that are needed for the induction of adipogenesis, chondrogenesis, or myogenesis. In this review, we highlight recent data elucidating molecular mechanisms that indicate that pericytes can be tissue-specific precommitted MSC precursors in vivo and that this precommitment is a major driving force behind MSC differentiation.

Tooth Bioengineering and Regenerative Dentistry.

Over the past 100 y, tremendous progress has been made in the fields of dental tissue engineering and regenerative dental medicine, collectively known as translational dentistry. Translational dentistry has benefited from the more mature field of tissue engineering and regenerative medicine (TERM), established on the belief that biocompatible scaffolds, cells, and growth factors could be used to create functional, living replacement tissues and organs. TERM, created and pioneered by an interdisciplinary group of clinicians, biomedical engineers, and basic research scientists, works to create bioengineered replacement tissues that provide at least enough function for patients to survive until donor organs are available and, at best, fully functional replacement organs. Ultimately, the goal of both TERM and regenerative dentistry is to bring new and more effective therapies to the clinic to treat those in need. Very recently, the National Institutes of Health/National Institute of Dental and Craniofacial Research invested $24 million over a 3-y period to create dental oral and craniofacial translational resource centers to facilitate the development of more effective therapies to treat edentulism and other dental-related diseases over the next decade. This exciting era in regenerative dentistry, particularly for whole-tooth tissue engineering, builds on many key successes over the past 100 y that have contributed toward our current knowledge and understanding of signaling pathways directing natural tooth and dental tissue development-the foundation for current strategies to engineer functional, living replacement dental tissues and whole teeth. Here we use a historical perspective to present key findings and pivotal advances made in the field of translational dentistry over the past 100 y. We will first describe how this process has evolved over the past 100 y and then hypothesize on what to expect over the next century.

A Mouse Model to Study Reparative Dentinogenesis.

Different animal models have been introduced recently to study the process of reparative dentinogenesis in response to injury-induced pulp exposure. Using a mouse model is advantageous over other animal models since mice can be genetically manipulated to examine specific cellular pathways and lineage trace the progeny of a single cell. However, enabling a standardized molar damage in mice is demanding due to the small size of the teeth compared to the available dental instruments. Here we describe a reproducible and reliable in vivo model that allows us to study dentinogenesis in the first maxillary mouse molar.

Tooth agenesis: from molecular genetics to molecular dentistry.

Tooth agenesis may originate from either genetic or environmental factors. Genetically determined hypodontic disorders appear as isolated features or as part of a syndrome. Msx1, Pax9, and Axin2 are involved in non-syndromic hypodontia, while genes such as Shh, Pitx2, Irf6, and p63 are considered to participate in syndromic genetic disorders, which include tooth agenesis. In dentistry, artificial tooth implants represent a common solution to tooth loss problems; however, molecular dentistry offers promising solutions for the future. In this paper, the genetic and molecular bases of non-syndromic and syndromic hypodontia are reviewed, and the advantages and disadvantages of tissue engineering in the clinical treatment of tooth agenesis are discussed.

Regulation of Reactionary Dentine Formation.

During the treatment of dental caries that has not penetrated the tooth pulp, maintenance of as much unaffected dentine as possible is a major goal during the physical removal of decayed mineral. Damage to dentine leads to release of fossilized factors (transforming growth factor-ß [TGF-ß] and bone morphogenic protein [BMP]) in the dentine that are believed to stimulate odontoblasts to secrete new "tertiary" dentine (reactionary dentine). This is formed on the pulpal surface of existing dentine and rethickens the dentine. We have previously shown that activation of Wnt/ß-catenin signaling is pivotal for tooth repair in exposed pulp injury, and the pathway can be activated by small-molecule GSK-3 antagonists, resulting in enhanced reparative dentine formation. Here, we use a nonexposed pulp injury model to investigate the mechanisms of reactionary dentine formation in vivo, using small molecules to modulate the Wnt/ß-catenin, TGF-ß, and BMP pathways. We found that a local increase of Wnt activation at the injury site enhances reactionary dentine secretion. In addition, inhibition of TGF-ß, BMP, or Wnt pathways does not impede reactionary dentine formation, although inhibition of TGF-ß and/or BMP signaling does result in more disorganized, nontubular reactionary dentine. This suggests that Wnt/ß-catenin signaling plays no major role in the formation of reactionary dentine, but in common with reparative dentine formation, exogenous elevation of Wnt/ß-catenin signaling can enhance tertiary dentine formation. Release of latent TGF-ß or BMPs from dentine is not required for the deposition of mineral to form reactionary dentine but does play a role in its organization.

Fish scale development: Hair today, teeth and scales yesterday?

A group of genes in the tumour necrosis factor signalling pathway are mutated in humans and mice with ectodermal dysplasias--a failure of hair and tooth development. A mutation has now been identified in one of these genes, ectodysplasin-A receptor, in the teleost fish Medaka, that results in a failure of scale formation.

Primary enamel knot cell death in Apaf-1 and caspase-9 deficient mice.

During molar development, apoptosis occurs in a well-characterised pattern suggesting several roles for cell death in odontogenesis. However, molecular mechanisms of dental apoptosis are only poorly understood. In this study, Apaf-1 and caspase-9 knockouts were used to uncover the engagement of these members of the apoptotic machinery during early tooth development, concentrating primarily on their function in the apoptotic elimination of primary enamel knot cells. Molar tooth germ morphology, proliferation and apoptosis were investigated on frontal histological sections of murine heads at embryonic days (ED) 15.5, the stage when the primary enamel knot is eliminated apoptotically. In molar tooth germs of both knockouts, no apoptosis was observed according to morphological (haematoxylin-eosin) as well as biochemical criteria (TUNEL). Morphology of the mutant tooth germs, however, was not changed. Additionally, knockout mice showed no changes in proliferation compared to wild type mice. According to our findings on knockout embryos, Apaf-1 and caspase-9 are involved in apoptosis during tooth development; however, they seem dispensable and not necessary for proper tooth shaping. Compensatory or other mechanisms of cell death may act to eliminate the primary enamel knot cells in the absence of Apaf-1 and caspase-9.

Mesenchymal Cell Community Effect in Whole Tooth Bioengineering.

In vitro expanded cell populations can contribute to bioengineered tooth formation but only as cells that respond to tooth-inductive signals. Since the success of whole tooth bioengineering is predicated on the availability of large numbers of cells, in vitro cell expansion of tooth-inducing cell populations is an essential requirement for further development of this approach. We set out to investigate if the failure of cultured mesenchyme cells to form bioengineered teeth might be rescued by the presence of uncultured cells. To test this, we deployed a cell-mixing approach to evaluate the contributions of cell populations to bioengineered tooth formation. Using genetically labeled cells, we are able to identify the formation of tooth pulp cells and odontoblasts in bioengineered teeth. We show that although cultured embryonic dental mesenchyme cells are unable to induce tooth formation, they can contribute to tooth induction and formation if combined with noncultured cells. Moreover, we show that teeth can form from cell mixtures that include embryonic cells and populations of postnatal dental pulp cells; however, these cells are unable to contribute to the formation of pulp cells or odontoblasts, and at ratios of 1:1, they inhibit tooth formation. These results indicate that although in vitro cell expansion of embryonic tooth mesenchymal cells renders them unable to induce tooth formation, they do not lose their ability to contribute to tooth formation and differentiate into odontoblasts. Postnatal pulp cells, however, lose all tooth-inducing and tooth-forming capacity following in vitro expansion, and at ratios >1:3 postnatal:embryonic cells, they inhibit the ability of embryonic dental mesenchyme cells to induce tooth formation.

Non-syndromic severe hypodontia caused by a novel frameshift insertion mutation in the homeobox of the MSX1 gene.

OBJECTIVE: Inherited congenital anomalies in tooth number, particularly hypodontia are relatively common. Although substantial progress has been made that permits a better understanding of the causes of tooth agenesis, overall knowledge of the phenotype:genotype correlations in this anomaly are still lacking. The aim in this study was to identify the causal gene mutation(s) in a family of two sisters with severe hypodontia (oligodontia) including 2nd premolars and 1st and 3rd molars, using whole exome sequencing (WES). METHODS: WES was performed using in-solution hybridization, followed by massively parallel sequencing. RESULTS: A frameshift insertion of 7 basepairs (GCAAGTT) in the homebox of MSX1 gene located in the exon 2 in heterozygous state has been identified in both sisters (NM_002448:exon2:c.572_573ins GCAAGTT: p.F191fs). CONCLUSION: We conclude that this frameshift mutation in the homeodomain (which plays an essential role in DNA binding) of MSX1 gene is responsible for tooth agenesis in this family. This expands the phenotype-genotype correlation associated with MSX1 mutations.

The evolution of WT1 sequence and expression pattern in the vertebrates.

WT1 is a Wilms' tumour predisposition gene, encoding a protein with four C-terminal Kruppel-type zinc fingers, which is also a major regulator of kidney and gonadal development. To pinpoint key regulatory domains involved in development and evolution of the vertebrate genitourinary system, we have isolated WT1 orthologues from all gnathostome classes. Partial nucleotide sequence from chick, alligator, Xenopus laevis and zebrafish reveals extensive conservation. Both the zinc fingers and the transregulatory domain exhibit a high level of similarity in all the species examined. However, of the two alternatively spliced regions only one, the three amino acid KTS insertion between zinc fingers 3 and 4, is found in species other than mammals. The 17 amino acid insertion at the C-terminal end of the transregulatory domain is present only in mammals. Residues with reported human pathological mutations are also unaltered across species, underlining their structural significance. Studies in chick and alligator reveal that the mammalian intermediate mesoderm expression pattern is conserved in birds and reptiles. A wider role in mesodermal differentiation is suggested by expression in some paraxial and lateral mesoderm derivatives.