ABSTRACT
OBJECTIVES: To identify the molecular etiology of distinct dental anomalies found in eight Thai patients and explore the mutational effects on cellular functions. MATERIALS AND METHODS: Clinical and radiographic examinations were performed for eight patients. Whole exome sequencing, mutant protein modelling, qPCR, western blot analysis, scratch assays, immunofluorescence, confocal analysis, in situ hybridization, and scanning electron micrography of teeth were done. RESULTS: All patients had molars with multiple supernumerary cusps, single-cusped premolars, and a reduction in root number. Mutation analysis highlighted a heterozygous c.865A>G; p.Ile289Val mutation in CACNA1S in the patients. CACNA1S is a component of the slowly inactivating L-type voltage-dependent calcium channel. Mutant protein modeling suggested that the mutation might allow leakage of Ca2+ or other cations, or a tightening, to restrict calcium flow. Immunohistochemistry analysis showed expression of Cacna1s in the developing murine tooth epithelium during stages of crown and root morphogenesis. In cell culture, the mutation resulted in abnormal cell migration of transfected CHO cells compared to wildtype CACNA1S, with changes to the cytoskeleton and markers of focal adhesion. CONCLUSIONS: The malformations observed in our patients suggest a role for calcium signaling in organization of both cusps and roots, affecting cell dynamics within the dental epithelium.
ABSTRACT
The cranial base exerts a supportive role for the brain and includes the occipital, sphenoid and ethmoid bones that arise from cartilaginous precursors in the early embryo. As the occipital bone and the posterior part of the sphenoid are mesoderm derivatives that arise in close proximity to the notochord and floor plate, it has been assumed that their development, like the axial skeleton, is dependent on Sonic hedgehog (Shh) and modulation of bone morphogenetic protein (Bmp) signalling. Here we examined the development of the cranial base in chick and mouse embryos to compare the molecular signals that are required for chondrogenic induction in the trunk and head. We found that Shh signalling is required but the molecular network controlling cranial base development is distinct from that in the trunk. In the absence of Shh, the presumptive cranial base did not undergo chondrogenic commitment as determined by the loss of Sox9 expression and there was a decrease in cell survival. In contrast, induction of the otic capsule occurred normally demonstrating that induction of the cranial base is uncoupled from formation of the sensory capsules. Lastly, we found that the early cranial mesoderm is refractory to Shh signalling, likely accounting for why development of the cranial base occurs after the axial skeleton. Our data reveal that cranial and axial skeletal induction is controlled by conserved, yet spatiotemporally distinct mechanisms that co-ordinate development of the cranial base with that of the cranial musculature and the pharyngeal arches.
Subject(s)
Bone and Bones/embryology , Gene Expression Regulation, Developmental , Hedgehog Proteins/metabolism , Signal Transduction , Skull/embryology , Animals , Bone Morphogenetic Proteins/genetics , Bone Morphogenetic Proteins/metabolism , Bone and Bones/metabolism , Chick Embryo , Chickens , Embryo, Mammalian/metabolism , Hedgehog Proteins/genetics , Mesoderm/metabolism , Mice , Skull/metabolismABSTRACT
Whether snakes evolved their elongated, limbless bodies or their specialized skulls and teeth first is a central question in squamate evolution. Identifying features shared between extant and fossil snakes is therefore key to unraveling the early evolution of this iconic reptile group. One promising candidate is their unusual mode of tooth replacement, whereby teeth are replaced without signs of external tooth resorption. We reveal through histological analysis that the lack of resorption pits in snakes is due to the unusual action of odontoclasts, which resorb dentine from within the pulp of the tooth. Internal tooth resorption is widespread in extant snakes, differs from replacement in other reptiles, and is even detectable via non-destructive µCT scanning, providing a method for identifying fossil snakes. We then detected internal tooth resorption in the fossil snake Yurlunggur, and one of the oldest snake fossils, Portugalophis, suggesting that it is one of the earliest innovations in Pan-Serpentes, likely preceding limb loss.
Subject(s)
Tooth Resorption , Tooth , Animals , Biological Evolution , Fossils/diagnostic imaging , Snakes/anatomy & histology , Reptiles/anatomy & histology , Tooth/diagnostic imaging , PhylogenyABSTRACT
Despite their importance to oral health, the mechanisms of minor salivary gland (SG) development are largely unexplored. Here we present in vivo and in vitro analyses of developing minor SGs in wild type and mutant mice. Eda, Shh and Fgf signalling pathway genes are expressed in these glands from an early stage of development. Developing minor SGs are absent in Eda pathway mutant embryos, and these mice exhibit a dysplastic circumvallate papilla with disrupted Shh expression. Supplementation of Eda pathway mutant minor SG explants with recombinant EDA rescues minor SG induction. Supplementation with Fgf8 or Shh, previously reported targets of Eda signalling, leads to induction of gland like structures in a few cases, but these fail to develop into minor SGs.
Subject(s)
Ectodysplasins/metabolism , Recombinant Proteins/pharmacology , Salivary Glands, Minor/embryology , Signal Transduction/physiology , Animals , DNA Primers/genetics , Ectodysplasins/genetics , Fibroblast Growth Factor 8/metabolism , Fibroblast Growth Factor 8/pharmacology , Genotype , Hedgehog Proteins/metabolism , Hedgehog Proteins/pharmacology , Histological Techniques , In Situ Hybridization , Mice , Mice, Mutant Strains , Polymerase Chain Reaction , Recombinant Proteins/metabolism , Salivary Glands, Minor/drug effectsABSTRACT
Hypohidrotic ectodermal dysplasia (HED) is characterized by defective ectodermal organ development. This includes the salivary glands (SGs), which have an important role in lubricating the oral cavity. In humans and mice, HED is caused by mutations in Ectodysplasin A (Eda) pathway genes. Various phenotypes of the mutant mouse Eda(Ta/Ta), which lacks the ligand Eda, can be rescued by maternal injection or in vitro culture supplementation with recombinant EDA. However, the response of the SGs to this treatment has not been investigated. Here, we show that the submandibular glands (SMGs) of Eda(Ta/Ta) mice exhibit impaired branching morphogenesis, and that supplementation of Eda(Ta/Ta) SMG explants with recombinant EDA rescues the defect. Supplementation of Edar(dlJ/dlJ) SMGs with recombinant Sonic hedgehog (Shh) also rescues the defect, whereas treatment with recombinant Fgf8 does not. This work is the first to test the ability of putative Eda target molecules to rescue Eda pathway mutant SMGs.
Subject(s)
Ectodysplasins/metabolism , Hedgehog Proteins/metabolism , Salivary Glands/metabolism , Animals , Ectodysplasins/genetics , Edar Receptor/genetics , Edar Receptor/metabolism , Edar-Associated Death Domain Protein/genetics , Edar-Associated Death Domain Protein/metabolism , Genotype , Hedgehog Proteins/genetics , In Situ Hybridization , Mice , Mice, Mutant Strains , Morphogenesis/genetics , Morphogenesis/physiology , Organ Culture Techniques , Salivary Glands/embryology , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
Organs throughout the body develop both asymmetrically and symmetrically. Here, we assess how symmetrical teeth in reptiles can be created from asymmetrical tooth germs. Teeth of lepidosaurian reptiles are mostly anchored to the jaw bones by pleurodont ankylosis, where the tooth is held in place on the labial side only. Pleurodont teeth are characterized by significantly asymmetrical development of the labial and lingual sides of the cervical loop, which later leads to uneven deposition of hard tissue. On the other hand, acrodont teeth found in lizards of the Acrodonta clade (i.e. agamas, chameleons) are symmetrically ankylosed to the jaw bone. Here, we have focused on the formation of the symmetrical acrodont dentition of the veiled chameleon (Chamaeleo calyptratus). Intriguingly, our results revealed distinct asymmetries in morphology of the labial and lingual sides of the cervical loop during early developmental stages, both at the gross and ultrastructural level, with specific patterns of cell proliferation and stem cell marker expression. Asymmetrical expression of ST14 was also observed, with a positive domain on the lingual side of the cervical loop overlapping with the SOX2 domain. In contrast, micro-CT analysis of hard tissues revealed that deposition of dentin and enamel was largely symmetrical at the mineralization stage, highlighting the difference between cervical loop morphology during early development and differentiation of odontoblasts throughout later odontogenesis. In conclusion, the early asymmetrical development of the enamel organ seems to be a plesiomorphic character for all squamate reptiles, while symmetrical and precisely orchestrated deposition of hard tissue during tooth formation in acrodont dentitions probably represents a novelty in the Acrodonta clade.
Subject(s)
Bone Development/physiology , Jaw/physiology , Lizards , Odontogenesis/physiology , Tooth/physiology , Animals , Lizards/anatomy & histology , Lizards/physiologyABSTRACT
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.
Subject(s)
Bone Morphogenetic Proteins/physiology , Genes, Homeobox , Homeodomain Proteins/genetics , Incisor/embryology , Molar/embryology , Odontogenesis , Transcription Factors/genetics , Animals , Body Patterning , Bone Morphogenetic Protein 4 , Carrier Proteins , Culture Techniques , Gene Expression Regulation, Developmental , Homeodomain Proteins/physiology , MSX1 Transcription Factor , Male , Mandible/embryology , Mesoderm/metabolism , Mesoderm/transplantation , Mice , Oncogene Proteins/genetics , Proteins/metabolism , Proteins/pharmacology , Signal Transduction , Tooth Germ/embryologyABSTRACT
Tuftelin is an acidic protein expressed at very early stages of mouse odontogenesis. It was suggested to play a role during epithelial-mesenchymal interactions, and later, when enamel formation commences, to be involved in enamel mineralization. Tuftelin was also detected in several normal soft tissues of different origins and some of their corresponding cancerous tissues. Tuftelin is expressed in low quantities, and undergoes degradation in the enamel extracellular matrix. To investigate the structure and function of tuftelin, the full length recombinant human tuftelin protein was produced. The full length human tuftelin cDNA was cloned using Gateway recombination into the Bac-to-Bac system compatible transfer vector pDest10. This vector adds a hexahistidine tag to the N-terminus of the expressed protein, enabling one-step affinity purification on nickel column. The recombinant human tuftelin protein was transposed into the bacmid and expressed in Spodoptera frugiperda (Sf9) insect cells. The yield of the purified, his-tagged recombinant full length human Tuftelin (rHTuft+) was 5-8 mg/L culture. rHTuft+ was characterized by SDS-PAGE, Western blot, ESI-TOF spectrometry, restriction mapping and MS/MS sequencing. The availability of the purified, full length recombinant human tuftelin protein opened up the possibility to investigate novel functions of tuftelin. Application of rHTuft+ agarose beads onto embryonic mouse mandibular explants caused changes in the surrounding epithelial cells, including morphology, orientation and spatial organization. Further studies using DiI labeling, revealed that rHTuft+, placed on the tooth germ region, brought about recruitment of adjacent embryonic mesenchymal cells. These findings support the hypothesis that tuftelin plays an important role during embryogenesis.
Subject(s)
Baculoviridae/genetics , Dental Enamel Proteins/metabolism , Recombinant Proteins/metabolism , Spodoptera/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Dental Enamel Proteins/chemistry , Dental Enamel Proteins/genetics , Dental Enamel Proteins/pharmacology , Female , Histocytochemistry , Humans , Male , Mandible/drug effects , Mandible/embryology , Mandible/growth & development , Mass Spectrometry , Mice , Microspheres , Molecular Sequence Data , Peptide Mapping , Phosphorylation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Tandem Mass SpectrometryABSTRACT
The electrical impedance tomography (EIT) image reconstruction problem is ill posed and spatially variant. Because of the problem's ill-posed nature, small amounts of measurement noise can corrupt reconstructed images. The problem must be regularized to reduce image artifacts. In this paper, we focus on the spatially variant characteristics of the problem. Correcting errors due to spatial variance should improve reconstruction accuracy. In this paper, we present methods to normalize the spatially variant image reconstruction problem by equalizing the point spread function (PSF). In order to equalize the PSF, we used the reconstruction blurring properties obtained from the sensitivity matrix. We compared three mathematical normalization schemes: pixel-wise scaling (PWS), weighted pseudo-inversion (WPI) and weighted minimum norm method (WMNM) to equalize images. The quantity index (QI), defined as the integral of pixel values of an EIT conductivity image, was considered in investigating spatial variance. The QI values along with reconstructed images are presented for cases of two-dimensional full array and hemiarray electrode topologies. We found that a spatially invariant QI could be obtained by applying normalization methods based on equalization of the PSF using conventional regularized reconstruction methods such as truncated singular value decomposition (TSVD) and WMNM. We found that WMNM normalization applied to WMNM regularized reconstruction was the best of the methods tested overall, for both hemiarray and full array electrode topologies.
Subject(s)
Image Processing, Computer-Assisted/methods , Models, Biological , Tomography/methods , Electric Impedance , Humans , Image EnhancementABSTRACT
Neuronal signaling is known to be required for salivary gland development, with parasympathetic nerves interacting with the surrounding tissues from early stages to maintain a progenitor cell population and control morphogenesis. In contrast, postganglionic sympathetic nerves arrive late in salivary gland development to perform a secretory function; however, no previous report has shown their role during development. Here, we show that a subset of neuronal cells within the parasympathetic submandibular ganglion (PSG) express the catecholaminergic marker tyrosine hydroxylase (TH) in developing murine and human submandibular glands. This sympathetic phenotype coincided with the expression of transcription factor Hand2 within the PSG from the bud stage (E12.5) of mouse embryonic salivary gland development. Hand2 was previously associated with the decision of neural crest cells to become sympathetic in other systems, suggesting a role in controlling neuronal fate in the salivary gland. The PSG therefore provides a population of TH-expressing neurons prior to the arrival of the postganglionic sympathetic axons from the superior cervical ganglion at E15.5. In culture, in the absence of nerves from the superior cervical ganglion, these PSG-derived TH neurons were clearly evident forming a network around the gland. Chemical ablation of dopamine receptors in explant culture with the neurotoxin 6-hydroxydopamine at early stages of gland development resulted in specific loss of the TH-positive neurons from the PSG, and subsequent branching was inhibited. Taken altogether, these results highlight for the first time the detailed developmental time course of TH-expressing neurons during murine salivary gland development and suggest a role for these neurons in branching morphogenesis.
Subject(s)
Neurons/cytology , Submandibular Gland/embryology , Sympathetic Nervous System/cytology , Tyrosine 3-Monooxygenase , Animals , Basic Helix-Loop-Helix Transcription Factors/physiology , Humans , Mice , Neurons/enzymologyABSTRACT
Tooth germs undergo a series of dynamic morphologic changes through bud, cap, and bell stages, in which odontogenic epithelium continuously extends into the underlying mesenchyme. During the transition from the bud stage to the cap stage, the base of the bud flattens and then bends into a cap shape whose edges are referred to as "cervical loops." Although genetic mechanisms for cap formation have been well described, little is understood about the morphogenetic mechanisms. Computer modeling and cell trajectory tracking have suggested that the epithelial bending is driven purely by differential cell proliferation and adhesion in different parts of the tooth germ. Here, we show that, unexpectedly, inhibition of cell proliferation did not prevent bud-to-cap morphogenesis. We quantified cell shapes and actin and myosin distributions in different parts of the tooth epithelium at the critical stages and found that these are consistent with basal relaxation in the forming cervical loops and basal constriction around enamel knot at the center of the cap. Inhibition of focal adhesion kinase, which is required for basal constriction in other systems, arrested the molar explant morphogenesis at the bud stage. Together, these results show that the bud-to-cap transition is largely proliferation independent, and we propose that it is driven by classic actomyosin-driven cell shape-dependent mechanisms. We discuss how these results can be reconciled with the previous models and data.
Subject(s)
Cell Proliferation , Molar/growth & development , Odontogenesis , Tooth Germ/growth & development , Animals , Female , Gene Expression Regulation, Developmental , Mesoderm , Mice , Morphogenesis , PregnancyABSTRACT
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.
Subject(s)
Anodontia/genetics , Odontogenesis/genetics , Tissue Engineering/trends , Animals , Anodontia/complications , Anodontia/therapy , Dentistry/trends , Forecasting , Humans , Mouth Abnormalities/complications , Mouth Abnormalities/genetics , Syndrome , Tooth Germ/physiologyABSTRACT
Electrical impedance tomography (EIT) is particularly well-suited to applications where its portability, rapid acquisition speed and sensitivity give it a practical advantage over other monitoring or imaging systems. An EIT system's patient interface can potentially be adapted to match the target environment, and thereby increase its utility. It may thus be appropriate to use different electrode positions from those conventionally used in EIT in these cases. One application that may require this is the use of EIT on emergency medicine patients; in particular those who have suffered blunt abdominal trauma. In patients who have suffered major trauma, it is desirable to minimize the risk of spinal cord injury by avoiding lifting them. To adapt EIT to this requirement, we devised and evaluated a new electrode topology (the 'hemiarray') which comprises a set of eight electrodes placed only on the subject's anterior surface. Images were obtained using a two-dimensional sensitivity matrix and weighted singular value decomposition reconstruction. The hemiarray method's ability to quantify bleeding was evaluated by comparing its performance with conventional 2D reconstruction methods using data gathered from a saline phantom. We found that without applying corrections to reconstructed images it was possible to estimate blood volume in a two-dimensional hemiarray case with an uncertainty of around 27 ml. In an approximately 3D hemiarray case, volume prediction was possible with a maximum uncertainty of around 38 ml in the centre of the electrode plane. After application of a QI normalizing filter, average uncertainties in a two-dimensional hemiarray case were reduced to about 15 ml. Uncertainties in the approximate 3D case were reduced to about 30 ml.
Subject(s)
Image Processing, Computer-Assisted/methods , Tomography/methods , Abdomen/anatomy & histology , Algorithms , Electric Conductivity , Electrodes , Hemorrhage , Phantoms, ImagingABSTRACT
BACKGROUND: Although as humans we lose our tails in the second month of embryonic development, a persistent tail is a prominent structural feature of most adult vertebrates. Indeed, the post-anal tail is part of the definition of a chordate. The internal organization of the developing tail--with neural tube, notochord and paired somites--is the same as that of the main body axis, so it can be expected that the mechanism of tail formation has a close relationship to that of the vertebrate body plan as a whole. Despite this, almost nothing is known about how tails arise. RESULTS: We present evidence to show that the tail bud of Xenopus laevis arises as the result of interactions between distinct zones of tissue at the posterior of the embryo at the neurula stage. These tissue interactions were demonstrated by manipulations of exogastrulae, which normally form no tail, and by transplantation experiments performed on the neural plate of stage 13 neurulae, whereby embryos with supernumary tails were produced. CONCLUSIONS: We propose a new model of tail bud determination, termed the NMC model, to explain the results we have obtained. In this model, the tail bud is initiated by an interaction between two territories in the neural plate and a posterior mesodermal territory.
Subject(s)
Tail/embryology , Animals , Culture Techniques , Embryonic Induction , Gastrula , Mesoderm/cytology , Models, Biological , Neural Crest/cytology , Tissue Transplantation , Xenopus laevisABSTRACT
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.
Subject(s)
Apoptosis/physiology , Apoptotic Protease-Activating Factor 1/deficiency , Caspase 9/deficiency , Dental Enamel/physiology , Animals , Cell Division/physiology , Dental Enamel/embryology , Epithelial Cells/cytology , Mesoderm/physiology , Mice , Mice, Knockout , Molar/embryology , Molar/physiology , Proliferating Cell Nuclear Antigen/analysis , Tooth Germ/anatomy & histology , Tooth Germ/embryologyABSTRACT
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.
Subject(s)
Dental Pulp Cavity/abnormalities , Ectodysplasins/genetics , Molar/abnormalities , Molar/embryology , Tooth Abnormalities/genetics , Tooth Root/abnormalities , Tooth Root/embryology , Adolescent , Animals , Child , Humans , Male , Mice , Odontogenesis/genetics , Phenotype , Signal Transduction , X-Ray MicrotomographyABSTRACT
Salivary glands are essential for the maintenance of oral health by providing lubrication and antimicrobial protection to the mucosal and tooth surfaces. Saliva is modified and delivered to the oral cavity by a complex multifunctional ductal system. During development, these ducts form as solid tubes, which undergo cavitation to create lumens. Apoptosis has been suggested to play a role in this cavitation process along with changes in cell polarity. Here, we show that apoptosis occurs from the very earliest stages of mouse salivary gland development, much earlier than previously reported. Apoptotic cells were observed in the center of the first epithelial stalk at early-stage embryonic day 12.5 (E12.5) according to both TUNEL staining and cleaved caspase 3 immunofluorescence. The presumptive lumen space was highlighted by the colocalization of a predictive lumen marker, cytokeratin 7. At E14.5, as lumens start to form throughout the glands, apoptotic expression decreased while cytokeratin 7 remained positive. In vitro inhibition of all caspases in E12.5 and E13.5 salivary glands resulted in wider ducts, as compared with the controls, and a defect in lumen formation. In contrast, no such defect in lumen formation was observed at E14.5. Our data indicate that apoptosis is involved during early stages of gland formation (E12.5 onward) and appears important for shaping the forming ducts.
Subject(s)
Apoptosis/physiology , Morphogenesis/physiology , Salivary Ducts/embryology , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Caspase 3/analysis , Caspase 3/drug effects , Caspase Inhibitors/pharmacology , Cell Polarity/physiology , Embryonic Development/physiology , Epithelium/embryology , In Situ Nick-End Labeling , Keratin-7/analysis , Mice , Organ Culture Techniques , Salivary Ducts/drug effects , Submandibular Gland/embryologyABSTRACT
Fas (CD95/APO-1) belongs to the TNF receptor (TNFR) family. Fas ligand binding followed by Fas-receptor oligomerisation leads to formation of a death-inducing signal complex starting with recruitment of the Fas-adapter protein (FADD). Components of this initiation complex (Fas, Fas-L, FADD) were correlated with apoptotic cells, detected by specific DNA fragmentation and morphological criteria. Apoptotic cells can be detected throughout the embryonic development of molar teeth. Restricted temporospatial distribution suggests several important roles for apoptosis in tooth morphogenesis. However, the mechanisms employed in dental apoptosis remain unclear. Frontal sections of the field vole at stage 13.5-15.5 of embryonic development were exploited to investigate and correlate location of Fas, Fas-ligand, FADD molecules and apoptosis in developing first molars by immunohistochemistry. During these stages the primary enamel knot appears and is gradually terminated by apoptosis. Initially, apoptotic cells were demonstrated in the most superficial layer of the dental lamina. The number of TUNEL-positive cells expanded from late bud to cap stages. Restricted areas of apoptotic cells were found in the stalk and primary enamel knot. Fas, Fas-L and FADD were co-localised, particularly in the primary enamel knot, and the stalk, correlating with the occurrence of apoptosis in these areas. Fas-L, however, was also found in proliferating parts of the developing tooth germ, such as in the cervical loops. Interestingly, FADD molecules were also observed in areas, where Fas protein was not detected. According to the immunohistochemical data, Fas-mediated signalling may have a triggering or enhancing role in dental apoptosis. This remains to be functionally confirmed.
Subject(s)
Arabidopsis Proteins/metabolism , Arvicolinae/embryology , Enamel Organ/metabolism , Fatty Acid Desaturases/metabolism , Odontogenesis/physiology , Signal Transduction/physiology , fas Receptor/metabolism , Animals , Apoptosis/physiology , Arabidopsis Proteins/analysis , Arvicolinae/metabolism , Fatty Acid Desaturases/analysis , Immunohistochemistry , In Situ Nick-End Labeling , Molar , fas Receptor/analysisABSTRACT
Xerostomia, or chronic dry mouth, is a common syndrome caused by a lack of saliva that can lead to severe eating difficulties, dental caries and oral candida infections. The prevalence of xerostomia increases with age and affects approximately 30% of people aged 65 or older. Given the large numbers of sufferers, and the potential increase in incidence given our aging population, it is important to understand the complex mechanisms that drive hyposalivation and the consequences for the dentition and oral mucosa. From this study we propose the Fgf10 +/- mouse as a model to investigate xerostomia. By following embryonic salivary gland development, in vivo and in vitro, we show that a reduction in Fgf10 causes a delay in branching of salivary glands. This leads to hypoplasia of the glands, a phenotype that is not rescued postnatally or by adulthood in both male and female Fgf10 +/- mice. Histological analysis of the glands showed no obvious defect in cellular differentiation or acini/ductal arrangements, however there was a significant reduction in their size and weight. Analysis of saliva secretion showed that hypoplasia of the glands led to a significant reduction in saliva production in Fgf10 +/- adults, giving rise to a reduced saliva pellicle in the oral cavity of these mice. Mature mice were shown to drink more and in many cases had severe tooth wear. The Fgf10 +/- mouse is therefore a useful model to explore the causes and effects of xerostomia.
Subject(s)
Fibroblast Growth Factor 10/genetics , Xerostomia/genetics , Animals , Disease Models, Animal , Drinking Behavior , Female , Fibroblast Growth Factor 10/metabolism , Heterozygote , Male , Mice, Transgenic , Salivary Glands/embryology , Salivary Glands/pathology , Tissue Culture Techniques , Tongue/pathology , Xerostomia/pathologyABSTRACT
c-Fos homozygous mice lack osteoclasts with a failure of the teeth to erupt and with an arrest of root development. Here, we characterize the defects associated with the failure in root development and the loss of the tooth-bone interface, and we investigate the underlying causes. We show that, while homozygous c-Fos mice have no multinucleated osteoclasts, heterozygous mice have a reduction in the number of osteoclasts with a reduction in the tooth-bone interface during development and subtle skeletal defects postnatally. In the homozygous mutants bone is found to penetrate the tooth, particularly at the apical end, physically disrupting the root forming HERS (Hertwig's epithelial root sheath) cells. The cells of the HERS continue to proliferate but cannot extend downward due to the presence of bone, leading to a loss of root formation. Tooth germ culture showed that the developing tooth invaded the static bone in mutant tissue, rather than the bone encroaching on the tooth. Although c-Fos has been shown to be expressed in developing teeth, the defect in maintenance of the tooth-bone interface appears to be driven solely by the lack of osteoclasts, as this defect can be rescued in the presence of donor osteoclasts. The rescue suggests that signals from the tooth recruit osteoclasts to clear the bone from around the tooth, allowing the tooth to grow, form roots, and later erupt.