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The objective of this study was to investigate molecular etiologies of oral exostoses and dental anomalies in 14 patients from eight families.Oral and radiographic examinations were performed on every patient. Whole exome and Sanger sequencing were performed on DNA of the patients, the unaffected parents and unaffected siblings. LRP6 mutant proteins were modeled and analyzed.Five mutations in LRP6, including four missense (p.Glu72Lys, p.Lys82Asn, Tyr418His, and p.Ile773Val) and one nonsense mutation (p.Arg32Ter), were identified. These mutations have not been reported to be associated with dental anomalies or oral exostoses. Oral features included a variety of oral exostoses (7 of the 14 patients), root defects (6 of the 14 patients), and tooth agenesis (5 of the 14 patients). Less common dental anomalies included microdontia, tooth fusion, odontomas, and mesiodens. Analysis of the protein models of the five LRP6 mutations shed light on their likely impact on LRP6 protein structure and function.Fourteen patients with five LRP6 mutations, including two recurrent mutations and three novel ones, are reported. Our study shows for the first time that mutations in LRP6 are associated with mesiodens, fusion of teeth, odontomas, microdontia, long roots, molars with unseparated roots, and taurodontism.

Objectives The primary enamel knot (PEK) is a population of cells that shows spatio-temporal restricted apoptosis during tooth development. It has been shown that caspase-9 and Apaf-1 are essential for apoptosis in the PEK as well as the central caspase-3. Caspase-7, as another executioner member in the caspase machinery, is considered to have caspase-3 like properties. Design The aim of this study was to detect caspase-7 activation during molar tooth development with a special focus on the cells of the PEK and to correlate the expression with the pattern of apoptosis and caspase-3 activation. Apoptosis in the PEK was investigated in caspase-7 deficient mice to examine the functional consequence of loss of this specific caspase. In addition, odontoblasts and ameloblasts, which are known to undergo cell death during their secretory and maturation stages, were investigated. Results Cleaved caspase-7 was found in the apoptotic region of the PEK, however, caspase-7-deficient mice still possessed apoptotic cells in the PEK in a similar distribution to the wild type. Caspase-7 is therefore not essential for apoptosis in the PEK. Notably, cleaved caspase-7-positive cells were found at later stages in odontoblasts and ameloblasts, but expression did not correlate with apoptosis in these tissues. Conclusions The results indicate a non-essential apoptotic role of caspase-7 in the PEK apoptosis but suggest also possible non-apoptotic functions for caspase-7 in tooth development.

Laser capture microdissection (LCM) uniquely allows the selection of specific cell populations from histological sections. These selected cells are then catapulted into a test tube without any contamination from surrounding tissues. During the last ten years, many significant results have been achieved, particularly at the level of DNA and RNA where amplification techniques are available. However, where amplification procedures are difficult, the benefits of LCM diminish. To overcome such difficulties, a novel approach, combining laser capture microdissection and flow cytometry, has been tested here for detection of apoptosis and proliferation in tissue bound cell populations without any amplification steps. The mouse cap stage molar tooth germ was used as a model. At the centre of the inner enamel epithelium, the primary enamel knot is a clearly defined apoptotic population with minimal proliferation, flanked by the highly proliferative cervical loops on each side. Thus within the tooth germ epithelium at this stage, two distinct populations of cells are found side by side. These populations were selected by laser capture microdissection and then analysed by flow cytometry for apoptosis and proliferation. Flow cytometric results correlated well with immunohistochemical findings, demonstrating the success and sensitivity of this combined procedure.

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.

Objective Salivary gland (SG) development is based on branching morphogenesis, in which programmed cell death has been proposed to play a role in cell signalling and organ shaping. In the mouse salivary gland apoptosis has been suggested to play a key role in lumen formation, removing the central cells of the epithelial stalks. Here we analyse the expression of several anti- and pro-regulators of apoptosis during human SG development in a range of developmental stages. Design Foetal SGs obtained from the University of Sao Paulo were analysed by immunohistochemistry to assess the expression of apoptosis-associated proteins: caspases (caspase-6, -7, -9 and cleaved caspase-3), Bcl-2 family members (Bax, Bak, Bad, Bid, Bcl-2, Bcl-x and Bcl-xL), Survivin (BIRC5), Cytochrome C and Apaf-1. Results Nuclear expression of Bax and Bak was identified in presumptive luminal areas at initial stages, while Bcl-xL showed the most relevant anti-apoptotic activity. Caspase-6, -7 and -9 were expressed during all stages, while interestingly cleaved caspase-3 showed no prominent expression, indicating that caspase-7 is the main effector. Apoptosome complex components Apaf-1 and Cytochrome C, as well as survivin were all positive in developing glands. Conclusions The particular expression pattern of several apoptotic regulators in human SG development suggests the existence of a fundamental role for apoptosis during duct formation. The absence of Bad and Bid expressions indicates that the instrinsic pathway is more active then the extrinsic during human gland formation. The subcellular localisation of intrinsic-apoptosis proteins correlated with apoptotic activity, but also suggested additional non-apoptotic functions.

Summary 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.

Replacement teeth in reptiles and mammals develop from a successional dental lamina. In monophyodont (single generation) species such as the mouse, no successional lamina develops. We have selected a reptilian monophyodont species - the Veiled Chameleon (Chamaeleo calyptratus) - to investigate whether this is a common characteristic of species that do not have replacement teeth. Furthermore, we focus on the sequence of tooth initiation along the jaw, and tooth attachment to the bones.Embryos of the Veiled Chameleon were collected during the first 6 months of incubation (from the 5th to 24th week) at 7-day intervals.After five weeks of incubation, an epithelial thickening was present as a shallow protrusion into the mesenchyme. A week later, the epithelium elongated more deeply into the mesenchyme to form the dental lamina. The formation of all tooth germs along the jaw was initiated from the tip of the dental lamina. Development of a successional dental lamina was initiated during the pre-hatching period but this structure became markedly reduced during juvenile stages. MicroCT analysis showed the presence of a heterodont dentition in young chameleons with multicuspid teeth in the caudal jaw area and simpler monocuspid teeth rostrally. Unlike the pleurodont teeth of most reptilian species, chameleon teeth are acrodontly ankylosed to the bones of the jaw. Odontoblasts produced a layer of predentine that connected the dentine to the supporting bone, with both tooth and bone protruding out of the oral cavity and acting as a functional unit.Chameleons may provide new and useful information to study the molecular interaction at the tooth-bone interface in physiological as well as pathological conditions.