Biomedical subjects

P T Sharpe

Publications and source records attributed to P T Sharpe.

At least 19 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

CD 95 mediated apoptosis in embryogenesis: implication in tooth development.

INTRODUCTION: Understanding of apoptotic mechanisms involved in tissue shaping is of particular interest because of possible targeted modulation of the development of organ structures such as teeth. Research of CD 95 mediated apoptosis has been focused particularly on cell death in the immune system and related disorders. However, CD 95 mediated apoptosis is also involved in embryogenesis of many organs as the kidney, the lung, the intestine and tissue networks such as the nervous system. DESIGN: Narrative review. RESULTS: This review briefly summarizes the current knowledge of CD 95 mediated apoptosis in embryogenesis with possible implication in tooth development. CD 95 receptor and CD 95 ligand are found at early stages of tooth development. The data suggest some positive correlations with dental apoptosis distribution, particularly in the primary enamel knot where apoptosis occurs during elimination of this structure. CD 95 deficient (lpr) adult mouse tooth phenotype, however, did not show any alterations in final tooth pattern and morphology. CONCLUSION: To date studies of apoptotic machinery during tooth development show spatial localization of many of the components together with precise and localized timing of cell death. There is still much to be learned about the regulation and importance of apoptosis in tooth development. Nevertheless, the involvement of apoptotic regulatory mechanisms interplaying with other molecules participates to the cellular cross-talk in developing tissues, which opens possible targeted modulations as suggested, e.g. for future molecular dentistry.

Animals↗

PubMed2005

Cell lineage of primary and secondary enamel knots.

Recent research indicates that control of cusp morphology involves a signalling center at the heart of the developing tooth germ, known as the enamel knot. The primary enamel knot forms in both incisors and molar tooth germs at the cap stage of tooth development. Secondary and tertiary enamel knots only develop in molar tooth germs. These sit at the sites of future cusp tips from the early bell stage of tooth development. In studies describing the relationship between the primary and secondary enamel knots, it is often assumed that there is a cellular continuity between these structures, such that cells from the primary enamel knot physically contribute to the secondary enamel knots. We have devised a method whereby the developing tooth germ can be cultured in frontal slices with the enamel knot visible. The fate of the primary enamel knot cells can then be followed by 1,1', di-octadecyl-3,3,3',3',-tetramethylindo-carbocyanine perchlorate (DiI) labeling. Using this method, no cells of the primary enamel knot were seen to move toward the developing secondary enamel knots. Thus, although the primary and secondary enamel knots have a close molecular and functional relationship in molar development, they are not actually derived from the same cells.

Animals↗

PubMed2005

Organized tooth-specific cellular differentiation stimulated by BMP4.

Mammalian teeth develop on the oral surface of the first pharyngeal arch by a series of reciprocal interactions between epithelial and mesenchymal cells. The embryonic first pharyngeal arch oral epithelium is able to induce tooth formation when combined with mesenchymal cells from the second pharyngeal arch, a region devoid of tooth development. Second pharyngeal arch mesenchyme is thus competent to form teeth if provided with the correct signals. First-arch oral epithelium expresses several signaling molecules that could be potential inducers of tooth development, including BMP4. The addition of BMP4 to intact second-arch explants resulted in the development of organized structures containing layers of cells that express marker genes of tooth-specific cells, odontoblasts and ameloblasts. Thus, although overt tooth development did not occur, BMP4 has the ability to stimulate organized differentiation of epithelial- and mesenchymal-derived dental-specific cells from non-dental primordia.

Ameloblasts↗

PubMed2004

The activation level of the TNF family receptor, Edar, determines cusp number and tooth number during tooth development.

Mutations in members of the ectodysplasin (TNF-related) signalling pathway, EDA, EDAR, and EDARADD in mice and humans produce an ectodermal dysplasia phenotype that includes missing teeth and smaller teeth with reduced cusps. Using the keratin 14 promoter to target expression of an activated form of Edar in transgenic mice, we show that expression of this transgene is able to rescue the tooth phenotype in Tabby (Eda) and Sleek (Edar) mutant mice. High levels of expression of the transgene in wild-type mice result in molar teeth with extra cusps, and in some cases supernumerary teeth, the opposite of the mutant phenotype. The level of activation of Edar thus determines cusp number and tooth number during tooth development.

Animals↗

PubMed2004

Death in the life of a tooth.

Programmed cell death (apoptosis) constitutes an important mechanism in embryonic development. Although there is substantial evidence for essential roles of apoptosis in organ shaping and controlling of cell number, the mechanisms of these processes are poorly understood. The regulation of cell proliferation to form tooth buds of the appropriate size and at the correct positions must involve a balance between cell division and cell death. Apoptosis has been suggested to play both passive and active roles in bud formation and morphogenesis and in reduction of the dental lamina, as well as silencing of the enamel knot signaling centers. The location of apoptotic cells during tooth development has been described and suggests their temporospatial roles. Unfortunately, there is little functional evidence on these roles, and the aim of this review is to highlight areas where apoptosis may play key roles in odontogenesis.

Animals↗

PubMed2004

Opg, Rank, and Rankl in tooth development: co-ordination of odontogenesis and osteogenesis.

Osteoprotegerin (OPG), receptor activator of nuclear factor-kappaB (RANK), and RANK ligand (RANKL) are mediators of various cellular interactions, including bone metabolism. We analyzed expression of these three genes during murine odontogenesis from epithelial thickening to cytodifferentiation stages. Opg showed expression in the thickening and bud epithelium. Expression of Opg and Rank was observed in both the internal and the external enamel epithelium as well as in the dental papilla mesenchyme. Although Rankl expression was not detected in tooth epithelium or mesenchyme, it was expressed in pre-osteogenic mesenchymal cells close to developing tooth germs. All three genes were detected in developing dentary bone at P0. The addition of exogenous OPG to explant cultures of tooth primordia produced a delay in tooth development that resulted in reduced mineralization. We propose that the spatiotemporal expression of these molecules in early tooth and bone primordia cells has a role in co-ordinating bone and tooth development.

Alveolar Process↗

PubMed2004

Stem-cell-based tissue engineering of murine teeth.

Teeth develop from reciprocal interactions between mesenchyme cells and epithelium, where the epithelium provides the instructive information for initiation. Based on these initial tissue interactions, we have replaced the mesenchyme cells with mesenchyme created by aggregation of cultured non-dental stem cells in mice. Recombinations between non-dental cell-derived mesenchyme and embryonic oral epithelium stimulate an odontogenic response in the stem cells. Embryonic stem cells, neural stem cells, and adult bone-marrow-derived cells all responded by expressing odontogenic genes. Transfer of recombinations into adult renal capsules resulted in the development of tooth structures and associated bone. Moreover, transfer of embryonic tooth primordia into the adult jaw resulted in development of tooth structures, showing that an embryonic primordium can develop in its adult environment. These results thus provide a significant advance toward the creation of artificial embryonic tooth primordia from cultured cells that can be used to replace missing teeth following transplantation into the adult mouth.

3T3 Cells↗

PubMed2002

Analysis of cDNAs from a mouse embryo tooth library: identification of novel genes during tooth development.

Signaling molecules, their receptors, and target genes from pathways and networks regulate the development of the tooth from initiation through cell differentiation. In order to identify genes involved in odontoblast and ameloblast differentiation, we constructed a cDNA library from E19.5 mouse molars. In this work, we report the partial cDNA sequences of 10 noncharacterized genes and we show cell expression of the transcripts on mouse embryo molars by in situ hybridization.

Animals↗

PubMed2001

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.

Animals↗

PubMed2001

Developmental genetics and early hominid craniodental evolution.

Although features of the dentition figure prominently in discussions of early hominid phylogeny, remarkably little is known of the developmental basis of the variations in occlusal morphology and dental proportions that are observed among taxa. Recent experiments on tooth development in mice have identified some of the genes involved in dental patterning and the control of tooth specification. These findings provide valuable new insight into dental evolution and underscore the strong developmental links that exist among the teeth and the jaws and cranium. The latter has important implications for cladistic studies that traditionally consider features of the skull independently from the dentition.

Animals↗

PubMed2001

An enhancer sequence directs LacZ expression to developing pharyngeal endoderm in transgenic mice.

The murine Hoxc-6 homeobox gene comprises three exons with two distinct promoters (PRI and PRII) located 9 kb apart. To characterise the PRII promoter, a region 3 kb upstream of the transcription start site was sequenced, and an Antananapedia-like consensus binding sequence was found (Coletta et al., 1991). A LacZ reporter gene construct, containing three copies of this sequence, directs highly specific expression in cells forming pharyngeal endoderm in transgenic mice. Expression was first detected in a few individual anterior endoderm cells at E7.5, which increase in number up to E9.5, where expression was clearly visible in the pharyngeal endoderm. Expression of the endodermal genes HNF3beta, Pax-9, Shh, and Nkx2.5 showed colocalization with the LacZ-positive cells in the foregut and pharyngeal endoderm. This novel enhancer provides a means of tracking the morphogenetic movement of endodermal cells fated to form the foregut.

Animals↗

PubMed2001

Sonic hedgehog regulates epithelial proliferation and cell survival in the developing tooth germ.

Shh expression is highly restricted to the future sites of tooth development during the initiation of odontogenesis. This suggests a role for Shh as a proliferative factor, as localized epithelial thickenings invaginate to form a tooth bud. We have investigated this role by blocking Shh signaling between E10.5 and E12.5 in murine mandibular processes using a 5E1 blocking antibody and the PKA activator Forskolin. This results in down-regulation of Ptc, a principle target of Shh signaling. The effects of inhibition varied with developmental time. At E10.5, tooth development was arrested as epithelial thickenings and the numbers of teeth developing were considerably reduced. Inhibition at E12.5 produced localized apoptosis in the epithelium at the tip of the tooth buds, although some teeth were able to develop. Thus, Shh has dual roles in early odontogenesis, first in bud formation by stimulating epithelial proliferation, and second in the development of cap-stage tooth germs by increasing epithelial cell survival.

Animals↗

PubMed2001

Neural crest and tooth morphogenesis.

Mammalian teeth develop from two types of cells: stomodeal ectoderm, which forms ameloblasts, and cranial neural-crest-derived (ecto) mesenchyme cells, which form odontoblasts and cementoblasts. These two cell types, juxtaposed in the developing oral cavity, interact to control the entire process of tooth initiation, morphogenesis, and cytodifferentiation. Cell-cell signaling pathways and their target nuclear factors have been identified as key mediators of the progressively complex exchange of information between ectoderm and ectomesenchyme. The constantly changing direction of the reciprocal signaling and cell responses between ectoderm and ectomesenchyme enables cells to monitor their relative spatial positions and differentiated states continuously. The least understood of the early processes in tooth development are morphogenesis and patterning. From a seemingly uniform layer of oral ectoderm and underlying mass of ectomesenchyme, different types (shapes) of teeth develop in different positions. Tooth type is determined very early in development, before the overt onset of morphogenesis. Thus, the early ectoderm-ectomesenchyme cell interactions must in some way either create or respond to positional differences in the jaw primordia.

Activins↗

PubMed2001

The role of effectors of the activin signalling pathway, activin receptors IIA and IIB, and Smad2, in patterning of tooth development.

The gene for activin betaA is expressed in the early odontogenic mesenchyme of all murine teeth but mutant mice show a patterning defect where incisors and mandibular molars fail to develop but maxillary molars develop normally. In order to understand why maxillary molar tooth development can proceed in the absence of activin, we have explored the role of mediators of activin signalling in tooth development. Analysis of tooth development in activin receptor II and Smad2 mutants shows that a similar tooth phenotype to activin betaA mutants can be observed. In addition, we identify a novel downstream target of activin signalling, the Iroquois-related homeobox gene, Irx1, and show that its expression in activin betaA mutant embryos is lost in all tooth germs, including the maxillary molars. These results strongly suggest that other transforming growth factor beta molecules are not stimulating the activin signalling pathway in the absence of activin. This was confirmed by a non-genetic approach using exogenous soluble receptors to inhibit all activin signalling in tooth development, which reproduced the genetic phenotypes. Activin, thus, has an essential role in early development of incisor and mandibular molar teeth but this pathway is not required for development of maxillary molars.

Activin Receptors, Type II↗

PubMed2001

Antagonistic regulation of Dlx2 expression by PITX2 and Msx2: implications for tooth development.

The transcriptional mechanisms underlying tooth development are only beginning to be understood. Pitx2, a bicoid-like homeodomain transcription factor, is the first transcriptional marker observed during tooth development. Because Pitx2, Msx2, and Dlx2 are expressed in the dental epithelium, we examined the transcriptional activity of PITX2 in concert with Msx2 and the Dlx2 promoter. PITX2 activated while Msx2 unexpectedly repressed transcription of a TK-Bicoid luciferase reporter in a tooth epithelial cell line (LS-8) and CHO cell line. Surprisingly, Msx2 binds to the bicoid element (5'-TAATCC-3') with a high specificity and competes with PITX2 for binding to this element. PITX2 binds to bicoid and bicoid-like elements in the Dlx2 promoter and activates this promoter 45-fold in CHO cells. However, it is only modestly activated in the LS-8 tooth epithelial cell line that endogenously expresses Msx2 and Pitx2. RT-PCR and Western blot assays reveal that two Pitx2 isoforms are expressed in the LS-8 cells. We further demonstrate that PITX2 dimerization can occur through the C-terminus of PITX2. Msx2 represses the Dlx2 promoter in CHO cells and coexpression of both PITX2 and Msx2 resulted in transcriptional antagonism of the Dlx2 promoter. Electrophoretic mobility shift assays demonstrate that factors in the LS-8 cell line specifically interact with PITX2. Thus, Dlx2 gene transcription is regulated by antagonistic effects between PITX2, Msx2, and factors expressed in the tooth epithelia.

Animals↗

PubMed2000

Wnt/Shh interactions regulate ectodermal boundary formation during mammalian tooth development.

Interactions between the Wnt (wingless) and hedgehog signaling pathways were first described as playing a role in establishing boundaries between ectodermal cells in Drosophila segmentation. During the initiation of mammalian tooth development, boundaries that distinguish oral from dental ectoderm must be formed to correctly position the sites of tooth formation. We describe a reciprocal relationship between the expression of Wnt-7b in presumptive oral ectoderm and Shh in presumptive dental ectoderm in mouse embryos that mark boundaries between these cells with different developmental fates. By using a murine retrovirus to ectopically express Wnt-7b in presumptive dental ectoderm in mandibular arch explants, we show that Shh expression in the ectoderm and Ptc expression in the underlying ectomesenchyme are down-regulated, and tooth development is subsequently arrested. This suggests that Wnt-7b acts to repress Shh expression in oral ectoderm, thus maintaining the boundaries between oral and dental ectodermal cells. Implantation of beads soaked in Shh protein into Wnt-7b-infected explants resulted in complete rescue of tooth development, confirming that the repressive action of Wnt-7b specifically affects Shh signaling.

Animals↗

PubMed2000

Inhibition of Wnt signaling by exogenous Mfrzb1 protein affects molar tooth size.

Wnt extracellular signaling molecules have essential roles as regulators of cell proliferation, migration, differentiation, and in epithelial-mesenchymal interactions involved in tissue morphogenesis. Frizzled integral membrane proteins have been shown to function as receptors for Wnt signaling molecules. Vertebrates also produce secreted proteins related to Frizzled receptors, Frizzled-related proteins (FRPs), which contain the cysteine-rich domain of Frizzleds and appear to function as Wnt antagonists. Tooth development is regulated by a reciprocal series of epithelial-mesenchymal interactions, and many Wnt signaling pathway genes are expressed in the developing tooth at these sites. Here we report the expression of one FRP gene, Mfrzb1, in the rostral mesenchyme of the mandibular primordium. Using explant cultures, we show that expression of Mfrzb1 in the mandibular mesenchyme is under the control of signals derived from the overlying epithelium. Bead implantation experiments in vitro show that FGF8 induces Mfrzb1 expression, whereas BMP4 and SHH proteins have no effect. We studied the effect of ectopic MFrzb1 protein on the developing tooth germs by transplanting explants treated with Mfrzb1 protein into renal capsules, and found it to retard tooth development. This suggests that Wnt signaling is required early in tooth germ formation and that interference with signaling via addition of an antagonist results in retarded development and formation of smaller teeth.

Animals↗