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Biomedical subjects

Paul T Sharpe

Publications and source records attributed to Paul T Sharpe.

At least 19 recordsLinked to original sources
PubMed2005

Expression of the Hedgehog antagonists Rab23 and Slimb/betaTrCP during mouse tooth development.

The sonic hedgehog signalling peptide has been demonstrated to play an important role in the growth and patterning of several organs including the tooth. Inappropriate activation of Shh signalling in the embryo causes various patterning defects and complex regulation of this pathway is important during normal development. A growing list of diverse antagonists have been identified that restrict Shh signalling in the embryo, however, only Ptc1, Gas1 and Hip1 have been studied during tooth development. We have examined the expression pattern of the putative antagonists Rab23 and Slimb/betaTrCP during early murine odontogenesis and find that these molecules are expressed in the developing tooth. Interestingly, Rab23 demonstrates contrasting expression domains in the incisor and molar dentition during the cap stage, being restricted to the mesenchymal compartment of molar teeth and the epithelium of the enamel knot in incisor teeth. These findings provide the first evidence of distinct regulatory pathways for Shh in teeth of different classes.

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PubMed2005

The Ectodysplasin and NFkappaB signalling pathways in odontogenesis.

Hypohidrotic ectodermal dysplasia (HED) is a congenital disorder affecting organs of ectodermal origin including teeth, hair and sweat glands. Defects in Ectodysplasin (tabby), Edar (downless) and Edar associated death domain (Edaradd) (crinkled) cause HED in both humans and mice. Ectodysplasin is a tumour necrosis factor (TNF) superfamily member whose downstream signalling is transduced by the inhibitor of kappaB kinase (IKK) complex and inhibitors of kappaB (IkappaB) to activate the transcription factor NFkappaB. NFkappaB signalling is involved in a wide range of cellular processes and at each stage the different family members must be tightly regulated for each function. Recent data have demonstrated the importance of this signalling pathway in odontogenesis, particularly in the formation of cusps. Here we review recent advances in our understanding of Ectodysplasin/NFkappaB signalling in tooth development and in particular the central role of the IKK complex.

Animals↗
PubMed2005

Tissue engineering of teeth using adult stem cells.

Tooth development, a process which occurs in the developing embryo, involves the reciprocal and sequential signalling between epithelial and mesenchymal tissue of the developing first branchial arch. The oral epithelium produces the first inductive signals for odontogenesis at around E10.0, which trigger off a cascade of events that result in the formation of a tooth. We have engineered a tooth in vitro by harnessing the basic principles of odontogenesis and the inductive capability of the oral epithelium of the developing embryo. We replaced the mesenchymal portion of the developing mandibular primordium with aggregates of stem cells from embryos as well as stem cells taken from adult mice. The cell aggregates were covered with embryonic epithelium from E10.0 mouse embryos to form recombinant explants. In vitro culture of these recombinant explants resulted in the induction of early tooth marker genes in the cell aggregates, indicating that the cells were able to respond to the odontogenic signals produced by the oral epithelium. In vivo culture of explants resulted in the induction of Dspp within the cell aggregates indicating that tooth tissue was present. Three recombinant explants, where the cell aggregates consisted of adult bone marrow cells, produced teeth. To determine whether the oral cavity would be able to sustain the growth of an implanted tooth germ, E14.5 molar rudiments were implanted into the diastema region of the maxilla of adult mice. The resulting teeth appeared to be normal in size and were connected to the underlying bone. These experiments are an indication that it is possible to induce odontogenesis and engineer a tooth using adult cells of non-dental origin. They also indicate that developing tooth germs could be successfully implanted into the gingiva of patients.

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PubMed2005

The stomach mesenchymal transcription factor Barx1 specifies gastric epithelial identity through inhibition of transient Wnt signaling.

Inductive interactions between gut endoderm and the underlying mesenchyme pattern the developing digestive tract into regions with specific morphology and functions. The molecular mechanisms behind these interactions are largely unknown. Expression of the conserved homeobox gene Barx1 is restricted to the stomach mesenchyme during gut organogenesis. Using recombinant tissue cultures, we show that Barx1 loss in the mesenchyme prevents stomach epithelial differentiation of overlying endoderm and induces intestine-specific genes instead. Additionally, Barx1 null mouse embryos show visceral homeosis, with intestinal gene expression within a highly disorganized gastric epithelium. Barx1 directs mesenchymal cell expression of two secreted Wnt antagonists, sFRP1 and sFRP2, and these factors are sufficient replacements for Barx1 function. Canonical Wnt signaling is prominent in the prospective gastric endoderm prior to epithelial differentiation, and its inhibition by Barx1-dependent signaling permits development of stomach-specific epithelium. These results define a transcriptional and signaling pathway of inductive cell interactions in vertebrate organogenesis.

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PubMed2004

Hierarchy revealed in the specification of three skeletal fates by Sox9 and Runx2.

Across vertebrates, there are three principal skeletal tissues: bone, persistent cartilage, and replacement cartilage. Although each tissue has a different evolutionary history and functional morphology, they also share many features. For example, they function as structural supports, they are comprised of cells embedded in collagen-rich extracellular matrix, and they derive from a common embryonic stem cell, the osteochondroprogenitor. Occasionally, homologous skeletal elements can change tissue type through phylogeny. Together, these observations raise the possibility that skeletal tissue identity is determined by a shared set of genes. Here, we show that misexpression of either Sox9 or Runx2 can substitute bone with replacement cartilage or can convert persistent cartilage into replacement cartilage and vice versa. Our data also suggest that these transcription factors function in a molecular hierarchy in which chondrogenic factors dominate. We propose a binary molecular code that determines whether skeletal tissues form as bone, persistent cartilage, or replacement cartilage. Finally, these data provide insights into the roles that master regulatory genes play during evolutionary change of the vertebrate skeleton.

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PubMed2004

Regionalisation of early head ectoderm is regulated by endoderm and prepatterns the orofacial epithelium.

The oral epithelium becomes regionalised proximodistally early in development, and this is reflected by the spatial expression of signalling molecules such as Fgf8 and Bmp4. This regionalisation is responsible for regulating the spatial expression of genes in the underlying mesenchyme. These genes are required for the spatial patterning of bone, cartilage orofacial development and, in mammals, teeth. The mechanism and timing of this important regionalisation during head epithelium development are not known. Using lipophilic dyes to fate map the oral epithelium in chick embryos, we show that the cells that will occupy the epithelium of the distal and the proximal mandible primordium already occupy different spatial locations in the developing head ectoderm prior to the formation of the first pharyngeal arch and neural crest migration. Moreover, the ectoderm cells fated to become proximal oral epithelium express Fgf8 and this expression requires the presence of endoderm. Thus, the first fundamental patterning process in jaw morphogenesis is controlled by the early separation of specific areas of ectoderm that are regulated by ectoderm-endoderm interactions, and does not involve neural crest cells.

Animals↗
PubMed2004

Restriction of sonic hedgehog signalling during early tooth development.

The signalling peptide encoded by the sonic hedgehog gene is restricted to localised thickenings of oral epithelium, which mark the first morphological evidence of tooth development, and is known to play a crucial role during the initiation of odontogenesis. We show that at these stages in the murine mandibular arch in the absence of epithelium, the Shh targets Ptc1 and Gli1 are upregulated in diastema mesenchyme, an edentulous region between the sites of molar and incisor tooth formation. This ectopic expression is not associated with Shh transcription but with Shh protein, undetectable in the presence of epithelium. These findings suggest that, in diastema mesenchyme, restriction of Shh activity is dependent upon the overlying epithelium. This inhibitory activity was demonstrated by the ability of transplanted diastema epithelium to downregulate Ptc1 in tooth explants, and for isolated diastema mesenchyme to express Ptc1. A candidate inhibitor in diastema mesenchyme is the glycosylphosphatidylinositol-linked membrane glycoprotein Gas1. Gas1 is normally expressed throughout mandibular arch mesenchyme; however, in the absence of epithelium this expression was downregulated specifically in the diastema where ectopic Shh protein was identified. Although Shh signalling has no effect upon Gas1 expression in mandibular arch mesenchyme, overexpression of Gas1 results in downregulation of ectopic Ptc1. Therefore, control of the position of tooth initiation in the mandibular arch involves a combination of Shh signalling at sites where teeth are required and antagonism in regions destined to remain edentulous.

Animals↗
PubMed2004

Intergenic enhancers with distinct activities regulate Dlx gene expression in the mesenchyme of the branchial arches.

The vertebrate Dlx genes, generally organized as tail-to-tail bigene clusters, are expressed in the branchial arch epithelium and mesenchyme with nested proximodistal expression implicating a code that underlies the fates of jaws. Little is known of the regulatory architecture that is responsible for Dlx gene expression in developing arches. We have identified two distinct cis-acting regulatory sequences, I12a and I56i, in the intergenic regions of the Dlx1/2 and Dlx5/6 clusters that act as enhancers in the arch mesenchyme. LacZ transgene expression containing I12a is restricted to a subset of Dlx-expressing ectomesenchyme in the first arch. The I56i enhancer is active in a broader domain in the first arch mesenchyme. Expression of transgenes containing either the I12a or the I56i enhancers is dependent on the presence of epithelium between the onset of their expression at E9-10 until independence at E11. Both enhancers positively respond to FGF8 and FGF9; however, the responses of the reporter transgenes were limited to their normal domain of expression. BMP4 had a negative effect on expression of both transgenes and counteracted the effects of FGF8. Furthermore, bosentan, a pharmacological inhibitor of Endothelin-1 signaling caused a loss of I56i-lacZ expression in the most distal aspects of the expression domain, corresponding to the area of Dlx-6 expression previously shown to be under the control of Endothelin-1. Thus, the combinatorial branchial arch expression of Dlx genes is achieved through interactions between signaling pathways and intrinsic cellular factors. I56i drives the entire expression of Dlx5/6 in the first arch and contains necessary sequences for regulation by at least three separate pathways, whereas I12a only replicates a small domain of endogenous expression, regulated in part by BMP-4 and FGF-8.

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PubMed2004

Neural crest contribution to mammalian tooth formation.

The cranial neural crest cells, which are specialized cells of neural origin, are central to the process of mammalian tooth development. They are the only source of mesenchyme able to sustain tooth development, and give rise not only to most of the dental tissues, but also to the periodontium, the surrounding tissues that hold teeth in position. Tooth organogenesis is regulated by a series of interactions between cranial neural crest cells and the oral epithelium. In the development of a tooth, the epithelium covering the inside of the developing oral cavity provides the first instructive signals. Signaling molecules secreted by the oral epithelium 1) establish large cellular fields competent to form a specific tooth shape (mono- or multicuspid) along a proximodistal axis; 2) define an oral (capable of forming teeth) and non-oral mesenchyme along a rostrocaudal axis; and 3) position the sites of future tooth development. The critical information to model tooth shape resides later in the neural crest-derived mesenchyme. Cranial neural crest cells ultimately differentiate into highly specialized cell types to produce mature dental organs. Some cranial neural crest cells located in the dental pulp, however, maintain plasticity in their developmental potential up to postnatal life, offering new prospects for regeneration of dental tissues.

Animals↗
PubMed2004

Traf6 is essential for murine tooth cusp morphogenesis.

Ectodermal appendages such as skin, hair, teeth, and sweat glands are affected in patients with hypohidrotic (anhydrotic) ectodermal dysplasia (HED). It has been established that mutations in the tumor necrosis factor (TNF) superfamily of molecules, i.e., ectodysplasin (EDA), EDA receptor (EDAR), and EDAR-associated death domain (EDARADD; the intracellular adaptor for EDAR), are responsible for several forms of HED in humans and mice. We show here by in situ hybridisation that another TNF family (orphan) receptor, TROY (also known TAJ, TAJ-alpha, TRADE, and TNFRSF19), is strongly coexpressed with Edar in the epithelial enamel knot signalling centres that are believe to regulate cuspal morphogenesis during murine tooth development. Traf6 is known to function as an intracellular adaptor protein for Troy and examination of Traf6 mutant mice revealed abnormalities in molar teeth that are similar but more severe than those produced by mutations in Eda signalling molecules. This finding suggests that, in additional to ectodysplasin, another TNF pathway involving Troy/Traf6 is involved in molar tooth cusp formation and identifies an essential role for a Traf in tooth development. Developmental Dynamics 229:131-135, 2004.

Animals↗
PubMed2004

Ror2 knockout mouse as a model for the developmental pathology of autosomal recessive Robinow syndrome.

Robinow syndrome (RS) is a human dwarfism syndrome characterized by mesomelic limb shortening, vertebral and craniofacial malformations and small external genitals. We have analyzed Ror2(-/-) mice as a model for the developmental pathology of RS. Our results demonstrate that vertebral malformations in Ror2(-/-) mice are due to reductions in the presomitic mesoderm and defects in somitogenesis. Mesomelic limb shortening in Ror2(-/-) mice is a consequence of perturbed chondrocyte differentiation. Moreover, we show that the craniofacial phenotype is caused by a midline outgrowth defect. Ror2 expression in the genital tubercle and its reduced size in Ror2(-/-) mice makes it likely that Ror2 is involved in genital development. In conclusion, our findings suggest that Ror2 is essential at multiple sites during development. The Ror2(-/-) mouse provides a suitable model that may help to explain many of the underlying developmental malformations in individuals with Robinow syndrome.

Animals↗
PubMed2004

TNF signalling in tooth development.

Mammalian tooth development has served as an excellent model system to investigate the intricate, interactive mechanisms of patterning, morphogenesis and cytodifferentiation during organogenesis. Teeth develop from interactions between epithelium and neural crest-derived (ecto)mesenchyme that are largely mediated by ligand-receptor signalling. It is well-established that signalling molecules of the Bmp, Fgf, Wnt and Hedgehog families, are involved at multiple stages of tooth development. Recently, however, a specific role for molecules belonging to the TNF-family of ligands in tooth morphogenesis has been identified, suggesting that this pathway, acting to activate NF-kappaB, has played an important role in the development and evolution of tooth number and shape.

Animals↗
PubMed2004

A dual role for Ikk alpha in tooth development.

IKK alpha is a component of the I kappa B kinase (IKK) complex that plays a key role in the activation of NF-kappa B. In Ikk alpha mutant mice and mice expressing a transdominant negative mutant of I kappa B alpha (cI kappa B alpha Delta N), molars have abnormal cusps, indicating that Ikk alpha is involved in cusp formation through the NF-kappa B pathway. However, Ikk alpha mutant incisors also have an earlier phenotype where epithelium evaginates outward into the developing oral cavity rather than invaginating into the underlying mesenchyme. A similar evagination of epithelium was also observed in whisker development, suggesting that Ikk alpha contributes to the direction of epithelial growth during the early stages of development in many ectodermal appendages. Since cI kappa B alpha Delta N mice have normal incisor epithelial invagination, Ikk alpha's role appears to be NF-kappa B independent. Changes in Notch1, Notch2, Wnt7b, and Shh expression found in incisor epithelium of Ikk alpha mutants suggest that this NF-kappa B-independent function is mediated by Notch/Wnt/Shh signaling pathways.

Animals↗
PubMed2003

Normal and abnormal dental development.

Teeth are vertebrate organs that arise from complex and progressive interactions between an ectoderm, the oral epithelium and an underlying mesenchyme. During their early development, tooth germs exhibit many morphological and molecular similarities with other developing epithelial appendages, such as hair follicles, mammary and salivary glands, lungs, kidneys, etc. The developing mouse tooth germ, which is an experimentally accessible model for organogenesis, provides a powerful tool for elucidating the molecular mechanisms that control the development of these organs. Dentition patterning also provides a unique model for understanding how different shapes of teeth arise in different regions of the jaws. We review here the main signalling networks mediating the epithelial-mesenchymal interactions involved in tooth morphogenesis and patterning.

Animals↗
PubMed2003

Notch2 protein distribution in human teeth under normal and pathological conditions.

Notch signaling is essential for the appropriate differentiation of many cell types during development and, furthermore, is implicated in a variety of human diseases. Previous studies have shown that although the Notch1, -2, and -3 receptors are expressed in developing and injured rodent teeth, Notch2 expression was predominant after a lesion. To pursue the role of the Notch pathway in tooth development and disease, we have analyzed the expression of the Notch2 protein in embryonic and adult wounded human teeth. During the earlier stages of tooth development, the Notch2 protein was expressed in the epithelium, but was absent from proliferating cells of the inner enamel epithelium. At more advanced stages, Notch2 was expressed in the enamel-producing ameloblasts, while it was absent in mesenchyme-derived odontoblasts that synthesize the dentin matrix. Although Notch2 was not expressed in the pulp of adult intact teeth, it was reexpressed during dentin repair processes in odontoblasts and subodontoblastic cells. Transforming growth factor beta-1, which stimulates odontoblast differentiation and hard tissue formation after dental injury, downregulated Notch2 expression in cultured human dental slices, in vitro. These observations are consistent with the notion that Notch signaling is an important element in dental physiological and pathogenic conditions.

Adult↗
PubMed2003

Tooth and jaw: molecular mechanisms of patterning in the first branchial arch.

The mammalian jaw apparatus is ultimately derived from the first branchial arch derivatives, the maxillary and mandibular processes, and composed of a highly specialised group of structures. Principle amongst these are the skeletal components of the mandible and maxilla and the teeth of the mature dentition. Integral to the development of these structures are signalling interactions between the stomodeal ectoderm and underlying neural crest-derived ectomesenchymal cells that populate this region. Recent evidence suggests that in the early mouse embryo, regionally restricted expression of homeobox-containing genes, such as members of the Dlx, Lhx and Gsc classes, are responsible for generating early polarity in the first branchial arch and establishing the molecular foundations for patterning of the skeletal elements. Teeth also develop on the first branchial arch and are derived from both ectoderm and the underlying ectomesenchyme. Reciprocal signalling interactions between these cell populations also control the odontogenic developmental programme, from early patterning of the future dental axis to the initiation of tooth development at specific sites within the ectoderm. In particular, members of the Fibroblast growth factor (Fgf), Bmp, Hedgehog and Wnt families of signalling molecules induce regionally restricted expression of downstream target genes in the odontogenic ectomesenchyme. Finally, the processes of morphogenesis and cellular differentiation ultimately generate a tooth of specific class. Many of the same genetic interactions that are involved in early tooth development mediate these effects through the activity of localised signalling centres within the developing tooth germ.

Animals↗
PubMed2003

Expression of TNF-receptor-associated factor genes in murine tooth development.

Tumor necrosis factor receptor-associated factors (TRAFs) belong to a family of intracellular adaptor proteins that mediate signaling downstream of various cell surface receptors. We carried out comparative in situ hybridization analysis of five Traf genes Traf1, Traf2, Traf3, Traf4 and Traf6 during murine odontogenesis from the formation of the epithelial thickening to the early bell stage. Traf2, Traf3 and Traf6 showed weak expression in the thickened epithelium. Expression of Traf1, Traf2 and Traf6 were observed in the outer edges of the bud epithelium whereas Traf3 was strongly expressed at the tip of the bud epithelium. Expression of Traf1, Traf4 and Traf6 were detected in the dental papilla mesenchyme. Traf2 showed restricted expression in the internal enamel epithelium of the bell stage while expression of Traf1, Traf3, Traf4 and Traf6 were observed in both the internal and the external enamel epithelium. During early odontogenesis, all five genes show dynamic spatiotemporal expression patterns.

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