Martyn T. Cobourne

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.

Alignment efficiency of Damon3 self-ligating and conventional orthodontic bracket systems: a randomized clinical trial.

INTRODUCTION The aim of this study was to compare the efficiency of mandibular tooth alignment and the clinical effectiveness of a self-ligating and a conventional preadjusted edgewise orthodontic bracket system. METHODS A multicenter randomized clinical trial was conducted in 2 orthodontic clinics. Sixty-two subjects (32 male, 30 female; mean age, 16.27 years) with mandibular incisor irregularities of 5 to 12 mm and a prescribed extraction pattern including the mandibular first premolars were randomly allocated to treatment with Damon3 self-ligating (Ormco, Glendora, Calif) or Synthesis (Ormco) conventionally ligated brackets. Fully ligated 0.014-in nickel-titanium archwires were used first in both groups, followed by a sequence of 0.014 x 0.025-in and 0.018 x 0.025-in nickel-titanium, and 0.019 x 0.025-in stainless steel. Study casts were taken at the start of treatment (T1), the first archwire change (T2), and the placement of the final 0.019 x 0.025-in archwire (T3). Cephalometric lateral skull and long-cone periapical radiographs of the mandibular incisors were taken at T1 and T3. RESULTS No significant difference was noted (P >0.05) in initial rate of alignment for either bracket system. Initial irregularity influenced subsequent rate of movement, but sex, age, and appliance type were statistically insignificant. Alignment was associated with an increase in intercanine width, a reduction in arch length, and proclination of the mandibular incisors for both appliances, but the differences were not significant. Incisor root resorption was not clinically significant and did not differ between systems. CONCLUSIONS Damon3 self-ligating brackets are no more efficient than conventional ligated preadjusted brackets during tooth alignment.

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.

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.

Periodic stripe formation by a Turing mechanism operating at growth zones in the mammalian palate

We present direct evidence of an activator-inhibitor system in the generation of the regularly spaced transverse ridges of the palate. We show that new ridges, called rugae, that are marked by stripes of expression of Shh (encoding Sonic hedgehog), appear at two growth zones where the space between previously laid rugae increases. However, inter-rugal growth is not absolutely required: new stripes of Shh expression still appeared when growth was inhibited. Furthermore, when a ruga was excised, new Shh expression appeared not at the cut edge but as bifurcating stripes branching from the neighboring stripe of Shh expression, diagnostic of a Turing-type reaction-diffusion mechanism. Genetic and inhibitor experiments identified fibroblast growth factor (FGF) and Shh as components of an activator-inhibitor pair in this system. These findings demonstrate a reaction-diffusion mechanism that is likely to be widely relevant in vertebrate development.

Gas1 is a modifier for holoprosencephaly and genetically interacts with sonic hedgehog

Holoprosencephaly (HPE) is a clinically heterogeneous developmental anomaly affecting the CNS and face, in which the embryonic forebrain fails to divide into distinct halves. Numerous genetic loci and environmental factors are implicated in HPE, but mutation in the sonic hedgehog (Shh) gene is an established cause in both humans and mice. As growth arrest-specific 1 (Gas1) encodes a membrane glycoprotein previously identified as a Shh antagonist in the somite, we analyzed the craniofacial phenotype of mice harboring a targeted Gas1 deletion. Gas1(-/-) mice exhibited microform HPE, including midfacial hypoplasia, premaxillary incisor fusion, and cleft palate, in addition to severe ear defects; however, gross integrity of the forebrain remained intact. These defects were associated with partial loss of Shh signaling in cells at a distance from the source of transcription, suggesting that Gas1 can potentiate hedgehog signaling in the early face. Loss of a single Shh allele in a Gas1(-/-) background significantly exacerbated the midline craniofacial phenotype, providing genetic evidence that Shh and Gas1 interact. As human GAS1 maps to chromosome 9q21.3-q22, a region previously associated with nonsyndromic cleft palate and congenital deafness, our results establish GAS1 as a potential locus for several human craniofacial malformations.

Familial human hypodontia--is it all in the genes?

The congenital absence of teeth is one of the commonest developmental abnormalities seen in human populations. Familial hypodontia or oligodontia represents an absence of varying numbers of primary and/or secondary teeth as an isolated trait. While much progress has been made in understanding the developmental basis of tooth formation, knowledge of the aetiological basis of inherited tooth loss remains poor. The study of mouse genetics has uncovered a large number of candidate genes for this condition, but mutations in only three have been identified in human pedigrees with familial hypodontia or oligodontia: MSX1, PAX9 and AXIN2. This suggests that these conditions may represent a more complex multifactorial trait, influenced by a combination of gene function, environmental interaction and developmental timing. Completion of the human genome project has made available the DNA sequence of the collected human chromosomes, allowing the localisation of all human genes and, ultimately, determination of their function. Therefore it is likely that our understanding of this complex developmental process will continue to improve, not only during normal development but also when things go wrong.

Abnormal Hair Development and Apparent Follicular Transformation to Mammary Gland in the Absence of Hedgehog Signaling

Summary We show that removing the Shh signal tranducer Smoothened from skin epithelium secondarily results in excess Shh levels in the mesenchyme. Moreover, the phenotypes we observe reflect decreased epithelial Shh signaling, yet increased mesenchymal Shh signaling. For example, the latter contributes to exuberant hair follicle (HF) induction, while the former depletes the resulting follicular stem cell niches. This disruption of the niche apparently also allows the remaining stem cells to initiate hair formation at inappropriate times. Thus, the temporal structure of the hair cycle may depend on the physical structure of the niche. Finally, we find that the ablation of epithelial Shh signaling results in unexpected transformations: the follicular outer root sheath takes on an epidermal character, and certain HFs disappear altogether, having adopted a strikingly mammary gland-like fate. Overall, our study uncovers a multifaceted function for Shh in sculpting and maintaining the integrity and identity of the developing HF.

Making up the numbers: The molecular control of mammalian dental formula

Teeth develop in the mammalian embryo via a series of interactions between odontogenic epithelium and neural crest-derived ectomesenchyme of the early jaw primordia. The molecular interactions required to generate a tooth are mediated by families of signalling molecules, which often act reiteratively in both a temporal and spatial manner. Whilst considerable information is now available on how these molecules interact to produce an individual tooth, much less is known about the processes that control overall tooth number within the dentition. However, a number of mouse models are now starting to provide some insight into the mechanisms that achieve this. In particular, co-ordinated restriction of signalling molecule activity is important in ensuring appropriate tooth number and there are different requirements for this suppression in epithelial and mesenchymal tissues, both along different axes of individual jaws and between the jaws themselves. There are a number of fundamental mechanisms that facilitate supernumerary tooth formation in these mice. A key process appears to be the early death of vestigial tooth primordia present in the embryo, achieved through the suppression of Shh signalling within these early teeth. However, restriction of WNT signalling is also important in controlling tooth number, with increased transduction being capable of generating multiple tooth buds from the oral epithelium or existing teeth themselves, in both embryonic and adult tissues. Indeed, uncontrolled activity of this pathway can lead to the formation of odontogenic tumours containing multiple odontogenic tissues and poorly formed teeth. Finally, disrupted patterning along the buccal-lingual aspect of the jaws can produce extra teeth directly from the oral epithelium in a duplicated row. Together, all of these findings have relevance for human populations, where supernumerary teeth are seen in association with both the primary and permanent dentitions. Moreover, they are also providing insight into how successional teeth form in both embryonic and post-natal tissues of the jaws.

Perception of discomfort during initial orthodontic tooth alignment using a self-ligating or conventional bracket system: a randomized clinical trial

The aim of this study was to compare the degree of discomfort experienced during the period of initial orthodontic tooth movement using Damon3 self-ligating and Synthesis conventional ligating pre-adjusted bracket systems. Sixty-two subjects were recruited from two centres (32 males and 30 females; mean age 16 years, 3 months) with lower incisor irregularity between 5 and 12 mm and a prescribed extraction pattern, including lower first premolar teeth. These subjects were randomly allocated for treatment with either bracket system. Fully ligated Damon3 0.014-inch Cu NiTi archwires were used for initial alignment in both groups. Following archwire insertion, the subjects were given a prepared discomfort diary to complete over the first week, recording discomfort by means of a 100 mm visual analogue scale at 4 hours, 24 hours, 3 days, and 1 week. The subjects also noted any self-prescribed analgesics that were taken during the period of observation. Data were analysed using repeated measures analysis of variance. There were no statistically significant differences in perceived discomfort levels between the two appliances; discomfort did not differ at the first time point and did not develop differently across subsequent measurement times. Overall, this investigation found no evidence to suggest that Damon3 self-ligating brackets are associated with less discomfort than conventional pre-adjusted brackets during initial tooth alignment, regardless of age or gender.