Australian Dental Journal

The official journal of the Australian Dental Association

Australian Dental Journal 2014; 59:(1 Suppl): 1–12 doi: 10.1111/adj.12160

The dentition: the outcomes of morphogenesis leading to variations of tooth number, size and shape AH Brook,*† J Jernvall,‡ RN Smith,§ TE Hughes,* GC Townsend* *School of Dentistry, The University of Adelaide, South Australia, Australia. †Institute of Dentistry, Queen Mary University of London, United Kingdom. ‡Institute of Biotechnology, University of Helsinki, Finland. §School of Dentistry, University of Liverpool, Liverpool, United Kingdom.

ABSTRACT The clinical importance of variations of tooth number, size and shape is seen in many dental disciplines. Early diagnosis allows optimal patient management and treatment planning, with intervention at an appropriate time to prevent complications in development and so reduce later treatment need. Understanding the process of dental morphogenesis and the variations in outcomes is an important contribution to the multidisciplinary clinical team approach to treatment. Tooth number, size and shape are determined during the initiation and morphogenetic stages of odontogenesis. The molecular evidence of repetitive signalling throughout initiation and morphogenesis is reflected clinically in the association of anomalies of number, size and shape. This association has been statistically modelled from epidemiological evidence and confirmed by 2D and 3D measurement of human dental study casts. In individuals with hypodontia, the teeth that are formed are smaller than the population mean and often show reduced and simplified shape. In contrast, in individuals with supernumerary teeth, the other teeth are larger than average and may show an enhanced shape. Clinical observations in humans and studies of laboratory animals gave rise to the concept of morphogenetic fields within the dentition. The findings, which can also be considered as reflecting gene expression territories, have been developed to incorporate field, clone and homeobox theories. The clinical distribution of developmental anomalies tends to follow the pattern of these fields or territories. Improved care for patients with these anomalies will come not only from utilizing a multidisciplinary clinical team but also by expanding the approach to include other relevant scientific disciplines. Keywords: Morphogenesis, supernumeraries, hypodontia, megadontia, microdontia.

INTRODUCTION The clinical importance of variations of tooth number, size and shape is seen in many dental disciplines, particularly paediatric dentistry, orthodontics, restorative dentistry and oral surgery. Early diagnosis allows optimal patient management and treatment planning, with intervention at an appropriate time to prevent complications in development and so reduce later treatment need. An example is the removal of a supernumerary tooth which would otherwise interfere with the eruption of an underlying permanent tooth. Understanding the process of morphogenesis and the variations in the outcomes is an important contribution to the multidisciplinary clinical team approach to treatment. Variation in outcome in a developmental process, such as the formation of the dentition, enables adaptation to different environments. Tooth number, size and shape are determined during the initiation and mor© 2014 Australian Dental Association

phogenetic stages of dental development. The molecular evidence of repetitive signalling throughout initiation and morphogenesis is reflected clinically in the association of anomalies of number, size and shape. This association has been statistically modelled from clinical and epidemiological evidence and confirmed by 2D and 3D measurement of human dental study casts. In individuals with hypodontia, the teeth that are present are smaller than the population mean and often show reduced and simplified shape. In contrast, in individuals with supernumerary teeth, the other teeth are larger than average and may show an enhanced shape. In the sections that follow we set out key findings concerning the early developmental stages of teeth, including the concepts of morphogenetic fields and gene expression territories. These are related to the prevalence, clinical features and associations of human clinical findings concerning the variations of number, size and shape. These developmental and 1

AH Brook et al. clinical aspects are then related and a unifying model incorporating these aspects is developed. The aetiology of these variations is also discussed. An overview follows of further analytical and investigative approaches that are advancing knowledge in the area and the conclusion relates all the new developments considered in this paper to the exciting future of clinical dentistry. This understanding is important to enhance the diagnosis and treatment planning for these conditions which in their more severe presentations require extensive, long-term care from multidisciplinary teams. Developmental aspects – initiation and morphogenesis In the previous section of this special issue the development of the dentition as a complex adaptive system is considered1 and the molecular and cellular interactions that regulate tooth initiation, morphogenesis and differentiation are detailed.2 Under this heading, we consider briefly those aspects of initiation and morphogenesis that are particularly relevant to exploring variations in tooth number, size and shape. The teeth are initiated from the dental lamina. They form from the epithelium and underlying mesenchyme, regulated by inductive interactions between these tissues. The molecular interactions involve a series of reiterative actions between specific signalling molecules, receptors and transcription factors,3 a number of which are summarized in Fig. 1. Figure 2 includes the distribution of these factors in the epithelium and the mesenchyme, and incorporates additional factors. In determining tooth regions within the dental lamina, Fgf and Bmp influence the location of mesenchymal expression of Pax9, a paired box transcription factor.4 Pax9 is stimulated by Fgf8 and inhibited by Bmp2 and Bmp4 influencing the site of tooth buds.5 However, Pitx2 and Shh are also present at this stage and tooth germs still develop in the same locations in Pax9 knockout mice.6 The Dlx homeobox genes are also important in early patterning of the dental

Fig. 1 Diagram of some of the signalling molecules, receptors and transcription factors identified for the initiation and morphogenesis of tooth germ development. Data derived from Galluccio et al.3 2

field.6–9 In addition, the Bmp antagonist Sostdc1 and the Wnt co-receptor Lrp 4 provide extracellular communication between mesenchymal and epithelial cells based on the integration of Wnt and Bmp pathways during regulation of tooth number.10 The action of transcription factors is necessary for initiation and progression beyond the initiation stage.11 Signals from the epithelium regulate expression of the transcription factors Msx1, Pax9 and Runx2. Msx1 is induced by Bmp and Fgf, while Pax9 and Runx2 are induced by Fgf.11 Bmp4 and Msx1 regulate one another in a positive feedback loop in the dental mesenchyme.12,13 If any one of these transcription factors is absent in knockout mice, tooth development may be arrested at the bud stage14,15 but different members of the same family, e.g. Msx1 and Msx2, may compensate for each other when one is inactivated. Msx 1 and Osr2 act antagonistically in the patterning of the tooth morphogenetic field by controlling the expression and spatial distribution of mesenchymal odontogenic signals along the buccolingual axis.15 In early tooth morphogenesis the critical role of timing is evidenced in the interaction of Pax9, Msx1 and Bmp4. For example, during initiation, Pax9 is not a transcriptional regulator of Msx1 at E12.5 days in utero in the mouse. However, at E13.5 days, Pax9 begins to induce Msx1 expression and then Pax9 and Msx1 proteins can induce Bmp4 expression.16 This emphasizes the importance of timing and critical periods in the developmental process. As tooth morphogenesis advances, the primary and secondary enamel knots control the development of crown dimensions and cusp formation. While the enamel knot expresses growth stimulatory signals, its cells remain non-proliferative.17 These non-dividing cells stimulate proliferation of both the surrounding cells and the mesenchymal dental papillae.18 The repeated activation and inhibition of signalling is related to differential growth and folding within the tooth germ and determines dimensions and cusp patterns. The shape of the tooth crown results during cap and bell stages when there is rapid proliferation of cells related to folding of the epithelium to form cusp shapes.6 In the enamel knots, apoptosis has been suggested as a mechanism controlling the duration of signalling17,19 and the expression of Bmp4 in the enamel knot cells is associated with their apoptosis.6 Function of the enamel knot as a signalling centre begins to be affected by apoptosis in the late cap to early bell stages.20,21 Cessation of activity in the enamel knot is linked to the expression of the cyclin-dependent kinase inhibitor p21 induced by Bmp4. Having undergone apoptosis at the late cap stage, the primary enamel knot is no longer detected at the © 2014 Australian Dental Association

The dentition: outcomes of morphogenesis

Fig. 2 Diagram of the progressive development of each tooth during initiation and morphogenesis stages, relating the macroscopic variations in number, size and shape to the molecular and cellular/tissue stages at which they arose. Epithelium – yellow; Mesenchyme – blue. Cellular and molecular aspects derived from http://bite-it.helsinki.fi/

bell stage. Secondary enamel knots develop at the sites of cusps in teeth with multiple cusps. They produce signalling molecules stimulating proliferation of nearby cells, leading to folding of the inner enamel epithelium and subsequent multiple cusp formation. Apoptosis in the enamel knot plays an important role in regulating tooth size and shape,22 and different dimensions are affected differently. The major role of the enamel knots in determining tooth size and shape is considered further in the section on further analytical and investigative approaches. Clinical aspects: prevalence, appearance and associations Prevalence The prevalence of these variations shows a range of values between the primary and permanent dentitions, between the genders and between ethnic groups. The frequency in the primary dentition is lower for all variations except for double teeth (geminated and fused teeth). In an epidemiological study in the UK, the prevalences in Table 1 were © 2014 Australian Dental Association

Table 1. Prevalence of six variations of tooth number, size and shape found in an epidemiological study of 741 three to five-year-old (primary) and 1115 eleven to fourteen-year-old (permanent) schoolchildren in Slough, UK23

Supernumeraries Hypodontia Invaginated teeth Double teeth Megadontia Microdontia

Primary (%)

Permanent (%)

0.8 0.3 0.1 1.6 0.0 0.5

2.1 4.4 4.1 0.1 1.1 2.5

found.23 Similar findings have been reported by other workers. While supernumerary teeth are uncommon in the primary dentition (0.2–0.8%) with no significant gender distribution yet described, they occur more frequently in the permanent dentition (1.5–3.8%) with males affected approximately twice as frequently as females.24–26 Hypodontia, the congenital absence of one or more teeth, is unusual in the primary dentition (0.1–1.5%) 3

AH Brook et al. with no significant gender difference, most often occurring in the maxillary lateral incisor region.23 In the permanent dentition, excluding the third molars, a prevalence between 3.5% and 7.0% has been found for most populations with a gender ratio of the order of males to females 1:1.5.24,27,28 Findings for hypodontia of third molars range from 9% to 37%. The lower second premolars are congenitally absent in 3–4% of patients, the upper lateral incisors in 1–2.5% and the upper second premolars in 1–2%.3,27,29 Megadontia is rare in the primary dentition, but has been reported in 1.1% of children in the permanent dentition from an epidemiological study of 1115 school children aged 11–14 years23 (Table 1). The permanent upper central incisors and lower second premolars were the teeth particularly involved. The prevalence of microdontia in the primary dentition ranges from 0.2% to 0.5%,23 affecting both upper and lower incisors. In the permanent dentition, many studies have concentrated on the upper lateral incisors, with findings of 0.5% to 3.1%. Females are affected more often than males. In patients with severe hypodontia, microdontia can affect all tooth types. When considering traits related to shape, as with those of number and size, they exhibit a quasicontinuous mode of variation or threshold dichotomy.1 Such traits do not form below a phenotypic realization threshold, but vary continuously along a range of expression once a threshold is exceeded.30 Carabelli cusp on molars is a good example of this and the prevalence found in a particular study will depend on the level at which the ‘diagnosis’ is set along the continuum. A male bias in Carabelli trait expression is expected given that sex differences are greater in crown size than in intercuspal distances,31 and it has been found that males are more likely to express Carabelli trait than females.32 Similarly invaginations are a quasi-continuous trait. They are classified according to specific degrees of severity of the trait.33 The range of prevalences reported is from 1% to 5% with a male to female ratio of 2:1.23 The teeth most commonly involved are the upper lateral incisors but cases are recorded in all tooth types. Important for treatment planning is that invaginations are often bilaterally symmetrical. ‘Double teeth’ have been described under a variety of titles – fusion, gemination, dichotomy, synodontia, schizodontia, connation. Many of these terms imply a particular mode of origin that at present cannot be reliably determined and, therefore, a neutral term such as double teeth is preferred.34 The prevalence of double teeth in the primary dentition ranges from 0.5% to 4.5% and in the permanent dentition from 0.1% to 0.3%. The overall frequency of anomalies in the permanent dentitions that follow on from primary 4

dentitions with double teeth is approximately 50%. This is another important finding for clinicians. Clinical features and associations Supernumerary teeth exhibit a great variety of shapes including conical, tuberculate, supplemental and odontome like. They are found in every region of the permanent dentition, most frequently in the maxillary incisor region. Approximately 75% of permanent supernumeraries fail to erupt and are diagnosed radiographically, sometimes when they are impeding the eruption of another tooth. Most commonly, only one supernumerary is present in a dentition; less frequently there are two supernumeraries, while three or four supernumerary teeth are rare. Supernumeraries in the primary dentition are followed by an anomaly in the permanent dentition in approximately 50% of cases. When accurate measurements are made, supernumerary teeth are associated with an increase in the tooth size of other teeth, with the differences greater in the mesiodistal than buccolingual crown dimensions.35–37 Using 2D image analysis it was shown that when a supernumerary tooth was present in the upper anterior region, there was a gradient effect from the site of the supernumerary, with upper and lower incisors showing the greatest difference in size from controls. The teeth adjacent to supernumeraries may exhibit changes in shape37 with the upper central incisor being more affected than the upper lateral incisor, supporting a local field effect38 (Fig. 3; Table 2). Supernumerary teeth are found associated with megadontia, double teeth and invaginations (Fig. 4). Hypodontia of primary teeth is followed in 75% of patients by agenesis of permanent teeth in the same region. In the permanent dentition, the congenitally

Fig. 3 Clinical photograph of a patient with an erupted midline supernumerary, showing the large size of the other teeth. The size and shape of the maxillary central and lateral incisors and the mandibular lateral incisors are particularly affected. This is shown in Table 2. © 2014 Australian Dental Association

The dentition: outcomes of morphogenesis Table 2. Average mesiodistal crown dimensions of permanent teeth (in mm) in patients with supernumeraries compared with control group37 Supernumerary

Control

9.05** 7.10* 8.01* 10.41 5.70 6.16* 7.06 10.55

8.68 6.84 7.84 10.15 5.51 5.97 6.89 10.40

Upper central Upper lateral Upper canine Upper 2nd molar Lower central Lower lateral Lower canine Lower 2nd molar

Table 3. Data from an epidemiological study of the permanent dentition in 1115 schoolchildren showing the highly statistically significant association between hypodontia and microdontia in this population sample24

Microdontia No microdontia Total

Hypodontia

No hypodontia

Total

9 40 49

19 1047 1066

28 1087 1115

Chi-squared value (with Yates’ correction) = 46.1, df = 1, p < 0.001.

*Significant differences in the mean values at p < 0.05. **Significant differences in the mean values at p < 0.01.

Fig. 5 A patient with hypodontia of the upper left lateral incisor and microdontia of the upper right lateral incisor. The upper central incisors also show a reduction in shape from the average.

Fig. 4 A patient with a supernumerary upper left lateral incisor, a megadont/double tooth upper right central incisor and generalized large tooth size.

absent teeth are most frequently those at the end of the morphogenetic fields.39 The number of missing teeth varies from one to the complete dentition, referred to as anodontia. The frequency with which these different degrees are found is compatible with a tail of the distribution in a quasi-continuous model, i.e. one or two teeth are missing commonly, while very severe hypodontia only occurs rarely.39 There is substantial evidence that hypodontia is associated with significantly smaller than average tooth size throughout the dentition (Table 3).24,28,35,36,40 The more severe the hypodontia, the greater the reduction in tooth size.40 Associated with hypodontia and the small tooth size are changes in shape. Again, the greater the number of missing teeth and the smaller the formed teeth, the more evident is the different shape with tapering crowns from the incisal/occlusal surfaces towards the cemento-enamel junction, more rounded contours and reduction in cusp number in posterior teeth (Figs. 5 and 6). These clinical findings have been confirmed by 2D and 3D analysis of dental study models.35,40,41 © 2014 Australian Dental Association

Fig. 6 A patient with severe hypodontia and marked tapering of the lower incisors and canines into a conical shape.

Relating developmental and clinical aspects The outcome of the developmental process is a dentition, seen as a phenotype. Valuable findings have emerged from animal as well as human studies in relating phenotype to genotype. Therefore, where relevant, the findings from animal models are considered alongside those from human studies, while acknowledging the limitations of extrapolating from experiments on animals. The human clinical phenotype reflects the progressive nature of the developmental process, with different teeth of a given tooth type forming and maturing at different times. The time gradient tends to follow 5

AH Brook et al. the spatial gradient from mesial to distal in each tooth region. Therefore, within a given tooth type, there will be teeth at different developmental stages. Similarly, between different tooth types, there will be some overlap in development, but also different developmental stages at a given point of time. The complexity of these time and space parameters of dental development is reflected in the complexity of the clinical phenotypes of dental anomalies of number, size, form and structure. These phenotypes need to be considered in the order of the stage of the developmental process at which they have occurred. In keeping with the multilayered nature of the process, the clinical outcome relates to evidence of the tissue changes and to the molecular genetic/epigenetic/environmental interactions.1 In addition, the multidimensional outcomes for each tooth reflect more than the influences of the molecular factors that would be considered if each tooth developed in isolation. The interactions of the developing tooth germs for nutrition and space reflected in their position in morphogenetic fields also affect the clinical phenotype.1,24,32 A unifying model for the developmental and clinical findings The molecular evidence of repetitive signalling throughout initiation and morphogenesis is reflected in the association of anomalies of number, size and shape seen together clinically in the same dentition (Figs. 3–6). These associations have been validated by laboratory measurement of human dental study models.36–38,42 A model has been previously published which incorporated the clinical and epidemiological findings for variation in tooth number and size.24 Here the model is developed further to include tooth shape in the light of molecular evidence and further laboratory studies of clinical material (Fig. 7). This model is based on the underlying continuous distribution of tooth size and shape, and the quasi-continuous nature of the variations in tooth number. The prevalence of the different degrees of severity of supernumeraries and hypodontia is compatible with a quasi-continuous distribution, as are the variations in tooth size and shape associated with these variations in number. It incorporates the gender differences in tooth size, reviewed in the clinical aspects section above, by having separate curves for males and females. With the thresholds added on the curves it also accounts for the higher prevalences of hypodontia and microdontia in females and the higher prevalences of megadontia and supernumeraries in males. The model also includes the associations between anomalies. At one end of the distribution are hypodontia and small 6

Fig. 7 A unifying aetiological model which incorporates the clinical and epidemiological findings for variations in tooth size, shape and number.

and tapering teeth and at the other are supernumeraries and large teeth, with some variations in shape. The shading between thresholds indicates that as teeth are nearer to the extremes of size, their shape tends also to change to a greater degree. This accords with the findings from animal models of cusp development and human studies of Carabelli trait and molar cusps.30,31,43,44 The reiterative nature of the molecular interactions between factors in the epithelium and mesenchyme throughout initiation and morphogenesis is also in agreement with the model. Many of the same genes are active at different stages throughout initiation and morphogenesis (Fig. 7). Thus, for tooth number, size and shape, as well as in patients with a PAX9 mutation, there is hypodontia, microdontia and tapering, rounded tooth shape.35 The aetiology of variations in number, size and shape The aetiology of these variations within the population is multifactorial with evidence of chromosomal, polygenic, single gene and major environmental influences in this complex aetiology; different factors may exert a major influence in different individuals.24 Mutations in MSX1, PAX9, AXIN2, EDA, EDAR and WNT10A have been identified in families with non-syndromic hypodontia.2,45–48 Msx1 and Pax9 are co-expressed in dental mesenchyme at the bud and cap stages. Many human mutations of PAX9 have been reported in hypodontia families, of which a number are missense mutations.3 The DNA binding and transcriptional ability of Pax9 proteins are affected differently in each different mutation.49 Missense mutations of Pax9 affecting the paired domain do not disrupt the physical interaction with Msx1 while, in the G51S mutation, synergistic cooperation with Msx1 is decreased or abolished in Bmp4/Luciferase assays. Functional regulation of Pax9 by homeobox proteins is necessary for early tooth development. In the G51S mutation, the phenotype presents as moderate to severe hypodontia, yet there is intact © 2014 Australian Dental Association

The dentition: outcomes of morphogenesis DNA binding and increased transcriptional activation but lack of responsiveness to modulation by Msx1. In general, the more severe phenotypes, with more congenitally absent teeth, relate to haploinsufficiency of Pax9 while those with mild or moderate hypodontia are associated with hypomorphic alleles.49 Differences in the patterns of hypodontia have been seen, with those having mutations of MSX1 particularly showing congenitally absent anterior teeth and those with PAX9 mutations predominantly affecting posterior teeth. Mutations in MSX1 are also associated with orofacial clefting, with and without hypodontia, and with Witkop syndrome.50–52 Mutations in WNT10A were found in 56% of patients in a study of isolated hypodontia.53 This is in accordance with the major role of Wnt in the initiation of tooth formation shown in transgenic mice.54,55 Inhibition of Wnt signalling can cause hypodontia and over-expression can give rise to supernumerary teeth. Similarly, inhibition of the EDA pathway leads to hypodontia while stimulation of EOA expression in mice can induce the formation of extra teeth.56,57 The frequency of congenital absence of individual teeth also relates to their position in the different morphogenetic fields (Fig. 8).32 In the permanent dentition, the third molars, second premolars, upper lateral incisors and lower central incisors are the most frequently absent teeth.39 These findings come from population and multiple clinical case studies. They probably reflect a general influence in hypodontia patients to reduced tooth tissue formation that results in lesser size and morphological changes in the earlier forming teeth in a field, i.e. affecting morphogenesis, while the later forming teeth at the end of the field have earlier effects on development, i.e. affecting initiation, and so fail to progress beyond the bud stage. While this is the general pattern in a population, in some individuals and families with hypodontia the pattern is different. In some, the congenitally absent teeth may be concentrated in the anterior region, while in others it is principally the molar region which is affected. Regional influences within the dentition are shown in a study of the distribution of congenitally absent teeth in 200 individuals with hypodontia. If one third molar is congenitally absent, the frequency of other third molars also being congenitally absent is much greater than expected by chance.39 Regional effects on tooth size were also seen in four ethnic groups; while Chinese had the largest teeth overall, this effect was seen predominantly in the anterior regions, while European and North American white Caucasians had larger molars than Chinese.58 Sometimes a mutation in a major single gene, e.g. PAX9, shows different degrees of hypodontia, microdontia and shape change in different members of the same family (Table 5).35 Thus, when considering the © 2014 Australian Dental Association

Fig. 8 A diagram of the frequency of congenitally missing teeth at each site in a group of patients.39 The arrows external to the dental arches indicate the direction of the morphogenetic fields. The later forming teeth in each field are more frequently missing than the first formed teeth.

aetiology in an individual or family, a number of factors may also be involved. The effect on tooth size in individuals having hypodontia is seen throughout the dentition. All teeth that develop in hypodontia subjects tend to be smaller than those in control groups when measured by classical manual techniques and by image analysis.36,40 This is also in accordance with the aetiological model proposed (Fig. 7).24,42 An additional finding is that different dimensions of individual teeth, e.g. mesiodistal and buccolingual crown diameters, are influenced to different degrees in hypodontia subjects compared to controls. Thus, in the family with a mutation of PAX9, the mesiodistal dimensions of the formed teeth were smaller to a different degree than the buccolingual dimensions.35 A further finding in this PAX9 family was that different teeth were affected to different extents. The reduction in size was greatest in permanent canines in the hypodontia family members with PAX9 mutation.35 7

AH Brook et al. Table 4. The distribution of congenitally missing teeth in a family with a mutation of PAX9.35 The dark stars show the teeth missing. This illustrates the variation of the number of missing teeth in family members having the same mutation of PAX9. Affected family member II:3 is the third sibling in the second generation (II) while III:6 is the sixth sibling in the third generation

Affected family members

right quadrants

left quadrants

molars premolars can incisors

incisors can premolars molars

3

2

1

2

1

1

2

1

1

2

1

1

2

1

2

3

3

2

1

2

1

1

2

1

1

2

1

1

2

1

2

3

II:3

II:4

III:2

III:5

III:6

IV:1

Table 5. The mean mesiodistal dental crown dimensions (in mm) of patients with varying degrees of hypodontia, severe (6+ teeth missing), moderate (3–5), mild (1–2), and their unaffected relatives. These are compared to a control group. The table shows decreasing tooth size as there is increasing hypodontia. A finding of major importance is that the relatives with a full complement of teeth had highly statistically significant smaller teeth than controls40 Severe Upper central Upper 1st premolar Lower 1st premolar

Moderate

Mild

Unaffected relative

Control

7.80***

8.24***

8.43***

8.30***

9.26

6.43***

6.44***

6.72***

6.81***

7.37

6.63**

6.72**

6.82**

7.11**

7.56

*** p < 0.001 ** p < 0.01.

Supporting evidence for the influence of multiple factors in hypodontia comes from studies of families with severe hypodontia. In families with a member who had six or more missing permanent teeth, excluding the third molars, those family members with complete dentitions had teeth that were statistically significantly smaller than controls (Table 4).36,40 8

In patients with supernumerary teeth, different effects on the whole dentition are observed compared with those seen in patients with hypodontia. A series of population studies show males more frequently have supernumerary teeth than females, as well as larger teeth than females.24 In patients with supernumerary teeth, the other teeth in the dentition tend to be larger than those of controls.34,37 This difference is seen in the whole dentition, but there is a gradient effect on the degree of difference. Thus, when the supernumerary tooth is in the upper central incisor region, the incisor teeth in the maxilla and mandible are the teeth that show the greatest difference in size. Image analysis measurements of the maxillary central incisors adjacent to the supernumerary show an effect on shape, as well as size, with the teeth having a more barrel-shaped outline from the labial view than controls. From studies of the mouse dentition it is suggested that mutations affecting Fgf, Eda, Bmp, Runx2, Apc, Shh and b-catenin are related to the occurrence of supernumerary teeth.10,57,59–62 Activation of b-catenin or ablation of Apc, an inhibitor of Wnt signalling, in embryonic mouse oral epithelium results in supernumerary teeth. The oral epithelium in adult mice remains responsive to b-catenin gain-of-function or APC loss-of-function and is still able to form new © 2014 Australian Dental Association

The dentition: outcomes of morphogenesis Table 6. The prevalence of hypodontia, microdontia and supernumerary teeth in samples of RomanoBritons and modern Britons of European ancestry.69 The differences are reflected in the separate curves for each population in Fig. 9 Romano-British (%)

Modern British (%)

39.0 13.0

12.7 4.4

6.4

2.5

1.2

2.1

Hypodontia (3rd molars) Hypodontia (except 3rd molars) Microdontia (except 3rd molars) Supernumeraries

teeth.59 In the mouse, supernumerary teeth may develop from vestigial tooth buds that are present in the incisor region and in the diastema between the incisors and molars. These vestigial buds usually undergo apoptosis, but if degeneration by apoptosis does not occur a supernumerary tooth or element is formed.63 Supernumerary incisor and molar teeth, as well as fused and large molar teeth, develop in Lrp4 and Sostdc1 deficient mice. Mice lacking the transcription factor Osr2, develop supernumerary teeth lingual to their normal molar teeth.15 Initiation of these supernumerary teeth is related to aberrant thickening of the oral epithelium lingual to the first molar tooth buds.15 Bmp4 induces expression of Sostdc1 whose inactivation was associated with fused first and second molars as well as supernumerary teeth in mice.60,64 Disruption of the antagonistic balance between Msx1 and Osr2 may underlie the hypodontia noted in Msx1 null mice, the supernumerary teeth in Osr2 deficient mice, and the hypodontia observed in humans with Msx1 mutations.15 Other important factors from mouse studies include the effects of up-regulation and down-regulation of specific genes and the results at a histological and clinical level on tooth number, size and shape. Changes in the regulation of Shh are related to holoprosencephaly, hypodontia and supernumerary teeth.63 In the absence of Pax9, the mesenchymal expression of Msx1, Lef1 and Bmp4 is down-regulated and tooth

Fig. 9 The unifying aetiological model of Fig. 7 developed further to incorporate data for Romano-Britons.69 The solid lines represent modern Britons of European ancestry and the dotted lines Romano-Britons. It is suggested that the curves are shifted to the left in the Romano-Britons because of major environmental insults throughout the period of dental development.

development is arrested at the bud stage, resulting in hypodontia.4 Loss of Eda leads to a smaller primary enamel knot and decreased expression of signalling molecules, with the phenotypic outcome of smaller teeth with reduced cusp morphology and altered outline shape.65 Mouse studies also suggest Sostdc1 plays a major role in determining tooth size and shape. Sostdc1, a Bmp and Wnt antagonist, integrates proapoptotic and pro-survival signals from the enamel knot and determines the area of the Bmp signal.64 Mice deficient in Sostdc1 show an increased area of Shh expression and large primary enamel knots, leading to large molars with a mesiodistal crest connecting cusps and supernumerary teeth.60 Supernumerary teeth and increased molar size and complexity are also seen with expression of an Edar receptor promoted by K14.66 In EVC mutant mice the morphology of molars was markedly affected with changes in the relative size and shape of first and second molars.67 The relationship between development and anomalies of number, size and shape of teeth in human studies reflects the reiterative patterns seen in mouse models of signalling of the ectodermal–mesenchymal interactions during tooth germ initiation and morphogenesis. Epigenetic events relating to the spatial arrangement of cells and the timing of the interactive signalling may explain differences in tooth number,

Fig. 10 A diagram of the major influences during the long development process from genotype to phenotype of the tooth. Changes in one or more of these major factors leads to increased variation. © 2014 Australian Dental Association

9

AH Brook et al. size and shape, as well as dental asymmetry in monozygotic twins.68 Similar findings for tooth number, size and shape in modern human populations were gained in studies of Romano-Britons. Females had smaller teeth than males and had a higher frequency of hypodontia and microdontia; males had larger teeth and a higher frequency of supernumerary teeth and megadontia (Table 6).69,70 The teeth of the Romano-Britons were smaller than modern Britons, possibly reflecting major environmental effects such as poor nutrition, ingestion of high levels of toxin (lead) and recurrent infections.69 These findings are compatible with the multifactorial model and provide an example of the interaction of genetic and environmental factors. The curves of the RomanoBritons are incorporated in Fig. 9 as dotted lines. They are similar curves to modern Britons of European ancestry, but the distributions are moved to the left, probably by these major environmental insults present throughout development. Figure 10 is a diagram incorporating the different factors, genetic/epigenetic/environmental, that influence the special and temporal development of the tooth and result in the erupted tooth, the final phenotype. Further analytical and investigative approaches A gene network model has been formulated to reflect the development of mammalian teeth from the cap stage to the early bell stage.43 The resultant crown shapes approximate to the morphology found in the mammals studied and the intermediate stages correspond to the correct temporal spacing in the stages and known expressions of the genes incorporated in the model. This model predicts co-variation among such variables as tooth size, intercuspal distance and cusp size. A key factor in the model is the signalling activity of the enamel knots. In a study of Carabelli cusp expression it was found that this model’s predictions were supported both across and within individuals. By comparing right-left pairs of upper first molars on dental study casts of orthodontic patients, it was shown that small variations in developmental timing or in the spacing of enamel knots could affect cusp pattern.30 A computational model of tooth development summarizes mathematically the basic genetic and cellular interactions that regulate tooth shape development.71 This developmental model produces a phenotype with 3D morphology from the parameters of each individual. The model has reproduced the morphological variation seen in natural populations and creates a genotype-phenotype map of how development leads to adult morphology and its variation.71 Another approach to the study of shape variation is geometric morphometric analysis.72 It is geometric in that it provides a set of rules for analysing data of 10

positions, distances and angles in 3D representation of objects. The morphometric component is the mathematical quantification of the object.73–75 Shape is the key concept of geometric morphometric analysis; it is defined as all the geometric information that remains when location, scale and rotational effects are filtered out from an object.76 Geometric morphometric analysis attempts to measure subtle differences in shape, e.g. in premolars and molars, and so examine different contributions from aetiological factors.41,77–81 In examining objects, an important issue is landmark reliability.82 Geometric morphometric analysis has been applied to early Pleistocene hominin teeth from China compared with samples from Africa, Asia, Europe and with modern humans to investigate possible evolutionary relationships.83 An additional approach to identifying further genetic mutations involved in variations of number, size and shape is whole exome sequencing.84 The approach has been used in other craniofacial developmental conditions such as craniosynotosis.85 CONCLUSIONS The variations considered in this paper are seen on a daily basis in clinical practice. Early diagnosis allows optimal patient management and treatment planning. In many instances, this will be by a multidisciplinary team. Intervention at appropriate times can sometimes reduce complications and the amount and complexity of future treatment. Understanding the process of development and the aetiological factors is also important clinically when discussing the condition, including aetiology, and the possible treatment with patients and their family. Advancing care for patients with these anomalies will come from both multidisciplinary team care and from research in a range of scientific disciplines. Future clinical practice will involve personalized, precision care, based on an individual’s genetic profile. DISCLOSURE The authors have no conflicts of interest to declare. REFERENCES 1. Brook AH, Brook O’Donnell M, Hone A, et al. General and craniofacial development are complex adaptive processes influenced by diversity. Aust Dent J 2014; doi:10.1111/adj.12158 [Epub ahead of print]. 2. Thesleff I. Current understanding of the process of tooth formation: transfer from the laboratory to the clinic. Aust Dent J 2013; doi: 10.1111/adj.12102 [Epub ahead of print]. 3. Galluccio G, Castellano M, La Monaca CL. Genetic basis of non-syndromic anomalies of human tooth number. Arch Oral Biol 2012;57:918–930. © 2014 Australian Dental Association

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Address for correspondence: Professor Alan Brook School of Dentistry The University of Adelaide Adelaide SA 5005 Email: [email protected] © 2014 Australian Dental Association