Morphogenetic fields within the human dentition: A new,clinically relevant synthesis of an old concept§

Grant Townsend a,e,*, Edward F. Harris b,e, Herve Lesot c,d,e, Francois Clauss c,d, Alan Brook e

aSchool of Dentistry, The University of Adelaide, Adelaide, South Australia 5005, AustraliabDepartment of Orthodontics, College of Dentistry, University of Tennessee, 875 Union Avenue, Memphis, TN 38163, USAc INSERM U595, Faculty of Medicine, 11, rue Humann, 67085 Strasbourg, Franced Faculty of Dentistry, Louis Pasteur University, 67085 Strasbourg Cedex, Francee International Collaborating Centre in Oro-facial Genetics and Development, The University of Liverpool, Liverpool L69 3GN, UK

a r c h i v e s o f o r a l b i o l o g y 5 4 s ( 2 0 0 9 ) s 3 4 – s 4 4

a r t i c l e i n f o

Article history:

Accepted 25 June 2008

Keywords:

Dental development

Clone model

Homeobox code

Patterning

Hypodontia

Supernumeraries

Twins

a b s t r a c t

This paper reviews the concept of morphogenetic fields within the dentition that was first

proposedbyButler (Butler PM. Studiesof themammalian dentition.Differentiation of thepost-

canine dentition. Proc Zool Soc Lond B 1939;109:1–36), then adapted for the human dentition by

Dahlberg (Dahlberg AA. The changing dentition of man. J Am Dent Assoc 1945;32:676–90;

Dahlberg AA. The dentition of the American Indian. In: Laughlin WS, editor. The Physical

Anthropology of the American Indian. New York: Viking Fund Inc.; 1951. p. 138–76). The clone

theory of dental development, proposed by Osborn (Osborn JW. Morphogenetic gradients:

fields versus clones. In: Butler PM, Joysey KA, editorsDevelopment, function and evolution of teeth.

London: Academic Press, 1978. p. 171–201), is then considered before these two important

concepts are interpreted in the light of recent findings from molecular, cellular, genetic and

theoretical and anthropological investigation. Sharpe (Sharpe PT. Homeobox genes and

orofacial development. Connect Tissue Res 1995;32:17–25) put forward the concept of an

odontogenic homeobox code to explain how different tooth classes are initiated in different

parts of the oral cavity in response to molecular cues and the expression of specific groups of

homeobox genes. Recently, Mitsiadis and Smith (Mitsiadis TA, Smith MM. How do genes make

teeth to order through development? J Exp Zool (Mol Dev Evol)2006; 306B:177–82.) proposed that

the field, clone and homeobox code models could all be incorporated into a single model to

explain dental patterning. We agree that these three models should be viewed as comple-

mentary rather than contradictory and propose that this unifying view can be extended into

the clinical setting using findings on dental patterning in individuals with missing and extra

teeth. The proposals are compatible with the unifying aetiological model developed by Brook

(Brook AH. A unifying aetiological explanation for anomalies of tooth number and size. Archs

Oral Biol 1984;29:373–78) based on human epidemiological and clinical findings. Indeed, this

new synthesis can provide a sound foundation for clinical diagnosis, counselling and manage-

ment of patients with various anomalies of dental development as well as suggesting hypoth-

eses for future studies.

# 2008 Elsevier Ltd. All rights reserved.

§ This Supplement arises from a series of papers given at an International Workshop on Oral Growth and Development held in Liverpoolon November 26–28, 2007.

* Corresponding author at: School of Dentistry, The University of Adelaide, Adelaide, South Australia 5005, Australia. Tel.: +61 8 83035968;fax: +61 8 83033444.

avai lable at www.sc iencedi rec t .com

journa l homepage: www. int l .e lsev ierhea l th .com/ journals /arob

E-mail address: grant.townsend@adelaide.edu.au (G. Townsend).

0003–9969/$ – see front matter # 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.archoralbio.2008.06.011

a r c h i v e s o f o r a l b i o l o g y 5 4 s ( 2 0 0 9 ) s 3 4 – s 4 4 S35

1. Field and clone theories

The concept of a morphogenetic field emerged in the 19th

century, though the molecular-genetic basis for patterned

series of skeletodental elements (e.g., vertebrae, ribs, teeth)

were unknown. Ordered form was a common observation in

numerous aspects of biology,8 but the governing principles, let

alone the causes, were virtually unknown.9

The accumulation of paleontological findings, along with

information on extant species, made it clear that the

dentitions of higher vertebrates were (1) heterodont—com-

posed of multiple tooth types, and (2) organised into

morphogenetic fields. For example, Bateson,10 amassed

extensive data on meristic variation, including dental exam-

ples primarily of hypo- and hyperdontia. Bateson’s far-ranging

compilations showed that the variable dental elements

occurred at the later-forming end of a field and that these

terminal teeth were metrically and morphologically most

variable within a field.

The Zeitgeist of developmental biology in the early 20th

century was strongly influenced by Spemann’s embryological

organizing centers (Spemann11; De Robertis12). In 1934 Huxley

and De Beer combined the study of morphology with the

morphogenetic field concept in their seminal work The

Elements of Experimental Embryology, which closely preceded

Butler’s1 description of fields and morphogenetic variations in

the mammalian dentition.

Weiss13 was among the leaders to reaffirm the interpre-

tative value of the field concept. Butler1 postulated that

morphogenetic fields can account for the way in which teeth

within a particular class formed meristic series, i.e., with each

tooth displaying similarities to others nearby because of the

influence of a common field but with graded differences

because of its position. Currently these dental elements are

considered to result from the repetition of ‘developmental

modules’.14,15 Butler’s phylogenetic study of the mammalian

dentition was wholly observational, but he inferred several

features of the changes that meshed well with the biological

features of field gradients, notably (a) a size gradient within

each field, (b) a gradient of morphological complexity, from the

complex pole tooth to the simpler variable tooth, and (c) a

gradient of variability, with less size and shape variability in

the pole tooth and increasing variability away from the pole.

Documenting such field gradients was important because the

observations substantiated that teeth exist within morpho-

genetic gradients as defined by the criteria established by

Huxley and De Beer16 and Weiss.14 The phylogenetic patterns

do not always match the ontogenetic situations, however;

while incisors have lost teeth distally-to-mesially, the number

of premolars has been reduced mesially-to-distally.

Butler17 later suggested that teeth were evolving as a part of

a system rather than as individual organs. He postulated two

different effects: a ‘meristic’ effect, influencing the number

and spacing of teeth, and a ‘field’ effect produced by

substances or signals that controlled their differentiation

and final shapes. He added that the order of development of

teeth also represents a field effect.18

Dahlberg2,3 adapted Butler’s1 concepts to the human

dentition and proposed that there was a field of influence

operating on each of the tooth classes, i.e., incisors, canines,

premolars and molars. Each field was thought to have its

strongest effect on the anterior or key tooth within a class. The

more distally placed teeth, which generally develop later than

those more mesially placed, were observed to show greater

phenotypic variation. Interestingly, Dahlberg2,3 did not define

fields within the primary dentition and, without any com-

ment, he added a premolar field to Butler’s1 three-field

paradigm. In contrast, Butler19 later argues that premolars

represent modified anterior members of a permanent molar

field, with the primary second molar displaying similar

morphology to the permanent first molar located directly

distal to it.

Osborn4 proposed that a single clone of pre-programmed

cells led to the development of all the teeth within a particular

class. For example, a molar clone of cells was postulated to

induce the dental lamina to initiate molar development. As the

clone of cells grew distally, tooth buds were formed,

surrounded by zones of inhibition that prevented other teeth

developing until the migrating clone had moved on suffi-

ciently. More recent work invokes a reaction-diffusion model

in the region of a presumptive tooth, where activators induce

placode formation while negative regulators are higher in

interplacodal regions, which prevents tooth formation and,

thus, accounts for the orderly spacing of teeth.20

The demonstration that isolated presumptive first molar

tissue explants could continue to grow and form all three

molar teeth in their normal sequence, provided strong

experimental support for the clone model.21 However, the

clone model does not provide an explanation for how the

dentition develops as a whole, with different tooth classes

displaying different shapes. Furthermore, the inclusion of the

phrase ‘‘fields versus clones’’ in the title of Osborn’s original

paper4 has led many to suppose that the field and clone

models are competing, mutually exclusive concepts. Recent

findings about the roles of signalling molecules and the

expression of homeobox genes during dental development

indicate that such a distinction is unwarranted.6 Indeed, the

field and clone models can be viewed as complementary to

each other.

2. The odontogenic homeobox code

The odontogenic homeobox code model explains how dental

patterns can be generated from different domains of expression

of homeobox genes in neural crest derived ectomesench-

yme.5,22 While the code was initially developed from studies of

the mouse dentition in which there are only incisor and molar

teeth, it has been extended to explain how canines and

premolars could also be produced by overlapping domains of

gene expression.22 Experimental studies have also shown that

by modulating certain signalling molecules it is possible to alter

homeobox gene expression domains in the ectomesenchymal

tissue and to modify tooth number, size, shape and differentia-

tion.23,24 A schematic representation of how dental patterning

can be produced by an odontogenic homeobox code has been

provided recently by Cobourne and Mitsiadis.25 They describe

how an ‘‘inter-mixing’’ of genes expressed by ectomesenchyme

of the first branchial arch can lead to the establishment of

different morphogenetic fields. Patterns are established by

a r c h i v e s o f o r a l b i o l o g y 5 4 s ( 2 0 0 9 ) s 3 4 – s 4 4S36

signals from the ectoderm that induce specific domains of

homeobox gene expression in the ectomesenchyme. This

patterning is plastic initially but over time it becomes fixed

into the ‘memory’ of the ectomesenchymal cells. It is these

specific domains that are postulated to provide the molecular

information needed to specify different tooth shapes.25

There is evidence that the nature of the molecular

signalling in the upper and lower jaws may vary. The dental

formula is the same in both arches in mice and in humans, but

the shape and morphologies of the homologous teeth in the

two jaws are each distinctive one from the other.26 Biochem-

ical signalling differences have been demonstrated in the

mouse for Dlx genes27,28 and also for activin/follistatin,29

although it is not known how neural crest derived cells

migrating into the developing maxillary and mandibular

regions develop the ability to respond differently to ectoder-

mal signaling. Reports of apparently independent genetic

determination of maxillary and mandibular dentitions, based

on tooth size data derived from twins,30 are consistent with

the molecular evidence.

3. Recent perspectives of the field, clone andhomeobox models

Apart from the role of homeobox genes in determining the

location of different fields within the developing dentition, it

has been shown that expression of the signalling molecule

ectodysplasin (EDA) is important in defining the size of dental

fields. Studies using transgenic mice have shown that

alteration in tooth number occurs when EDA, its receptor

(EDAR), or the intracellular adaptor protein EDAR-binding

death domain adaptor (EDARADD) are disrupted. It has been

proposed that if there is over-production of the EDA ligand, or

the receptors for EDA are over-expressed, then the size of the

molar field in mice increases, resulting in the formation of

supernumerary teeth.31 In transgenic mice, the supernumer-

ary teeth develop mesial to the first molar, in the diastema

region that is present normally between the incisor and molar

segments. The supernumeraries usually have a reduced cusp

pattern and do not resemble normal mouse molars. We would

raise the question whether these ‘supernumeraries’ actually

arise from tooth germs that would normally undergo

apoptosis during development. In contrast to over-expression

of EDAR and its association with extra teeth, loss of EDAR

signalling leads to missing molars.32

Peterkova et al.33 have identified five different morpho-

types in the mandibular molar region of tabby/EDA mice and

reported that these result from abnormal segmentation of the

epithelial compartment of the dental lamina along a mesio-

distal axis. Increased apoptosis has been noted in the mesial

aspect of the dental epithelium of affected mice, and this

seems to be a consequence of impaired tooth development

associated with the defective segmentation process.34

In humans, apart from the X-linked form of ectodermal

dysplasia (XLHED) that involves mutation of the ectodysplasin

gene ED1 (locus Xq12-q13.1), autosomal forms of the condition

have also been described.35,36 These autosomal dominant and

recessive forms of HED are linked to mutations of EDAR at the

2q11-q13 locus or EDARADD at the 1q42.2-43 locus.

The phenotypes of autosomal dominant and recessive

forms of HED, linked to EDAR mutation, are similar to the

XLHED phenotype, including hypohidrosis, hypotrichosis and

oligodontia, which reflects the common developmental

patterns of many ectodermal derivatives.37,20 However, some

differences are evident. The skin and hair phenotypes

associated with the autosomal forms of HED seem to be

milder than those in XLHED.38 Craniofacial dysmorphic

features observed in autosomal HED also seem to be less

severe than in XLHED, with mild frontal bossing and malar

flattening.35 Moreover, dental and craniofacial phenotypic

manifestations are less severe in autosomal dominant forms

than in autosomal recessive HED.36 As for the dental

phenotype of autosomal HED, hypodontia, often severe, with

varying degrees of primary and permanent tooth agenesis is

evident, associated with anomalous conical incisors and

canines (Fig. 1).

The X-linked form of HED constitutes the majority of HED

phenotypes. Nevertheless, the ED1 mutation is not system-

atically identified in patients with HED: only 65.4% in a group

of patients exhibiting the HED phenotype,39 other loci being

mutated, such as EDAR (in 25% of non-ED1 HED) and, less

often, EDARADD.36,40

Dental phenotypic variability is observed in autosomal HED

(as shown in Fig. 1) and is probably due to genetic hetero-

geneity.38 Indeed, different non-sense mutations distributed

along the 11 exons of the EDAR gene have been identified and

are associated with varying phenotypic expression of auto-

somal HED.36 These loss-of-function mutations lead to

alteration of EDAR trimerisation and partial or total loss of

EDA-EDAR and EDAR-EDARADD molecular interactions.38 An

absence or reduced activation of NF-kB during embryogenesis

and abnormal cell signaling are the consequences of the EDAR

mutations. The same molecular mechanisms are involved in

EDARADD mutations leading to complete or partial abrogation

of NF-kB activation in both autosomal dominant and recessive

mutations.40

Line41 has emphasised that the concept of morphogenetic

fields should not be limited to the expression of a single gene

or its protein product but rather include consideration of how

various genetic influences, modulated by epigenetic effects,

affect development of the dentition. By studying variation in

the number of missing teeth in families with mutations of the

MSX1 and PAX 9 genes, Line has attempted to clarify the

relative influence of these two genes within the human

dentition, referring to their influences as ‘‘relative molecular

morphogenetic fields’’. Variations in patterns of symmetry of

missing teeth within individuals were also used to gain insight

into the effects of these two genes on the developmental

stability of dental fields. Line found that the morphogenetic

fields associated with MSX1 and PAX9 were not confined to a

single tooth class, nor did there seem to be a simple gradient

pattern. He interpreted these findings as reflecting an

interaction between different signalling molecules. Although

there was some evidence that mutations in MSX1 and PAX9

could increase the extent of asymmetry of tooth agenesis, no

consistent pattern was identified. These findings are all

consistent with the multifactorial aetiology as proposed by

Gruneberg,42 Brook,43,7 Chosack et al.,44 Brook et al.45 and

others.

Fig. 1 – Dental phenotypes associated with an autosomal form of hypohidrotic ectodermal dysplasia (HED), linked to EDAR

mutation. Clinical phenotype associated with a 10-year-old boy includes oligodontia (agenesis of mandibular permanent

incisors and maxillary lateral incisors) and conical-shaped canines (a). A panoramic radiograph (b) of another patient (7

years old) with autosomal HED shows mandibular anodontia and maxillary oligodontia. There is agenesis of the primary

right central incisor, lateral incisors, left canine and first primary molars with presence of only one dysmorphic permanent

right central incisor germ and permanent first molars (b). Clinical (c) and radiographic (d) appearance of another patient (5

and 1/2 years old) with an autosomal dominant form of HED. Agenesis of maxillary and mandibular primary incisors, first

molars and second maxillary right primary molar is evident. There is also agenesis of all permanent tooth germs, except for

the permanent maxillary and mandibular first molars.

a r c h i v e s o f o r a l b i o l o g y 5 4 s ( 2 0 0 9 ) s 3 4 – s 4 4 S37

Much less is known about the epidemiology or genetic

factors influencing supernumerary teeth (Fig. 2). Brook7

proposes that the addition of ‘‘extra’’ teeth represents the

antithesis of hypodontia. With supernumerary teeth, other

teeth tend to be larger and morphologically more complex.

Recent work45,46 also suggests that the tempo of tooth

formation is depressed with hypodontia; we speculate that,

conversely, supernumerary teeth may be associated with

faster tempos as occurs among human ethnic groups with

varying tooth sizes and morphologies.47 Supernumerary teeth,

notably the addition of eumorphic teeth, show appreciable

ethnic variation,48 and they tend to cluster in families. The

extension of a tooth class by replicating another tooth at

the variable end of tooth field gradient also corresponds to

the model of reiterative signaling – repeating the engrained

‘developmental module’ used to develop the prior teeth in the

same class13,49,15 – and may represent phenomena seen with

EDA and EDAR.31 However, this would not immediately

explain mesiodens and paramolar supernumeraries.

Mitsiadis and Smith6 have described a synthesis to explain

dental patterning, referred to as cooperative genetic interac-

tion (CGI), which combines the field, clone and odontogenic

homeobox models. Their concept is built on evidence that

neural crest derived mesenchymal cells are influenced by

signals from the oral epithelium to express homeobox

containing genes.50,51 All three elements – the ‘‘clones’’ of

neural crest derived cells, the homeobox containing genes in

the mesenchyme, and the signalling molecules released by the

oral epithelium – are considered to play important roles in

patterned tooth development. For example, it is proposed that

mutations in genes in the mesenchymal cell clones could

affect their cellular proliferation, leading either to agenesis or

reduction in size of a tooth. A key feature of this model is its

time-dependent nature, not only in terms of the importance of

the timing of release of various signalling molecules from the

oral epithelium and the subsequent diffusion gradients that

are established, but also in terms of the temporal expression of

different combinations of homeobox genes from the prolifer-

ating neural crest derived mesenchymal cells.

4. Phenotypic variation, key teeth andheritability estimates

In adapting Butler’s field concept to the human permanent

dentition, Dahlberg2,3 referred to the most stable tooth in each

field as the ‘key’ or ‘pole’ tooth. These key teeth in the

maxillary dentition were identified as the central incisor, the

canine, the first premolar and the first molar. A similar pattern

was identified in the mandibular dentition, except that the

lateral incisor was identified (without any comment) as the

key tooth in the incisor region. Univariate analyses of dental

crown size, including variability (based on coefficients of

variation) and asymmetry (based on quantitative measures of

fluctuating asymmetry), have tended to conform with

expected patterns.52 Furthermore, the observed patterns of

Fig. 2 – Panoramic radiographs of two American Black

adolescents with a developing fourth molar in each

quadrant. These supernumerary teeth are reduced in size,

but exhibit differentiation into multiple occlusal cusps.

These extensions of the molar fields could conceivably

involve differences in these subjects’ ectodysplasin, as

described by Tucker and Sharpe.31

a r c h i v e s o f o r a l b i o l o g y 5 4 s ( 2 0 0 9 ) s 3 4 – s 4 4S38

phenotypic variation seem to be generally consistent with the

relative amount of time that each developing tooth germ

spends in the soft tissue phase prior to calcification. For

example, the permanent maxillary incisors appear to differ-

entiate at similar times, but initiation of mineralisation of the

lateral incisor occurs later than the central whereas crown

completion is slightly earlier for the lateral than the central.53

Therefore, the developing maxillary lateral incisor spends a

relatively longer period in its soft tissue stage prior to

calcification than the maxillary central incisor, during which

time epigenetic and environmental factors may influence its

final size and shape. Indeed, Keene54 has provided a useful

general concept, which he referred to as the morphogenetic

triangle, that describes the relationships between initiation of

proliferation, initiation of differentiation and crown comple-

tion during odontogenesis. This, the dynamic development of

a tooth is seen in the continued growth among formative

cusps so the spatiotemporal relationships continue to change

until dentinogenesis bridges the cusps and ‘petrifies’ the

occlusal phenotype.

Using a multivariate statistical approach, Harris55 has

shown that most of the variation in mesiodistal and

buccolingual crown dimensions of human permanent teeth

is shared, with the majority of the shared variation being

associated with tooth type (i.e., whether teeth are incisors,

canines, premolars and molars), and very little attributable to

tooth position (i.e., ‘key’ or ‘pole’ tooth versus distal tooth

within a particular class). Harris notes that his finding in

relation to tooth type is intuitively appealing as it conforms

with the concept of heterodonty, with four basic morphoge-

netic fields corresponding to incisors, canines, premolars and

molars as proposed by Dahlberg. However, the data used for

this analysis and for most other multivariate analyses of this

type56,57 are traditional crown diameters, i.e., maximum

mesiodistal and buccolingual diameters, and whether similar

patterns would be found if other phenotypes were used, e.g.,

crown heights, intercuspal distances and angles, or peri-

meters, areas and volumes is a research question for future

studies using new measurement techniques.

Similarly, studies aimed at disclosing patterns in estimates

of heritabilities for dental crown size within the human

dentition have also been based on mesiodistal and buccolin-

gual crown diameters. In these studies it has generally been

assumed that the key tooth within each morphogenetic dental

field should display the highest heritability. In contrast, the

non-key teeth, often those more distally positioned within a

class, would be expected to show lower heritabilities. Some

researchers have reported trends in heritability estimates that

conform with this pattern58 but others have been unable to

find any such trends.59

There are at least two reasons why these inconsistent

findings are not unexpected. Firstly, we have seen that there is

a complicated series of epigenetic and morphogenetic events

involved in odontogenesis, apparently without any over-

riding genetic control mechanism. Temporo-spatial variations

in signalling pathways during development can lead poten-

tially to different outcomes (or phenotypes), and it seems

likely that the longer a tooth or component of a dental crown

remains in its soft tissue phase, prior to crown mineralisation,

the more opportunity exists for phenotypic variations to be

expressed.60,61 In these circumstances, it is unlikely that

statistically significant differences in estimates of heritability

for final crown size or shape will be discerned. Secondly, most

previous genetic studies of human tooth size have been based

on traditional measures of overall crown size. These measures

represent composites of the various components of a dental

crown and are rather crude representations of complicated

morphologies. Interestingly, heritability estimates for inter-

cuspal distances of molar teeth, which may be more

biologically meaningful phenotypes than overall crown

measures, are only moderate in magnitude.62 Given that

secondary enamel knots63,64 that form during molar odonto-

genesis will determine sites of future cusp tips, the moderate

heritability values obtained for intercuspal distances are

consistent with the important role of self-organizing, sequen-

tial epigenetic processes in enamel knot formation during

odontogenesis.

5. Genotypes, phenotypes and developmentalbiology

Jernvall and Jung49 have highlighted the value of multiple

approaches to the study of dental development, with devel-

opmental genetics, mathematical modelling, and population

genetics being used to link development with both micro- and

macro-evolution. They explain how the repeated activation of

the same set of genes, or a so-called ‘developmental module’,

accounts for the cumulative variation observed in later-

developing molar cusps. Salazar-Ciudad and Jernvall65 have

provided a mathematical model to demonstrate that the

a r c h i v e s o f o r a l b i o l o g y 5 4 s ( 2 0 0 9 ) s 3 4 – s 4 4 S39

morphology of mammalian teeth can be predicted by

integrating experimental data on gene interactions and

growth into a ‘‘morphodynamic mechanism’’, thereby linking

genotype to phenotype. Jernvall et al.66 make use of a

topographic method, Geographic Image Systems (GIS), to

show that molecular pre-patterning can predict the arrange-

ment of cusps more than a day in advance, with subtle

heterotopic shifts being postulated to play an important role

during evolution in producing new cuspal arrangements.

More recently, Salazar-Ciudad et al.67 have suggested that

developmental mechanisms used for generating patterns can

be grouped into three categories: cell autonomous mechan-

isms; inductive mechanisms; and morphogenetic mechan-

isms. The first category is postulated to include those

mechanisms by which cells become arranged into patterns

without interacting with other cells. The second category

refers to mechanisms by which cells communicate with one

another to produce patterning through reciprocal or hier-

archical alteration of phenotypes. The third category refers to

mechanisms that can lead to changes in patterns through cell

interactions that do not involve phenotypic changes in cells.

Of particular relevance to morphogenetic fields, inductive and

morphogenetic mechanisms can be combined either mor-

phostatically, in which the inductive mechanisms occur first,

or morphodynamically, in which both types of mechanisms

interact continuously. Salazar-Ciudad et al.67 suggest that the

mammalian dentition provides a good example of a develop-

mental system that employs morphodynamic mechanisms,

with induction and morphogenesis taking place concurrently

and interdependently. They also explain how morphody-

namic mechanisms use spatial epigenetic information pre-

sent in the emerging phenotype at each stage of the

developmental process to influence later development. In

this sense, morphodynamic mechanisms exhibit dependency

on the so-called ‘intermediate phenotype’.

Salazar-Ciudad et al.67 avoid using the term ‘morphoge-

netic field’ because it presupposes the concept of prospective

cell fate. They prefer to use the term ‘gene expression

territory’. They emphasise that morphodynamic mechanisms

involve interaction between cells along a developmental

trajectory that is continually interacting with a changing

molecular and geometric microenvironment. With this per-

spective of dental development, it becomes more apparent

that the field and clone models are complementary inter-

pretations of the same observations; they are mutually

supportive rather than opposing concepts. This perspective

is also entirely consistent with recent findings of discordant

dental development in monozygotic twin pairs.68,69

6. Genetics, epigenetics and environment: theMZ co-twin model

While molecular geneticists have tended to focus on methyla-

tion and acetylation of DNA when referring to epigenetics,

there is growing recognition of the need for a broader

interpretation of the term.70,71 We use the term ‘epigenetics’

in the broad sense, as proposed originally by Waddington,72,73

to refer to processes by which genotype gives rise to

phenotype. This broader view enables description of those

interactive processes that occur between cells at the local

tissue level during dental development as epigenetic events, in

addition to those that may operate directly on DNA. Molenaar

et al.74 referred to epigenetic influences as a ‘‘third source of

developmental differences’’ that can account for phenotypic

variation in development, in addition to genetic and environ-

mental factors. This third source is comprised of non-linear

epigenetic events that can cause variability at all phenotypic

levels and that these epigenetic influences result from

autonomous developmental processes with ‘‘emergent self-

organizing properties.’’ The role of epigenetic factors in the

broad sense during odontogenesis has been discussed by Lesot

et al.75 while local epigenetic influences, including cell–cell

and cell–matrix interactions and their effects on asymmetrical

growth and differential cell proliferation during dental

development have also been described.76

Interestingly, Eaves et al.77 have pointed out that Mole-

naar’s concepts can be interpreted in terms of chaos theory.

They have shown through simulations that chaotic and near-

chaotic processes may reduce the strength of correlations

between twin pairs. By simulating a simple non-linear model

behaving chaotically, Eaves et al.77 found that ‘‘small

variations in initial conditions (e.g., small quantitative

differences between MZ twins at the molecular level such

as the degree of methylation of a particular gene in a

particular tissue) will have consequences at the phenotypic

level which look like occasion-specific environmental

effects.’’ The findings of Eaves and colleagues are particularly

relevant when attempting to explain differences in expres-

sion of missing and extra teeth between monozygotic co-

twins with the same genotypes.

Eaves et al.77 provide theoretical support for our view that

minor variations in local epigenetic events during dental

development, probably relating to the spatial arrangement of

odontogenic cells and/or the timing of the signalling events

between them, can produce distinct differences in dental

features between monozygotic (MZ) twin pairs who have been

confirmed to have the same genotypes.67,68,78 These differ-

ences can include discordances in the number and/or location

of congenitally missing teeth or differences in the number of

supernumerary teeth between MZ co-twins.

7. Stepping into the clinical setting

The approaches of Line41 and Mitsiadis and Smith6 provide an

important step forward in explaining how the field, clone and

homeobox code concepts can all be used as a foundation for

understanding how phenotypic patterning is established in

the human dentition. We now provide some additional

perspectives based on our own research to position these

new concepts within a clinical context that ranges from minor

anomalies of the size and shape of teeth through to more

severe anomalies, including missing and extra teeth.

The most common clinical presentation involving missing

teeth is hypodontia of only one or a few teeth, commonly the

third molars, second premolars or maxillary lateral incisors.

On the other hand, these generalities are based on the

numerous studies of peoples of European extraction,79 and the

limited data from other ethnic groups suggest the frequencies

Fig. 3 – Dental panoramic radiographs (a and b) and

maxillary clinical views (c and d) of a pair of monozygotic

twins affected by X-linked hypohidrotic ectodermal

dysplasia (XLHED) with a mutation of the ED1 gene, coding

for ectodysplasin (EDA) morphogenetic factor.

a r c h i v e s o f o r a l b i o l o g y 5 4 s ( 2 0 0 9 ) s 3 4 – s 4 4S40

and patterns may differ.80–82 When considering supernumer-

ary teeth, the most common example is the mesiodens which

occurs in the anterior midline region of the maxilla. A

multifactorial model linking tooth size and number can

account for the different patterns of expression of tooth size,

hypodontia and supernumerary teeth observed in males and

females.7,43,45,83–85 The relatives of individuals with missing or

extra teeth have also been found to be more likely to also

display missing or extra teeth, supporting the concept of an

underlying genetic predisposition to hypodontia or super-

numerary teeth. We believe that such a multifactorial model,

with multiple genetic, epigenetic and environmental influ-

ences, provides the best explanation for our observations

involving missing and extra teeth in MZ twin pairs.

Molecular studies, primarily in mice, show that congeni-

tally missing teeth can arise from diverse developmental

causes. Tooth development can be interrupted at, at least,

three stages. First, the ectoderm may not have the capacity to

induce bud-formation in the underlying mesenchyme.20,86

Second, missing or mutant signals of a mesenchymal

transcriptional factor such as Msx1 or Pax9 can arrest

development prior to enamel knot formation.87 Third, devel-

opment can be arrested shortly after the initiation of

dentinogenesis, with subsequent apoptosis. This latter stage

corresponds to the incipient formation of lateral incisors in

mice,88,89 where these teeth never fully develop in this species.

Similarly tooth germs anterior to the first molar (i.e.,

premolars and canines) in mice undergo apoptosis.34

To illustrate the dental variability between MZ co-twins, a

pair of twins diagnosed with a mutation of the ED1 gene are

described, both showing oligodontia but with different

patterns of missing teeth (Fig. 3). One of the MZ twins (a)

showed mandibular anodontia, whereas the other exhibited a

severe mandibular hypodontia with the presence of a

mandibular left permanent molar (b). The distribution of

maxillary teeth again illustrates variability in the severity of

the phenotype. Only permanent maxillary canines and the left

first molar are evident for the first MZ twin, whereas maxillary

canines and the contra-lateral right permanent first molar are

present in the second MZ twin (b), together with the left

maxillary central incisor and a retained maxillary second right

primary molar.

The dental phenotypic variability between these XLHED co-

twins could be accounted for by different local epigenetic and

environmental factors interacting with the EDA mutation, as

described in the multifactorial theory. The dental phenotype

of the second XLHED MZ twin (b) suggests residual capacity

for initiation of odontogenesis, allowing the development of

a permanent maxillary central incisor and a permanent

mandibular molar. However, the phenotypic variability

observed shows that other epigenetic mechanisms, besides

genetic factors, represented mainly by the odontogenic

homeobox code and signaling molecules such as FGF, BMP,

Shh, and EDA-NF-kappaB,90,91 are also important for initiation

of odontogenesis and tooth morphogenesis.

The strong link between tooth number, size and shape is

illustrated by the dental phenotype of these XLHED patients.

Microdontia of canines (Fig. 3c and d), dysmorphic cone-

shaped incisors (d) and canines (c and d) and molar root fusion

and shape anomalies (a and b) are associated with the

phenotype of oligodontia and also linked to the ED1 mutation

and molecular alterations of the EDA-NF-kappaB signalling

pathway.92

By superimposing thresholds on an underlying distribution

of tooth size, it is possible to explain the relationship between

tooth size and the presence or absence of teeth. For example,

missing teeth will tend to occur below a certain threshold for

tooth size. The prevalence of missing teeth will be greater in

females who have smaller teeth, on average, than males. At

the other extreme of the distribution, where tooth size is

larger, another threshold is reached above which extra teeth

will tend to occur. The prevalence of extra teeth will tend to be

greater in males who have larger teeth, on average, than

females. These relationships are summarised in the model of

Brook (Fig. 4). It is also helpful to remember that the

‘thresholds’ actually are zones rather than well-demarcated

lines because of spatiotemporal effects of the person’s genetic

background and dynamics of his environment.93,94

Although the model is a theoretical one, it has direct

relevance to clinical practice. It emphases that common

anomalies such as missing and extra teeth represent part of a

continuous spectrum of inter-related dental phenotypes that

are influenced by a combination of genetic, epigenetic and

environmental factors. Furthermore, when linked to the

Fig. 4 – Model based on normal distributions with

superimposed thresholds to explain the relationships

between anomalies of tooth number and size after Brook.7

a r c h i v e s o f o r a l b i o l o g y 5 4 s ( 2 0 0 9 ) s 3 4 – s 4 4 S41

concept of morphogenetic fields, this conceptual model

enables clinicians to predict which teeth are most likely to

be affected, e.g., third molars, second premolars and maxillary

lateral incisors display hypodontia most commonly and are

also most often affected by certain other anomalies of

development. Such a framework provides a valuable basis

for clinical diagnosis, treatment planning and counselling.

Apart from the value to the clinician of appreciating that there

are links between tooth size, shape and number within the

dentition, reflecting underlying developmental processes,

there are many other dental phenotypes that have been

shown to be associated with congenitally missing teeth.94

These include delayed formation and eruption of other teeth,

malposition and ectopic eruption of teeth, taurodontism,

rotation of teeth, short roots and simplified morphology.95–101

Furthermore, more generalised anomalies in the craniofacial

region have also been shown to be associated with missing

teeth, including alterations in dental arch form, presence of

Class II division 2 craniofacial type and sella turcica

bridging.44,102,103 A recent report has even linked tooth

agenesis to colo-rectal cancer.104

These several associations among size and shape of

structures certainly are important clinically, but they also

reflect the integrated development of skeletodental com-

plexes, both at the molecular41 and phenotypic levels. Indeed,

Bateson10 described various examples of how nearby teeth are

themselves affected when there is hypo- or hyperdontia,

which often parallel the situations that have occurred

phylogenetically.1,105 Understanding co-variations can help

clinically as well as in the search for common developmental

etiologies.

8. Finding the genes for dental development:implications for future practice

To date, molecular-genetic studies in humans have concen-

trated mainly on locating the genes associated with missing

teeth. Mutations in three genes, MSX1, PAX9 and AXIN2104,106–

109 have been shown to be associated with familial cases of

severe hypodontia (where many teeth are missing). The

families selected for these studies had pedigrees consistent

with an autosomal dominant mode of inheritance although

variations in the number and position of teeth missing have

been noted. However, there are more than 300 genes that

appear to be involved in dental development,110 and a number

of them could be candidates for missing and extra teeth.

Furthermore, as we have indicated in the previous section of

this review, the most common clinical presentation relating to

missing teeth is simple hypodontia where only one or a few

teeth are missing rather than many, so identifying the causes

of common hypodontia is of clinical merit.

Given that there appears to be a link between the size and

shape of teeth, and hypodontia or supernumerary teeth, we

propose that there is likely to be a group of genes that exert

pleiotropic effects on multiple dental phenotypes, accounting

for their observed co-variation. Just how many genes are

involved remains to be seen, but it is possible that it may be a

relatively small number. Support for this view is provided by

Kangas et al.111 who have shown that dental characters in

mice seem to be non-independent. So too, tooth crown

dimensions as well as morphological traits are positively

intercorrelated within and among tooth types.57,112 Increasing

the levels of expression of just one gene can lead to increases

in cusp number, altered cusp shape and position, develop-

ment of longitudinal crests on teeth, and increases in tooth

number.

Rather than a simple monogenic mode of inheritance

alone, we consider that a multifactorial model7,45,83 – with

genetic, epigenetic and environmental influences – provides

the best explanation for our observations involving hypodon-

tia and supernumerary teeth in MZ twin pairs, as well as

different phenotypic expressions in MZ co-twins with ecto-

dermal dysplasia. Such a model, with superimposed thresh-

olds linking tooth size, morphology and number, provides a

plausible explanation for why MZ co-twins, who have the

same genotypes, may display different dental phenotypes.

The MZ twin pairs may have genotypes that place them near to

a threshold for either missing or extra teeth, but variations in

local epigenetic events during odontogenesis may determine

on which side of the threshold they fall.

As we have mentioned previously, and as is reported in

other papers in this special issue of Archives, measures of tooth

size that have been reported most commonly in the past have

been maximum mesiodistal and buccolingual crown dia-

meters. Often these measurements have been recorded from

dental casts with hand-held calipers that cannot provide

accuracies better than 0.1 mm. Although there have been

attempts to describe tooth size and shape using other

variables, including cusp areas, perimeter and area and

volumes,60,85,113 these types of data have rarely been used

in genetic studies. With the advances in image capturing

techniques, including 3D laser systems, we are now in a

position to define new dental phenotypes that should give

clearer insights into the nature and causes of variation in the

dentition.114

Genome-wide association studies (GWAS) are currently

being used to identify genes linked to various common

diseases, including coronary heart disease, hypertension,

diabetes and arthritis, and it is planned to use a similar

approach to identify key genes involved in dental develop-

ment using a suite of newly defined dental phenotypes in both

a r c h i v e s o f o r a l b i o l o g y 5 4 s ( 2 0 0 9 ) s 3 4 – s 4 4S42

2D and 3D.115 Although very important, the genetic aspects are

not sufficient to explain fully how various dental anomalies

arise in individuals. This is where further exploration of

epigenetic factors will be essential.

Already researchers are beginning to study epigenetic

biomarkers in an attempt to explain the reasons for observed

differences between MZ twin pairs.116 At this stage the focus is

on trying to determine the extent of differences in global

genomic DNA methylation levels but it is likely that more

specific analyses will be developed soon. Once these

approaches aimed at the level of DNA are refined further,

and our understanding of the nature of the epigenetic

influences at a local tissue level improves, we should be able

to provide a clearer picture of how genetic, epigenetic and

environmental factors influence human dental development

and lead to phenotypic patterning within the dentition.

Acknowledgements

The support of the following funding agencies is gratefully

acknowledged: National Health and Medical Research Council,

Canberra, Australian Dental Research Foundation, National

Institute of Health, USA, Wellcome Trust, UK.

Disclosures

Competing interests: None declared.

Funding: National Health and Medical Research Council of

Australia, Australian Dental Research Foundation, National

Institute of Health, USA, INSERM UMR595, Wellcome Trust,

UK, National French Reference Centre for Oral Manifestations

of Rare Diseases.

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