1. The Genetics of Tooth Development and its Effects
Introduction
The growth and development of teeth is an incredibly complex process, involving the
coordination of numerous genes, proteins, and signalling pathways. The failure of teeth to grow
or develop properly can result in patients having too many or too few teeth, as well as a variety
of other disorders. These dental abnormalities can place severe financial, psychological, or
physical burdens on patients and as such understanding the genetic basis behind these disorders
is essential. Despite the differences in size, shape, number, and location, the genetic etiology of
dentition is well conserved across vertebrates and physical abnormalities in growth and
development will often appear similar between species. Because the genetics of dentition are so
well conserved, vertebrate models such as mice and zebrafish can be used to study normal and
abnormal dental growth in order to provide insight into the human model. Several key dental
pathways in vertebrates have been discovered, including the Hedgehog, TGFβ, Wingless, and
FGF signalling pathways. The numerous genes that regulate and are involved in these pathways
have been implicated in the coordination and control of human dentition. This research has
equipped medical professionals with the knowledge to better treat dental abnormalities and
improve current therapies to make them better for the future.
Overview of Human Dentition

2. Teeth are a vertebrate invention, and over millions of years they have taken on a variety
of different shapes, sizes, and locations within the vertebrate mouth. However, despite these
differences, all vertebrate teeth are similar in that they contain a central pulp cavity encased by
the mineralized tissue dentine and capped with either enamel or enameloid (Klein et al. 2013).
Human beings are diphyodont, which means they develop two sets of teeth. The first set, often
referred to as the “primary”, “baby”, or “deciduous” set, often appear around six months after
birth, and are comprised of about twenty teeth. The second set, which appears after the baby
teeth fall out, is typically comprised of 32 teeth: 16 teeth on the upper jaw (referred to as the
maxilla) and 16 teeth on the lower jaw (referred to as the mandible). The human mouth is
typically divided into four quadrants: upper right, upper left, lower right, and lower left. Humans
typically have two incisors, one canine, two premolars (also known as bicuspids), and three
molars per quadrant (Fig. 1) (Tucker and Sharpe 2004; Klein et al 2013). Teeth begin to form at
approximately seven weeks in the human embryo and on the 11th day in mice embryos, and is
characterized by a thickening of the oral epithelium. Tooth development is then guided by series
of interactions, known as epithelial-mesenchymal transitions (EMT) in which the oral epithelium
and the neural-crest-derived mesenchyme continuously communicate until a tooth forms or
“erupts” (Thesleff 2006).
Stages of Human Tooth Development
Tooth development coincides with the formation of the head, a process that occurs in the
earliest stages of embryonic development (Wilkie and Morris-Kay 2001). Two main groups of
cells, cranial neural crest (CNC) and mesenchymal, help to direct the formation of teeth in the

3. developing embryo (Fig. 2). Proliferating CNC cells eventually give rise to almost all head
structures, including the tissue around future oral cavities, a single frontonasal process, and a
maxilla and mandible. All the muscles in the head are formed by mesenchymal cells, which are
also derived from the neural crest; as development advances these cells will continuously interact
to give rise to a completed head and face, and the teeth will form from the maxillary and
mandibular processes (Kouskoura et al. 2010). The formation of the bones and teeth are part of
a series of epithelial mesenchymal transitions (EMTs), or “dialogue” between the neural crest-
derived mesenchymal cells and the epithelium, which covers all of the oral cavity and face. The
epithelium is comprised of two distinct cell lineages, the basal cells contacting the basement
membrane, and a group of loosely arranged cells known as the stellate reticulum (Melton 2013).
These EMTs are orchestrated by groups of proteins that are the products of specific genes
involved in tooth formation. These proteins direct various groups of cells to grow and divide,
undergo cell death (apoptosis) or differentiate into specific types of cells, such as chondrocytes,
odontoblasts, or osteoblasts, among others. Additional molecules, such as signalling molecules
and transcription factors, also help to regulate the formation of tooth development (Kouskoura et
al. 2010).
Signalling Molecules and Transcription Factors in Tooth Formation and Development
Signalling molecules incorporate a wide class of chemical substances that include
hormones, neurotransmitters, or cytokines, among others. These molecules are secreted by cells
and bind to specific receptors on other cells to activate molecular pathways that regulate
transcription factors (TFs). These TFs can bind directly to DNA to regulate gene expression by

4. activating or repressing genes that control cell behaviour. Many of the signalling molecules that
control tooth formation and development are growth factors, which are a group of steroid or
peptide hormones that help to stimulate cellular proliferation, growth, or differentiation (Kapadia
et al. 2007). There are four main families of growth factors involved in human dentition and
orofacial development: the Hedgehog (HH) family, the Transforming Growth Factor beta
(TGFβ) family, the Fibroblast Growth Factor (FGF) family, and the Wingless (WNT) family.
Many of these growth factors are conserved among vertebrates, and mutations in any of these
factors can lead to mutations that are phenotypically similar between species. This underlines
the essential role of these factors in the proper development of the human dentition (Kouskoura
et al. 2010).
The Hedgehog Signalling Pathway
The main member of the Hedgehog (HH) family is Sonic Hedgehog (SHH); mutations in
this gene or at various points in this signalling pathway are most commonly associated with
abnormal tooth or orofacial development. SHH is expressed at all stages of tooth development
and is vital for proper tooth formation, tooth size, and crown formation (Dassule et al. 2000). It
also plays an important role in epithelial cell proliferation and differentiation into ameloblasts
(Takahashi et al. 2007), as mice with a mutated SHH receptor displayed delayed tooth eruption
and root development (Nakatomi et al. 2006). Mice with overexpressed SHH develop cleft
palates and incomplete tooth formation (Cobourne et al. 2009). In humans, SHH is expressed in
the maxillary and frontonasal processes during development; mutations in this growth factor
leads to holoprosencephaly, or the failure of the embryonic forebrain to develop into two distinct

5. hemispheres (Roessler et al. 1996). In embryonic chicks, decreased SHH expression leads to
facial clefts that are analogous to cleft palates in humans. Overexpression of SHH in chicks can
cause a widening of the space between the eyes, known as hypertelorism in humans (Hu and
Helms 1999).
The Transforming Growth Factor Signalling Pathway
The TGFβ family of growth factors are also expressed at all stages of tooth development,
and includes the Activin and BMP (Bone Morpogenetic Protein) signalling molecules. The
TGFβ signalling pathway has been shown to play a vital role in craniofacial development and lip
formation. Inactivation of the TGFβ receptor gene (TGFβR2) caused cranial defects and cleft
palates in mice (Ito et al. 2003; Wurdak et al. 2005); disruption of a TGFβ receptor (ALK5) in
mice also caused clefts in the upper lip of mice (Li et al. 2008). Mutations in members of the
BMP family also altered the facial development of mouse embryos. BMP2 and BMP4 are
expressed at various stages of tooth development in particular regions of the maxillary and
mandibular processes (Francis-West et al. 1994). Mutations or overexpression of BMP causes
clefts in the upper lip in mice (Ashique et al. 2002). Mutations in Noggin, an antagonist of
BMP, can lead to incisors without enamel, or mandibular or maxillary molars with an altered
crown shape or reduced number of roots (Plikus et al. 2005).
The Fibroblast Growth Factor Signalling Pathway

6. There are 25 members of the FGF family, including seven major receptors in humans:
FGFR1b, FGFR1c, FGFR2b, FGFR2c, FGFR3b, FGFR3c, and FGFR4. The FGF signaling
pathway is involved in cell stimulation and proliferation in the mesenchyme and facial
epithelium (Kouskoura et al. 2010). Members of the FGF family are also expressed during the
development of palatal shelves; mutations during this development can result in cleft palates,
especially mutations in either FGF10 or FGFR2b (Rice et al. 2004). In mice, progression of
tooth development and tooth formation and elongation require FGF signalling (Celli et al. 1998;
Ota et al. 2007). In humans, mutations in FGF23 can cause Autosomal Dominant
Hypophosphataemic Rickets (ADHR), leading to enlarged pulp chambers of teeth and
hypomineralized dentine (Pereira et al. 2004).
The Wingless Signalling Pathway
In humans, the WNT family includes 19 members that act through ten different receptors
(Frizzled 1-10). The WNT signalling molecules are expressed in the early stages of embryonic
craniofacial development, specifically at the initiation sites of teeth in the oral epithelium, the
initiation sites of bone formation in the mandible and maxilla, and in the mesenchyme of palatal
shelves. Expression of these molecules usually occurs in conjunction with expression of HH
and TGFβ signalling pathways (Silva-Palvia et al. 2010. In mice, inactivation of the WNT
pathway can lead to a cleft palate or reduced facial growth (Juriloff et al. 2006). Specific
reduction of WNT expression in the mesenchyme during tooth formation can result in smaller
teeth in mice (Sarkar and Sharpe 2000). In humans, mutations in the WNT3 gene can cause

7. tetra-amelia, in which patients are missing all four limbs and can also develop a cleft lip
(Niemann et al. 2004).
Key Genes Involved in Human Dentition
There are over 200 genes that are known to be expressed during tooth development. The
genes most commonly associated with oligodontia are PAX9 (paired box gene 9), MSX1
(muscle segment homeobox 1), AXIN2 (axis inhibition protein 2), and EDA (ectodysplasin A)
(Ruf et al.2013). Agenesis of the permanent dentition is one of the most common dental
abnormalities, and has a prevalence ranging from 1.6% to 9.6% in the general population (Mitsui
et al. 2014). However, mutations in these different genes have a different impact on agenesis
patterns; mutations in PAX9 usually causes agenesis in molars (Stockton et al. 2000; Das et al.
2002), while MSX1 mutations affect premolars (Vieira et al. 2004; Kim et al. 2006). Patients
with AXIN2 mutations are commonly missing both molars and premolars (Lammi et al. 2004),
and patients with defective EDA genes are usually missing their incisors (Tao et al. 2006; Ayub
et al. 2010).
PAX9
PAX9 is one of the major genes associated with non-syndromic tooth agenesis, primarily
affecting the molars (Bergendal et al. 2011). It is expressed during embryonic development and
is required for the mesenchymal expression of Lef1, Msx1, and Bmp4. Mice that are have a null
Pax9 display tooth arrest at the bud stage as well as the secondary cleft palate. The mice also
exhibit skeletal abnormalities in their limbs and facial area (Peters et al. 1998). Mitsui et al.
(2014) identified two mutations in the evolutionarily-conserved paired domain of PAX9; a three

8. base pair deletion (c.73-75 delATC) and a missense mutation (C146T). PAX9 mutations in
humans can lead to posterior tooth agenesis inherited in an autosomal dominant fashion, as is
also the case with mutations in the MSX1 gene (De Coster et al. 2009).
MSX1
In addition to causing non-syndromic tooth agenesis, MSX1 mutations can also cause
cleft lip and/or cleft palate (Jumlongras et al. 2001). Mutations in this gene have also been
implicated in breast and ovarian cancer (Chalothorn et al. 2008; Stockton et al. 2000).
AXIN2
Mutations in AXIN2 can cause autosomal-dominant oligodontia (Callahan et al. 2009).
The gene encodes the axis inhibition protein 2, which helps to stabilize beta-catenin, a protein
that helps in cell-cell adhesion. Patients with mutations in this gene were also shown to have a
predisposition for breast and colorectal cancer (Lammi et al. 2004; Marvin et al. 2011).
EDA
Ectodysplasin A is the ligand encoded by the EDA gene; mutations in this gene are the
most common cause of ectodermal dysplasia in the teeth, hair, skin, and sweat glands (Kere et al.
1996; Monreal et al. 1998). This is also common in patients with mutations in their EDAR
(EDA receptor) gene, or the EDARADD (EDAR-associated death domain) gene (Headon et al.
2001; Mikkola 2009). Mutations in these two genes have also been linked to cases of isolated
hypodontia (Tao et al. 2006; Song et al. 2009).
Stages of Tooth Development

9. In the developing embryo, tooth formation is usually divided into three stages, the
initiation, bud, and bell stages. The earliest sign of the initiation stage begins with a distinction
between the vestibular and dental lamina. A cleft forms between the two, and the vestibular
lamina directs formation of the lips and cheeks, while the dental lamina cells direct formation of
the developing jaw and teeth (Melton 2013). Tooth formation begins when the dental laminae
form odontogenic bands, which are strips of thickened oral epithelium that delineate the future
tooth rows (Stock 2007). After the oral epithelium thickens, it begins to grow into the
mesenchyme which then condenses, leading to the formation of a tooth bud. The epithelium
then continues to grow into the mesenchyme, eventually wrapping around it to form a “cap” and
then a “bell-stage tooth germ”. Eventually, the epithelium completely encases the mesenchyme,
known as the “late-bell stage”. Cytodifferentiation occurs during these bell stages, and cells can
either become amelobalsts, which produce the hard casing of enamel that covers the teeth, or
odontoblasts, which produce dentin, which forms the soft pulp of the tooth below the enamel
(Fig. 3). During the bell and cap stages, dental mesenchyme growth and epithelial
morphogenesis are coordinated and controlled by signals produced by a specific group of
densely packed, non-proliferating epithelial cells, known as the “enamel knot” (Fig. 4). This
signalling center is formed at the center of the tooth germ at the beginning of the cap stage and is
then eliminated by apoptosis (Klein et al. 2013). Further differentiation to form the basic pattern
of dentition of molars, premolars, canines, and incisors, is also determined in the early embryo,
before any visible signs of tooth development. The areas delineating the molars and incisors
work through proximal-distal and rostral-caudal patterning on the lower and upper jaws. The
combination of proximal-distal and rostral-caudal expression patterns have been termed the

10. “odontogenic homeobox code”; it helps to guide both mouse and human dentition in the
developing embryo (Tucker and Sharpe 2004).
Human Dentition Abnormalities
Dental abnormalities can be caused by a variety of factors; gene mutations can lead to
changes in protein expression and/or proper functioning of a signalling pathway. Environmental
factors can also play a role in gene expression and protein functioning. Genetic abnormalities of
teeth can be divided in three ways. First, there may be an abnormality in the number or shape of
the teeth. If a patient is missing up to six permanent teeth, this is referred to as hypodontia. If a
patient is missing more than six permanent teeth, it is referred to as oligodontia. In both cases
the third molars are excluded from the total of permanent teeth, as these are commonly missing
in over 20% of patients. If a patient is missing all of their teeth, it is known as anodontia. All
three cases are together referred to as tooth agenesis. Abnormal tooth shape can include an
enlargement of the tooth, known as taurodontism, or fusing of two teeth together. Secondly, it is
important to know whether the abnormality is part of a condition, i.e. is syndromic, or whether it
occurred on its own. Third, the mode of inheritance of the abnormality must be determined; it
can occur randomly or by a recessive or multifactorial mutation. The genetic etiologies of these
abnormalities will be discussed below.
Hypodontia
There are over 80 different syndromes in which hypodontia occurs. It can also occur as
part of a mutation or as a non-syndromic familial form. The non-syndromic form can occur as

11. part of an autosomal recessive (Ahmad et al. 1998) autosomal dominant (Alvesalo and Portin
1969) or sex-linked trait (Erpenstein and Pfeiffer 1967). Hypodontia is most common in the
permanent rather than the primary set (Matalova et al. 2008), and the most common missing
teeth among Caucasians are the mandibular second premolars (4.2%), maxillary lateral incisors
(2.3%), and maxillary second premolars (2.2%) (Polder et al. 2004). There is also a 3:2 female
to male ratio of hypodontia prevalence, although the reasons for this are not well understood
(Brook 1975).
Sporadic hypontia has also been known to occur, and can be a result of environmental or
genetic factors, or both (Schalk-Van Der Weide et al. 1993; Vastardis 2000). Generally, when
hypodontia occurs the missing tooth is the most distal in its group. For example, if a molar is
missing it is almost always the third molar (Klein et al 2013). Environmental factors that may
cause or contribute to missing teeth can include radiation, surgery on the jaw, trauma on the jaw,
or early removal of primary teeth, among others. The genetic factors leading to hypodontia are
less well known, but mutations in PAX9 have been known to cause both sporadic hypodontia and
oligondontia.
Familial, non-syndromic hypodontia has been linked to a variety of mutations and in
many cases is thought to be an extremely complex multifactorial condition. The most common
mode of inheritance in families is autosomal dominant with varying degrees of expressivity and
incomplete penetrance. Some genes that have been linked to familial hypodontia are MSX1,
PAX9, AXIN2, WNT10A, and EDA. Mutations in MSX1 were found in a family that all had
missing third molars and second premolars, despite having a normal primary dentition. Many of
the affected family members were also missing their mandibular first molars, maxillary first
premolars, a single lower central incisor or one or both lateral incisors (Vastardis et al. 1996).

12. A family that also had missing molars was shown to have autosomal dominant
hypodontia due to a frameshift mutation in the PAX9 gene (Stockton et al. 2000). Since then, a
number of PAX9 mutations have been associated with types of hypdontia that usually affects the
molars (Nieminen et al. 2001; Das et al. 2003). AXIN2 and WNT10A, which are part of the
WNT signaling pathway, have also been shown to cause hypodontia when mutated. Eleven
members of a Finnish family that were missing at least eight permanent teeth were shown to
have mutations in AXIN2 (Lammi et al 2004), and it is now knows that about 56% of patients
who had isolated hypodontia also had a mutation in their WNT10A gene (van den Boogaard et al.
2012).
Syndromic Hypodontia
As mentioned above, hypodontia can occur as part of a syndrome, and there are over 80
syndromes currently known to be associated with hypodontia. A full list of syndromes is
available at the Online Mendelian Inheritance in Man (OMIM,
http://www.ncbi.nlm.nih.gov/omim). This paper will discuss Van der Woude syndrome (VWS,
OMIM #119300), Rieger syndrome (OMIM #601542), oral-facial-digital syndrome type I
(OFD1, OMIM #311200) ectodermal dysplasia (ED), and holoprosencephaly (HPE; OMIM #
236100) because they are among the most common and well studied the syndromes.
VWS is most commonly characterized by a cleft palate (CP), hypodontia, and lip
sinuses/pits (Leck and Aird 1984. On average it occurs every 1 in 40,000 live or still born births
(Burdick 1986). 77.8% of patients who had cleft palates and lip sinuses also developed
hypodontia, with the second premolar being the most common missing tooth (Ranta and Rintala

13. 1982). The syndrome is autosomal dominant and is caused by mutations in the IRF6 gene
(interferon regulatory factor 6); mutations in this gene can also cause popliteal pterygium
syndrome (PPS), which may also cause webbing of fingers and toes (Lees et al. 1999).
Rieger syndrome is also inherited in an autosomal dominant fashion with a prevalence of
1 in 200,000 in the population. Its most common symptoms include hypodontia and ocular
abnormalities (Shields et al 1985). Hypodontia can occur in either the permanent or primary
dentition, and the most common missing teeth are the upper second molars, lower second
premolars, or central incisors. Tooth shape and size may also be affected, leading to smaller,
conical- or peg-shaped teeth, or taurodontism (Dressler et al. 2010).
OFD1 is caused by mutations in the gene OFD1, which is responsible for the formation
of primary cilium. Because it is an X-linked disorder, OFD1 is lethal in males and affects only
females (Klein et al. 2013). Lower lateral incisors are missing in over 50% of patients, although
hyperdontia and enamel dysplasia were also commonly seen (Toriello and Franco 2007).
Ectodermal dysplasia can be either X-linked (the most common), autosomal recessive, or
autosomal dominant. The X-linked form, known as X‐linked hypohidrotic ED, or XLHED
(OMIM #305100). This disorder is caused by a mutation in the EDA gene, which functions in
the TNF signaling pathway and helps to regulate NFKB1, a gene important for odontogenesis
(Ohazama and Sharpe 2004). People with XLHED usually only develop about nine permanent
teeth, with males showing severe oligodontia in both their primary and permanent dentitions,
whereas females usually have milder phenotypes due to X-inactivation. Women who are carriers
have a 60-80% chance of developing oligodontia (Cambiaghi et al 2000; Lexner et al. 2007).
HPE can be dominantly or recessively inherited, and is caused by mutations in the SHH

14. (Sonic hedgehog) gene, which is essential for normal dentition (Lami et al. 2013). The dental
manifestations of this syndrome can vary from severe (which includes hypodontia and defects of
the forebrain and mid-face), mild (evidenced by a solitary median maxillary control incisor,
SMMCI), or a patient may show no symptoms at all (El-Jaick et al. 2007). SMMCI has also
been linked to several other disorders, including velocardiofacial syndrome, VACTERL
association, Duane retraction syndrome, ED, and CHARGE syndrome (Oberoi and Vargervik
2005).
Abnormalities in the Formation and Eruption of Teeth
In addition to abnormal numbers of teeth, the formation and eruption times of teeth may
be cause by gene mutations as a result of certain syndromes, including Apert syndrome and
oculofaciocardiodental (OFCD) syndrome. In Apert syndrome, teeth fail to erupt through the
thickened gingival tissues. This can result in swelling in the maxillary arch and abnormalities in
the shape of incisors and molars (Kaloust et al. 1997; Peterson and Pruzansky 1974). Mutations
in the Fgf (Fibroblast growth factor) gene family are known to be the causes of these symptoms,
although the exact mechanisms have yet to be elucidated.
Abnormalities in Size and Shape of Teeth
About 5% of the population has a discrepancy between the sizes of teeth in their upper
and lower jaws. These abnormalities are usually the result of an abnormal “cap-bell” or
morphodifferentiation stage of tooth development. The most affected teeth are the second

15. premolars and the upper lateral incisors. If the patient also has hypodontia, the upper lateral
incisors will appear peg-shaped. A discrepancy between jaw and tooth size is also common, and
in the cases of normal jaw size and smaller teeth, there will often be spaces between the teeth,
termed tremata (Peterka et al. 1996; Klein et al. 2013).
Teeth can also fuse during development, resulting in teeth joined at the dentin, but with
separate pulp chambers. Teeth can also “germinate”, resulting in two teeth sharing a common
pulp chamber. Taurodontism is a condition that results in an enlarged tooth pulp chamber caused
by a constriction of the cementoenamel junction resulting in abnormal growth of the epithelial
root sheath (Klein et al. 2013). Rates of the syndrome vary widely amongst populations, with
incidence rates as low as 0.5-3.2% in Caucasian Americans (Blumberg et al. 1971), and as high
as 46.4% in young adult populations in China (Macdonald-Jankowski and Li 1993).
Taurodontism is also seen as a symptom of many disorders, including a frequency of over 50%
in patients with VWS (Nawa et al. 2008).
Hyperdontia
Supernumerary teeth, or hyperdontia, occurs most commonly on the permanent dentition
(1.5-3.5%) and less often on the primary dentition (0.3-0.8%) (Brook 1974). A supernumerary
tooth is any tooth that is found in addition to the permanent teeth anywhere on the dental arch
and usually occurs as the result of abnormalities during the initiation and proliferation stages of
dentition. It occurs most commonly in the midline of the maxillary known as the mesiodens
(Klein et al 2013). About twice as many males as females develop single supernumerary teeth
(Kantor et al. 1998), and that ratio jumps to 3:1 in cases of multiple supernumerary teeth (Gibson

16. 1979). Most supernumeraries (>90%) will occur in the upper jaw, and of these 25% will erupt.
Sometimes the teeth will become impacted and must be removed (Moore et al. 2002). The
condition is rarely inherited, but there have been reported cases of both autosomal dominant and
recessive forms of inheritance, as well as X-linked inheritance (Cassia et al. 2004). However,
the genetic underpinnings of supernumerary teeth is currently unknown, although several
hypotheses have been proposed. Liu (1995) proposed that supernumerary teeth form due to a
dichotomy of the tooth bud; Scheiner and Sampson (1997) proposed that it may be caused by
hyperactivity in the dental lamina.
Syndromes Associated with Hyperdontia
A variety of syndromes have been linked to hyperdontia, including Gardner syndrome
and cleidocranial dysplasia Gardner syndrome is a syndrome variation of familial adenomatous
polyposis (FAP; OMIM #175100); both are caused by mutations in the APC protein, which
helps to regulate the WNT signaling pathway (Barth et al. 1997). Hyperdontia is fairly common
among individuals with Gardner syndrome, as well as a variety of other dental problems,
including distorted tooth morphology, unerupted or impacted teeth, or fused molar roots, among
others (Butler et al. 2005).
Cleidocranial dysplasia (CCD, OMIM #119600) is the condition most commonly
associated with hyperdontia, although it is one of the rarest (prevalence of 1 in 1,000,000). It is
an autosomal dominant disease caused by mutations in the RUNX2 gene. This gene
transcriptionally regulates osteoblast differentiation (Otto et al. 2002), and as such more than
90% of CCD patients have dental anomalies, including hyperdontia, delayed eruption of teeth,

17. and/or enamel hypoplasia. Supernumerary teeth may also develop asymmetrically, or in some
cases patients do not have any missing or supernumerary teeth (Golan et al. 2003).
Oral Clefts
An oral cleft is caused by a tissue discontinuity as the result of a fissure or fissures in the
lip and/or palate area. It has a prevalence of about 1 in 750 live births, although this figure is
higher in patients of Native American or Asian ancestry (Croen et al. 1998). Babies born with a
cleft lip (CL), cleft palate (CP), or a cleft lip and palate (CLP) can encounter several problems,
including problems speaking and eating, as well as abnormal facial or dental development
(Hodgkinson et al. 2005). Genetic models, especially mice, have been especially useful in
identifying the genes and environmental factors that can cause CP/CLP, and a variety of other
disorders caused by abnormal human dentition.
Genetic Models of Tooth Growth and Development
A variety of vertebrate genetic models, including the mouse the zebrafish, have been used
to study tooth growth and development (Thesleff 2006; Klein et al. 2013). These models have
provided insight into tooth development in humans, and have allowed scientists and dentists to
begin developing modes of treatments for a variety of dental problems.
Mouse Dentition

18. Mice have become the main model for studying the genetic and cellular basis of tooth
development, and most of what scientists know about human dentition is based on the mouse
model (Fig. 5). Although human dentition is more complex than that of mice, the basic
mechanisms that drive tooth development are highly conserved and thus extremely similar in the
two species. Mice have a much simpler permanent dentition than humans, consisting of just
three molars at the rear of the mouth and one incisor at the front in each quadrant. In between
these two types of teeth is a toothless region referred to as a “diastema”. Although simplified,
this mouse model allows for the study of two extremely important areas of tooth development:
tooth regeneration and tooth suppression (Klein et al 2013).
Because mouse incisors grow continuously throughout their lifetimes, they are a known
source of stem cell differentiation and development that shares many similarities with human
regeneration systems. Incisor cellular renewal was first discovered in adult rats by Smith and
Warshawsky (1975); since then, the genes that control this continual regeneration and
differentiation have been identified. The source of this regeneration was a population of slowly
dividing epithelial cells located at the proximal end of the incisor in an area known as the labial
cervical loop (LaCL). It was then determined that these epithelial cells were able to differentiate
into enamel-secreting ameloblasts through the control of TGF-β/BMP and FGF signaling, and
that adult stem cell were able to differentiate through SHH signaling. However, until recently
the mechanisms for controlling the homeostasis of these cells was not well understood. Biehs et
al. (2013) was able to determine that adult stem cells in the LaCL expressed Bmi1, a proto-
oncogene that is a known regulator of differentiation and renewal of stem cells in humans (Jiang
et al. 2009). In mice, Bmi-1 is able to regulate the growth and differentiation of stem cells in the
LaCL through two distinct mechanisms: repression of Ink4a/Arf expression, which allows for

19. stem cell self-renewal, and the suppression of Hox genes, which has been observed in other
systems, including the house fly Drosophila (Park et al. 2003).
The diastema, or toothless region in the mouse mouth, has also become important for
understanding the cellular nature of hypodontia. The diastema contains several rudimentary
teeth primordia whose growth and development have been increasingly suppressed throughout
evolution (Klein et al. 2013). This suppression serves as an excellent model for analyzing the
genetic mechanisms that underlie tooth development failure in humans. Tooth suppression in
mice is maintained by apoptosis, or programmed cell death (PCD). PCD can help to eliminate
nonfunctional or harmful cells, but it is also a normal part of cellular development in an
organism. In mice, apoptosis helps to suppress vestigial tooth buds throughout the diastema in
the early stages of mouse odontogenesis. In the embryo stage, small primordia appear within the
diastema and disappear within 24 hours via apoptosis; this helps to establish the incisor-diastema
boundary and prevent supernumerary teeth. Larger vestigial teeth next to the molars also
undergo partial apoptosis and are reduced and reshaped so they can be incorporated into the first
molar (Peterkova et al. 2003).
Proximal-Distal Patterning
During the early stages of development, three important proteins control the
differentiation of the molar and incisor fields. Fgf8 and Fgf9 (fibroblast growth factors 8 and 9)
are expressed laterally (proximally) throughout the potential molar area, and BMP4 (bone
morphogenetic protein 4) is expressed medially (distally) throughout the potential incisor area.
How these proteins come to be expressed in these areas during development is not currently well
understood (Tucker and Sharpe 2004). However, it is known that Fgf8 and Fgf9 control the

20. homeobox proteins Barx1 (BarH-like homeobox 1) and Dlx2 (Distal-less homeobox 2). BMP4
negatively regulates expression of Barx1 while positively regulating expression of Msx1 and
Msx2 (Msh-like 1 and 2 homeoboxes) (Fig. 6). Homeobox genes (also known as homeotic
genes) are transcriptional regulators that help to regulate normal patterns of growth during
embryonic development. These genes contain a highly conserved sequence of DNA known as
the “homeobox sequence” that contains 183 nucleotides that encodes a 61 amino acid motif
known as the homeodomain. This domain contains a conserved helix-loop-helix motif that
allows it to bind DNA and activate or repress target genes (Murthi 2014). During the early
stages of mouse dentition, BARX1 and DLX2 are restricted to the molar region, while MSX1
and MSX2 are only expressed in the incisor region, and these four homeobox genes help to
regulate growth and development of the molar and incisor teeth in the mouse jaw during
embryonic development. Discovery of these genes and the process of their control was
determined by studies that knocked-down or altered the expression of the homeobox genes
(Tucker and Sharpe 2004). Tucker et al. (1998) placed Noggin protein (an antagonist that blocks
BMP signalling) in the distal mesenchyme, Msx1 expression (which is positively regulated by
BMP4) is lost, and Barx1 expression (which is normally repressed by BMP4) is increased. This
resulted in the formation of a molar tooth in the area where an incisor would normally form.
Similarly, mice with knockouts of Dlx1 and Dlx2 did not develop upper molars, but their incisors
developed normally. Addition of Dlx5 and Dlx6 can help to rescue this mutation (Thomas et al.
1997). In addition, it is now known that FGF8/9 and BMP4 are mutually antagonistic, meaning
that they work to block the expression of each other (Fig. 6). This means that when the Noggin
antagonist is expressed to block BMP4, Fgf8/9 is now free to be expressed in the distal region.
Other proteins, such as Islet1, are part of a positive feedback loop with BMP4 where expression

21. of one increases expression of the other, and so on. Overexpression of Islet1 leads to a increase
in the expression of BMP4 and subsequent decrease in the expression of Fgf8/9 and Barx1,
which is positively regulated by Fgf8. Expression of Fgf8 is controlled by the homeobox gene
Pitx2. BMP4 repression can be induced by overexpression of Pitx2 at very high levels, whereas
expression of Fgf8 can be maintained at low levels of Pitx2.
Rostral-Caudal Patterning
The oral-aboral (also known as the rostral-caudal) axis is also under control of signals
from the epithelium (Fig 6). FGF8 helps to regulate the LIM homeobox genes Lhx6 and Lhx7.
Expression of these genes marks the areas where tooth buds will form and because their
expression is not repressed by BMP4 they are expressed throughout the mesenchyme. Another
homeobox gene, Gsc, is positively controlled by FGF8. Gsc is turned on after Lhx6/7 and it is
expressed everywhere Lhx6/7 are not, i.e. in the aboral mesenchyme. This area is found below
the teeth and contains many of the skeletal elements that help to support the teeth (Tucker and
Sharpe 2004). Therefore, in mice where Gsc has been knocked out teeth develop normally but
this skeletal structure is missing, resulting in teeth that are severely impaired (Rivera-Pérez et al.
1995; Yamada et al. 1995).
Control of Tooth Number
In mice as in humans, the determination of tooth size and number is still just beginning to
be understood. It is known that the size of the tooth field is directly proportional to tooth
number. The tooth field area is under the control of the ectodysplasin (EDA) family of
signalling molecules. The receptor of EDA is EDAR, and its intracellular adaptor protein is

22. EDARADD. When the function of any of these are disrupted in mice, tooth number is affected
(Sofaer 1969; Headon 2002). These gens were thus some of the first to provide insight to the
genetics of hypodontia and hyperdontia.
In mice, levels of EDA could be increased by a constitutively active receptor or
overexpression of the ligand. In both cases, the area of the molar tooth field expanded, and an
extra tooth grew next to the first molar. However, this extra tooth has a different shape from a
normal molar, and tends to be shaped more like a premolar tooth (Mustonen et al. 2003; Tucker
et al. 2004). Thus, it appears that the shape of a tooth is under the control of the particular set of
homeobox genes that is expressed in the particular area of the mesenchyme into which they
invaginate. Interestingly, mouse with Eda gene knockouts can be rescued with overexpression
of Eda if it is expressed early enough in development. However, if Eda is introduced after teeth
have already begun to form, then the defect cannot be rescued (Gaide and Schneider 2003).
While hypodontia is most commonly correlated with a loss of Eda or Edar and hyperdontia with
an overexpression of either of these genes, there have been observed cases of hypodontia
occurring after an increase of Edar (Tucker et al. 2004), and hyperdontia has been observed after
a loss of Edar or Eda (Grüneberg 1966; Headon et al. 2002). It therefore appears that control of
tooth number is a delicate mechanism that requires a precise balance of EDA signalling during
early development.
Formation of the Tooth Bud
Once the size of the tooth area and what types of teeth will form have been determined,
tooth buds can begin to form, marked by the invagination of the epithelium into the mesenchyme

23. (day E11 in mice). Within the epithelium are four areas of Shh (sonic hedgehog) expression;
these spots will mark the areas of the tooth buds of the developing molar and incisor tooth germs.
Wnt7b has the opposite pattern of expression of Shh, and overexpression of Wnt7b can arrest
tooth development by reducing levels of Shh; this phenotype can be rescued with the addition of
SHH (Tucker and Sharpe 2004).
Within the mesenchyme, the paired box gene Pax9 controls the position of the tooth
fields. Like Shh, it marks four areas which will become the future sites of invagination into the
mesenchyme. It works in conjunction with activin as the earliest positional markers of the
forming tooth germ. Like Barx1, Pax9 in negatively regulated by BMP4 and positively
regulated by FGF8. It is also repressed by BMP2, which belongs to the same family as BMP4.
Although many of the same genes are involved in controlling tooth bud formation, the different
times that they are expressed has a large impact on expression patterns (Tucker and Sharpe
2004). Control of Pax9 by FGF8 and BMP4 occurs after the boundaries of the molar field have
been established. Knockout of Pax9 allows tooth buds to form but growth arrest halts, leading to
tooth agenesis (Peters et al. 1998). Similarly, knockout of Msx1 will also lead to tooth agenesis.
Msx1 has a role in marking the early incisor region as well as development of the tooth bud in
the mesenchyme. Both MSX1 and BMP4 both act in a positive feedback loop, and although
knockdown of Msx1 does not lead to initial decrease in Bmp4 expression, later expression
disappears in the mesenchyme during development. However, the Msx1-/- phenotype can be
rescued by addition of BMP4 if it occurs before the “cap” stage (Zhang et al. 2000; Zhao et al.
2000; Bei et al. 2000).

24. Zebrafish Dentition
In recent years, other vertebrate species such as Zebrafish (Danio rerio) has been used as
a model to study human dentition as well as a variety of other aspects of human development.
Zebrafish were initially chosen as a model because they reproduce much more quickly are much
cheaper than mice, yet still share many similar genes with humans. Although zebrafish do not
have teeth in their oral jaws, they do have sets of teeth on the rearmost pharyngeal arch. These
teeth are continually regenerated throughout the life of the zebrafish and this regeneration has
been well documented, as well as the growth, development, patterning, and differentiation of
zebrafish teeth (Fig. 7) (Klein et al. 2013).
Differences between Zebrafish Dentition and other Vertebrates
Zebrafish dentition is remarkably similar to that of mammals with only a few notable
exceptions; the first is that tooth formation in mammals requires a downgrowth, or deep
invagination of the tooth into the mesenchyme, known as the dental lamina. Additionally,
mammalian teeth possess an “enamel knot” as described above, which is a central hub for much
of the signaling that controls tooth development (Stock 2007). Finally, Zebrafish are
polyphodont, which means that their teeth are continuously replaced their lifetimes. It is
estimated that a juvenile zebrafish loses its teeth once every eight days (Van der heyden et al.
2000). This cycle of tooth loss and regeneration is an artifact of the ancestors of the zebrafish,
while diphodonty of humans and monophodonty of mice are offshoots of these ancestors (Sire et
al 2002).

25. Some key differences between the mouse and zebrafish model is that zebrafish teeth
develop and are replaced very early in development, between 48 and 80 hours post-fertilization.
This allows teeth to develop before any mutations become lethal and makes it easier to study
tooth development in the young embryo. Additionally, because zebrafish teeth continuously
regenerate, this is more similar to the diphyodont nature of human teeth than the mouse model.
Thus, using the zebrafish as a model poses several advantages for studying human dentition
(Stock 2007; Klein et al. 2013).
Zebrafish tooth development can be divided into three partially overlapping stages:
initiation and morphogenesis, cytodifferentiation, and attachment. The first stage of tooth
development in the zebrafish, initiation, is marked by a thickening of the pharyngeal epithelium
with densely packed cells. A few mesenchymal cells are directed towards the epithelium and
make contact with the basal lamina; they are separated by a narrow space composed of thin
collagen fibrils. The morphogenesis stage is divided into four sub-stages: The early bud stage,
the late bud stage, the early bell stage and the late bell stage. During the early bud stage, there is
epithelial invagination of desmosome-linked cells. On the proximal side of the downgrowth the
cells are polarized but not extremely well aligned, giving the appearance of a smooth surface.
During cytodifferentiation the dentine and enameloid matrices are deposited along the epithelial-
mesenchymal interface. Then comes the attachment phase, during which bone attaches itself to
the matrix so teeth may begin to develop (Huysseune et al. 1998; Stock 2007).
Although zebrafish lack some key genes such as pax9, which is expressed early on in
mammalian tooth development, they have retained many key features, including Fgf, Bmp, and
Wnt signaling. As discussed above, these signaling pathways are crucial for normal tooth
development. In zebrafish, overexpression of Fgf ligands or downregulation of Bmp signaling

26. results in supernumerary teeth. Blocking Fgf signaling will lead to a growth arrest of primary
teeth (Jackman et al. 2004; 2013). Mutations of the Wnt pathway do lead to any noticeable
abnormalities in zebrafish (Wiweger et al. 2012), but zebrafish with mutations in the Lef1 gene
develop oligodontia (McGraw et al 2011). The zebrafish plasma membrane Ca2+ ATPase
(PMCA) SqET4 was shown to be required for bone mineralization of zebrafish teeth. Humans
have four genes that encode PMCAs (ATP2B1-4), and in mice PMCA1-null mice died in the
early stages of development (Go and Korzh 2013). N-cadherin, encoded by the gene cdh2, is
also known to be essential for zebrafish dentition. In zebrafish deficient for this gene, primary
tooth formation occurred but then stopped in the early stages of cytodifferentiation. In mice,
knockout of N-cadherin is embryonic lethal (Verstraeten et al. 2013).
Overall, the zebrafish has provided an excellent vertebrate model for the study of
dentition. Although more research needs to be done in this model, the many physical and genetic
similarities it shares with humans has proven the zebrafish to be an up-and-coming model for the
study of tooth development and formation.
Current and Future Therapies
Craniofacial abnormalities such as craniosynostosis and clefts and/or palates, can be
detected at 16 weeks of gestation or later (Ghi et al. 2002). The only way to treat clefts is to
close them surgically, although this cannot be done perfectly and sometimes results in defects in
growth in the upper face (Nollet et al. 2008). Stem cell therapy offers exciting new possibilities
for correcting CLP before they occur at birth. Between 5-12 weeks of pregnancy, stem cells free
of genetic abnormalities can be injected into the immature immune system of the embryo, where

27. they can help to correct the malformation (Jones and Trainor 2004). Stem cell therapy and tissue
engineering can also help to treat oral clefts in infants and children. During surgery to repair
CLP surgeons often face a shortage of tissue; stem cells from dental pulp and other tissues can
help to fix this problem and replace any tissue that has been lost and increase the time to
recovery (Panetta et al. 2008). Scar-free wound repair can also help minimize the scarring that
often occurs after CLP surgery. During healing and after CP closure, fibrocytes and stem cells
can differentiate into myofibroblasts because of the mechanical tension of the wound or various
growth factors. Myofibroblasts are cells with contractile capabilities, at the site of the wound
they can cause the skin to contract, leaving a visible scar. This can also lead to abnormal tooth
eruption and development, as well as restricted maxillary growth (Wijdeveld et al. 1991).
Treatment for scarring includes the use of growth factors to inhibit cell differentiation into
myofibroblasts or to reduce the contractile action of myofibroblasts. Apoptotic signals can also
be delivered to these cells to help reduce the effects of scarring (van Beurden et al. 2005).
Conclusion
It is evident that the proper development of teeth requires immense and sophisticated
coordination between a variety of genes, proteins, pathways, and signaling molecules. In
humans, four main dentition signaling pathways, Hedgehog, TGFβ, Wingless, and FGF, control
the majority of tooth growth and development. Transcription factors control gene expression,
which in turn regulates proteins and signaling molecules to help coordinate timing during
embryonic development. A variety of dental abnormalities can arise as a result of improper gene
functioning or developmental timing; these disorders can range from too many or too few teeth

28. to ectodermal dysplasia or cleft palates. Before scientists had an understanding of the genetic
etiology of dentition, it was difficult to provide treatment or preventative care. However, with
the emergence of vertebrate models such as the mouse and zebrafish, scientists have been able to
map the genetics of vertebrate dentition in a way that can be compared back to the human model
in order to understand the role of specific genes and the coordination of development timing.
This has also allowed dentists and surgeons to use biologically-based therapies such as stem cell
treatment and scar-free wound healing to help fix patient dental and facial abnormalities.
Continued research in vertebrate models and improvements in various therapies will help to
improve our understanding of human dentition and provide patients with the best possible dental
therapies for the future.

29. Figure 3
Figure 2
Figure 1

30. Figure 4
Figure 5

31. Figure 6

32. Figure 7

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