Genetics in Tooth Development Introduction The Molecular Program of Tooth Development Primary Epithelial Band Dental Lamina Vestibular Lamina Initiation of the Tooth Genes expressed during tooth development Developmental signals controlling the position and the number of tooth germs along the oral surface Homeobox code model Instructive Signals for Patterning Tooth Type Determination Regionalization of Oral and Dental Ectoderm Bud Stage Bud-to-Cap Transition Signaling centres Applied aspects

1. Genetics in Tooth
Development
Dr Purva Pihulkar
Senior Lecturer
Dr HSRSM Dental College and
Hospital,Hingoli,Maharashtra

2. INDEX
 Introduction
 The Molecular Program of Tooth Development
 Primary Epithelial Band
 Dental Lamina
 Vestibular Lamina
 Initiation of the Tooth
 Genes expressed during tooth development
 Developmental signals controlling the position and
the number of tooth germs along the oral surface

3.  Homeobox code model
 Instructive Signals for Patterning
 Tooth Type Determination
 Regionalization of Oral and Dental Ectoderm
 Bud Stage
 Bud-to-Cap Transition
 Signaling centres
 Applied aspects

4. INTRODUCTION
 Teeth, like all epithelial appendages, form via a
sequential and reciprocal series of inductive signals
transmitted between the epithelium and neural crest
derived mesenchyme.
 Each tissue layer instructs the other to differentiate in a
precisely determined manner leading to the formation of
highly specialized structures, such as incisors, canines,
premolars and molars.
 Each of these groups of teeth derives from different parts
of the oral epithelium and, depending on the species,
teeth can be formed from both endoderm and ectoderm
or from ectoderm only

5.  Animal and human studies that employ the tools of
contemporary molecular genetics have identified a
number of genes that act at specific stages of tooth
development and regulate its patterning and
differentiation process

6. However, to better understand morphogenesis, the
molecular signals that control cell growth, migration,
and ultimately cell fate and differentiation also must
be considered.

7.  The molecular aspect of tooth development is
interesting in that it shares many similarities with the
development of a number of other organs (e.g., lung
and kidney) and that of the limbs.
 Thus the tooth organ represents an advantageous
system in which to study not only its own
development but also developmental pathways, in
general

8.  The five major conserved signaling pathways that
intervene in these events are
 (1) Bone morphogenetic protein (BMP)
 (2) Fibroblast growth factor, (FgF)
 (3) Sonic hedgehog (Shh)
 (4) Wingless-related integration site (Wnt)
 (5) Ectodysplasin A (Eda).

9. The Molecular Program of Tooth
Development
 The signaling molecules mediating communication
between cells constitute one of the key groups of
molecules in this conserved toolbox.
 There are four major families of signal molecules
that are essential for cell communication in all
animals from flies to man as well as in all different
organs, including teeth.
 These are BMP (bone morphogenetic protein), FGF
(fibroblast growth factor), hedgehog, and Wnt

10.  In addition, ectodysplasin (Eda), an NFkB family
signal, plays key roles in the development of teeth
and other ectodermal appendages.
 The signals can be thought to constitute the
“language” of interacting cells, and they regulate
tooth development all the way from initiation to root
formation

11.  The toolbox also includes receptors for signals at the
cell surface, mediators transmitting the signal in
the cell, and transcription factors regulating gene
expression in the nucleus.
 The transcription factors are of special importance
because they regulate the fate of cells.

12.  In particular, specific combinations of transcription
factors can determine the identities of different cell
types. Knowledge of such transcription factor codes
is essential for cellular reprogramming in
regeneration studies.
 However, so far the “transcription factor codes” of
tooth- specific cells are not known.

13.  The reciprocal and sequential interactions between
dental mesenchyme and epithelium constitute the
core of the molecular program.
 The interactions are mediated by the conserved
signal molecules activating the expression of
specific transcription factors, which in turn
regulate the expression of numerous other genes
important for advancing morphogenesis and cell
differentiation in the developing tooth.

15. Primary Epithelial Band
 After about 37 days of development, a continuous
band of odontogenic epithelium forms around the
mouth in the presumptive upper and lower jaws.
 These bands are roughly horseshoe-shaped and
correspond in position to the future dental arches of
the upper and lower jaws

16.  A key feature of the initiation of tooth development is
the formation of localized thickenings or placodes
within the primary epithelial bands.
 Dental placodes are believed to initiate formation of
the various tooth families.

18.  The basic mechanisms and genes involved in the
formation and function of all placodes are similar.
 The balance between stimulatory (FGFs, Wnts)
and inhibitory signals (BMPs) is important in
determining the site of placodes

19.  Formation and growth of placodes is believed to
involve the transcription factor p63, tumor
necrosis factor (TNF), and ectodysplasin (Eda).

20. Dental Lamina
 On the anterior aspect of the down-growing dental
lamina, continued and localized proliferative activity
leads to the formation of a series of epithelial
outgrowths into the mesenchyme at sites
corresponding to the positions of the future
deciduous teeth

21.  Ectomesenchymal cells accumulate around these
outgrowths.
 From this point, tooth development proceeds in
three stages: the bud, cap, and bell.

22. image

23. Vestibular Lamina
 The vestibule forms as a result of the proliferation of
the vestibular lamina into the ectomesenchyme
soon after formation of the dental lamina.
 The cells of the vestibular lamina rapidly enlarge and
then degenerate to form a cleft that becomes the
vestibule between the cheek and the tooth-bearing
area

24. Initiation of the Tooth
 Odontogenesis is initiated first by factors resident in
the first arch epithelium influencing
ectomesenchyme but that with time this potential is
transferred to and is assumed by the
ectomesenchyme
 These experimental findings are mirrored by the
expression pattern of transcription and growth
factors in these tissues.

25. image

26.  The earliest histologic indication of tooth
development is marked by a thickening of the
epithelium where tooth formation will occur on the
oral surface of the first branchial arch
 The genes that are implicated in the ensuing
 To date, the earliest mesenchymal markers for tooth
formation are the LIM-homeobox (Lhx) domain
genes (transcription factors), Lhx-6 and Lhx-7

28.  Both of these genes are expressed in the neural
crest–derived ectomesenchyme of the oral portion of
the first branchial arch as early as day 9 of
gestation
 Experimental data demonstrate that the expression
of Lhx-6 and Lhx-7 results from a signaling
molecule originating from the oral epithelium of the
first branchial arch.

29.  If second arch mesenchyme is recombined with first
branchial arch oral epithelium, Lhx-6 and Lhx-7 will
be induced.
 However, if first branchial arch mesenchyme (which
expresses Lhx-6 and Lhx-7) is recombined with
second branchial arch epithelium, expression of both
genes will be downregulated quickly

30.  A prime candidate for the induction of Lhx genes is
secreted fibroblast growth factor-8 (Fgf-8); this
growth factor is expressed at the proper place and
time in the first branchial arch and is able to induce
Lhx-6 and Lhx-7 expression .

31. Genes expressed during tooth development

32. In terms of developmental signals , What
controls the position and the number of
tooth germs along the oral surface?
 The Pax-9 gene is one of the earliest mesenchymal
genes that define the localization of the tooth
germs.
 Pax-9 gene expression colocalizes with the exact
sites where tooth germs appear.
 Pax-9 is induced by Fgf-8 and is repressed by
bone morphogenetic proteins (BMP-2 and BMP-4).

33.  Fgf-8, Bmp-2, and Bmp-4 are expressed in
nonoverlapping areas, with Pax-9 being expressed
at sites where Fgf-8 is but Bmp is not.
 Number of other genes are also expressed in oral
epithelium at the same time.
 Whether they directly regulate the expression of
Fgf-8 or Bmps is not clear at this time.

34.  Little is known about the regulatory mechanisms of
signaling molecules, and untangling the network of
regulatory events can be difficult.
 At least 12 transcription factors are expressed in
odontogenic mesenchyme, and some have
redundant roles.
 To date, more than 90 genes have been identified
from the oral epithelium, dental epithelium, and
dental mesenchyme during the initiation of tooth
development.

35.  In mice, expression of Shh is localized to the
presumptive dental ectoderm at E11 and is thus
another good signaling candidate for tooth initiation .
 Mutations in Gli genes that are downstream
mediators of Shh action suggest a role in early tooth
development, because Gli2−/− and Gli3−/− double
mutant embryos do not produce any recognizable
tooth buds.

37.  Addition of Shh-soaked beads to oral ectoderm can
induce local epithelial cell proliferation to produce
invaginations that are reminiscent of tooth buds.
 Shh thus appears to have a role in stimulating
epithelial cell proliferation, and its local expression at
the sites of tooth development implicates Shh
signaling in tooth initiation

38.  Cbfa1, also referred to as Osf2, is a transcription
factor that plays a critical role during bone formation
.
 Its expression in dental mesenchyme is associated
with the early signaling cascades regulating tooth
initiation.
 It regulates key epithelial–mesenchymal
interactions that control advancing morphogenesis
and histodifferentiation of the enamel organ.

39.  Paired like homeodomain transcription factor 2 (Pitx-
2) is a key player in pattern formation and cell fate
determination during embryonic development.
 Pitx-2 is one of the earliest markers of tooth
development and continues to be expressed through
crown formation.

40.  It regulates early signaling molecules and
transcription factors necessary for tooth
development.
 Another factor is Lef-1, a member of the high-
mobility group family of nuclear proteins that
includes the T-cell factor proteins, known to be
nuclear mediators of Wnt signaling.

41.  Lef-1 is first expressed in dental epithelial
thickenings and during bud formation shifts to being
expressed in the condensing mesenchyme.
 Expression of several genes in ectomesenchyme
marks the sites of tooth germ initiation.
 These include Pax-9 and Activin-A, both of which are
expressed beginning around E11 in mice within small
localized groups of cells corresponding to where
tooth epithelium will form buds

42.  In the case of Pax-9, antagonistic interactions
between Fgf-8 and Bmp-4, similar to those found to
regulate Barx-1 expression, from oral ectoderm have
been shown also possibly to act to localize Pax-9
expression.

43. Tooth Type Determination

44.  The determination of specific tooth types at their
correct positions in the jaws is referred to as
patterning of the dentition.
 The determination of crown pattern is a remarkably
consistent process
 Two hypothetical models—the field and clone
models —have been proposed to explain how these
different shapes are determined, and evidence exists
to support both

46.  The field model proposes that the factors responsible for
tooth shape reside within the ectomesenchyme in distinct
graded and overlapping fields for each tooth family
 The fact that each of the fields expresses differing
combinations of patterning homeobox genes supports
this theory.
 The The homeobox code (field) model for dental
patterning is based on observations of the spatially
restricted expression of several homeobox genes in the
jaw primordial ectomesenchyme cells before E11.

47.  The early expression of Msx-1 and Msx-2 homeobox
genes before the initiation of tooth germs is
restricted to distal, midline ectomesenchyme in
regions where incisors (and canines in human
beings), but not multicuspid teeth, will develop,
whereas Dlx-1 and Dlx-2 are expressed in
ectomesenchyme cells where multicuspid teeth, but
not incisors (or canines), will develop.

48.  Expression of Barx-1 overlaps with Dlx-1 and Dlx-
2 and corresponds closely to ectomesenchymal cells
that will develop into molars
 The homeobox code model thus proposes that the
overlapping domains of the previously mentioned
genes provide the positional information for tooth
type morphogenesis

49.  Support for this model comes from the dental
phenotype of Dlx-1−/− and Dlx2−/− double-knockout
mice in which development of maxillary molar teeth
is arrested at the epithelial thickening stage.
 As predicted by the code model, incisor development
is normal in these mice; normal development of
mandibular molars (not predicted by the code)
results from functional redundancy with other Dlx
genes, such as Dlx-5 and Dlx-6, which are
expressed in ectomesenchyme in the mandibular
primordium.

50.  Further functional support for the code model comes
from misexpression of Barx-1 in distal
ectomesenchyme cells, which results in incisor tooth
germs developing as molars.
 Barx-1 expression is localized to proximal
ectomesenchyme (molar) by a combination of
positive and negative signals from the oral ectoderm

51.  FGF-8 localized in proximal ectoderm induces Barx-
1 expression, whereas BMP-4 in the distal ectoderm
represses Barx-1 expression.
 Expression of Barx-1 experimentally induced in distal
(presumptive incisor) ectomesenchyme by inhibition
of BMP signaling has the effect of repressing Msx
gene expression, which is induced in distal
ectomesenchyme by BMP-4.

52.  The transformation of incisors into molars thus may
require a combination of loss of “incisor” genes (Msx)
and gain of “molar” genes (Barx-1).
 It has also been reported that the transcriptional
regulator Isl1, a LIM homeodomain–containing
protein, plays a role in tooth formation and
patterning.

53.  On the other hand, the clone model proposes that
each tooth class is derived from a clone of
ectomesenchymal cells programmed by epithelium
to produce teeth of a given pattern .

54. Instructive Signals for Patterning
 Recombinations of incisor and molar epithelium with
mesenchyme from young mouse embryos (~ E10)
showed that when molar epithelium was recombined
with incisor mesenchyme, a molar tooth formed, and
when incisor epithelium was recombined with molar
mesenchyme, an incisor formed.

55.  This led to the conclusion that the epithelium was
responsible for determining the type and shape of a
tooth.
 Other recombinations with older embryos (~ E14),
however, produced different results, in which molar
epithelium recombined with incisor mesenchyme
resulted in incisor teeth and incisor epithelium
recombined with molar mesenchyme resulted in
molar teeth

56. Regionalization of Oral and Dental
Ectoderm
 This shows that the Wnt-7B gene represses Shh
expression in oral ectoderm and thus the boundaries
between oral and dental ectoderm are maintained by
an interaction between Wnt and Shh signaling
similar to ectodermal boundary maintenance in
segmentation in insects.

57. Bud Stage
 The bud stage is represented by the first epithelial
incursion into the ectomesenchyme of the jaw
 The epithelial cells show little if any change in shape
or function.
 The supporting ectomesenchymal cells are packed
closely beneath and around the epithelial bud.
 As the epithelial bud continues to proliferate into the
ectomesenchyme, cellular density increases
immediately adjacent to the epithelial outgrowth. This
process is classically referred to as a condensation
of the ectomesenchyme.

59. Bud-to-Cap Transition
 Molecularly, Msx-1 is expressed with Bmp-4 in the
mesenchymal cells that condense around tooth buds.
 Msx-1−/− embryos have tooth development arrested at
the bud stage, and Bmp-4 expression is lost from the
mesenchyme, suggesting that Msx-1 is required for Bmp-
4 expression.
 Bmp-4 is able to maintain Msx-1 expression in wild-type
tooth bud mesenchyme, indicating that Bmp-4 induces
its own expression via Msx-1.
 Tooth development can be rescued in Msx-1−/− embryos
by addition of exogenous BMP-4.

61.  Bmp-4 expressed in the bud mesenchyme is
required to maintain Bmp- 2 and Shh expression in
the epithelium.
 Loss of Bmp-4 expression in Msx-1 mutants is
accompanied by loss of Shh expression at E12.5,
which can be restored by exogenous BMP-4.
 Blocking SHH function with neutralizing antibodies
also results in loss of Bmp-2 expression, suggesting
Shh and Bmp-2 may be in the same pathway and
that down- regulation of Bmp-2 in Msx-1 mutants
may be downstream of the loss of SHH.

62.  Blocking SHH signaling using neutralizing
antibodies or forskolin shows that at E11 to E12,
SHH is required for dental epithelium proliferation to
form tooth buds, whereas blocking at E13 affects
tooth bud morphology, but these buds still can form
teeth.

63.  Another homeobox gene with a role in the bud-to-
cap transition is Pax- 9.
 Pax-9 is expressed in bud stage mesenchyme and
earlier in domains similar to Activin-βA and Msx-1 in
patches of mesenchyme that mark the sites of tooth
formation.
 Pax-9−/− mutant embryos have all teeth arrested at
the bud stage. Despite being coexpressed, early
Activin-βA expression

64.  Is not affected in Pax-9−/− embryos, and Pax-9
expression is not affected in Activin-βA−/− embryos.
 These two genes are essential for tooth
development to progress beyond the bud stage and
thus appear to function independently; however,
changes occur in expression of other genes, such as
Bmp-4, Msx-1, and Lef-1 in Pax-9−/− tooth bud
mesenchyme

65. Signaling centres
 There are three sets of transient signaling centers in
the dental epithelium that produce more than a
dozen different signaling molecules belonging to the
BMP, FGF, Shh, and Wnt families.
 First come the initiation knots, which appear in the
dental placode and initiate budding of the tooth
epithelium.

66.  Then appear the primary and secondary enamel
knots that initiate the bud-to-cap stage transition
and tooth crown formation.
 Precursor cells of these knots are first detected at
the tip of the tooth buds by expression of the p21
gene, followed shortly after by Shh.
 The primary enamel knots become visible
histologically as clusters of nondividing epithelial
cells in sections of molar cap-stage tooth germs .

68.  These clusters express genes for several signaling
molecules, including Bmp-2, Bmp-4, Bmp-7, Fgf-4, Fgf-9,
Wnt-10b, Slit-1, and Shh .
 Three-dimensional reconstructions of the expression of
these genes have revealed highly dynamic spatial and
temporal nested patterns in the enamel knot.
 On the whole, receptors for the enamel knot signals are
localized in the epithelial cells surrounding the enamel
knot. Each cap- stage molar tooth germ has a single
primary enamel knot that induces formation of secondary
enamel knots at the tips of the future cusps and thereby
regulate crown patterning.

69.  Fgf-4 and Slit-1 may be the best molecular markers
for enamel knot formation, because they have been
observed in both primary and secondary knots.

70.  In summary, the enamel knot represents an
organizational center that orchestrates cuspal
morphogenesis.
 The enamel knot shares many similarities with the
apical ectodermal ridge of developing limbs: both
consist of nondividing cells; both express Fgfs,
Bmps, and Msx-2; and both act as signaling
centers

71. Applied aspects
 Among the first genes in which mutations were
shown to cause tooth agenesis in mice and humans
were MSX1 and PAX9.
 These genes encode transcription factors, which
have essential functions in the mediation of BMP,
Wnt, and FGF signaling in early dental
mesenchyme.
 Tooth development is arrested at the bud stage in
Msx1 and Pax9 knockout mice, and in humans
heterozygous loss of function mutations in MSX1
and PAX9 genes cause oligodontia (defined as more
than six missing teeth excluding wisdom teeth).

72.  In addition to MSX1 and PAX9, the genes that have
been associated with nonsyndromic human
hypodontia (i.e., no defects in other organs) include
WNT10A, AXIN2, LRP6, GREM2, SPRY2, SPRY4,
and EDA.
 Notably all these genes encode signals or inhibitors
of signaling

73.  Human tooth agenesis is commonly associated with
congenital defects in other organs, most often with
ectodermal organs developing from the outer surface of
the embryo.
 Conditions that affect two or more ectodermal organs are
called ectodermal dysplasia.
 The most common of these is X-linked hypohidrotic
ectodermal dysplasia (HED), caused by mutations in the
EDA gene and characterized by oligodontia,hair loss, dry
mouth, and inability to sweat. Identical phenotypes result
from mutations in other components of the Eda signal
pathway, including the receptor EDAR and signal
mediator EDARADD

74.  The stimulation of Eda expression in transgenic mice
induced the formation of extra teeth as well as
mammary glands and stimulated the growth of hair,
nails, and salivary glands. The EDA pathway is
unique because it seems to be necessary, almost
exclusively, for the formation of teeth and other
ectodermal organs, unlike the other conserved signal
pathways, which have more widespread functions.

75.  Interestingly, WNT10A has come up as the most
common gene associated with human tooth
agenesis, and mutations in WNT10A have been
shown to account for more than half of the
nonsyndromic hypodontia cases.
 Based on mouse experiments, the Wnt pathway
appears to be the most upstream signal pathway
and the inducer of tooth initiation. The inhibition of
Wnt signaling by overexpressing the Wnt inhibitor
Dkk1 in transgenic mice prevents the formation of
tooth placodes, and the initiation of teeth fails.

76.  Conversely, when the Wnt pathway was
overactivated in the oral epithelium of transgenic
mouse embryos (β-catex3K14/+), dozens of teeth
were generated in succession.
 This may indicate that the capacity for continuous
tooth formation, which was lost in the mouse (and
humans) during evolution, could be unlocked by
increased Wnt signal activity in the oral
epithelium.

77.  There is already one potential treatment for the
prevention and cure of X-linked HED, the ectodermal
dysplasia syndrome caused by mutations in the EDA
gene
 Clinical trials are currently ongoing to test whether
neonatal injections of EDA protein can prevent
hypodontia and other congenital defects of human X-
linked HED.

78. THANK YOU!