RESEARCH ARTICLE 3189
Development 139, 3189-3199 (2012) doi:10.1242/dev.079558
© 2012. Published by The Company of Biologists Ltd
Ectodysplasin regulates activator-inhibitor balance in murine
tooth development through Fgf20 signaling
Otso Häärä1, Enni Harjunmaa1, Päivi H. Lindfors1, Sung-Ho Huh2, Ingrid Fliniaux1, Thomas Åberg1,
Jukka Jernvall1, David M. Ornitz2, Marja L. Mikkola1 and Irma Thesleff1,*
SUMMARY
Uncovering the origin and nature of phenotypic variation within species is the first step in understanding variation between species.
Mouse models with altered activities of crucial signal pathways have highlighted many important genes and signal networks
regulating the morphogenesis of complex structures, such as teeth. The detailed analyses of these models have indicated that the
balanced actions of a few pathways regulating cell behavior modulate the shape and number of teeth. Currently, however, most
mouse models studied have had gross alteration of morphology, whereas analyses of more subtle modification of morphology are
required to link developmental studies to evolutionary change. Here, we have analyzed a signaling network involving ectodysplasin
(Eda) and fibroblast growth factor 20 (Fgf20) that subtly affects tooth morphogenesis. We found that Fgf20 is a major downstream
effector of Eda and affects Eda-regulated characteristics of tooth morphogenesis, including the number, size and shape of teeth.
Fgf20 function is compensated for by other Fgfs, in particular Fgf9 and Fgf4, and is part of an Fgf signaling loop between epithelium
and mesenchyme. We showed that removal of Fgf20 in an Eda gain-of-function mouse model results in an Eda loss-of-function
phenotype in terms of reduced tooth complexity and third molar appearance. However, the extra anterior molar, a structure lost
during rodent evolution 50 million years ago, was stabilized in these mice.
INTRODUCTION
The regulation of organ form and complexity is a key question in
biology. The development of complex structures requires specific
sets of genes to be activated and/or inhibited in a correct temporal
and spatial pattern. Gene expression in all developing organs is
regulated by conserved signals belonging to well characterized
signaling molecule families such as Wnt, Fgf, Hedgehog (Hh),
Bmp and Tgf. These signaling pathways are also of central
importance for the morphogenesis of ectodermal appendages, such
as hair, teeth and exocrine glands. In addition, the ectodysplasin
(Eda) pathway, composed of the tumor necrosis factor-like ligand
Eda-A1 (hereafter Eda) and its receptor Edar, is crucial for
ectodermal organogenesis (Mikkola, 2009). Mutations in EDA and
other components of the EDA pathway cause human ectodermal
dysplasia syndromes characterized by impaired formation of
ectodermal organs (Mikkola, 2009). The phenotypes include
missing and abnormally shaped teeth, sparse hair, and impaired
function of various exocrine glands. As shown in hair and teeth,
Eda has a key function in the formation of ectodermal placodes,
which function as signaling centers during the initiation of
organogenesis (Mustonen et al., 2004). Recently, a similar role for
Eda was shown in the formation of fins and scales in zebrafish
(Harris et al., 2008), identifying a conserved function for Eda
signaling throughout vertebrate evolution.
Mammalian teeth are ecologically important in food processing,
and the type of diet that a species has adapted to eat is reflected in
their tooth morphology. Mammalian molars show a substantial
1
Developmental Biology Program, Institute of Biotechnology, University of Helsinki,
POB 56, 00014 Helsinki, Finland. 2Department of Developmental Biology,
Washington University School of Medicine, St Louis, MO 63110, USA.
*Author for correspondence ([email protected])
Accepted 10 June 2012
degree of phenotypic variation, which has been a long-standing
interest of evolutionary biologists (Osborn, 1888; Bateson, 1894).
Recently, genes and networks underlying dental variation have been
intensively studied (Salazar-Ciudad and Jernvall, 2010). In particular,
transgenic mouse models in which mutations in signaling pathways
have caused phenotypic alterations in dental characteristics have
provided insights into mechanisms possibly involved in evolutionary
changes of dentition (Järvinen et al., 2006; Kangas et al., 2004;
Kassai et al., 2005; Klein et al., 2006; Line, 2003).
Tooth shape characteristics are determined during embryonic
development. The mouse incisors and molars are initiated in
embryonic day (E) 11-12 embryos from ectodermal placodes that
invaginate into the mesenchyme, forming epithelial buds. The bud
grows and folds into a cap-shaped enamel organ surrounding the
mesenchymal dental papilla. A central structure in tooth
development, the primary enamel knot (pEK), is formed during the
bud-to-cap transition in the epithelium. The pEK is a signaling center
composed of non-proliferating cells (Jernvall et al., 1994) expressing
a set of secreted signaling molecules and their antagonists including
Shh, several Wnts, Bmps, Fgfs and follistatin (reviewed by Tummers
and Thesleff, 2009). In multicuspid teeth, the pEK induces the
formation of secondary enamel knots, which begin to form in mouse
molars at E15. The balance between activators and inhibitors has
been proposed to determine the spatial arrangement of the secondary
enamel knots (Salazar-Ciudad and Jernvall, 2002; Salazar-Ciudad
and Jernvall, 2010). The molecules secreted by the secondary enamel
knots regulate epithelial proliferation and differentiation to specify
the position and shape of the cusps and, thus, the shape of the tooth
crown. Experimentally manipulating the level of gene expression in
the enamel knots results in variation of molar morphologies. For
example, modulation of Shh, follistatin and Wnt signaling have been
shown to result in altered molar shapes and cusp patterns (Dassule
et al., 2000; Harjunmaa et al., 2012; Järvinen et al., 2006; Liu et al.,
2008; Wang et al., 2004).
DEVELOPMENT
KEY WORDS: Ectodysplasin, Edar, Fgf20, Tooth development, Evolution, Mouse
3190 RESEARCH ARTICLE
MATERIALS AND METHODS
Animals
K14-Eda mice (Mustonen et al., 2003) were maintained in C57Bl/6
background, and Eda–/– (Tabby) mice in B6CBA background (Pispa et al.,
1999). The generation of Fgf20Gal/Gal mice has been described previously
(Huh et al., 2012) and they were maintained on a mixed C57Bl/6J-129Sv/J
background. The appearance of the vaginal plug was taken as E0. The
embryos were staged by limb morphology.
Organ cultures
Embryonic teeth were cultivated in a Trowell-type culture as described
previously (Laurikkala et al., 2001; Närhi and Thesleff, 2010). The
following proteins (all from R&D Systems) were used: Fgf20, Fgf4, Fgf9
and bovine serum albumin (BSA). Beads were incubated in a protein
concentration of 100 ng/l for 1 hour at 37°C prior to use. The samples
were imaged under Olympus SZX9 or Olympus AX70 microscope. EdU
proliferation assay (Invitrogen) was performed according to the
manufacturer’s protocol using 1:1000 dilution and 1 hour EdU
incorporation at 37°C.
Promoter analysis and luciferase assay
A 10 kb sequence upstream of the translation initiation site of Fgf20, as
well as a 5 kb sequence spanning the first intron, were aligned with
corresponding human and rat Fgf20 sequences and analyzed for the
presence of potential NF-B responsive elements. CONSITE program
(http://asp.ii.uib.no:8090/cgi-bin/CONSITE/consite)
with
80%
conservation cut-off, window size 50 and score threshold 80% was used
for analysis. To test whether the regions containing the NF-B responsive
elements were responsive to Edar, three different luciferase reporter vector
constructs were generated (supplementary material Fig. S1). The luciferase
reporter assay was carried out as previously described (Fliniaux et al.,
2008), using pNF-B luc as a positive control (Koppinen et al., 2001).
Primer sequences are listed in supplementary material Table S1.
Histology, in situ hybridization and X-gal staining
The tissues were fixed in 4% paraformaldehyde (PFA) and taken through
an ethanol series (50%, 70%, absolute) and xylene into paraffin and
sectioned at 7 m. For whole-mount in situ hybridization (ISH), embryonic
tissues were fixed in 4% PFA for 24 hours at 4°C and dehydrated through
a methanol series (25, 50, 75, 100%). Whole-mount ISH was performed
with InSituPro robot (Intavis AG, Germany) and ISH on histological
sections as previously described (Fliniaux et al., 2008), using specific
probes for Runx2, Fgf3 and Fgf20 (D’Souza et al., 1999; Kettunen et al.,
2000). Digoxigenin-labeled probes were detected with BM Purple AP
Substrate Precipitating Solution (Boehringer Mannheim, Germany).
Radioactive ISH on paraffin sections was carried out according to standard
protocols using 35S-UTP labeling (Amersham) and probes specific to
Fgf20, Spry2, Spry4, Wnt10b, ectodin (Sostdc1 – Mouse Genome
Informatics), Shh and Edar (Åberg et al., 2004; Bitgood and McMahon,
1995; Laurikkala et al., 2001; Laurikkala et al., 2002; Sarkar and Sharpe,
1999; Zhang et al., 2001). X-gal staining was performed as described
previously (Häärä et al., 2011).
Quantitative RT-PCR
Lower molars were dissected from E13 and E14 Eda–/– mice. Four to six
molars from one side of the mandible were pooled and incubated for 4
hours in medium supplemented with 250 ng/ml recombinant Fc-Eda-A1
protein (Gaide and Schneider, 2003), whereas molars from the other side
of the mandible were maintained in control medium. For RNA isolation,
molars were harvested in 350 l of RNeasy (Qiagen) lysis buffer as
specified by the manufacturer. DNase-treated RNA (300 ng) was reverse
transcribed with Superscript III (Invitrogen), and quantitative PCR was
performed in a LightCycler480 (Roche). Expression of each gene was
normalized against Ranbp1. Primer sequences are listed in supplementary
material Table S1.
Morphometric analysis
For adult tooth shape analysis, the jaws were boiled in tap water, cleaned
and imaged (Olympus SZX9 microscope). Morphometric parameters were
measured with ImageJ. High-resolution three-dimensional laser-confocal
imaging has been described previously (Evans et al., 2007).
RESULTS
Fgf20 expression is confined to localized domains
in dental epithelium throughout tooth
morphogenesis and correlates with Edar
expression
In situ hybridization in E10-16 mouse embryos was used to map
Fgf20 expression during molar development (Fig. 1). At the onset
of tooth morphogenesis (E10-11) Fgf20 was expressed in the
thickened oral epithelium at the site of tooth initiation (Fig. 1A,B).
At the onset of epithelial budding, expression was restricted to the
signaling center in the tooth placode epithelium (E12) and
subsequently to the tip of the bud in the forming enamel knot (E13)
(Fig. 1C,D). Fgf20 expression was strongly localized in the fully
formed pEK at E14 (Fig. 1E), and subsequently in the secondary
enamel knots at E16 and E18 (Fig. 1F; data not shown).
Interestingly, this pattern of expression correlated with the
expression of Edar during molar development (Tucker et al., 2000;
Laurikkala et al., 2001).
DEVELOPMENT
Edar is expressed in the incisor and molar placodes as well as in
the primary and secondary enamel knots and is activated by
epithelially derived Eda (Laurikkala et al., 2001). Teeth are affected
in spontaneous Eda pathway loss-of-function mouse mutants
(Grüneberg, 1966; Pispa et al., 1999). Wild-type (WT) mice have,
in each half of the jaw, one incisor, a toothless diastema region and
three molars (m1-m3). Eda–/– mice have smaller teeth and reduced
cusp numbers, particularly in the first molar. Tooth number can also
vary and 17-55% of Eda–/– mice lack the third molar (m3) (Charles
et al., 2009; Grüneberg, 1966; Pispa et al., 1999; Kangas et al.,
2004) (our unpublished results). Inactivation of transcription factor
NF-B results in a phenotype that is very similar to the Eda
pathway loss-of-function mutants and a multitude of evidence
indicates that Eda signaling is mediated through NF-B (Häärä et
al., 2011; Mikkola, 2009; Schmidt-Ullrich et al., 2001).
Overexpression of Eda, under control of the keratin 14 promoter
(K14-Eda), leads to a subtle increase in molar complexity and the
formation of an extra molar anterior to the molars, in the location
of an ancestral premolar (Kangas et al., 2004; Mustonen et al.,
2003). Tooth characteristics of both Eda–/– and K14-Eda mice are
polymorphic in nature. By genetically manipulating Eda activity,
several tooth characteristics, including size, shape and number, can
be simultaneously affected.
To study the developmental functions of the Eda pathway in
ectodermal organogenesis, we searched for Eda-responsive genes by
microarray analysis (Fliniaux et al., 2008; Lefebvre et al., 2012). One
of the putative target genes identified was Fgf20, a member of the
fibroblast growth factor (Fgf) family. In this report, we have
validated the regulation of Fgf20 by Eda and show that molars in
Fgf20-null (Fgf20Gal/Gal) mice resemble Eda–/– molars. We provide
evidence indicating that Fgf4 and Fgf9, which are also expressed in
the pEK, function redundantly with Fgf20. Analysis of tooth
morphogenesis in K14-Eda;Fgf20Gal/Gal mutants indicated that
Fgf20 has an essential function in the Eda-mediated activatorinhibitor balance that regulates tooth size, number and cusp
patterning. These studies define a gene interaction network that
regulates dental variation and suggest that changes in the expression
level of Eda target genes, such as Fgf20, might be a mechanism for
the evolutionary adaptation of tooth number and shape.
Development 139 (17)
Fgf20 in tooth development
RESEARCH ARTICLE 3191
mice, the forming extra molar expressed Fgf20-Gal
(supplementary material Fig. S1). From these results, we conclude
that Fgf20 lies downstream of Eda signaling in tooth development.
To examine further the function of Eda/Edar/NF-B signaling in
activating Fgf20 transcription, we searched for putative NF-B
binding sites in Fgf20 promoter and intronic regions. Although
several potential NF-B binding sites were identified, in promoterreporter assays neither a 5.2 kb promoter region nor intron 1
sequences encompassing putative NF-B sites were responsive to
Edar (supplementary material Fig. S2), suggesting either that an
additional factor is needed to activate Fgf20 transcription, or that
Fgf20 is regulated through a distant enhancer sequence yet to be
identified.
Fgf20 expression is regulated by the Eda pathway
Microarray analysis revealed that Fgf20 is one of the most rapidly
induced genes by Eda in hair placodes and, thus, is a putative
transcriptional target of Eda (Fliniaux et al., 2008; Lefebvre et al.,
2012). To test whether Eda regulates Fgf20 in developing teeth, we
treated E13 Eda–/– molar buds with recombinant Eda protein (250
ng/ml) for 4 hours. This led to a ~4.5-fold increase in the Fgf20
mRNA levels compared with controls (Fig. 2A; n14; Wilcoxon
signed rank test, P<0.05). To validate this observation in vivo, we
compared the intensity of Fgf20 expression by whole-mount in situ
hybridization in the jaws of Eda–/–, WT and K14-Eda mice at E12,
E13 and E14. Fgf20 expression was significantly decreased in the
Eda–/– molars whereas the K14-Eda mice showed increased Fgf20
expression compared with WT mice (Fig. 2B-J). We also
confirmed the correlation of Fgf20 expression level with Eda
activity by analyzing Fgf20 expression using an Fgf20-galactosidase (Gal, encoded by lacZ) knock-in allele and
quantifying Gal staining in compound mutants of
Eda–/–;Fgf20+/Gal, Fgf20+/Gal and K14-Eda;Fgf20+/Gal mice at
E14.5, E15.5 and E16.5 (supplementary material Fig. S1).
Significant differences in the size of Gal staining domains were
observed between the mutants at all stages. In addition, in K14-Eda
Anteroconids are absent or smaller in developing
Fgf20Gal/Gal molars resembling Eda–/– molar
development
To study the developmental basis of tooth abnormalities in
Fgf20Gal/Gal mice, we analyzed embryonic molar development in
ex vivo cultures. Lower molars were dissected at E13 and cultured
Fig. 2. Fgf20 expression is regulated by
Eda. (A)Treatment of E13 Eda–/– mouse
molars with Eda protein upregulates Fgf20
transcription after 4 hours of culture (mean
± s.d.). *P<0.05. (B-J)Expression of Fgf20 at
E12 (B-D), E13 (E-G) and E14 (H-J) in
developing teeth of Eda–/–, WT and K14-Eda
mice. i, incisor; m, molar. Asterisk indicates
extra molar.
DEVELOPMENT
Fig. 1. Fgf20 expression in mouse molar development. (A-D)Fgf20
is expressed in the dental epithelium: at initiation (A,B), placode (C) and
bud (D). (E,F)At the cap stage, expression is concentrated in the
primary enamel knot (E) and at the bell stage in the secondary enamel
knots (F).
Fgf20Gal/Gal mice have smaller molars with mildly
altered anteroconid cusp morphology
To elucidate the role of Fgf20 in tooth development, we examined
the tooth phenotype in the lower jaw of Fgf20-null (Fgf20Gal/Gal)
mice. No differences were found between the teeth of heterozygote
Fgf20+/Gal and those of WT mice. The number of teeth was
normal in Fgf20Gal/Gal mice but the molars were smaller than in
WT littermates (Fig. 3A-B⬘, Eda–/– for comparison in 3C). Both
mesio-distal length (Fig. 3D) and bucco-lingual width (Fig. 3E)
were significantly reduced, and thus the crown area was
significantly reduced in all Fgf20Gal/Gal molars (n14 tooth rows)
compared with WT littermate controls (n15) (Fig. 3F; Student’s
t-test, P<0.001). The jaws of the Fgf20Gal/Gal and WT littermates
were the same size (Fig. 3G) with no apparent morphological
differences. The incisors of Fgf20Gal/Gal mice appeared similar in
size and shape as in littermate controls with no apparent enamel
defects (supplementary material Fig. S3).
We next analyzed the cusp patterns and morphology of molars (Fig.
3H, see 3A for corresponding cusp pairs). Although the overall cusp
pattern of the Fgf20 mutants appeared normal, the anterior cusps were
mildly affected: the mean distance (normalized to bucco-lingual width
of corresponding molar) between the first pair of cusps was reduced
(cusp pair 1 in Fig. 3H). The relative size of the molars (m2/m1 and
m3/m1) was similar in Fgf20Gal/Gal and WT mice (data not shown).
These results suggest that Fgf20 is involved in the regulation of tooth
size and can fine-tune the patterning of the anterior cusps.
3192 RESEARCH ARTICLE
Development 139 (17)
for seven days. The molars of Fgf20 heterozygotes (n18; not
shown) and WT littermates (n12) appeared to be similar before
and during culture (Fig. 4). Fgf20Gal/Gal molars were small
compared with WT (P<0.01), with the most anterior cusp, the
anteroconid, reduced in relative size (5/13) or lacking completely
(6/13). In addition, the posterior part of the molar, the talonid, was
represented by a single cusp instead of the normal two or three
(5/13). This reduction in size and cusp number is similar to what is
seen in cultured Eda-null molars (Harjunmaa et al., 2012), though
dissimilar in that the effect was not quite as severe, and that the
anteroconid was reduced more often than the talonid. In vivo
histological analysis supported the findings from organ culture
experiments. At E14, Fgf20Gal/Gal molars formed a cap, but this
was dome-shaped, thus resembling Eda–/– molars (Pispa et al.,
1999). At E18, Fgf20Gal/Gal molars appeared histologically
normal, but the shape of the anteroconid cusp varied more in
Fgf20Gal/Gal compared with control molars (supplementary
material Fig. S4). Additionally, the overall size of the molars was
smaller in Fgf20Gal/Gal mutants. At E14, the expression of wellcharacterized genes in tooth development, Edar and Wnt10b in the
pEK, and ectodin in mesenchyme and epithelium excluding the
pEK, were found to be patterned similarly in Fgf20Gal/Gal and WT
embryos (supplementary material Fig. S5). The embryonic analysis
is consistent with the adult Fgf20Gal/Gal tooth phenotype (Fig. 3)
and suggests a role for Fgf20 in the regulation of tooth size and
anteroconid cusp patterning.
Fgf4 and Fgf9 might have redundant functions
with Fgf20 during molar development
As the changes in the Fgf20-null tooth phenotype were relatively
subtle, we examined the potential redundancy between Fgf20
and other Fgf ligands. Fgf4 and Fgf9 are co-expressed with
Fgf20 in the pEK (Kettunen and Thesleff, 1998). We first studied
the effects of recombinant Fgf20, Fgf4 and Fgf9 proteins on
dental mesenchyme isolated from E13 WT molars. All three Fgfs
induced Fgf3 expression, whereas the control BSA beads did not
(Fig. 5A-D). Fgf4 and Fgf9 have previously been shown to
induce Runx2 expression, which is required for the induction of
Fgf3 in dental mesenchyme (Åberg et al., 2004). Also Fgf20
induced the expression of Runx2 (Fig. 5E,F). We next analyzed
the capacity of Fgf20 to promote proliferation of dental
mesenchymal cells, as previously reported for Fgf4 and Fgf9
(Kettunen et al., 1998). When isolated E13 mesenchyme was
cultured with Fgf20 beads, proliferation was induced in the cells
underlying and surrounding the bead (Fig. 5I,J). BSA beads had
no effect (Fig. 5G,H).
To analyze further the possible compensatory actions of Fgf9
and Fgf20 in vivo, we crossed Fgf9 and Fgf20 mutant mice to
create Fgf9–/–;Fgf20Gal/Gal compound mutants. Because Fgf9–/–
mice die at birth, we studied the embryonic development. We
analyzed the phenotype of E14.5 cap stage molars from serial
histological sections and the expression of Shh as a marker of the
pEK. The molars appeared morphologically normal but the enamel
knot length, measured on serial sections, indicated that the
Fgf9–/–;Fgf20Gal/Gal enamel knot was shorter compared with
controls (Fig. 5K-N), including Fgf9–/–;Fgf20+/Gal (P<0.01) and
Fgf20Gal/Gal genotypes (Fig. 5O; P<0.05; Mann-Whitney U-test
with Bonferroni correction). Hence, removal of both Fgf9 and
Fgf20 had a significant additive effect on enamel knot length.
Conditional deletion of Fgf4 function in dental epithelium with the
Nestin-Cre line did not affect either the shapes or sizes of adult
molars or the histological appearance of cap stage embryonic
molars (T.Å., X. Sun, G. Martin and I.T., unpublished). The
deletion of the floxed exon in cap stage molars was confirmed by
in situ hybridization.
As Fgf4 and Fgf9 are co-expressed in the enamel knot with
Fgf20 and Edar, we next analyzed by qRT-PCR whether they are
induced by Eda. However, in contrast to Fgf20 (Fig. 2A), Fgf4
(data not shown) and Fgf9 (Fig. 5P) expression were unaltered in
E13 molars after 4 hours Eda treatment. As Fgf4 expression level
was very low at E13, we analyzed Fgf4 expression also at E14 and
found no effect of Eda (Fig. 5P).
DEVELOPMENT
Fig. 3. Adult Fgf20Gal/Gal molars are small and show defects in anteroconid cusp patterning. (A-C)Molars in Fgf20Gal/Gal mice are smaller
compared with WT, resembling Eda–/– mice. The cusp pattern is affected in the anterior part of the first molar (dashed line). (D-G)All molars (m1,
m2, m3) are shorter (D) and narrower (E), and thus smaller in two-dimensional size (F) compared with WT teeth. The width of the whole jaw is
unaffected (G). (H)The distance (normalized to molar diameter) between the cusps in the first cusp pair (1) is shorter, whereas the distances in
other cusp pairs (2-7) show no differences compared with the WT molars (cusp pairs indicated in A). Mean ± s.d. *P<0.05, **P<0.01, ***P<0.001.
Fgf20 in tooth development
RESEARCH ARTICLE 3193
Inactivation of Fgf20 in K14-Eda mice leads to
reduced tooth size, altered tooth number and
aberrant cusp morphology
K14-Eda mice have a more complex cusp pattern in all molars
compared with WT mice, and, in addition, ~50% of individuals (in
the FVB background) have an extra molar anterior to the first
molar in the position of an ancestral premolar (Kangas et al., 2004;
Mustonen et al., 2003). To assess the role of Fgf20 in the
phenotype of K14-Eda mice, we generated compound K14Eda;Fgf20Gal/Gal mutants. Measurements of molar dimensions
indicated that m1, m2 and m3 were smaller in K14Eda;Fgf20Gal/Gal mice (n16) compared with K14-Eda littermates
(n16; one-way ANOVA with Bonferroni post hoc, P<0.001; Fig.
6). In K14-Eda;Fgf20+/Gal mice (n34) only m2 was statistically
significantly smaller compared with K14-Eda (P<0.01), but all
three molars were significantly larger compared with K14Eda;Fgf20Gal/Gal mice (P<0.001).
K14-Eda;Fgf20Gal/Gal mice showed a trend of decreasing
molar size towards the posterior molars compared with K14-Eda,
and thus had reduced m2/m1 and m3/m1 ratios (Fig. 6H,I; P<0.05).
K14-Eda;Fgf20+/Gal molars showed a similar, although milder,
trend. In addition to reduced absolute and relative size, a
characteristic feature of K14-Eda;Fgf20Gal/Gal molars was
reduced molar complexity with the absence of the extra cusps
typical of K14-Eda and with the buccal cusps becoming fused in
m1 and m2 (Fig. 6; supplementary material Fig. S6). The aberrant
development of cusps was visible prior to tooth eruption (E18),
excluding enamel defects as the cause of the reduced crown
complexity observed in adult K14-Eda;Fgf20Gal/Gal mice
(supplementary material Fig. S6). The cusp pattern of K14Eda;Fgf20+/Gal was more similar to K14-Eda than to K14Eda;Fgf20Gal/Gal. However, some of the extra cusps present in
K14-Eda were absent in K14-Eda;Fgf20+/Gal and the overall shape
of m1 and m2 resembled more WT than K14-Eda (supplementary
material Fig. S6). This suggests that partial loss of Fgf20 signaling
in K14-Eda might rescue the cusp pattern close to WT phenotype,
but the complete loss of Fgf20 signaling leads to highly reduced
cusp number and flattened shape of the existing cusps, resembling
Eda–/– molars.
The frequency of the anterior extra molar (EM) was increased
after removal or reduction of Fgf20 in the K14-Eda background:
50% of K14-Eda, but 76% of K14-Eda;Fgf20+/Gal and 88% of
K14-Eda;Fgf20Gal/Gal tooth rows had the EM (Fisher’s exact test,
P<0.05; Fig. 6I). Moreover, 31% of K14-Eda;Fgf20Gal/Gal mice
lacked m3 (P<0.05).
Altogether, K14-Eda;Fgf20Gal/Gal molars showed alterations in
number, size, shape and molar proportions compared with the
molars of K14-Eda mice. Thus, the loss of Fgf20 in the K14-Eda
background had a more severe and variable effect than its loss in
otherwise WT mice. The phenotype of K14-Eda;Fgf20+/Gal was
intermediate between K14-Eda and K14-Eda;Fgf20Gal/Gal for all
tooth characteristics measured.
DEVELOPMENT
Fig. 4. Fgf20Gal/Gal molars are reduced
in size and cusp number during
development. (A,B)In tissue culture
conditions, WT mouse molars (A) typically
form five cusps, lacking the most posterior
cusp, the hypoconulid. Eda–/– molars (B)
typically form only three cusps, lacking the
most anterior cusp, the anteroconid (white
arrow). The two most posterior cusps, the
talonid cusps (black arrows), are represented
by only one cusp. (C-E)Fgf20Gal/Gal molars
show a variety of phenotypes ranging from
WT-like five cusps (C) to Eda-null-like four
cusps (D). Most commonly, the anteroconid
is abnormally small (E) or missing completely.
(F)Measurement of tooth area at E13+7
days. Mean ± s.d. **P<0.01. NS, nonsignificant.
3194 RESEARCH ARTICLE
Development 139 (17)
Fig. 5. Fgf20 has similar effects on
embryonic dental mesenchyme as Fgf4
and Fgf9 and acts redundantly with Fgf9.
(A-D)Beads supplemented with Fgf4, Fgf9
and Fgf20, but not BSA, induce the
expression of Fgf3 in WT molar mesenchyme
after 24 hours. (E,F)Fgf20 induces Runx2
expression around the bead. Asterisk
indicates endogenous expression of Runx2 in
an island of osteogenic mesenchyme.
(G-J)Fgf20 protein stimulates cell
proliferation in isolated dental mesenchyme
(EdU-positive cells; red) after 24 hours of
culture, whereas BSA has no effect. The
position of the protein-releasing bead is
indicated (dashed line).
(K-N)Fgf9–/–;Fgf20Gal/Gal molars have
developed into cap stage at E14.5, similar to
WT control and Fgf9 and Fgf20 single
mutants, and express Shh in the enamel knot
(red). (O)However, the enamel knot is
significantly shorter in Fgf9–/–;Fgf20Gal/Gal
compared with single mutants and controls.
(P)Expression of Fgf4 and Fgf9 is not induced
after 4 hours Eda treatment in E13 (Fgf9) or
E14 (Fgf4) Eda–/– molars. Mean ± s.d.
*P<0.05, **P<0.01. NS, non-significant.
Kangas et al. (Kangas et al., 2004) who detected Shh positive foci
anterior to m1 Shh expression in all K14-Eda jaws, even though
only half of them developed extra molars. The requirement of Eda
upregulation for the AE to develop EM was clearly seen in Fgf20
X-gal staining in E15.5 molars (supplementary material Fig. S7).
In Fgf20Gal/Gal m1, an anterior extension of epithelial X-gal
activity was observed but this did not grow down to the
mesenchyme to form EM. However, in K14-Eda;Fgf20+/Gal and
K14-Eda;Fgf20Gal/Gal mice, the AE grew down to the
mesenchyme and was separated from m1 and gave rise to EM
(supplementary material Fig. S7).
Fgf20 regulates the expression of Spry2 and Spry4
The inhibitors of Fgf signaling sprouty 2 and sprouty 4 (encoded
by Spry2 and Spry4) are expressed at E14.5 in the dental
epithelium and mesenchyme, respectively (Zhang et al., 2001). As
loss of function of either Spry2 or Spry4 can result in the formation
of an extra anterior molar in the diastema (Klein et al., 2006), we
analyzed whether Spry expression is affected by Fgf20. Fgf20
beads, but not BSA beads, induced Spry2 and Spry4 in the
epithelium and mesenchyme, respectively (Fig. 8A-D). As loss of
function of either Spry2 or Spry4 induces the formation of the EM,
and because the same feature was observed in K14Eda;Fgf20Gal/Gal jaws, we hypothesized that Spry2 or Spry4
DEVELOPMENT
Fgf20 downregulation stabilizes the Shhexpressing foci in the anterior end (AE) of m1 but
Eda upregulation is required for their
development into EMs
In the mouse mutants developing EM, such as K14-Eda, Sostdc1–/–,
Sprouty2–/– and Sprouty4–/–, a Shh expression focus (also called
rudimentary bud or diastemal bud in the literature) is detected in
the AE of m1 at E13.5 or E14.5 (Kangas et al., 2004; Kassai et al.,
2005; Klein et al., 2006). To study the effect of Fgf20 signaling on
this Shh expression domain, we analyzed Shh expression by wholemount ISH in lower jaws of K14-Eda;Fgf20 mutant mice. Shh
expression was detected at E13.5 in ~30% of WT jaws, in ~50%
of Fgf20+/Gal and in all Fgf20Gal/Gal jaws studied (Fisher’s exact
test, P<0.05; Fig. 7). Similar to Fgf20-null embryos, in K14-Eda,
K14-Eda;Fgf20+/Gal and K14-Eda;Fgf20Gal/Gal embryos, the Shh
expression focus in the AE was stabilized in >90% of jaws
(P<0.01). At E14.5, this Shh expression was lost in WT mice but
sustained in >80% of Fgf20+/Gal and in all Fgf20Gal/Gal, K14Eda, K14-Eda;Fgf20+/Gal and K14-Eda;Fgf20Gal/Gal jaws (Fig.
7G-L; data not shown; P<0.05). The Shh domain was also enlarged
in K14-Eda;Fgf20Gal/Gal compared with K14-Eda embryos (Fig.
7N). From these observations we conclude that Fgf20 deletion
stabilizes the Shh foci in the AE, but is not sufficient to induce EM
formation. This is consistent with the previous observation by
Fgf20 in tooth development
RESEARCH ARTICLE 3195
might be aberrantly expressed in the AE of K14-Eda;Fgf20Gal/Gal
molars. To address this, we compared the expression of Spry2 and
Spry4 in tissue sections of K14-Eda and K14-Eda;Fgf20Gal/Gal
molars by in situ hybridization. The expression levels of both Spry2
and Spry4 were reduced in the AE of K14-Eda;Fgf20Gal/Gal m1,
and also in the middle of m1 (cap), compared with K14-Eda (Fig.
8E-L). Etv5 (also known as Erm), a direct target of Fgf signaling
(Roehl and Nüsslein-Volhard, 2001), was highly expressed in the
AE epithelium in the molars of the E14.5 K14-Eda;Fgf20Gal/Gal
mice but was downregulated in the mesenchyme compared with
K14-Eda (Fig. 8M,N). Reduced Spry2 expression in the epithelium
is thus not caused by reduced epithelial Fgf signaling, whereas
reduced Etv5 expression in the mesenchyme might be due to the
loss of Fgf20 function. From these results we conclude that Fgf20
can regulate the expression of Spry2 and Spry4 and that the
reduction in the expression levels of Spry2, and possibly Spry4, in
the K14-Eda;Fgf20Gal/Gal molars might facilitate the formation of
the EM.
DISCUSSION
Fgf20 is an important mediator of Eda signaling
in teeth
The mechanisms by which Eda regulates tooth size, shape and
number can be understood by identifying signaling pathways that
function downstream of Eda. Eda signaling is a key regulator of
morphogenesis of ectodermal organs in all vertebrates and
regulates the transcription of downstream genes via the
transcription factor NF-B. Here, we report that one of the Fgf
family ligands, Fgf20, is regulated by the Eda pathway in the
developing tooth. We demonstrated that Eda rapidly induced Fgf20
expression and that the expression levels of Fgf20 correlated with
Eda activity in vivo. The intimate link between Fgf20 and Eda was
further supported by the similarities of Fgf20Gal/Gal and Eda–/–
molar phenotypes. The polymorphism in cusp development is
milder in Fgf20Gal/Gal than in Eda–/– mice, but the tendency
towards a smaller or lacking anteroconid cusp is a shared feature
in both mutants. Taken together, our results indicate that Fgf20 is
a key mediator and is likely to be a direct target of Eda signaling.
Fgf20 is also regulated by Wnt/-catenin signaling (Chamorro et
al., 2005). The residual Fgf20 expression observed in Eda-null
teeth might, thus, be induced by the canonical Wnt pathway, which
is known to be active in tooth placodes and in the enamel knot
(Järvinen et al., 2006; Liu et al., 2008).
Although Eda/Edar/NF-B signaling is crucial for normal tooth
development, its target genes in teeth have remained elusive.
Candidate gene and microarray profiling studies on embryonic
mouse skin have shown that Eda signaling targets several
components of major signal pathways including Bmp, Wnt and Hh
(Mou et al., 2006; Pummila et al., 2007; Fliniaux et al., 2008;
Häärä et al., 2011; Lefebvre et al., 2012). The addition of the Fgf
DEVELOPMENT
Fig. 6. The number, size and shape
of K14-Eda molars are affected by
deletion of Fgf20. (A,B)K14-Eda
mice have an extra molar (EM)
anterior to m1 in ~50% of tooth
rows (A with an EM; B without an
EM, black arrow). (C,G)Inactivation of
one Fgf20 allele (K14-Eda;Fgf20+/Gal)
results in a slightly smaller m2,
without otherwise affecting the K14Eda phenotype (mean ± s.d.). (D-F)In
complete knockout of Fgf20 (K14Eda;Fgf20Gal/Gal), m1-m3 are smaller
and the cusp pattern is abnormal,
with reduced cusp number and
complexity. (H)Tooth size in K14Eda;Fgf20Gal/Gal mice shows a trend
towards increased reduction of more
posterior molars compared with K14Eda. (I)Also, the relative size of
molars within a tooth row is
changed: m2/m1 and m3/m1 are
altered in K14-Eda;Fgf20Gal/Gal mice
compared with K14-Eda (linear
regression of means). Upper table:
mean molar proportions (m2/m1 and
m3/m1) ± s.d. Lower table: Frequency
of the EM is increased in K14Eda;Fgf20+/Gal and, more clearly, in
K14-Eda;Fgf20Gal/Gal tooth rows
compared with K14-Eda mice. Also,
m3 is lost in 31% of the K14Eda;Fgf20Gal/Gal tooth rows.
*P<0.05, **P<0.01, ***P<0.001.
3196 RESEARCH ARTICLE
Development 139 (17)
pathway to the list of evolutionarily conserved signaling pathways
regulated by Eda underlines the function of Eda as a signal
pathway modulator in ectodermal organ formation.
Fgf20 acts redundantly with Fgf4 and Fgf9 and is
part of the Fgf signaling loop between the
epithelium and the mesenchyme
Fgfs regulate the morphogenesis of all organs and, typically,
epithelial and mesenchymal Fgfs function in signaling loops across
tissues (Kettunen et al., 2000; Sun et al., 2000; Klein et al., 2008;
Ornitz and Yin, 2012). Fgf20 belongs to a class of Fgfs that are
usually expressed in epithelium and that usually signal to
mesenchyme. Fgf20 is co-expressed in tooth placodes and/or
enamel knots with Fgf3, Fgf4, Fgf9 and Fgf15 (Åberg et al., 2004;
Kettunen and Thesleff, 1998; Porntaveetus et al., 2011), and we
showed that Fgf20 has similar effects on dental mesenchyme as
Fgf4 and Fgf9. We also showed that Fgf20 directly affects the
dental epithelium, similar to Fgf4, which was the first Fgf to be
discovered in the enamel knot (Niswander and Martin, 1992) and
has been suggested to regulate cusp formation (Jernvall et al.,
1994). The overlapping in vitro functions of Fgf20, Fgf4 and Fgf9
suggest redundancy between these ligands, as tooth phenotypes in
the Fgf20, Fgf4 and Fgf9 single null mutants are mild. The more
severe embryonic molar phenotype of the Fgf20;Fgf9 compound
mutant compared with single mutants directly demonstrates
redundant functions of the two Fgfs.
The coordinated growth of tooth compartments is at least
partially controlled by an Fgf signaling loop. Epithelial Fgfs
regulate the expression of mesenchymal Fgfs, such as Fgf3 and
Fgf10, which act redundantly to support growth and patterning of
molars mainly via epithelial Fgf receptor 2b (Fgfr2b) (Wang et al.,
2007; De Moerlooze et al., 2000). The molars are small in
Fgf3–/–;Fgf10+/– mice (Wang et al., 2007), similar to Fgf20Gal/Gal
reported here, suggesting that reduction of either epithelial or
mesenchymal Fgf signaling cause similar effects on tooth growth.
Fgf10 protein partially rescued the Eda–/– molar phenotype in vitro
(Pispa et al., 1999), which, together with our results, indicates that
one of the functions of Eda in tooth development is to sustain
and/or balance the Fgf signaling loop (Fig. 9).
The inhibitory functions of Spry2 and Spry4 on Fgf signaling are
thought to keep the diastema toothless (Klein et al., 2006), a
characteristic feature of mouse dentition for over 50 million years.
We showed that Fgf20 can induce the expression of Spry2 and
Spry4 and, importantly, that Spry2 and Spry4 levels were reduced
in the anterior end in K14-Eda;Fgf20Gal/Gal compared with K14Eda molars. Although still speculative, a scenario can be
envisioned in which Fgf20 would have a more prominent role in
regulation of Spry2 and Spry4 expression compared with other
Fgfs. Hence, loss of Fgf20 would in fact lead to increased Fgf
signaling activity in the anterior end of the molar bud. In support
of this hypothesis, expression of the Fgf target gene Etv5 was high
in the anterior end epithelium of K14-Eda;Fgf20Gal/Gal molars.
DEVELOPMENT
Fig. 7. Eda upregulation and Fgf20
inhibition independently stabilize the
foci of Shh signaling in the anterior end
of m1. (A-L)Shh localization in E13.5 and
E14.5 mandibles by whole-mount in situ
hybridization. In WT mice, a small dot of Shh
expression (blue, black arrow) is detected in
the anterior end (AE) of m1 in one third of
the E13.5 jaws (A). This Shh expression focus
has disappeared by E14.5 (G). However,
partial (Fgf20+/Gal, B,H) or complete
(Fgf20Gal/Gal, C,I) loss of Fgf20 stabilize the
Shh focus at E13-14.5. Eda upregulation
alone (K14-Eda, D,J) or combined to partial
(K14-Eda;Fgf20+/Gal, E,K) or complete (K14Eda;Fgf20Gal/Gal, F,L) loss of Fgf20 also
stabilize the Shh expressing foci.
(M)Percentage of Shh-expressing foci present
or absent in E13.5 half mandibles and the
number of samples. (N)In K14Eda;Fgf20Gal/Gal, the Shh foci are larger
compared with K14-Eda;Fgf20+/Gal at E14.5
*P<0.05, **P<0.01. Error bars represent
mean ± s.d.
Fgf20 in tooth development
RESEARCH ARTICLE 3197
Fig. 9. Integration of Eda/Edar/NF-B signaling in the enamel
knot with an Fgf signal loop regulates tooth crown
development. Schematic based on our findings and reported data.
Activation of Edar receptor by Eda in the enamel knot (EK) leads to
activation of NF-B and Fgf20 transcription. Fgf20, together with Fgf4
and Fgf9, moves to mesenchyme, induces the transcription of Runx2
and Fgf3 and stimulates cell proliferation. Fgf3 and Fgf10 signal back to
the epithelium and stimulate proliferation. Fgf20 induces Spry4
expression in the mesenchyme and Spry2 expression in the epithelium.
Spry4 and Spry2 inhibit Fgf signaling in the mesenchyme and
epithelium, respectively.
Fig. 8. Fgf20 regulates the expression of Spry2 and Spry4.
(A-D)E14.5 molar (m1) epithelium (A,B) or mesenchyme (C,D) cultured
with heparin beads soaked in Fgf20 recombinant protein or BSA. Fgf20
induces Spry2 (blue; B) in the epithelium and Spry4 in the mesenchyme
(D), whereas BSA does not (A,C). (E-L)Sections of E14.5 molar (m1)
from the anterior end (AE) and the largest area of the cap (as illustrated
in the schematic). Spry2 is expressed in the epithelium in the AE and
cap of K14-Eda molar (E,G) and is reduced in K14-Eda;Fgf20Gal/Gal
especially in AE (F,H). Spry4 expression is confined to mesenchyme (I,K),
and is reduced in AE in K14-Eda;Fgf20Gal/Gal (J,L) compared with K14Eda. (M,N)Etv5 expression is high in the AE of K14-Eda;Fgf20Gal/Gal in
the epithelium but reduced in the mesenchyme (N) compared with the
AE of K14-Eda (M). Dashed line indicates the margin of the epithelium.
Whether these effects are directly caused by loss of Fgf20, or are
partially due to alterations in the expression of mesenchymal Fgfs,
is currently not known. We propose that the combined effect of
high Eda and low Spry expression levels resulted in stabilization
of the EM in K14-Eda;Fgf20Gal/Gal lower jaws. As Fgf20 activity
appeared to inhibit the formation of the extra molar, other Eda
targets must stimulate it. The reported Eda-induced genes include
modulators of Bmp, Wnt and Hh pathways (Mikkola, 2009;
Lefebvre et al., 2012), all of which have been linked to EM
Fgf20 and Eda play important roles in the
activator-inhibitor balance regulating tooth
number, size and shape
A characteristic feature of K14-Eda;Fgf20Gal/Gal molars was the
increased reduction in size towards posterior molars and the
relatively frequent lack of m3. We suggest that these two
phenotypes are linked and can be explained by the inhibitory
cascade model (Kavanagh et al., 2007). The increased reduction in
size towards posterior molars indicates, according to this model,
increased inhibition in K14-Eda;Fgf20Gal/Gal tooth development
compared with K14-Eda. Moreover, 31% of K14Eda;Fgf20Gal/Gal tooth rows lacked m3, although K14-Eda and
Fgf20Gal/Gal mice do not lack m3 (Mustonen et al., 2003) (this
study). The result follows the predictions of the inhibitory cascade
model as a consequence of increased inhibition. Eda–/– mice lack
m3 at a frequency of 17-55%, depending on the genetic
background (Grüneberg, 1966; Pispa et al., 1999; KristenováCermáková et al., 2002). The removal of Fgf20 in Eda gain-offunction mice caused the m3 phenotype to resemble the Eda lossof-function phenotype. This highlights the importance of Fgf20 in
the activator-inhibitor balance that modulates the initiation and size
of posterior molars. The total effect of removal of Fgf20 in K14Eda was ‘anteriorization’ of dentition: stabilization of the extra
molar, and reduction or absence of m3. Thus, Fgf20 is likely to act
as an activator during tooth development. The proportional
reduction in tooth size towards posterior molars and reduced
DEVELOPMENT
stabilization (Tummers and Thesleff, 2009). We suggest that the
balance between the different Eda targets, i.e. those that promote
and those that inhibit extra molar formation, determines the
frequency of EM appearance.
complexity are characteristic features of faunivorous rodent taxa
(Kavanagh et al., 2007). Interestingly, both of these features were
observed in K14-Eda;Fgf20Gal/Gal mice, which suggests that tooth
characteristics regulated by Eda and Fgf20 might be, ultimately,
under the control of ecology and that Eda- and Fgf20-induced
variation might facilitate the microevolution of dentition.
Our work demonstrates how combinatorial genetic alteration
within one pathway during tooth development can lead to a wide
variety of phenotypes, causing subtle, but evolutionary important,
dental variation. The reduction of Fgf20 expression in Fgf20+/Gal
mice had no detectable effects on tooth development and the
complete loss of Fgf20 signaling (Fgf20Gal/Gal) lead to phenotypic
alteration of one characteristic (size), together with mild alteration
in another (anteroconid cusps). When Fgf20 loss of function was
combined with high Eda signaling, several tooth characteristics
(size, number and cusp pattern) were changed, some more
drastically than others, owing to imbalanced activation and
inhibition. In nature, mutations in the regulatory regions of Eda and
Fgf20 pathway genes could produce dental variation, the material
for natural selection. Interestingly, Eda signaling activity is under
natural selection in three-spined stickleback fish populations
producing variation in armor plates (Colosimo et al., 2005), and, in
humans, a specific allele of Edar is a major contributor to scalp hair
thickness in Asian populations (Fujimoto et al., 2008). We
demonstrated some mechanisms behind the Eda and Fgf20-induced
variation. Maintenance of the embryonic Shh expression foci in the
AE was supported by the loss of Fgf20 but completion of the
formation of the EM required Eda upregulation. Epitheliumderived Fgf20 modulates mesenchymal cell proliferation and
induces the expression of Fgf3, which reciprocally signals to the
epithelium. By this mechanism at least, Fgf20 could regulate size
of the molars and cusp development. Moreover, we connected Eda
to this Fgf loop by demonstrating that Eda induces Fgf20
expression in the epithelium. Our results indicate that other
epithelial Fgf ligands, Fgf4 and Fgf9 in particular, may compensate
for the loss of Fgf20 explaining the relatively mild phenotype of
Fgf20Gal/Gal dentition. The other targets of Eda probably include
important inhibitors, as, according to the inhibitory cascade model,
the characteristic molar proportions of K14-Eda;Fgf20Gal/Gal
mice resulted from increased inhibition.
Acknowledgements
We thank Nobuyki Itoh for support during the initiation of the project; Gail
Martin and Xin Sun for the generation of Fgf4 conditional mutant mouse; and
Raija Savolainen, Riikka Santalahti, Merja Mäkinen and Tuomas Kankaanpää
for technical assistance.
Funding
The project was supported financially by Viikki Doctoral Programme in
Molecular Biosciences, Academy of Finland, and Sigrid Juselius Foundation.
Generation of Fgf20Gal mice was aided by National Institutes of Health
support grants [P30DC04665, P30DK052574 and P30AR057235] at
Washington University, and a generous contribution from Edward and Linda
Ornitz. Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.079558/-/DC1
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Fgf20 in tooth development