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Effect of eda Loss of Function on Upper Jugal Tooth Morphology.

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THE ANATOMICAL RECORD 292:299–308 (2009)
Effect of eda Loss of Function on Upper
Jugal Tooth Morphology
iPHEP, CNRS UMR 6046, Faculté SFA, Université de Poitiers, 40 avenue du recteur
Pineau, Poitiers Cedex, France
Molecular Zoology Team, Institut de Génomique Fonctionnelle de Lyon, Université de
Lyon, CNRS, INRA, UCB Lyon 1, IFR128 Lyon Biosciences, Ecole Normale Supérieure de
Lyon, 46 allée d’Italie, Lyon Cedex 07, France
Department of Teratology, Institute of Experimental Medicine, Academy of Sciences CR,
Prague, Czech Republic
European Synchrotron Radiation Facility, 6 rue Jules Horowitz, Grenoble Cedex, France
The Tabby/eda mice, which bear a loss of function mutation for the
eda (ectodysplasinA) gene, are known to display developmental anomalies
in organs with an ectodermal origin. Although the lower jugal (cheek)
teeth of Tabby/eda mice have been extensively studied, upper teeth have
never been investigated in detail. However, this may help us to further
understand the function of the eda gene in tooth development. In this
work, the shape and size of both the crown and the radicular system
were studied in the Tabby/eda mice upper jugal teeth. To deal with the
high morphological variability, we defined several morphotypes based on
cusp numbers and position. Statistical tests were then performed within
and between the different morphotypes to test the correlation between
tooth size and morphology. Our analysis reveals that, as in lower teeth,
eda is necessary to segment the dental lamina into three teeth with the
characteristic size and proportions of the mouse. Nevertheless, since
strong effects are observed in heterozygous upper teeth while lower are
only mildly affected, it seems that the upper jaw is more sensitive than
the lower jaw to the loss of eda function. Modifications in cusp number
and the abnormal crown size of the teeth are clearly linked, and our
results indicate a role of eda in cusp patterning. Moreover, we found that
the Tabby mutation induces variations in the dental root pattern, sometimes associated with hypercementosis, suggesting a newly uncovered
role played by eda in root patterning and formation. Anat Rec, 292:299–
308, 2009. Ó 2008 Wiley-Liss, Inc.
Key words: Tabby/eda mouse; Mus musculus; dentition; jugal
teeth; morphology
Grant sponsor: ANR; Grant name: Quenottes, Ministry of
Education, Youth, and Sports of the CR; Grant number: COST
B23.002; Grant sponsor: Grant Agency of the CR; Grant number:
304/05/2665; Grant sponsors: Poitou-Charentes and Rhône-Alpes,
The Fondation pour la Recherche Médicale, The Fondation
Singer-Polignac), The COST B-23 Action (Brussels, EU).
*Correspondence to: Cyril Charles, iPHEP, UMR CNRS 6046, 40
avenue du recteur Pineau, 86022 Poitiers Cedex, France. Tel: 1335-49-45-40-55. Fax: 133-5-49-45-40-17. E-mail: cyril.charles@univÓ 2008 WILEY-LISS, INC. (or) Laurent Viriot, iPHEP, UMR CNRS 6046, 40 avenue
du recteur Pineau, 86022 Poitiers Cedex, France. Tel: 133-5-49-4540-55. Fax: 133-5-49-45-40-17. E-mail:
Received 24 July 2008; Accepted 5 August 2008
DOI 10.1002/ar.20804
Published online 2 December 2008 in Wiley InterScience (www.
The mutant Tabby/eda mice display developmental
anomalies in organs with an ectodermal origin such as
hair, glands, teeth (Grüneberg, 1971), and palatal rugae
(Charles et al., 2007). The mouse Tabby syndrome is homologous to the X-linked hypohidrotic ectodermal dysplasia syndrome in humans (Weeks and Blecher, 1983;
Blecher, 1986; Srivastava et al., 1997). This syndrome is
caused by a mutation in the eda gene on X-chromosome
(Kere et al., 1996), which leads to a deficiency in the
ectodysplasin A (EDA) protein (Srivastava et al., 1997).
This protein initiates a signaling cascade, known as the
EDA pathway, which ultimately regulates the activity of
the NF-jB transcription factor (Courtney et al., 2005).
During early development of ectodermal organs, the
EDA pathway promotes placodal cell fate (Mustonen
et al., 2004) and would thereby have an impact on the
size of the forming organ. It has been demonstrated that
overexpression of eda leads to the development of wider
jugal teeth with an increased cusp number, and sometimes leads to a supernumerary tooth (Mustonen et al.,
2003, Kangas et al., 2004).
Tooth defects in Tabby/eda mice, including disruption
in tooth shape, size, and number, were reported by several authors who all noticed a high phenotypic variability
(Grüneberg, 1966; Sofaer, 1969a,b; Kristenova et al.,
2002; Mustonen et al., 2003; Kangas et al., 2004; Peterkova et al., 2005). To deal with this variability, Kristenova
et al. (2002) had to class the lower jugal teeth of the
Tabby/eda mice into five morphotypes. Additionally, since
the two to four lower jugal teeth of Tabby/eda mice cannot
be homologized with certainty to the three molars of wild
type mouse, they introduced a new nomenclature using
T1, T2, T3, for lower jugal tooth 1, 2, and 3 (numbering
from the most mesial jugal tooth). Further analysis of the
five morphotypes during embryogenesis suggested that
the phenotype could be explained by a defect in segmentation of the dental epithelium during early stages of
odontogenesis (Peterkova et al., 2002).
Only a few authors (Grüneberg, 1966; Sofaer, 1969a;
Cermakova et al., 1998) have described the upper jugal
teeth of the Tabby/eda mouse and with only general
descriptions, mainly concerning size variation. We believe
that studying in detail the upper tooth row of Tabby/eda
mice might help to further illuminate the role of the eda
gene in tooth development. We thus studied variations of
the upper jugal dentition in homo/hemizygous and in heterozygous Tabby/eda specimens, both for the crown and
the root apparatus. We defined five main morphotypes in
heterozygous and two in homo/hemizygous mutant specimens. These various morphotypes revealed themselves to
be linked with opposed length variations of the first and
second jugal teeth. We tested the relationship between
tooth size and occlusal morphology to see if the crown size
variations could be linked with modifications of cusp size
or number. Taken together, our analyses support the idea
that the eda gene is involved in the segmentation of the
upper dental lamina and in the cusp number and
arrangement, but also supports an uncovered role in root
patterning and formation.
Tabby Mice
Breeding pairs of four different stocks of Tabby/eda
mice corresponding to three independent alleles of the
eda gene were purchased from the Jackson Laboratory
(Bar Harbor, Maine, USA) from 1994 to 2001:
C3H/HeJ-Ta5J/1 (stock number JR1232)
C57BL/6J-Aw-J-Ta6J/1 (stock number JR0338)
C57BL/6J-Aw-J-Ta1/1Tfm (stock number JR0569)
B6CBACa-Aw-J/A-Ta/0 (stock number JR0314)
The Ta allele is a null allele resulting from a 2 kb deletion in the 50 part of the eda gene. The Ta6J allele is
also expected to be a null allele, as a base pair deletion
results in a translation of an EDA protein truncated of
its functional domains. The Ta5J allele is not yet characterized. The Tabby/eda mutant and wild type (WT) mice
were generated by inbreeding (Kristenova et al., 2002;
Kristenova-Cermakova et al., 2002) in the Institute of
Experimental Medicine ASCR (Prague, Czech Republic).
Mice phenotype/genotype was identified according to
external morphological criteria, which made possible
the distinction between heterozygous, homo/hemizygous
Tabby/eda mice, and WT specimens. Those criteria are
mainly the yellowish color of the coat and the bald spot
behind ears for homozygous (2/2) and hemizygous
Tabby/eda mice (2/Y or 2/0) and the striping of the coat
for heterozygous specimens (1/2). Homozygous and
hemizygous Tabby/eda mice exhibited the identical
external traits and thus cannot be distinguished from
one another. These animals were offspring of crosses
between homo/hemizygous, hemizygous or heterozygous
females with their hemizygous or WT male siblings. The
WT mice were generated by further inbreeding of wild
type (phenotypically normal, non-Tabby) animals derived
from the same stock.
The sample composed of 205 mice, 85 heterozygous
females (Ta/1), 23 homo/hemizygous Tabby/eda females
(Ta/Ta, Ta/0), 57 hemizygous Tabby/eda males (Ta/0),
and 40 control (WT) females. For each specimen, left
and right tooth rows have been studied independently.
The age of the specimens ranged from 15 postnatal days
to adults. Mice were killed by cervical dislocation and
their heads fixed in a series of graded ethanols 70%–
96%. The experimental protocol was designed in compliance with the recommendation of the European Economic Community (86/609/CEE) for the care and use of
laboratory animals. All of the animals’ treatment satisfied the requirement of the Institutional Review Board.
Observation and Imaging of Dental Rows
After mandible dislocation, jugal teeth were examined
using a Leica MZ 16 stereomicroscope. Based on the
number and arrangement of the cusps, various morphotypes were defined for upper tooth rows, and specimens
were ranked according to these morphotypes. Among the
entire sample, 14 upper jaws were selected as a representative panel covering the totality of observed morphologies to study in detail not only the shape of the
crown but also the dental root number and position. The
28 selected upper tooth rows were imaged using X-raysynchrotron microtomography at the European Synchrotron Radiation Facility (ESRF, Grenoble, France),
beamline ID19 and BM5, with a monochromatical beam
at energy of 25 keV. X-ray synchrotron microtomography
has been demonstrated to bring very high-quality
results for accurate imaging of small teeth (Tafforeau
et al., 2006) and allows nondestructive virtual extraction
of teeth roots (Chaimanee et al., 2006). A cubic voxel of
7.46 mm was used. All 3D renderings and virtual slices
were performed using VGStudiomax software.
Various statistical tests were used to verify the significance of differences in tooth size between the different
morphotypes. Width and length of jugal teeth were
measured from digitized pictures using Optimas software. A preliminary step was to test differences of morphotype occurrence between the various mouse strains.
For that purpose, we used a v2 test to compare the morphotype observed repartition with the theoretical repartition to see if the four strains were similar. Then, to
determine which morphotype displayed the same tooth
size, we used ANOVA and the post-hoc Scheffé test
(Winer et al., 1991). This test compares the tooth size
mean for each morphotype and determines which morphotypes are statistically different from the others. The
Scheffé test was preferred to the Tukey test because of
the unequal number of specimens in the various morphotypes. The jugal tooth row length was measured for
specimens exhibiting all jugal teeth fully erupted (65%
of specimens). We also used ANOVA to test the homogeneity of jugal tooth row length among the different morphotypes. Finally, we tested the parallelism of the linear
regression of the tooth size measurements among the
various groups using a test of parallelism adapted from
the Student’s t test. The t value was calculated as follows (Dagnelie, 1998):
t 5 |b1 2 b2|/H(r2Y.x (1/(RSSx)1 1 1/(RSSc)2)), with b1
and b2 the slope coefficient of the two considered linear
regression, r2Y.x the common, pooled, estimate of the residual variance, and RSS the relative residuals sum of
As for lower teeth (Kristenova et al., 2002), the uncertain homology of teeth between WT and Tabby/eda specimens led us to adopt a nomenclature using Tx, x
designating the rank of the teeth (T1 for the first upper
jugal tooth).
We had four different tabby strains at our disposition,
corresponding to three different mutations in the eda
gene (see Materials and Methods section for details).
However, we did not find any statistical difference in
morphotype occurrence between the various mouse
strains when comparing observed and theoretical distributions (P > 0.1). We also tested the strain effect on
tooth size using an ANOVA and found no statistically
significant effect (P > 0.7). The interstrain variability is
thus negligible when compared with the intrastrain one.
So, we considered all specimens together regardless of
the mouse strain. This improved the number of studied
Tabby/eda specimens, and therefore the reliability of our
measurements and statistical values.
Various Morphotypes of Upper
Jugal Tooth Rows
Wild type mice. The upper jugal dentition of wild
type (WT) mice counts for three upper molars (M1, M2,
and M3). The cusps of the M1 are linked by three trans-
Fig. 1. Wild-type morphology, terminology used to name the main
cusps of upper jugal teeth (terms from Miller, 1912). The red arrow
indicates a supplementary cusp observed in one WT specimen. M,
mesial; D, distal; L, lingual; V, vestibular. Scale bar: 1 mm.
versal chevron-shaped crests (Fig. 1A). The most mesial
chevron links together the three first cusps t1 (lingual),
t2 (central), and t3 (vestibular) according to Miller
(1912). Similarly, the second crest composed of t4, t5,
and t6 cusps, and the distal crest links only two cusps,
t8 and t9 (Fig. 1A). The M2 shows the same general
arrangement except that the mesial crest is limited to
the t1 (absence of t2 and t3). M3 exhibits an isolated t1
and a cusp circle resulting from the two distal cusp rows
One of the spontaneous variations from the classical
crown pattern was the occurrence of a tiny t3 cusp in
10% of the second molars. A rare second spontaneous
variation was the presence in one specimen of a tiny
supplementary cusp in the mesio-vestibular corner of
the M1 (arrowed in Fig. 1B). The radicular part of the
M1 (Fig. 1C) composed of three rounded roots (one
mesial, one vestibular, and one disto-lingual). The M2
also exhibits three roots (one rounded mesio-lingual, one
rounded disto-lingual, and one elliptic vestibular). The
M3 variably exhibits two (one mesial and one distal) or
three (two mesial and one distal) roots.
Heterozygous Tabby/eda mice. Five major morphotypes, based on both the number and arrangement of
cusps, were defined in heterozygous specimens. We considered independently the right and the left tooth rows
in each specimen and found that nearly half (46%) of
the heterozygous mice do not exhibit the same morphotype on both sides (but the five morphotypes occurred in
the same proportion on both sides). Nevertheless, tooth
rows were always located at the same position at the
left and right sides of the skull.
Morphotype Heterozygous 0 (MT He0) was the most
frequent (34%) among the heterozygous tooth rows. It is
indistinguishable from WT (Fig. 2A).
Fig. 2. Morphotypes defined on the heterozygous Tabby mice. A:
WT; B: MT He1; C: MT He2; D: MT He3; E: MT He4. The red arrows
indicate some specifics detailed in the text. M, mesial; D, distal; L, lingual; V, vestibular. Scale bar: 1 mm.
Morphotype Heterozygous 1 (MT He1) was observed
in 20% of upper tooth rows. In this morphotype, T1
displayed mild defects on the vestibular side when compared with the WT (Fig. 2B). The major systematic
defect was the occurrence of a supplementary cusp distal
to the t6 (t6b), which appeared either small or as large
as a main cusp (arrowed in Fig. 2A). Its presence was
not linked with the disappearance of the t9 as these two
cusps could occur in a same tooth (data not shown).
However, the t9 was often reduced or even sometimes
absent. Occasionally, another supplementary minute
cusp can occur between t3 and t6 cusps. The T2 crown
was similar to the WT M2. The T3 showed a decrease in
the cusp number associated with a reduction of tooth
size. The T1 radicular system was similar to that of the
WT M1. On the contrary, the root number of T2 and T3
differed from that observed for WT M2 and M3. Indeed,
T2 can exhibit only one or two roots instead of three.
The remaining roots were then not rounded, but displayed more complex shapes, suggesting that they might
result from a fusion of roots (Fig. 2B). The T3 displayed
either one or two roots.
Morphotype Heterozygous 2 (MT He2) was found in
9% of tooth rows. The T1 of the MT He2 showed a vertical and rounded t2 cusp and a reduction of both the t3
and t9 cusps (arrowed in Fig. 2C). The T2 was grossly
similar to the WT M2, except for a reduction or even disappearance of the t9 cusp. Moreover, in a few tooth rows
(5% of He2), we found the supplementary tiny t3 cusp
on T2 mesio-vestibular corner that is also occasionally
found in WT M2. The T3 displayed either two or three
cusps. The mesial root of the T1, located just below the
t2 cusp, was more vertical than in WT M1 and was
sometimes fused with the lingual root. As for MT He1,
the number and shape of roots was variable in T2 and
reduced to one in T3.
Morphotype Heterozygous 3 (MT He3) was the least
common morphotype with a frequency of 8.2%. It was
characterized by a reduced T1 (Fig. 2D). This reduced
tooth corresponds was considered as a supernumerary
by Sofaer (1969b). The T1 notation, always used here to
describe the first jugal tooth, leads to homologize the
small mesial tooth in type He3 with the T1 of other morphotypes. We, however, keep the T1 notation for this
tooth as we saw intermediary sizes between the T1 of
MT He3 and T1 of the other morphotypes, especially MT
He4. Jugal tooth rows composed of either three or four
teeth. The T1 occlusal side displayed only one to three
distinct cusps. The T2 was the largest tooth of the row
and exhibited a t3 cusp in 91% of specimens and always
a reduced t9. The T3 was smaller than the T2 and exhibited five cusps. Of note, T2 and T3 in MT He3 looks like
T1 and T2, respectively, in MT He2 (Fig. 2D). When present, a bicuspidate T4 was smaller than the T3. The T1
was single-rooted (with a root inclined toward the mesial
direction), whereas the T2 displayed two roots similar to
the MT He2 T2. The T3 had three roots and the T4 only
Morphotype Heterozygous 4 (MT He4) corresponded
to 25% of observed tooth rows (Fig. 2E). It was characterized by a disruption of the t4-t5 interconnection in
T1, and the appearance of a new mesio-distal connection between the t2 and t4 cusps by a lingual crest.
This later connection was sometimes prolonged until
the t8 cusp, resulting in a crest which occupied the
entire lingual border of the tooth. Like T1, the T2 can
exhibit a disruption of the t4-t5 interconnection. In 47%
of MT He4 specimens, the T2 also showed a crest connecting t1 and t3 cusps. The T3 displayed three cusps.
The radicular system of the T1 generally composed of
three roots. However, the vestibular root tended to split
into two different roots (Fig. 2E). The T2 displayed two
roots, the elliptic mesial one resulted from the fusion of
two rounded roots. The T3 exhibited three rounded
Other rare morphologies represented only 4% of the
observed tooth rows (Fig. 3). The first case showed the
appearance of both a t1bis cusp associated to an extra
root on the T1 (arrowed in red in Fig. 3A). A vestibular
extra cusp (arrowed in white) occurred between t3 and
t6 cusps. The mesial cusps occupied abnormal positions:
t1 was distally displaced while both the t1bis and t3
were closer to the t2, resulting in an acute triangular
configuration (Fig. 3A). Another specimen exhibited a T1
morphologically close to the T1 of the MT He4 with a
duplication of t5-t6 enclosing an extralingual cusp
(arrowed in Fig. 3B). A third exceptional case displayed
a tooth row similar to that of MT He3 with fused T1 and
T2 crowns and roots (Fig. 3C). The radicular complex of
both the T1 and the first crest of the T2 were grouped
into a unique root.
Of note, whatever the morphotype occurring in the
two sides of heterozygous asymmetric specimens, the
Fig. 4. Morphotypes defined on the homo/hemizygous Tabby
mice. A: MT Ho1; B: MT Ho2; C and D: Radicular patterns observed
in homozygous Tabby mice. The red arrows indicate the occlusal surface specifics detailed in the text. M, mesial; D, distal; L, lingual; V,
vestibular. Scale bar: 1 mm.
cusps. Based on these characters, MT Ho1 and MT Ho2
were very close to the MT He4 defined for heterozygous
Tabby/eda mice. The MT Ho1 and MT Ho2 sometimes
occur on the same specimen, resulting in an asymmetric
configuration. Apart from these occlusal traits, two different root patterns were observed for T1 in both Ho1
and Ho2. In the first pattern (Fig. 4C), mesial and lingual roots were fused and not connected to the vestibular one. In the second pattern (Fig. 4D), the three roots
were fused and met in the center of the tooth.
In Tabby/eda mice, the radicular complex sometimes
displayed hypercementosis (e.g., Fig. 2B–D). The amount
of cementum that covered the root dentine was highly
variable from one specimen to another.
Fig. 3. Rare morphologies of heterozygous Tabby mice. Arrows
referred to supplementary cusps, roots, and abnormal connection indicated in the text. M, mesial; D, distal; L, lingual; V, vestibular. Scale
bar: 1 mm.
mesial and distal extremities of the jugal tooth rows are
located at the same mesio-distal position on the skull.
Homo/hemizygous Tabby/eda mice.
In all
homo/hemizygous specimens, the T1 was characterized
by a reduction of vestibular cusps, a disruption of the t4t5 interconnection and the appearance of a new mesiodistal connection between t2 and t4 cusps by a lingual
crest. Two major morphotypes were defined, mainly differing by the T1 morphology (Fig. 4A,B). In the MT Ho1
(69% of tooth rows), T1 exhibited only one central cusp
in the mesial area (t2) linked to the t4 by a long semicircular lingual crest (Fig. 4A). In the second morphotype
MT Ho2, two cusps (t1 and t4 arrowed on Fig. 4B) were
still present in the lingual area and attached to a lingual semicircular crest connected to the t2. In both morphotypes, T2 systematically lacked the t9 cusp, and a
lingual crest similar to the one observed on the T1 was
sometimes present. The T3 displayed either two or three
Classes of Morphotypes
To further characterize the different morphotypes, we
used measurements of the total jugal tooth row and of
the first jugal tooth. Measurements of the tooth row
length revealed two groups (Fig. 5). The first group composed of WT and a part of the heterozygotes (MT He0
and He1), which have a normal tooth row length and
are morphologically very close to wild type. The second
included the other heterozygous (MT He2, He3, and
He4) and all of the homo/hemizygous Tabby/eda tooth
rows, which all have a reduced tooth row length. These
associations were statistically significant and we found
no difference between the measurements of jugal tooth
row length in WT, MT He0, and MT He1 mice (ANOVA, F
5 2.83, P 5 0.07) or between MT He2, MT He3, MT He4,
MT Ho1, and MT Ho2 (ANOVA, F 5 1.08, P 5 0.37).
Measurements of the first jugal tooth (Fig. 6) confirmed homogeneity of the first group containing WT,
MT He0, and MT He1 (Scheffé test, threshold value
0.05). Tooth rows in this group displayed the largest T1,
with mild or no cusp defects. Measurements of the first
jugal tooth also allowed the division of the second group
into three subgroups. (1) MT He2 group: The T1 was
shorter than M1 of WT in relation with a more vertical
Fig. 5. A: Length of the upper jugal tooth rows for the different morphotypes. Black lines indicate the
range of variation of each morphotype. B: Proportion of the various jugal teeth of the tooth row (mean values). Black: first jugal tooth; dark gray: second jugal tooth; clear gray: third jugal tooth; white: fourth jugal
Tooth Size Among the Jugal Tooth Row
Fig. 6. First jugal tooth morphologies and first jugal tooth size
modifications. Each morphotype is represented by the mean value
and the standard deviation of the mean. The ellipses regroup morphotypes exhibiting close morphologies for the first upper cheek tooth.
mesial area. The T2 was also shorter than wild type M2
(see Fig. 5b); (2) MT He4, MT Ho1, and MT Ho2 group:
In this homogeneous group (Scheffé test, threshold value
0.05), the T1 was still shorter, displaying strongly
reduced mesial and vestibular areas and a lingual crest.
The T2 is of normal or even increased size as compared
with M2 (Fig. 5b); (3) MT He3 group: The T1 consisted of
a minute tooth composed of one, two, or three cusps.
The T2 is clearly longer than M2 (Fig. 5b).
Kavanagh et al. (2007) proposed a model to explain
size relationships within the tooth row (the ‘‘inhibitory
cascade model’’). In this model, sequential development
along with cumulative inhibition explain the relative
size of the mouse molars: the first molar develops first
and inhibits the second molar, which as a consequence is
smaller, and the third molar, which cumulates inhibition
of both the first and the second, is even smaller. To further evaluate changes in the relative size of the molars
in Tabby mice (Fig. 5), we used a graph that represents
the cumulative nature of the ‘‘inhibitory cascade model’’
(Fig. 7). We represented the size of a tooth as a function
of the cumulated size of the previous teeth. On this
graph, each wild-type tooth row is represented by two
points: one linking M2 with M1 size, and the second linking M3 with M11M2 size. On the same graph, we can
incorporate the Tabby 3-teeth row, but also the 4-teeth
row, with three possible relationships: T2 and T1, T3 and
T11T2, and lastly, T4 and T11T21T3. Importantly, in
this last case, the three points were found aligned, showing that the same cumulative relationship as the one
linking T1 and T2 size to T3 size can predict T4 size, and
thus supported this representation.
We can consider three different regressions: WT (Fig.
7, line Œ), MT He 0-1 (Fig. 7, line ), and MT He2-3-4
plus MT Ho1-2 Tabby (Fig. 7, line Ž). The position of
these lines on the graph is mainly linked to the T1/T2 ratio. Line 2 is slightly shifted and line 3 is strongly
shifted toward the left. This reflected the unusual T1/T2
ratio in Tabby morphotypes (especially in the more
affected morphotypes, He2-3-4 and Ho1-2), as already
shown. Then, the slope of these lines is related to the T3
size relative to the T2 size (for a 3-teeth row, the slope
coefficient would be: size of T3/size of T2-1). First, we
noted that lines 1, 2, and 3 had very close slope values.
Fig. 7. Relationship between the size of a tooth and the total size
of the more mesial jugal teeth. The black line represents the linear
regressions: Œ for WT specimens,  for MT He0 and MT He 1 specimens, Ž for MT He2, MT He 3, MT He 4, and homo/hemizygous
Tabby specimens. Measurements indicate similar tooth proportion on
the entire jugal row among MT He2-3-4 and homo/hemizygous Tabby
specimens and similar T2, T3 and when present T4 proportions among
all of the Tabby mice (parallelism of the linear regression  and Ž).
We used a test of nonparallelism to see if differences
were statistically significant. We found a statistically relevant nonparallelism (Pnonparallelism > 0.05) between the
regression lines 1 and 2 (WT versus He0-1), and 1 and 3
(WT versus He2-3-4-Ho1-2), but not between 2 and 3
(light tabby phenotype He0-1 versus strong tabby phenotype He2-3-4-Ho1-2; Pnonparallelism < 0.05). Thus, between
the two groups of Tabby morphotypes, the T2 2 T3 proportions are not significantly different. In conclusion,
the T1/T2 proportions strongly differ between homo/hemizygous and MT He2-3-4 specimens on the one hand,
and the MT He0-1 specimens on the other hand, yet the
T2 2 T3 proportions are similar among all of the Tabby
specimens and only slightly differ from the WT condition
(slope values are close even if statistically different).
Correlation Between Tooth
Morphology and Size
The tooth measurements showed a higher variability
in heterozygous than in homo/hemizygous Tabby/eda
mice. The Coefficient of variation of the T1 length was
2.9 for the WT (interval of confidence 2.5–3.4), 16.6 (IC
14.8–18.8) for heterozygous, and 4.5 (IC 3.9–5.4) for
homo/hemizygous Tabby/eda specimens. This size variability accompanied the morphological diversity among
the heterozygous specimens, suggesting that there is a
relation between tooth size and tooth shape. To test this
relation, the next step consisted of comparing the variations of cusp number with tooth size among the heterozygous sample because this genotype exhibits the most
important morphological variations. Results presented
in the Fig. 8 showed that for each tooth, the size clearly
increased with the number of cusps.
Fig. 8. Relationships between teeth size and cusps number in heterozygous Tabby mice in red: first jugal tooth (T1), in blue: second jugal tooth (T2), in black: third jugal tooth (T3). The verticals lines represent the range of size variation for each number of cusps. For each
tooth, the black line represents the linear regression.
Natural Variability of Upper Jugal
Teeth in WT Mice
Among the WT mouse sample, two main variations
have been found: (1) a supplementary cusp mesial to the
first chevron of the M1; this has already been described
in Mus setulosus but occurs less frequently in Mus musculus (Misonne, 1969); and (2) a mesial cingulum in 10%
of the M2. This has also been reported by Cermakova
et al. (1998) in WT and ICR mice.
Morphological Diversity of the Upper Jugal
Tooth Row in Tabby/eda Mutant Mice
Previous studies on Tabby/eda mouse dentition mainly
documented a reduction of the jugal tooth size and a
decrease in the cusp number (Grüneberg, 1966; Sofaer,
1969a; Miller, 1978; Cermakova et al., 1998). Our study
also revealed these two general trends. However, among
heterozygotes, we observed very few specimens with
larger crown size and higher cusp number. These latter
correspond to the ‘‘Incomplete twinning’’ specimens
described by Grüneberg (1966). At least one of these
teeth (Fig. 3C) can clearly be considered as the result of
an abnormal fusion between the first two teeth of the
MT He3.
Among heterozygous Tabby/eda mice, 54% of the tooth
rows display a normal length and a morphology similar
(MT He0) or close (MT He1) to the WT one. It corresponds to tooth rows in which the effect of the Tabby
mutation is very small. However, the mild modifications
observed between the crown shapes of these two morphotypes might reflect an effect of the eda gene on cusp
number and arrangement, which could be compared
with the changes that can be observed between two
related taxa in nature (Misonne, 1969).
The rest of the heterozygous and homozygous tooth
rows all have a reduced length, with an opposed
variation in tooth length between T and T . MT He3 is
characterized by a T1, which is smaller than the T2.
This is a reversed situation as compared with the wild
type. MT He2 may be interpreted as a MT He3 with an
aborted development of the most mesial tooth. Such a
situation in front of the future T1 was previously
observed during the development of the lower tabby
tooth row (Peterkova et al., 2002). Here, it could have
provoked the vertical disposition of the t2 cusp in the T1
of MT He2 and the change in the orientation of its
underlying mesial root (Fig. 2). As for the lower jaw,
both the aborted tooth of MT He2 and the T1 of MT He3
might be interpreted as a ‘‘revival’’ of tooth rudiments
found in wild type mouse (Viriot et al., 2002; Peterkova
et al., 2005; Klein et al., 2006). However, developmental
data will be necessary to test this possibility.
Finally, the last heterozygous morphotype (MT He4),
corresponding to about 25% of the heterozygous specimens displays similar features to those of the two homozygous morphotypes (MT Ho1 and MT Ho2), i.e., the
reduction of vestibular cusps and the occurrence of a lingual cingulum. Grüneberg (1966), Sofaer (1969a), and
Miller (1978) considered that this mesial cingulum is a
typical feature of Tabby/eda mice. However, it has been
observed in WT and ICR mice. In the latter, the mesial
cingulum is tiny and usually smaller than the mesial
crest observed in Tabby/eda mice. We thus state that the
occurrence of a mesial cingulum is not characteristic of
the Tabby mutants, but the bigger size of this cingulum
is a specificity of Tabby/eda mice. Of note, the reduction
of the total tooth row in these three morphotypes is
mainly attributed to the reduction of the T1, which is
nevertheless still meanly 25% longer than the T2.
The Tabby Mutation Putatively Modifies the
Segmentation of the Upper Dental Lamina
The various morphotypes defined for lower jugal tooth
rows have been related to differences in the segmentation of dental epithelium during determination of tooth
boundaries; although the total antero-posterior length of
the dental epithelium is similar, the length of the particular tooth primordia differs between the jaws, even in
the same embryonic head (Peterkova et al., 2002, 2005).
Moreover, Kristenova et al. (2002) described an opposed
size variation between the first and second lower jugal
teeth associated with various morphotypes. In our study
of Tabby/eda upper tooth rows, we also found that the
limits between the different teeth are located at variable
mesio-distal positions despite similar row length, revealing a similar modification in the segmentation of the
upper dental lamina. We also found that the size of a
tooth depends on the size of the more mesial jugal teeth
(Fig. 7). The occurrence of a fourth tooth in some Tabby
heterozygous specimens is then a consequence of the
reduced size of the three mesial ones, teeth continuing
to develop according to the observed relationship until
the tooth size falls under the minimal size (Fig. 7).
Interestingly, this size relationship might be explained
by the recent ‘‘inhibitory cascade model’’ proposed by
Kavanagh et al. (2007). These authors proposed that the
developing first molar exerts an inhibitory effect on the
second molar, and that this inhibitory effect will remain
as strong as the first molar development continues. This
model could explain the tooth size relationships observed
in Tabby heterozygous mice as well as the number of
teeth. The observed proportions of the various teeth
among the jugal tooth row could thus directly result
from the variability of size of the first tooth (T1) among
heterozygous Tabby mice. This T1 size variability might
itself be the consequence of a variable amount of EDA
protein in heterozygous Tabby specimens.
Relationship Between Tooth Size
and Morphology
Measurements emphasized a link between cusp number and crown size in T1, T2, and T3 (Fig. 8). According
to the ‘‘patterning cascade mode of cusp development’’
published by Jernvall (2000), the succession of the signaling centers, and consequently the number and position of cusps, is linked to the crown size. This relationship had previously been observed for the lower teeth of
Tabby mice (Kangas et al., 2004) and holds true here for
upper Tabby jugal teeth (Fig. 8). As the number of cusp
is clearly linked to the tooth size, one could think that
the morphological modifications could only be the result
of a primary effect on the tooth size, which consecutively
provokes modifications of the cusp pattern. This is
clearly happening in Tabby mutants. However, we do
not think this is the only explanation, essentially for two
reasons: first, eda gene is not only expressed in early
stages of tooth formation but also at the time of cusp formation (Laurikkala et al., 2001). Second, in MT He4,
Ho1, and Ho2, T2 is about the normal size of WT mice
M2, but the cusp pattern is nevertheless strongly
affected. Rather, we think that the effect on tooth morphology is a consequence of both a primary effect on
tooth size and a direct effect on cusp formation. Finally,
Jernvall and Thesleff (2000) explained the Tabby/eda jugal tooth morphology by closer locations of cusp tips.
Our results show that the whole morphological variability observed among Tabby/eda jugal dentition is not only
related to closer cusp positions but also to new cusp
arrangements and occurrence of neoformed cusps.
Comparison of the Tabby/eda Phenotype
Between Lower and Upper Tooth Rows
For lower tooth rows, Cermakova et al. (1998) found
only very minor defects in heterozygotes and still a
small percentage of wild type tooth rows among homozygotes. This situation contrasts with the situation in
upper tooth row, where 46% of heterozygous and 100%
of homozygous tooth rows were abnormal. It is interesting to further compare the morphotypes we described in
both hetero- and homozygotes with the one described for
the homozygous lower tooth row (Kristenova et al.,
2002; Peterkova et al., 2002). Class I lower tooth rows
have T1 shorter than T2. Thus, they may be analogous
to MT He3 upper tooth rows. In Class IIa, the first tooth
aborts, and adult T1 is longer than T2. Class IIa might
thus correspond to MT He2 for upper tooth row. Finally,
in Class IIb, T1 develops first and is longer than T2.
Class IIb might then correspond to MT He4, Ho1, and
Ho2 for upper tooth row. Taken all together, this comparison suggests that the effect of the eda mutation on
the upper and lower tooth row is rather similar, but that
the upper tooth row development is more sensitive to
eda loss of function. Differences in the developmental
control between upper and lower jugal teeth have already been suggested by Shimizu et al. (2004) after a
study of size variation in first upper and lower molars of
SMXA recombinant inbred strains of mice. Several
mutations lead to a complete loss of molars on one jaw
while molars on the opposite jaw develop normally (see
dlx12/2,dlx22/2 double mutant, Qiu et al., 1997 or
activinbA2/2 mutant, Ferguson et al., 1998). The case of
eda mutants provides a novel and more subtle example
of differences in the genetic control of upper and lower
tooth row development.
Associated Modifications of the
Radicular Complex
We observed modifications of the root pattern in
Tabby/eda mice. Since root formation is consecutive to
crown formation, an abnormal crown size specification
could very logically disrupt the root patterning program.
Except for some rare specimens showing an increase in
both crown size and number of roots (Fig. 3), the major
tendency in Tabby/eda is a decrease in the crown size
associated with a decrease in the number of roots. Notably, Grüneberg (1966) described a unique root for the
second upper jugal tooth, whereas we observed specimens displaying one, two, or three roots. However, the
position of these roots and the enlarged shape of the
unique root of other specimens indicate that the unique
root results from the fusion of the three normal ones
(Fig. 4C,D).
Importantly, however, we also often observed variations in the number of roots for similar crown size, suggesting a more direct perturbation of root patterning.
Moreover, the observation of Tabby/eda specimens with
a higher amount of cementum than the normal condition
could indicate a role of eda not only in root patterning
but also in the rhizogenesis itself. Very interestingly,
another gene implicated in the EDA pathway, edar, was
very recently found in a subtractive hybridization screen
for genes specifically involved in the molar multiroot rhizogenesis versus the incisive single root pseudo-rhizogenesis (Xing et al., 2007). Together with our results,
these data strongly suggest that the EDA pathway plays
a role in root patterning and formation.
We show that, as in the lower jaw, eda function is necessary during development to obtain a correct tooth row
length and a correct segmentation of this tooth row.
However, the upper jaw seems more sensitive to Tabby/
eda heterozygoty than the lower jaw. Moreover, we have
indications that, apart from determining tooth crown
size, which is linked to cusp and root number, eda may
play a more direct role in cusp and root patterning and
The authors thank J.-J. Jaeger and V. Lazzari for comments concerning this study. They also thank J.
Baruchel and ID 19 staff of the European Synchrotron
Radiation Facility of Grenoble. They acknowledge P.
Kristenova for collecting the material; L. Foley-Ducrocq
for language revision; the reviewers for their comments;
C. Noël and G. Florent for administrative guidance.
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loss, morphology, effect, eda, upper, toots, function, jugal
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