COMPARISON OF THE TOOTH SHAPE AND SIZE IN TABBY AND NON · TABBY MICE

Cermakova P., M. Peterka. 1. Capkovli, J. Ture~kova. 1. V. Ruch, H. Lesot. R. Pe te rko v Ii: Comparison of the Tooth Shape and Size in Tabby and NOll-tabby Mice. Acta vet. Brno 1998,67: 3-14. Basic anatomical and embryological investigations of the dental disorder in tabby mice were performed some 20-30 years ago. In order to bridge the gap in research activity in this field and as a prerequisite for future developmental studies, the dental characteristics of the tabby mice were updated in a stock presently available. Qualitative and quantitative parameters of the functional teeth were determined in 50 males and females of different phenotypes segregated from the stock of tabby mice. A parallel investigation was made in a common laboratory mouse (ICR stock). In ICR mice. the body weight was two times higher and this was reflected in the cheek teeth which were significantly larger (but similar in shape) when compared to the wild type non-tabby controls. Among tabby homozygous and hemizygous mice, at least one incisor was absent in 50% of females. and in 70% of males where predominance of the right side was apparent. In these groups the mean length and width of the cheek teeth were significantly reduced compared to the corresponding wild type controls, despite similar body weight. Changes in crown pattern. including also reduction or absence of cusps. resulted in characteristic morphology of the cheek teeth. In contrast to the earlier literature, duplication of an incisor or an explicit supernumerary tooth in the cheek region were not found in the present tabby collection and the heterozygous specimens were less affected. Gelle. mUTatioll. syndrome. allomaly. development Congenital decreases or increases in tooth number (hypodontia, hyperodontia) and size (microdontia, macrodontia) are well recognised clinical features in dental pathology. The most frequent disturbance is hypodontia, which is more common in humans than in other species. It affects about 7% of the human population (exclusive of agenesis of the third molars, which occurs in 10-25% of population). (Jorgenson 1980). In patients with orofacial clefts, congenital tooth agenesis increases to 10-40% (Poyry and Ranta 1985). Hypodontia may be associated with microdontia. Hyperodontia and macrodontia are much less frequent (Ra vn 1971). . Dental anomalies have been reported in more than 25 genetic syndromes exhibiting autosomal dominant, autosomal recessive or X-linked heredity. The dentition is severely affected in ectodermal dysplasias. The classic variety hypohidrotic (anhidrotic) ectodermal dysplasia includes hypodontia (or even anodontia); the existing teeth exhibit smaller size and modified shape (Salmon and Lindenbaum 1978; Jorgenson 1980; Crawford et al. 1991). The hypohidrotic ectodermal dysplasia is considered to be homologous to the Address for correspondence: Ml"Dr. Renata Peterkova. CS~. InstJ[utc: of Experimental ~kJidne. Academ) of Sdences of the Czech Republ ic Videflsk:i 1083, 1-/.2:W Prague 4. Czech Republic Phone: +420-2-4751232 Fa" +~20·2-~752604 E-maiL repete@biomed.ca~.cz

mouse X-linked tabby (Ta) syndrome (Weeks 1983;Blecher 1986).In comparison with non-mutant mice, Ta carriers exhibit characteristic defects of hair, exocrine glands, and teeth (Griine berg 1971;Green 1981a).The incisors may be hypoplastic, fused or absent, the third molar may be absent and the first and second molars are reduced in size and their shape is simplified.A supernumerary tooth may be present in front of the upper or lower molars.Macrodontia of the first molar may occur (Griineberg 1965(Griineberg ,1966;;Sofaer I 969ab, 1979;Mill e r 1978).Disturbance of the epithelio-mesenchymal interactions in the tabby mouse has been suggested to explain the developmental defects of various epithelial derivatives including the dentition (Miller 1978).The tabby mouse, therefore, represents a valuable model to analyse some of the mechanisms involved in abnormal development of teeth and of other epithelial-derived structures.
Basic postnatal and prenatal investigations on the tabby teeth were performed some 20-30 years ago by Griineberg (1965Griineberg ( , 1966)), Sofaer (l969ab, 1975Sofaer (l969ab, , 1979) ) and Mi II er (1978).As a prerequisite for future developmental studies focusing on the aetiopathogenesis of tooth defects in tabby mice, the characteristics of their postnatal dentition were determined in a tabby stock available commercially at present.Qualitative and quantitative tooth parameters were determined and compared in various phenotypes segregated from a stock of the tabby mouse.These results were confronted with findings of a parallel study in the common laboratory mouse (ICR stock) and with earlier literature on the pattern of tabby teeth.This knowledge will provide essential background for the design, completion and interpretation of future tooth developmental studies in the tabby mutants.

Mice
Three groups of mice were investigated: tabby mutant males and females, their non-tabby (wild-type) counterparts and ICR mice (Table 1-4).The tabby phenotypes of the mice were determined according to external anatomical criteria (Green 198Ia).

I. Tabby mutant mice
The animals were segregated from the inbred tabby line B6CBACa-AW-J/A-TalO (the breeder pairs were purchased from the Jackson Laboratory, U.S.A): (TalTa) homozygous females XIX (TalO) hemizygous females XlO (TaI+) heterozygous females XIX (TalO) hemizygous males XIY These animals were successors of inbred crossings between tabby females (TalTa.TalO, or TaI+) with tabby (TalO) or wild type (+/0) males.The Ta-homozygous females and Ta-hemizygous males and females exhibited an identical phenotype (except for reproductive organs).For this reason the TalTa and TalO females were joined in a unit group indicated as Ta-homozygous/hemizygous females.

Control non-mutant mice
Control mice were generated by inbreeding of wild-type (phenotypically normal.non-tabby) brothers and sisters of mutant animals.The male and female successors were harvested as representatives of the genetic background for Ta allele: (+/+) homozygous female XIX (+/0) hemizygous female XlO (+/0) hemizygous male XIY All these animals exhibited a normal.non-tabby phenotype and are indicated as wild-type (WT) males or females in the text.The females (+/+) and (+/0).that could not be distinguished anatomically from external features.were combined in a unit group of WT females.

ICRmice
The specimens were obtained from random-bred crossings between the ICR (Velaz.C.R.) males and females.

Material preparation
Ten animals from each genotype/phenotype subgroup (Table 1-4) were collected from 1995 till 1997.Only one male and/or female of a given phenotype was harvested from each litter during postnatal days 24-26 (day of birth = day 0).At that time, functional occlusion of the first and second molars should have been achieved (Cohn 1957).The specimens were killed by ether inhalation and weighed.Where necessary, the identification of males or females was confirmed by dissection of internal genital organs.The heads of animals were fixed in 96% ethanol and the lower jaw isolated by careful dissection under a stereo-loupe (Zeiss).The upper and lower teeth were counter-stained with hematoxylin.

Morphological evaluation
Identification of the cheek teeth as the first (M 1), second (M2) and third (M3) molar, and evaluation of their cusp pattern (Plate I., Fig. 1A, Plate II., Fig. 10) were made according to morphological criteria (0 a u n t 1955).Where alteration of crown shape prevented explicit identification, the cheek teeth were identified as the first, second and third tooth in the mesio-distal sequence (Plate II., Fig. IF).
Variable features of MI (Orlineberg 1965;Sofaer 1969c) were also taken into account: the small cusp near the base of cusp I, the extra cusp or a ridge between B2 and B3 cusps  (Sofaer 1969c).
In the second upper molar, the presence of a "rampart'; (formed by the cusps B I and L1 interconnected by a transversal ridge -Orli n e berg 1966) was investigated (Plate I., Fig. lA,C).

Morphometry
The crown size of the cheek teeth (Tab.1-4, Fig. 2 and 3) was measured using a stereo-loupe equipped with an ocular micrometer at 42x magnification.The maximum mesio-distal length and the maximum buccolingual width were measured in each crown parallel to the occlusal plane.In addition, the maximum total length was determined for M I +M2 (tooth I +2).For information (without further statistical processing), third molars were measured in which the largest part of the crown had already emerged into the oral cavity (Table 1-4).All measurements were standardised and the subjective error of measurement was determined to be nonsignificant.

Statistics
The results were compared between the right and left contralateral dental quadrants in each subgroup of males or females of an identical phenotype using the ttest (paired two sample for means).Except for specific cases.where a significant right/left difference was found, the right and left \'alues were combined in one sample group for further testing: Sex differences in tooth size were evaluated between males and females of identical phenotype by means of the two sample t-test.This test was also applied to comparison of the tooth size between different subgroups of mice and for evaluation of differences in body weight.The t-test was also employed to test any eventual influence of mother phenotype (Ta-heterozygous or Tahomozygous/hemizygous) on tooth size in their Tahomozygous/hemizygous daughters.
The statistical significance was determined with respect to the usual limits P<0.05 and P<O.OI.

Incisors
In both ICR and Taheterozygous mice, all incisors were present, well fonned and in correct occlusion.Among WT mice, an inclination was observed for one or both contra-lateral incisors in the upper jaw of 10% of males and 20% of females.In the latter group, an associated inclination of the lower incisors was also apparent.
An incisor was absent in 70% ofTa-hemizygous males: The right lower and upper incisor was absent in 40% and 30% of animals.respectively.On the left side, the incisor was missing in 10% of upper or lower jaws.In Ta-homozygous/hemizygous females, the lower incisor was absent in 30% of right and in 20% of left quadrants, and the upper one was absent in 10% of left quadrants.Both upper or lower contra-lateral incisors were absent in 10% of males or females, respectively.
Absence of an incisor was associated both with malocclusion and with inclination of the remaining teeth in the incisor region: in some cases, the incisor exhibited a smaller diameter (not evaluated quantitatively).

Cheek teeth Tooth number
In all ICR, WT and Taheterozygous mice, the molar teeth could be identified on the basis of morphological criteria (Gaunt 1955), (Plate I.-III., Fig. I).In ICR mice, eruption of the third molar into the oral cavity was observed in 80% of upper quadrants in males or females, and in 70% and 80% of lower quadrants in males and females, respectively.In WT mice, the third molar was exposed to the oral cavity in 55% of male and in 45% of female upper quadrants, and in 40% and 35% of lower quadrants in males and females, respectively.In the Ta-heterozygous females, the third molar could be detected in the oral cavity aspect in 90% of upper and 75% of lower dental quadrants.

b) Sex differences
In comparison with corresponding females, the ICR males exhibited longer upper M2 teeth (P<0.05) and a larger total length for M 1 +M2 teeth in both the upper and lower jaws (P<O.OI); a wider upper M2 tooth (P<0.05) was found in WT males.We found no differences in tooth sizes between tabby males and females (Tab.1-4, Fig. 2 and 3).

c) ICR and WT mice
The tooth dimensions were significantly smaller (P<O.OI) in WT specimens than in the ICR strain, except for a less significant difference (P<O.05) in the width of the left upper MI in males and M2 in females.The width of the upper M2 in males did not differ (Tab. 1 and 2, Fig. 2 and 3).

d) Various phenotypes of the tabby mice
When compared to the WT males, the Ta-hemizygous males possessed significantly smaller teeth: the lower second cheek tooth was shorter (P<0.05) and all remaining parameters were smaller (P<O.OI).Similarly, all tooth parameters were significantly smaller (P<O.O I) in Ta-homozygouslhemizygous than in WT females except for the length of the lower second cheek tooth.In contrast, the Ta-heterozygous females did not differ frqm the WT females except for the shorter upper M 1 (P<0.05)and shorter upper and lower M2 (P<O.Ol), (Tab.2-4, Fig. 2 and 3).
All morphometric parameters were significantly smaller (P<O.OI) in teeth from the Tahomozygouslhemizygous than from Ta-heterozygous females, except for the length of the upper M2 and the lower second cheek tooth (Tab.3 and 4, Fig. 2 and 3).
In the lower quadrants, the most mesially situated cheek tooth was longer than its distal neighbour in 50% ofTa-hemizygous males and in 45% ofTa-homozygouslhernizygous females.
e) The same phenotype offsprings of phenotypically different mothers Two samples of Ta-homozygouslhemizygous females were ranked according to their Ta-heterozygous or Ta-homozygouslhemizygous mothers.Analysis of the tooth size in these two samples showed a difference related only to the width of the lower M2 (P<0.05):teeth were wider in daughters of Ta-heterozygous mothers.

Body weight
The mean body weight of the ICR males or females (19.2 g or 16.7 g, respectively) was two times higher than in all remaining groups of the inbred mice.Among the inbred mice, there was no significant difference in weight between either the WT males and Ta-hemizygous males (9.73 g and 8.04 g. respectively), or between WT females and Ta-homozygouslhemizygous females (8.53 g and 7.59 g, respectively).The only significantly higher body weight was found in Ta-heterozygous females (11.4 g), when compared to ei ther Tahomozygouslhemizygous females (7.59 g) -P<O.O I, or WT females (8.53 g) -P<0.05.

Discussion
In contrast to the XlO condition in humans (Turner's syndrome), the x/a mice are anatomically normal and fertile (Ashworth eta\. 1991;Omoe andEndo 1994).Forthis reason both XIX and XlO mice could be used in breeding and their offspring collected in the tabby as well as in non-tabby (wild type) groups.
In the present study, a parallel evaluation of tooth morphology and size was performed in a common random-bred laboratory mouse -ICR stock which has been used for a recent revision of tooth morphogenesis in normal mouse embryos (Lesot et al. 1996;Peterkova et al. 1996;Tureckova et al. 1996;Viriot et al. 1997).It is known that non-inbred mice are generally more robust than mice of an inbred strain (Green 1981 b).Indeed, the body weight was two times higher in the random-bred ICR mice than in all the remaining groups of inbred animals (WT, Ta-homozygous/hemizygous, Taheterozygous).The bigger size of the whole body can explain the existence of significantly larger (although morphologically similar) teeth in the ICR strain, when compared to those in non-mutant (WT) controls of the tabby mice.
The anomalies in the dentition of the tabby mice and some aspects of their tooth development have been described previously by Grtineberg (1965Grtineberg ( , 1966)), Sofaer (1969ab, 1975Sofaer (1969ab, , 1979) ) and Miller (1978).Miller (1978) found a higher frequency of incisor anomalies in the upper jaw in Ta-hemizygous males, whilst S ofaer (1969) reported the lower jaw to be more affected.We found nearly the same frequency of missing incisors in both jaws in Ta-hemizygous males, with a predominance on the right side; the Ta-homozygous/hemizygous females were less affected, and predominantly in the lower jaw.These differences can be explained by the fact that expression of a phenotypic feature is influenced by the genetic background on which a mutant gene finds itself.The stock backgrounds differ in their ability to favour the appearance of tooth variability, or anomaly induced by a mutant gene (Grtineberg 1965;Sofaer 1969bc, 1979Sofaer and MacLean 1970).In the present study, the stock of the tabby mice B6CBACa-A W -JI A-TalO was used.The genetic background of mice carrying the Ta gene was heterogenous in Grlineberg' s study (Grlineberg 1966); the strains A andlor JV were employed by S ofaer (l969b, 1975, 1979) and the strain C3Hf was used by Miller (1978).
The findings reported here illustrate a decrease in the crown size and reduction or even disappearance of cusps in the cheek teeth of Ta-homozygous/hemizygous animals.These results are in agreement with earlier data (Grlineberg 1966;Sofaer 1969b;Miller 1978).In contrast to Sofaer (l969b), however, the pattern of tabby dental affection was not well maintained in the present Ta-heterozygotes.Besides shape parameters, Sofaer (1975Sofaer ( ,1979) also evaluated the maximum mesio-distallength in all cheek teeth in the Taheterozygous females and the length of the first molars in control males.Compared to the present Ta-heterozygous females, Sofaer (1979) reported conspicuously shorter upper M 1, M2 and lower M 1 teeth in heterozygotes, while the length of the first molars in male controls (S ofaer 1975) was similar to our control data.A frequent difference in the present Ta-heterozygotes from their wild-type controls was the extra cusp interposed in the former group between B2 and B3, or the ridge connecting B2 with B3 cusps of the first upper molar.Grlineberg (1966) considered these features as common minor variants not specifically related to the tabby.Both the extra cusp and the ridge have been described as variations regularly present in A and BALB/c strains of mice, respectively (Grlineberg 1965).A question remains, however, as to why the manifestation of this feature was ~avoured in the heterozygous specimens?Although the "rampart" of the upper M2 situated In front of cusp 2 (Grtineberg 1966) has been considered to be a specific feature of the tabby dentition (Grlineberg 1966;Sofaer 1969ab;Miller 1978), this structure was also .found in non-tabby wild type (WT) mice and it was frequently present in ICR speCImens.
We failed to find in the present collection a macrodontia in place of the first molar, or four cheek teeth explicitly documenting the existence of a supernumerary tooth in front of the molars.The identification of a supernumerary tooth in tabby mice is complicated in case of problems with identification of the molars themselves -because they are conspicuously reduced in size, show changes in the cusp pattern and exhibit a putative absence of the third molar.Sofaer (I 969b) assumed as supernumerary the most mesial cheek tooth, whose size is smaller than the size of the tooth adjacent distally.The author himself, however, did not consider this criterion to be ideal (Sofaer 1969b).According to such a criterion, a "supernumerary" tooth was present in about one half of the lower jaw quadrants in our Tahomozygous/hemizygous specimens.Prenatal studies should help to elucidate not only the problem of identification of the supernumerary tooth, but also mechanisms involved in the aetiopathogenesis of other dental abnormalities in the tabby mice.
and WT mice In phenotypically normal ICR and WT mice, variation in tooth morphology was observed: An extra cusp was present near the base of cusp I of the upper MI in 2590 of teeth in ICR males or females and in 10% of teeth in WT females.The tips (enamel free areas) of B3 and 3 in the upper M I were connected in 70% of male and 509C of female teeth in ICR stock (Plate I., Fig.I A), and in 30% of teeth in WT males or females.The small extra cusp between Ll and B 1 appeared in 10% and 20% of the lower first molars in I CR males and females, respectively (Plate II., Fig. lD).The small cusp B I of the second upper or lower molar was variable in size in phenotypically normal mice -ICR and WT.The B I cusp was absent in 20% and 5% of the second upper molars in WT males and females, respectively.The ,,rampart" of the upper M2 was well fonned or at least suggested in 65% and 40% of teeth in ICR males and females, respectively (plate I.,Fig.IA), and in 15% of male and 10% of female teeth in WT mice.b) Ta-heterozygous mice Fig. I C):Reduction in the cusp I, a strong reduction or diminution ofLl and B I, and an absence of B3.In the place of the former cusps I, Ll and L2, a unit ridge was fonned.The cusps B2 and 2 were closer aligned, whilst the interconnection between 2 and L2 was disrupted.In the second upper molar, the B3 cusp was absent.the interconnection between 2 and L2 was suppressed and the L2 cusp moved distally.A small accessory cusp interposed between Ll and L2 (Fig.IC) or a ridge interconnecting Ll with L2 was found in most cases.The "rampart" (Fig.I C) was present in 60% of teeth.In the lower jaw, two or three teeth were present (Plate II., Fig.IF).The most mesially situated tooth exhibited variable shape showing one or more cusps on its occlusal surface.The occlusal surface of the second tooth was regularly fonned by two transversal ridges; suggesting a fusion of B2+L2 and B3+L3 cusps.The third, most distally locate d tooth (when present) possessed a transversal row of two cusps mesially (or a ridge suggesting a fusion between them); at its distal end, a single cusp or a transverse ridge was apparent (Plate II., Fig.I F).

Fig_ 2 .
Fig_ 2. Mean length and width of the upper cheek teeth of males and females in ICR and in different phenotype/genotype subgroups ofWT (wild type) and Ta (tabby) mice.The dark, grey or white column represents mean value for the respective first, second or third molars (right + left).Bar-standard deviation, mm -millimeters.
Fig. 3. Mean length and width of the lower cheek teeth of males and females in ICR and in different phenotype/genotype sub~ups of~ (wild type) and Ta (tabby) mice.The dark, grey or white column represent mean value for the first, second or third.molars (nght + le~), res~tively (in ICR, WT and Ta-heterozygous mice), or for the first, second and third cheek teeth (nght + left), respec!lvely (m Ta-homozygouslhemizygous mice).Bar __ standard deviation.mm --millimeters.

Table I
Mean length and width of the cheek teeth in maxilla and mandible of ICR males and females

Table 2
Mean length and width of the cheek teeth in maxilla and mandible of WT (wild type) males and females

Table 3
Mean length and width of the cheek teeth in maxilla and mandible of Ta (tabby) homozygouslhemizygous males and females -the tirst.secondandthirdcheek tooth.respectively.found in the cheek region in 60% of mandibles (Plate II., Fig.IF), but only 2 teeth occurred in the other specimens.The presence of one or four cheek teeth was not observed.

Table 4
Mean length and width of the cheek teeth in maxilla and mandible of mm -millimeters.SD -standard deviation.rox -maxilla.rod -mandible.L -left side.R -right side.00males.22females 1.2. 3 -the first.second and third molar.respectively