Absence of third molars occurs in some inbred strains of mice (CBA and A) in which these teeth are small, and in the mutant Crooked-tail (Cd/Cd) in which all the molars and the lower incisors are reduced in size. Both in CBA and in Cd/Cd mice, the teeth which will later be absent are represented by tooth germs at first. On the 6th and 5th day after birth respectively, these tooth germs are arrested in the ‘cap’ stage or a little before. They do not invaginate to form a ‘bell’ and ultimately regress. The mechanism of this regression has not been elucidated.

The normal mouse has three molars in each jaw. Of these, the third is by far the smallest. Indeed, it is not constant, and its absence in wild mice from widely separated localities has been reported by various authors (Barrett-Hamilton, 1910; Searle, 1958; Deol, 1958; Harland, 1958; Herold & Zimmermann, 1960; Berry, unpublished). Absence of third molars occurs in varying frequencies in most mouse populations which have been adequately sampled, and usually the upper rather than the lower third molars are more strongly affected. Absence of third molars (predominantly in the lower jaw) occurs frequently (about 18 per cent.) in the CBA/Gr inbred strain of mice (Grüneberg, 1951) and less commonly (about 3 per cent.) in the A/Gr strain (Searle, 1954a), but it is virtually absent from the C57BL/Gr strain of mice. These inter-strain differences are evidence that the condition is, to some extent at least, under genetical control. A genetic analysis carried out by Grüneberg (1951, 1952) showed that absence of third molars is a quasi-continuous character, i.e. underlying the discontinuous variant ‘absence of third molars’ is a continuous variation involving the size of the third molars when present. If the size of the third molars falls below a certain threshold, they tend to be absent altogether. The difference in third molar size between the strains CBA and C57BL, like most metrical characters, is determined by multiple genes with additive effects.

Whereas the genetic basis of the condition is thus fairly well understood, no information is available concerning its mode of development: in the case of a tooth which is absent later in life, is there no tooth germ at all, or is there a tooth germ which is arrested in development or which regresses completely? These and related questions have been investigated in the CBA and C57BL strains and in crosses derived from them. The analysis is complicated by the fact that affected animals tend to occur in ‘bunches’ in certain litters, presumably as the result of precipitating causes which affect the litter as a whole. It is thus necessary to sample a number of litters before one can be reasonably certain that, at a given stage in development, the anomaly is not yet present. This difficulty is almost absent in the case of the mutant Crooked-tail (Cd/Cd’, Morgan, 1954); in this mutant, the lower incisors are much reduced, a variant which will not be further considered in this paper; however, Grüneberg (unpublished) discovered that such animals, in our present stock, nearly always lack several of the third molars. To broaden the inquiry, the embryology of absence of third molars was also investigated in that mutant.

The development of the third molar was studied in 160 mice from the CBA/Gr strain ranging in age from birth to 18 days. As the affected animals tend to occur in ‘bunches’, several litters had to be examined at certain critical stages before it could be said that the anomaly had not yet made its appearance. As the development of the tooth takes place almost entirely in postnatal life, no embryos have been examined. The controls (32 mice) were taken from the C57BL/Gr strain and from C57BL×CBA crosses (F1 animals). From the Crooked stock 12 Cd/Cd mice, along with 12 normal (10+/+; 2 Cd/+) litter mates, were used. The details of the material are given in Table 1.

Table 1.

Number of mice used in histological study

Number of mice used in histological study
Number of mice used in histological study

For histological study, the heads were fixed in Bouin’ s fluid and decalcified in 3 per cent, nitric acid in 70 per cent, alcohol. After neutralization with 5 per cent, sodium sulphate they were washed in running water overnight and then dehydrated and embedded in celloidin and paraffin. The part of the skull with the third molars was then sectioned in the transverse plane at 10 or 12’. The sections were stained with Ehrlich’ s haematoxylin and eosin.

The incidence of the anomaly in the Crooked stock was studied in 34 alizarin preparations (16 Cd/Cd, 13 Cd/+, 5 +/+) and 81 papain preparations (14 Cd/Cd, 13 Cd/+, 54 +/+).

The left molars from both jaws were measured by means of a measuring microscope in 25 mice from each of the three inbred strains A, C57BL, and CBA. Each tooth was measured antero-posteriorly as well as bucco-lingually (mean of two measurements in each case). Similar measurements were also made on 10 Cd/Cd, 7 Cd/+, and 10 +/+ mice, except that in Cd/Cd mice teeth from both sides were measured.

Measurements of molars in Cd and inbred mice

The 30 Cd/Cd mice examined in the form of alizarin-stained and papainmacerated preparations all had at least one third molar missing, while their 59 normal and 26 Cd/+ litter mates had all teeth present. The majority of the homozygotes had no third molars at all, and there were only three animals with just one tooth missing (Table 2). Altogether the 30 Cd/Cd mice had only 18 third molars between them. The incidence of the anomaly is thus much higher than in the CBA strain. Another important difference is that, whereas the upper and lower jaws are about equally affected in Cd/Cd mice, in the CBA strain the anomaly is far commoner in the lower jaw. Unlike the CBA strain, there is a (just significant) difference between the sexes, ♀ ♀ being rather less severely affected than ♂♂ there is no difference between right and left.

Table 2.

Absence of third molars in Cd/Cd, Cd/+ and +/+ mice (+ = present’, — = absent)

Absence of third molars in Cd/Cd, Cd/+ and +/+ mice (+ = present’, — = absent)
Absence of third molars in Cd/Cd, Cd/+ and +/+ mice (+ = present’, — = absent)

Dental measurements of the three inbred strains, of the Cd stock, and of a few animals with pituitary dwarfism (dw/dw’, Snell, 1929) and with pygmy (pg/pg;King, 1950) are given in detail in Table 3, together with their standard deviations. As a measure of the area of the crown, the product of the two linear measurements is given in Table 4.

Table 3.

Mean bucco-lingual and antero-posterior diameters (mm.) and their standard deviations of the molars in different types of mice studied

Mean bucco-lingual and antero-posterior diameters (mm.) and their standard deviations of the molars in different types of mice studied
Mean bucco-lingual and antero-posterior diameters (mm.) and their standard deviations of the molars in different types of mice studied
Table 4.

Comparison of the projection area (in mm.2) of the molar crowns of the left side

Comparison of the projection area (in mm.2) of the molar crowns of the left side
Comparison of the projection area (in mm.2) of the molar crowns of the left side

The results for the three inbred strains are in general agreement with the findings of Grüneberg (1951) and Searle (1954a), except that in the present material there is a greater fluctuation of tooth size in the C57BL strain. This may be due to accidents Of sampling, since only 25 animals were used in the present study.

The Cd gene clearly reduces the size of all the molars. The effect is comparatively slight in the heterozygotes, but severe in the homozygotes. The reduction is proportionately greatest in the third molars which, presumably for this reason, tend to be absent in Cd/Cd, but not in Cd/+ mice. It will be remembered that in CBA and A mice, the reduction is confined to the third molars; indeed, in CBA mice the first molars are larger than normal.

The general reduction of molar size in Cd/Cd mice is probably at least in part accounted for by the fact that they are much smaller at birth and remain so ever after. It has been shown by Deol & Truslove (1957) that a reduction of birth weight in C57BL mice leads also to a diminution of the size of the teeth. Similarly, in pygmy (pg/pg) mice (Tables 3 and 4) all the molars are somewhat smaller than in normals. In pituitary dwarfism (dw/dw, Tables 3 and 4), by contrast, the molars are of practically normal size. This may be the exception which proves the rule: there is some evidence that the retardation of growth in these animals does not start until the critical phase for the growth of the teeth is over (de Beer & Grüneberg, 1940). Whereas it is thus clear that some, at least, of the reduction in size of the Cd/Cd molars is a consequence of their small size as such, this is probably not the whole story. The severe involvement of the lower incisors mentioned in the introduction strongly suggests that there is also a specific effect on the teeth themselves.

The development of the anomaly in CBA and Cd/Cd mice

In the following account of the development of the normal and abnormal third molars the terminology used is that of Noyes, Schour & Noyes (1948). The upper and lower teeth are dealt with as one group, for the differences between them are trivial.

In the new-born CBA mouse the third molar is in the early initiation stage (chemodifferentiation stage of Huxley, 1932) of development. A few cells in the basal layer of the dental lamina have become odontogenic and their multiplication has produced a slight thickening of the lamina. The tooth enters the ‘proliferation’ stage soon after birth, when the odontogenic cells divide rapidly to form the enamel organ. At the age of about 2 days the enamel organ has the appearance of a bud (‘bud’ stage). By the 5th day (Plate 1, figs. A, B) the continued activity of the odontogenic cells has enlarged the enamel organ greatly in relation to the dental lamina, and it looks like an inverted saucer (‘cap’ stage). Two distinct layers of cells can now be seen in the enamel organ : the layer forming the outer surface of the saucer is the outer enamel epithelium, and the one forming the inner surface the inner enamel epithelium.

Up to the age of 5 days the tooth germs of all four third molars are present in all CBA mice, and there is no evidence of any major abnormality in any of them. If tooth germs were absent at this stage we should have found about 13 affected animals among the 74 that were sectioned (Table 1), for the incidence of the anomaly in the CBA strain is about 18 per cent. It is thus very unlikely that it has been missed by chance. Nor is it likely that it has been missed on account of ‘bunching’, as these 74 animals came from 17 litters.

The first sign of abnormality in some CBA teeth appears on the 6th day (Plate 1, figs. C, D). The saucer-shaped enamel organ has now deepened and taken the shape of a bell (early ‘bell’ stage). The concavity enclosed by the inner epithelium has begun to be filled with mesenchyme cells. In some teeth, however, the enamel organ has stopped growing, and, what is more, has actually reverted to the ‘bud’ stage. This reversion is accompanied by a reduction in its size, and also in that of the dental lamina. The result is that the abnormal tooth at 6 days is less differentiated than the normal tooth at 5 days. The cells of the enamel epithelium appear to be normal on the whole, except that there is much less mitotic activity in the organ, and pycnosis occasionally occurs. The normal teeth in CBA mice at this stage do not differ from those in C57BL mice except in size (see below). The fact that the percentage of mice with abnormal teeth at this stage is almost the same as that of animals with missing third molars later on (about 19 and 18 per cent, respectively) indicates that we are dealing with the same entity in both cases. This assumption is further supported by the ‘bunching’ of the 4 abnormal animals: they were found in 2 litters out of the 5 examined (Table 1).

At the age of 7 days (Plate 1, figs. E, F) the space between the outer and inner enamel epithelia of the normal tooth has enlarged and the cavity enclosed by the enamel organ has deepened (late ‘bell’ stage). The mesenchymal tissue in this cavity has now developed into the dental papilla. In the abnormal teeth, on the contrary, the enamel organ has regressed still farther and is now no more than a thickening in the dental lamina, or at most a small ‘bud’ . The incidence of affected animals (3 out of 23) is in reasonable agreement with expectation.

At the age of 8 days (Plate 2, figs. G, H) the normal tooth has entered the histodifferentiation stage. The cells of the inner enamel epithelium have become columnar and are now called ameloblasts. Under the organizing influence of these some of the mesenchymal cells of the dental papilla change into odontoblasts (Glasstone, 1936, 1939). The formation of the dentino-enamel junction is also under way. In the abnormal teeth the enamel organ has completely disappeared. The dental lamina is greatly reduced and the few cells that remain in it show no mitotic activity. The number of affected mice (2 out of 23) is less than expected; however, the difference is not statistically significant, and both affected animals were found in one litter (out of the 4 sectioned).

In older stages (Plate 2, figs. I, K) the development of the teeth in unaffected CBA mice continues to proceed normally and resembles that in the C57BL strain in every respect. In affected animals the sites of teeth which have regressed can be identified by the reduced dental lamina, which can be seen even in 18-day-old mice, the oldest available.

In addition to the major changes which occur in those CBA third molars which undergo regression, the third molars which persist and complete their development are smaller than those of C57BL mice. This difference is clearly present at the 6-day stage when it can easily be seen in projection drawings of sections. It is almost certainly already present at the 5-day stage, but has not been seen at earlier stages. Needless to say, a small difference is not excluded; but accurate measurements would be very difficult to carry out and have not been attempted.

The development of the anomaly in Cd/Cd mice (Plate 2, figs. L, M) takes place in the same manner as in the CBA strain, except for minor differences. Cd/Cd mice are slightly behind their normal litter mates in the development of the third molars. At the age of 3 days (the earliest available) in normal mice of that stock the enamel organ has reached the ‘bud’ stage, while in Cd/Cd mice it is still in the early ‘bud’ stage. This difference persists up to the age of 8 days (the latest available) in those teeth which do not start regressing. The regression begins earlier in Cd/Cd, and lasts longer: beginning on the 5th day instead of the 6th as in CBA mice, it is not yet complete on the 8th day, when a small enamel organ (usually a small ‘bud’ ) can still be seen.

Both in CBA and in Cd/Cd mice, third molars which will later be absent are at first represented by tooth germs. These reach the ‘cap’ stage, but do not invaginate to form a ‘bell’ . In the absence of a dental papilla, further growth and development evidently cannot take place, and the tooth germ undergoes regression. The proliferative stage is thus not followed by morphogenesis nor, of course, by histodifferentiation.

As discussed in more detail by Griineberg (1951), there is a relationship between the size of third molars and their tendency to be absent. In strains in which these teeth are large (such as C57BL), they are always present; in strains in which the teeth are small (such as CBA and A), they tend to be absent. Litter mates tend to have third molars of similar size, but successive litters of the same (inbred) parents may differ considerably from each other. These striking intralitter correlations are due to environmental factors which act on the litter as a whole, lactational performance of the mother probably being the most important single factor responsible (Searle, 1954 b, c). In strains with large third molars, these ups and downs from litter to litter can be detected by measurement, but have no other known effects. In a strain with small third molars like CBA, a poor lactation leads to a further lowering of tooth size and often a whole ‘bunch’ of abnormal young in a litter. On the basis of these observations, Griineberg (1951) concluded that the basic factor underlying absence of third molars is the size of the tooth germ at some critical stage in development : if the tooth germ exceeds a certain minimum size, a tooth will be formed; if it falls below the threshold, the tooth will tend to be absent altogether.

The observations reported in this paper are compatible with Grüneberg’ s hypothesis. A reduction in size of the third molars is present in CBA mice at the 6-day stage, at which time some teeth take the path towards ultimate regression. This reduction in size is very probably present on the 5th day or even earlier and, if great enough, might tend to arrest the enamel organ at the ‘cap’ stage and lead to its eventual absorption.

However, the observations are also compatible with a different interpretation of the facts. Supposing a more specific mechanism ‘damages’ the tooth germ, then in some instances the ‘damage’ might be sufficient to lead to the regression of the tooth whereas in others it might merely make the tooth smaller (Schour & Massler, 1940). If so, the reduction in size is only the by-product of a specific lesion affecting the teeth, and absence of third molars would not simply be a threshold effect depending solely on the size of the tooth germ.

On balance, the threshold hypothesis appears more probable, as variations in lactational performance cause fluctuations in the size of the third molars in all stocks of mice whatever the mean size of these teeth in the stock may be. Lactational deficiency only leads to regression, however, in strains in which the third molars are normally small.

In the case of the CBA strain, Griineberg (1951) considered the possibility that the molars might be competing for a limited amount of dental lamina: the first molars being larger, the third molars smaller than normal. This suggestion is not supported by the present observations. The reduction in size of the CBA third molars does not seem to be present from the start, but apparently arises on the 5th day or possibly a little earlier. In Cd/+ and Cd/Cd mice, all the molars are reduced in size though the third one rather more than the others.

The histological preparations give no clear clue to the mechanism of regression of the third molars. On the 6th day, when the anomaly first makes its appearance, there is a distinct fall in the number of mitoses in the enamel epithelium; but there is no reason why this should lead to regression rather than to a mere arrest of growth. The number of pycnotic cells seen in the epithelium is hardly sufficient to account for it. There is no evidence that cells of the enamel organ migrate or revert to less specialized forms.

Presumably the ultimate cause of regression of the tooth is chemical in nature. Pourtois (1961) has recently made a very detailed study of the histochemistry of the teeth of mouse embryos of 10-19 days. An extension of this work to the postnatal period, and to abnormal development, may prove rewarding.

Le développement d’ une anomalie dentaire héréditaire chez la souris

Il arrive que les troisièmes molaires manquent chez certaines races sélectionnées de souris (CBA et A) chez lesquelles les dents sont petites, et chez le mutant ‘Crooked-tail’ (Cd/Cd) dont toutes les molaires et les incisives inférieures ont une taille réduite. Chez les souris CBA comme chez les Cd/Cd, les dents qui sont destinées à disparaître sont représentées d’ abord par des germes dentaires. Ceux-ci s’ arrêtent de croître respectivement le 6e jour et le 5e jour après la naissance, au stade de la ‘coiffe’ ou un peu avant. Ils ne s’ invaginent pas pour former des ‘cloches’ et finissent par régresser. Le mécanisme de cette régression n’ est pas connu.

I am deeply indebted to Professor H. Griineberg, F.R.S., who suggested this investigation and who interested himself in the work throughout; Professor Griineberg also kindly lent me the alizarin and papain preparations on which the dental measurements were carried out. I wish to express my thanks to Professor M. A. Rushton, who read the manuscript, and to Drs. M. S. Deol and Gillian M. Truslove for help in various ways. The photomicrographs were made by Miss June Denny, to whom I wish to express my appreciation.

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Photomicrographs of tooth germs of third molars of CBA (figs. A-K) and Crooked-tail (CdI Cd) mice (figs. L, M). Bouin fixation. Embedding by Peterfi’ s method. Sections 10 µ, H. & E. × 120.

Plate 1

Figs. A and B. Left upper and lower molars (‘cap’ stage), 5-day mouse, both normal (G 155)

Fig. C. Right lower molar (early ‘bell’ stage), 6-day mouse, normal (G 148).

Fig. D. Left lower molar of same mouse, abnormal.

Fig. E. Left lower molar (‘bell’ stage) of a 7-day mouse, normal (G 157).

Fig. F. Right lower molar of a 7-day mouse, abnormal (G 215).

Plate 1

Figs. A and B. Left upper and lower molars (‘cap’ stage), 5-day mouse, both normal (G 155)

Fig. C. Right lower molar (early ‘bell’ stage), 6-day mouse, normal (G 148).

Fig. D. Left lower molar of same mouse, abnormal.

Fig. E. Left lower molar (‘bell’ stage) of a 7-day mouse, normal (G 157).

Fig. F. Right lower molar of a 7-day mouse, abnormal (G 215).

Plate 2

Fig. G. Left lower molar of an 8-day mouse (histodifferentiation stage), normal (G 107).

Fig. H. Right lower molar of same mouse, abnormal.

Fig. I. Left upper molar of a 17-day mouse, normal (G 101).

Fig. K. Region of left upper molar of a 17-day mouse, abnormal; remnant of dental lamina indicated by arrow (G 100).

Fig. L. Left lower third molar of a Crooked-tail (Cd/ Cd) mouse, 8 days old; normal tooth (G 312).

Fig. M. Left lower third molar of a Crooked-tail (Cd/Cd) mouse, 8 days old; abnormal tooth germ (G 322).

Plate 2

Fig. G. Left lower molar of an 8-day mouse (histodifferentiation stage), normal (G 107).

Fig. H. Right lower molar of same mouse, abnormal.

Fig. I. Left upper molar of a 17-day mouse, normal (G 101).

Fig. K. Region of left upper molar of a 17-day mouse, abnormal; remnant of dental lamina indicated by arrow (G 100).

Fig. L. Left lower third molar of a Crooked-tail (Cd/ Cd) mouse, 8 days old; normal tooth (G 312).

Fig. M. Left lower third molar of a Crooked-tail (Cd/Cd) mouse, 8 days old; abnormal tooth germ (G 322).