ABSTRACT
The sex-linked gene tabby, Ta (Falconer, 1953), and two autosomal mimics of tabby, crinkled (cr, linkage group XIV) (Falconer, Fraser & King, 1951; King, 1956) and downless (dl, linkage group IV) (Mouse News Letter, 1960,1966) each produce a similar mutant syndrome involving the coat and dentition of the mouse. Studies on the coats of tabby and crinkled mice point to a timed gene effect causing suppression of formation of new hair follicles between and 17 days of gestation and again from birth onwards, with a reduction in the rate of growth of the follicles that do form (Falconer et al. 1951). Associated with this is a reduction in hair calibre and a lack of differentiation of the coat into hair types (Grüneberg, 1966 b). A model to explain the timed action of the tabby gene has been proposed by Dun (1959).
The teeth of tabby and crinkled mice have been described in detail by Grüneberg (1965, 1966a), and a comparative study of the effects of two alleles of tabby, Ta and Ta°, crinkled and downless, has been made by Sofaer (1969). In all mutant homozygotes and tabby hemizygotes incisors may be reduced or absent.The first and second molars are generally reduced and their morphology is characteristic. Third molars are often absent. The dentitions of heterozygotes for each of the genes may contain normal teeth, frankly mutant teeth, and teeth combining characteristics of both the normal and mutant phenotypes. All three types of tooth may be present in the same animal. A further feature of the heterozygote dentition is the rare occurrence of an additional molar tooth. Grüneberg(.1966a) has called this phenomenon’ twinning’ and has described three categories:
Overt twinning, where there are four molars in a row instead of the usual three. The normal first molar is represented by two twin teeth, the anterior of which tends to be the smaller.
Concealed twinning, which is similar to overt twinning except that the third molar is absent. There are therefore, only three teeth in the row, as in the normal mouse, but the first two can usually be diagnosed as twins with reasonable certainty on the basis of the appearance of twins in the overt cases.
Incomplete twinning, which is recognized by the presence of additional cusps and roots, and by anteroposterior elongation and pinching in of the sides of the first molar crown. In one case described the twins had separate crowns, but there was a single root that was common to both.
Grüneberg (1966a) suspected that twinning may also take place in homozygotes and tabby hemizygotes. Examples of twinning in homozygotes and hemizygotes, including incisor twinning, have been found both in the embryological material which will be described presently, and amongst the adult dentitions examined by Sofaer (1969).
The present investigation is concerned with the development of tabby (Ta) teeth only, with particular reference to the phenomenon of twinning. There is no reason to suppose that the development of the teeth of Tac, crinkled, or downless mice is fundamentally any different, so conclusions drawn here could be applied with equal confidence to all the genes. An attempt has been made to explain dental aspects of the syndrome in the light of what is known of the development of the coat so that both tooth and hair defects can be considered in terms of the principle of ‘unity of gene action’ (Grüneberg, 1943a).
MATERIALS AND METHODS
The A strain background has been found to favour the expression of incisor abnormality in tabby hemizygotes (Grüneberg, 1965), as well as the expression of molar abnormalities in heterozygotes (Sofaer, 1969). Material for sectioning was accordingly obtained as follows. A strain males mated to A strain females provided a control group of litters. A strain males mated to homozygous tabby females provided litters of mixed heterozygotes and tabby hemizygotes. The majority of homozygous tabby mothers were from stock, but a few were the result of one or two crosses to the A strain. It was originally intended to use these latter animals exclusively, but poor fertility made this impossible. The majority of litters examined were therefore heterozygous for the A strain background, but a few were nearly homozygous. There were no obvious differences between these two types of litter.
Animals were caged one male to a maximum of three females. No suckling females were used. Matings were examined for births and females were examined for vaginal plugs between 9 and 10 a.m. Material was collected between 10 a.m. and midday. The day on which a plug was found was regarded as day zero. Litters were collected at 2-day intervals from day 13 to day 29. Eight postpartum litters were used for which plug dates were not known. Birth was then taken as the criterion of age and was taken to have occurred at 20 days. (Of the 25 post-partum litters collected for which plug dates were known, one was born on day 18, twelve on day 19, eleven on day 20, and one on day 21). The ages of litters collected before birth were checked by examination of the external features of the embryos (Grüneberg, 1943 b). All animals of the A strain litters, but heterozygotes only of the mixed litters, were checked in this way.
Tabby hemizygotes and heterozygotes of 13-day litters were separated on the basis of presence or absence of the postorbital tubercles. These are the first signs of the developing postorbital vibrissae which are very nearly always absent in hemizygotes and present in heterozygotes. There was no difficulty in separating the two types at this stage. For classification of older individuals additional criteria were adopted: the degree of eruption of body hairs; the number of supraorbital vibrissae; and in post-partum litters, the sex of the individual. Although a postorbital fibre is rarely present in tabby hemizygotes at birth, Dun (1959) found that at 5 days after birth there is invariably a small, slow growing, atypical fibre at this site. Such fibres are lost in the hair of the fully grown coat. Fibres of this type were found in the present material but were easily distinguishable from those of heterozygotes. The additional use of the other criteria at this stage made the possibility of misclassification very remote.
All individuals were classified prior to fixation after examination under a dissecting microscope. The 13- and 15-day embryos were fixed whole. Seventeenday embryos were decapitated and the heads only were fixed. The classification of these embryos was checked again after fixation and prior to further processing. Individuals of 19 days and older were decapitated and the heads were skinned before fixation. Classification of these animals could therefore not be checked subsequently. There were very few cases where classification was in doubt. These were mainly instances where a postorbital fibre was present on one side but not on the other. Animals of this type were rejected. Examination of the prepared material, in the light of what is known to occur in adult animals, provided no evidence to suggest that any misclassification had been made.
All litters were fixed in Bouin’s fluid. Litters of 19 days and older were decalcified in 5 % nitric acid. All material was embedded in paraflin wax, serially sectioned at 10 μ in the sagittal plane, and stained with haematoxylin and eosin.
A total of 127 animals from sixty-five litters were prepared and examined. The numbers of each genotype sectioned at each stage are shown in Table 1.
RESULTS
These will be considered in four sections: incisors, lower first and second molars, upper first and second molars, and third molars. The findings in the control group were comparable with those of previous workers (Gaunt, 1955, 1956, 1961 ; Cohn, 1957; Hinrichsen, 1959; Hay, 1961).
1. Incisors
Heterozygotes showed no differences from the controls and are therefore not considered here.
At 13 days there was no definite difference between the tooth rudiments of Ta and control animals except that, on average, the Ta rudiments were probably a little smaller. At 15 days a definite difference was apparent. The Ta tooth germs were obviously smaller than the controls and were barely invaginated. (Fig. 1, compare A and B).
A.Control lower incisor at 15 days. The tooth germ is in the early bell stage with early differentiation of the internal and external enamel epithelia.
B.Tabby hemizygote lower incisor at 15 days. The downgrowth of labial lamina is comparable with the control, but almost no invagination of the tooth germ has taken place.
C.Control lower incisor at 19 days. Dentine formation has started and the preameloblasts are well differentiated.
D.Tabby hemizygote lower incisor at 19 days. An example of a more or less well differentiated tooth germ of abnormal size and shape.
E.Tabby hemizygote lower incisor at 19 days. A poorly differentiated example with degenerating internal enamel epithelium and abnormal odontoblasts.
F.Tabby hemizygote lower incisor region at 19 days, showing an undifferentiated lower incisor rudiment (indicated by the arrow).
G.Tabby hemizygote lower incisor at 27 days. A poorly differentiated example which has grown and maintained its structure.
H.Tabby hemizygote lower incisor region at 27 days, showing retained remnants of degenerating dental epithelium (indicated by the arrow).Tabby tooth development. I
A.Control lower incisor at 15 days. The tooth germ is in the early bell stage with early differentiation of the internal and external enamel epithelia.
B.Tabby hemizygote lower incisor at 15 days. The downgrowth of labial lamina is comparable with the control, but almost no invagination of the tooth germ has taken place.
C.Control lower incisor at 19 days. Dentine formation has started and the preameloblasts are well differentiated.
D.Tabby hemizygote lower incisor at 19 days. An example of a more or less well differentiated tooth germ of abnormal size and shape.
E.Tabby hemizygote lower incisor at 19 days. A poorly differentiated example with degenerating internal enamel epithelium and abnormal odontoblasts.
F.Tabby hemizygote lower incisor region at 19 days, showing an undifferentiated lower incisor rudiment (indicated by the arrow).
G.Tabby hemizygote lower incisor at 27 days. A poorly differentiated example which has grown and maintained its structure.
H.Tabby hemizygote lower incisor region at 27 days, showing retained remnants of degenerating dental epithelium (indicated by the arrow).Tabby tooth development. I
There was a striking difference in intensity of abnormality between upper and lower jaws. This is consistent with what has been found in fully formed dentitions. All the upper incisor germs looked as if they would have formed teeth. By contrast there was a wide range of expression in the lowers varying from near normality to degeneration. From 19 days three distinct categories of abnormal lower incisor germ were discernible:
More or less well differentiated though variable in size and shape (Fig. 1D, compare with control C).
Poorly differentiated (Fig. 1 E), in which the internal enamel epithelium showed signs of degeneration and where the odontoblasts were abnormal. No enamel but some dentine was formed. Such partially differentiated germs increased in size up to the latest stage examined (Fig. 1G).
Undifferentiated (Fig. 1F), in which the dental epithelium showed no sign of morphodifferentiation or further histodifferentiation and appeared to be undergoing degeneration. Epithelial remnants were retained up to the latest stage examined (Fig. 1H).
Table 2 shows the relative frequencies of these categories of abnormal lower incisor germ at different stages.
The numbers of Ta lower incisor germs in three categories of abnormality observed at different stages

In the upper jaw the relatively small size of the Ta germs was maintained at all stages and was associated with delayed histodifferentiation (compare Figs. 2 A and B).
A.Control upper incisor at 17 days.
B.Tabby hemizygote upper incisor at 17 days. The bell is smaller and histodifierentiation much less advanced than in the control.
C.Control lower first and second molar germs at 17 days.
D.Control lower first molar germ at 17 days, sectioned lingually to show the normal anterior extension of dental lamina (indicated by the arrow).
E.Tabby heterozygote at 17 days with the first molar sectioned lingually. There is a large bud of dental lamina anteriorly (indicated by the arrow).
F.The same example as in E, sectioned further buccally to show the maximum diameter of m1 and m2, which are smaller than in the control.
G.Tabby hemizygote at 17 days with the first molar germ sectioned lingually. There is an anterior bud of dental lamina showing some invagination (indicated by the arrow).
H.The same example as in G, sectioned further buccally to show the maximum diameter of m1 and m2,
A.Control upper incisor at 17 days.
B.Tabby hemizygote upper incisor at 17 days. The bell is smaller and histodifierentiation much less advanced than in the control.
C.Control lower first and second molar germs at 17 days.
D.Control lower first molar germ at 17 days, sectioned lingually to show the normal anterior extension of dental lamina (indicated by the arrow).
E.Tabby heterozygote at 17 days with the first molar sectioned lingually. There is a large bud of dental lamina anteriorly (indicated by the arrow).
F.The same example as in E, sectioned further buccally to show the maximum diameter of m1 and m2, which are smaller than in the control.
G.Tabby hemizygote at 17 days with the first molar germ sectioned lingually. There is an anterior bud of dental lamina showing some invagination (indicated by the arrow).
H.The same example as in G, sectioned further buccally to show the maximum diameter of m1 and m2,
It can therefore be concluded that, in Ta animals, growth and histodifferentiation of developing incisor germs may be retarded; that in more severely affected cases, found only in the lower jaw, the internal enamel epithelium is the first tissue to suffer degeneration ; and that in the most severely affected cases there is which are smaller than in the heterozygote shown in F, and much smaller than in the control.a complete lack of differentiation and epithelial growth very nearly, if not completely, ceases.
2. Lower first and second molars
Both the heterozygote and hemizygote groups showed differences from the controls and are therefore both considered here.
At 13 days there were no detectable differences between the tooth rudiments of Ta, Ta+ and control animals. At 15 days differences became apparent. At this and subsequent stages Ta tooth germs were generally smaller than the controls and more bulbous in shape. Small size was sometimes associated with delayed histodifferentiation. Similar but less severe abnormalities were present in some heterozygotes. Examples of interaction between developing first and second molars were observed. Poor development of m1 was sometimes associated with an enlarged m2 in which differentiation was sometimes more advanced than in the control m2. However, m2 never appeared to be as advanced as m1At no stage was there any evidence of division of the first molar germ into two in either Ta+ or Ta animals.
A feature of the control animals was a small extension of the dental lamina anteriorly from the point of origin of the first molar germ, and somewhat lingually (Figs. 2D; 3C). In a few 7h+ and Ta animals there was proliferation of this extension of lamina to form an epithelial downgrowth anterior to the developing mx(Figs. 2E, G). In some of these cases a supernumerary tooth germ was formed (Fig. 3D, G) and in others the epithelial downgrowth appeared to regress (Fig. 3F; Fig. 4 A, C). There was evidence of interaction between this epithelial downgrowth and the developing m1 and m2, whether or not a supernumerary germ was formed. The presence of a potential or developing supernumerary germ was associated with a small m1 and a small m2 (Fig. 2, compare F, H with control, C; Fig. 3, compare E, H with control, A, B). Cases of degenerating epithelial downgrowths showed signs of the same interaction though to a lesser extent (compare Fig. 4A, B with Fig. 3G, H, and control, A, B).
A.Control lower first molar at 19 days.
B.The same example as in A, sectioned further buccally and further posteriorly to show the maximum diameter of m2
C.Control lower first molar at 19 days sectioned lingually to show the normal anterior extension of dental lamina at this stage (indicated by the arrow).
D.Tabby heterozygote at 19 days with mxsectioned lingually. There is a small supernumerary germ anteriorly with its own laminal connexions.
E.The same example as in D, sectioned further buccally and further posteriorly to show the maximum diameter of ms1 and m2, which are smaller and less well differentiated than in the control.
F.The same animal as shown in D and E, but the opposite side. There is considerable epithelial downgrowth anteriorly (indicated by the arrow). Comparison with the opposite side suggests that this was an unsuccessful attempt to form a supernumerary tooth germ.
G.Tabby hemizygote at 19 days, showing the maximum diameter of an anterior supernumerary germ. The first molar is sectioned rather lingually.
H.The same example as in G, sectioned further buccally and further posteriorly to show the maximum diameter of mxand m2.
A.Control lower first molar at 19 days.
B.The same example as in A, sectioned further buccally and further posteriorly to show the maximum diameter of m2
C.Control lower first molar at 19 days sectioned lingually to show the normal anterior extension of dental lamina at this stage (indicated by the arrow).
D.Tabby heterozygote at 19 days with mxsectioned lingually. There is a small supernumerary germ anteriorly with its own laminal connexions.
E.The same example as in D, sectioned further buccally and further posteriorly to show the maximum diameter of ms1 and m2, which are smaller and less well differentiated than in the control.
F.The same animal as shown in D and E, but the opposite side. There is considerable epithelial downgrowth anteriorly (indicated by the arrow). Comparison with the opposite side suggests that this was an unsuccessful attempt to form a supernumerary tooth germ.
G.Tabby hemizygote at 19 days, showing the maximum diameter of an anterior supernumerary germ. The first molar is sectioned rather lingually.
H.The same example as in G, sectioned further buccally and further posteriorly to show the maximum diameter of mxand m2.
A.Tabby hemizygote at 19 days, showing an anterior downgrowth of dental lamina which suggests an unsuccessful attempt to form a supernumerary tooth germ.
B.The same example as in A, sectioned further buccally and further posteriorly to show the maximum diameter of mxand m2.
C.Tabby heterozygote at 21 days with the first molar sectioned lingually. There is a degenerating downgrowth of dental lamina anteriorly (indicated by the arrow).
D.Tabby hemizygote at 23 days, showing the laminal connexions of an anterior supernumerary with mr sectioned lingually. Dentine formation in these two teeth is about equally advanced.
E.The same example as in D, sectioned further buccally and further posteriorly to show the maximum diameter of m2, which is much smaller and less advanced than the m2 of the opposite side (see F), and possibly would not have progressed to form a tooth.
F.The same animal as in D and E, but the opposite side showing mx and a small m2. There was no sign of an attempt to form a supernumerary tooth germ anteriorly.
G.Tabby hemizygote at 25 days, showing the maximum diameter of m1 and m2. Enamel formation in m1 is much further advanced than in m2, but m1 is much smaller than m2. more normal (e.g. Fig.
A.Tabby hemizygote at 19 days, showing an anterior downgrowth of dental lamina which suggests an unsuccessful attempt to form a supernumerary tooth germ.
B.The same example as in A, sectioned further buccally and further posteriorly to show the maximum diameter of mxand m2.
C.Tabby heterozygote at 21 days with the first molar sectioned lingually. There is a degenerating downgrowth of dental lamina anteriorly (indicated by the arrow).
D.Tabby hemizygote at 23 days, showing the laminal connexions of an anterior supernumerary with mr sectioned lingually. Dentine formation in these two teeth is about equally advanced.
E.The same example as in D, sectioned further buccally and further posteriorly to show the maximum diameter of m2, which is much smaller and less advanced than the m2 of the opposite side (see F), and possibly would not have progressed to form a tooth.
F.The same animal as in D and E, but the opposite side showing mx and a small m2. There was no sign of an attempt to form a supernumerary tooth germ anteriorly.
G.Tabby hemizygote at 25 days, showing the maximum diameter of m1 and m2. Enamel formation in m1 is much further advanced than in m2, but m1 is much smaller than m2. more normal (e.g. Fig.
Whether the most anterior germ was a first molar or a supernumerary was decided after comparison of all the molar tooth germs on that side (e.g. Fig. 3D, E; Fig. 4D, E); of the affected side with the opposite side, which was generally
3D with F; Fig. 4E with F); and of the affected animal with others at the same stage (e.g. Fig. 3D, E with control, A, B).
It was considered that a supernumerary germ could never be larger or more advanced than the m1 it preceded, although after 19 days histodifferentiation in these two teeth appeared to be about equally advanced (Fig. 3D, G; Fig. 4D). It was also considered that m1 would always be in a more advanced state of histodifferentiation than m2. However, m1 and m2 were sometimes observed to be of almost equal size, and in one case the tooth taken to be m1 on the basis of the thickness of its enamel and dentine was considerably smaller than m2 (Fig. 4G).
Table 3 shows the total numbers of developing Ta+ and Ta first molars examined at different stages, the numbers of cases where proliferation of the anterior lamina without supernumerary tooth germ formation was observed, and the numbers of cases where a supernumerary germ had become established.
3. Upper first and second molars
Abnormalities of the upper molars were less striking than those of the lowers. Just as in the lowers, there was no evidence of division of a first molar germ into two. However, only one example of what appeared to be early supernumerary development was found (Fig. 5; compare C with control, A, B). Amongst individuals of the more advanced stages there were two examples of established supernumerary teeth (Fig. 5E, compare with control, D; Fig. 5F). These three cases were all in the Ta + group. No upper supernumeraries were observed in the Ta group.
A.Control upper first molar germ at 15 days, showing the maximum concavity of the bell.
B.The same example as in A, sectioned further buccally to show the maximum height of the buccal margin of the bell (indicated by the arrow).
C.Tabby heterozygote at 15 days, showing the maximum heights of the buccal margins of two bells (indicated by the arrows).
D.Control upper first and second molar germs at 19 days.
E.Tabby heterozygote at 19 days, showing an anterior supernumerary with m1 and m2. The total anteroposterior length of these three germs is similar to that of the normal m1 and m2 in D.
F.Tabby heterozygote at 23 days, showing an anterior supernumerary with m1, m2, and the rudiment of m3.
A.Control upper first molar germ at 15 days, showing the maximum concavity of the bell.
B.The same example as in A, sectioned further buccally to show the maximum height of the buccal margin of the bell (indicated by the arrow).
C.Tabby heterozygote at 15 days, showing the maximum heights of the buccal margins of two bells (indicated by the arrows).
D.Control upper first and second molar germs at 19 days.
E.Tabby heterozygote at 19 days, showing an anterior supernumerary with m1 and m2. The total anteroposterior length of these three germs is similar to that of the normal m1 and m2 in D.
F.Tabby heterozygote at 23 days, showing an anterior supernumerary with m1, m2, and the rudiment of m3.
The ‘rampart’ of the tabby upper second molar (Grüneberg, 1965) has been regarded as a reaction to the small size of m1. Figure 6A, B shows the difference in size between normal and tabby upper first molars at 17 days. The rampart starts as an anterior outgrowth which is first noticeable at 19 days (Fig. 6D; compare with control, Fig. 6C), and which subsequently becomes bent occlusally as it increases in size and as the space between m1 and m2 closes (Fig. 6F, H ; compare with control, E, G). The attempt at compensation therefore appears to be at least partially frustrated by lack of space.
A.Control upper first molar at 17 days.
B.Tabby hemizygote upper first molar at 17 days.
C.Control upper second molar at 19 days.
D.Tabby hemizygote upper second molar at 19 days, showing the first sign of the developing rampart (indicated by the arrow).
E.Control upper second molar at 21 days.
F.Tabby hemizygote upper second molar at 21 days, showing further development of the rampart (indicated by the arrow). Histodifferentiation appears to be a little more advanced than in the control.
G.Control upper second molar at 23 days.
H.Tabby hemizygote upper second molar at 23 days, showing further development of the rampart (indicated by the arrow). Histodifferentiation is more advanced than in the control
A.Control upper first molar at 17 days.
B.Tabby hemizygote upper first molar at 17 days.
C.Control upper second molar at 19 days.
D.Tabby hemizygote upper second molar at 19 days, showing the first sign of the developing rampart (indicated by the arrow).
E.Control upper second molar at 21 days.
F.Tabby hemizygote upper second molar at 21 days, showing further development of the rampart (indicated by the arrow). Histodifferentiation appears to be a little more advanced than in the control.
G.Control upper second molar at 23 days.
H.Tabby hemizygote upper second molar at 23 days, showing further development of the rampart (indicated by the arrow). Histodifferentiation is more advanced than in the control
4. Third molars
A difference between rudiments which were presumed to be destined for regression and those which looked as if they would form teeth started to be detectable at 25 days and was definite at 27 days. The rudiments which were destined for regression did not invaginate to form bells. No cases of regression were found in the controls, though absence of lower third molars does occur in the A strain at a low frequency. No bell was formed by any of the Ta m3 rudiments at 27 and 29 days. About half the Ta m3 rudiments had formed bells at these stages. Most of the 77z+ m3 and all of the Ta+ m3 rudiments had formed bells at these stages (Fig. 7). These findings are comparable with those of Grewal (1962), who demonstrated a similar embryological basis for the absence of third molars in CBA and crooked tail mice.
Lower third molars
A.Control rudiment at 25 days.
B. Tabby hemizygote rudiment at 25 days.
C.Control at 27 days.
D.Tabby hemizygote at 27 days.
E.Tabby heterozygote at 29 days.
F.Tabby hemizygote at 29 days. Upper third molars
G.Control rudiment at 25 days.
H.Tabby hemizygote rudiment at 25 days.
I.Control at 27 days.
J.Tabby hemizygote at 27 days.
K.Tabby heterozygote at 29 days.
L.Tabby hemizygote at 29 days.
Lower third molars
A.Control rudiment at 25 days.
B. Tabby hemizygote rudiment at 25 days.
C.Control at 27 days.
D.Tabby hemizygote at 27 days.
E.Tabby heterozygote at 29 days.
F.Tabby hemizygote at 29 days. Upper third molars
G.Control rudiment at 25 days.
H.Tabby hemizygote rudiment at 25 days.
I.Control at 27 days.
J.Tabby hemizygote at 27 days.
K.Tabby heterozygote at 29 days.
L.Tabby hemizygote at 29 days.
DISCUSSION
I. General observations
In tabby hemizygotes the general effect on the developing tooth germs appeared to be one of reduced rate of growth and delayed histodifferentiation. The effect on the lower incisors was the most severe. Sometimes no tooth at all was formed, and sometimes there was an intermediate condition where some dentine but no enamel was formed. In the case of the molars there was no evidence to suggest that enamel formation is ever prevented or that a first molar is ever completely suppressed. It did seem likely that complete suppression could be the rare fate of some lower second molar germs (Fig. 4E). Regression of third molar germs was a frequent occurrence. Similar but less severe effects were observed in the molars of some heterozygotes.
‘Overt twinning’ in the lower jaw was found to be produced by the de novo development of a supernumerary tooth from an overgrowth of a normal anterior extension of dental lamina. Direct evidence for this in the upper jaw was limited, though observations here were not inconsistent with the lower jaw findings. There was no evidence of division into two of any first molar germ, either upper or lower, at any stage. As the failure of third molar rudiments to form bells was observed many times it is reasonable to assume that ‘concealed twinning’ does occur. Examples of developing supernumerary teeth were found in both upper and lower jaws of heterozygotes, but in the lower jaw only of hemizygotes.
The picture formed is therefore one of a generalized partial suppression of growth and differentiation of dental epithelium with occasional localized points of abnormal overgrowth. The greatest variation was found in the lower molars. These will now be considered in more detail in the light of what is known of the development of the coat, and what has been observed in the fully formed dentition. A diagrammatic representation of the developmental sequence of relationships of the teeth of the lower jaw and the phases of hair follicle suppression is shown in Fig. 8.
The relationship of the developmental sequence of the lower teeth to the phases of hair-follicle formation and suppression. The arrows represent the time taken for the course of development of each tooth from the appearance of a definitive epithelial bud to the first appearance of calcified dentine, observations being made every 2 days from day 13. No example of a supernumerary tooth was found at the 21-day stage. The point of the S (supernumerary) arrow has been arrived at by interpolation from 19- and 23-day examples. I — incisor.
The relationship of the developmental sequence of the lower teeth to the phases of hair-follicle formation and suppression. The arrows represent the time taken for the course of development of each tooth from the appearance of a definitive epithelial bud to the first appearance of calcified dentine, observations being made every 2 days from day 13. No example of a supernumerary tooth was found at the 21-day stage. The point of the S (supernumerary) arrow has been arrived at by interpolation from 19- and 23-day examples. I — incisor.
The first period of hair follicle suppression, from to 17 days, is just that during which the first molar develops from a small bud of epithelium to an early stage of morphodifferentiation and histodifferentiation. At 17 days, the end of this suppression phase and the beginning of the phase of follicle formation, definite signs of overgrowth of the anterior extension of dental lamina were observed (Fig. 2E, G). At 19 days, towards the end of the follicle formation phase, the overgrowth had, in some instances, developed into a tooth germ in which histodifferentiation was almost as advanced, if not equally advanced, as in the first molar posterior to it (Fig. 3D, G). Subsequently, the various stages of histodifferentiation appeared to proceed together in the supernumerary and first molar germs.
2. Stabilization of length of the tooth row
The interpretation offered for these observations is based on the premise that there is a tendency for the length of the tooth row to be stabilized. Because of its retarded growth the first molar fails to occupy all the space allotted to it. As a consequence there is overgrowth of the dental lamina to form an additional tooth germ to take up the vacant space.
Perhaps it would be better to say that, at least during the developmental phase, the dental lamina is under a growth pressure which is inhibited when normally developing tooth germs have become established. Poor growth of developing germs would then result in a lesser degree of laminal inhibition which, if below a critical level, would allow the lamina to proliferate further. Such an explanation is consistent with the hypothesis that developing organs specifically inhibit like differentiation of the surrounding tissues (Rose, 1952, 1957). Evidence in support of this hypothesis has been provided by Saetren (1956) and by Clarke & McCallion (1959a, ó).
The formation of a supernumerary tooth at the anterior end of the molar row can therefore be regarded as a positive reaction to the small size of the developing tooth row, tending to restore it to its normal length. There is good evidence that in the reverse situation a negative response can also occur, namely the reduction in size and eventual complete suppression of the third molar with increasing size of the first two (Grüneberg, 1951; Grewal, 1962; Van Valen, 1962). Van Valen (1962) also cites evidence in favour of the existence of such size interactions in a number of different developing systems.
The first obvious signs of laminal overgrowth were observed at 17 days—the end of the suppression phase and the beginning of the follicle formation phase. During the follicle formation phase rapid development of the supernumerary germ occurred. It is difficult to avoid the conclusion that this reaction was a response to relaxation of suppression. Such relaxation would no doubt affect the first molar as well as the dental lamina, and this could explain why epithelial downgrowths which failed to produce supernumerary germs were found. A sudden increase in size of the first molar germ could presumably prevent a downgrowth from developing further. However, there must be some difference in sensitivity to the suppressive influence between the laminal cells and those of the first molar germ. If there were not, no supernumerary downgrowths would develop. It is suggested that this difference in sensitivity is associated with the degree of differentiation of the two groups of cells, the less well differentiated cells of the dental lamina being more ready to react by proliferation as relaxation of suppression becomes more complete. The fact that the differentiating internal enamel epithelium is the first tissue to suffer degeneration as abnormality of the lower incisors increases, is evidence in favour of such a differential sensitivity.
The second molars, also in a less differentiated state than the first, would similarly be likely to react more readily to a relaxation of suppression. This would then be the basis of the general size interaction observed between first and second molars, especially noticeable in cases where no supernumerary was present. More specifically, it would explain the origin of the rampart of m2. The existence of these size relationships in the fully formed dentition was recognized by Grüneberg, (1965).
If this interpretation of the observations is correct, then it is basically the size of the developing first molar at and before 17 days which controls the ultimate form of the whole molar row. The final size of the first molar is not a good indication of its status at 17 days, as recovery or further suppression could take place after 17 days and before its final form is decided by the onset of hard tissue formation. A slight difference in size between left and right first molars at 17 days could result in the successful formation of a supernumerary tooth germ on one side but the suppression of a potential counterpart on the other. Thus small differences in local conditions at a critical stage of development could be responsible for formidable asymmetry in the adult dentition.
3. Incomplete twinning
‘Overt twinning’ and ‘concealed twinning’ have already been discussed but no explanation has so far been given for ‘incomplete twinning’. If the extra teeth found in the first two cases arise independently, then in the third the rare composite teeth observed must be the consequence of fusion rather than of incomplete fission. Hitchin & Morris (1966) showed that fusion of the developing incisors of the dog, or connation as they called it, is related to the persistence of dental lamina between two adjacent incisor germs. Rapid growth of adjacent germs was thought to cause the external enamel epithelium to be stripped off the persisting interdental lamina. As a result, the stellate reticulum of the two germs becomes confluent, their internal enamel epithelia come into contact, and fusion takes place. In addition to connation of two incisors of the normal series there were examples of connation of a first incisor with a supernumerary tooth. Figure 9A shows a case already illustrated in a different section (Fig. 5E). The external enamel epithelium between the supernumerary and first molar has just become separated from the underlying dental lamina. This illustration is comparable with one of those of Hitchin & Morris, though in their case separation was more extreme.
A.Tabby heterozygote at 19 days (the same example as in Fig. 5E, in a different section). The external enamel epithelium between the supernumerary and first molar germs is becoming separated from the underlying lamina (indicated by the arrow).
B.Tabby hemizygote at 21 days. Transverse section through the upper right incisor region. There are two germs with their internal enamel epithelia in intimate contact.
C.The left side of the same animal as in B, showing a single incisor germ.
D.The same example and the same side as in B, but further posteriorly to show a connexion between the pulp cavities of the two germs.
E.A fully formed composite upper right incisor from a tabby homozygote at 4 weeks of age.
F.Complete separation between a supernumerary and an upper right incisor in a tabby hemizygote, viewed from the buccal surfaces.
G.Lingual view of a composite lower right first molar from a downless homozygote.
A.Tabby heterozygote at 19 days (the same example as in Fig. 5E, in a different section). The external enamel epithelium between the supernumerary and first molar germs is becoming separated from the underlying lamina (indicated by the arrow).
B.Tabby hemizygote at 21 days. Transverse section through the upper right incisor region. There are two germs with their internal enamel epithelia in intimate contact.
C.The left side of the same animal as in B, showing a single incisor germ.
D.The same example and the same side as in B, but further posteriorly to show a connexion between the pulp cavities of the two germs.
E.A fully formed composite upper right incisor from a tabby homozygote at 4 weeks of age.
F.Complete separation between a supernumerary and an upper right incisor in a tabby hemizygote, viewed from the buccal surfaces.
G.Lingual view of a composite lower right first molar from a downless homozygote.
It seems probable that separation of the external enamel epithelium from persisting interdental lamina would not only be a function of rapid growth of adjacent tooth germs, but also of their proximity. The more tightly squeezed together the developing germs the greater the likelihood of epithelial stripping. Fusion would then be more likely to occur in the presence of a supernumerary tooth, as a greater than normal number of germs are then growing and competing for room in a restricted space.
In trials made prior to the main investigation some of the material was sectioned transversely. Figure 9B-D are of a tabby hemizygote at 21 days.
Anteriorly on the left of the upper jaw there was a single normal incisor germ (Fig. 9C). Anteriorly on the right there were two incisor germs with their internal enamel epithelia in intimate contact (Fig. 9B). Further posteriorly on the right there was a connexion between the future pulp cavities of the two germs (Fig. 9D). The anterior end of a developing incisor is the first to form, whereas the posterior end is the youngest region where proliferation continues throughout life. The case just described must therefore have started out as two separate germs which fused subsequently. An example of a fully formed upper incisor of this sort is shown in Fig. 9E. It can be appreciated that once such a tooth has been subjected to wear the nature of its origin would be obscured. Figure 9 F shows a case where separation between supernumerary and normal incisor has been maintained.
A similar argument can be used to explain the origin of composite molars with separate crowns and common roots. The crown develops before the root, so if the crowns are separate and the root common there must originally have been two germs which fused after formation of the crowns was complete. Such a case is illustrated in Fig. 9G.
Further evidence for the origin of fusion being associated with restriction of space comes from the study of artificially induced malformations. Knudsen (1965a, b, 1966a,b) made a detailed study of the dental malformations associated with exencephaly induced in mice by teratogenic agents. There were various degrees of fusion of the two incisors within each jaw, and also intermediate cases where the future pulp cavities of the two germs were separate but their stellate reticulum was confluent. Upper incisor fusion was very much more common than lower incisor fusion. Ritter (1963) induced lower incisor fusion, and fusion of the lower molars of one side with those of the other, by Jf-radiation. These mandibular fusions were associated with mandibular micrognathy. Knudsen (1966a) reported on the molar malformations of exencephalic embryos. There were amazing cases of fusion of upper molar germs with lower molar germs on the same side. All these cases of fusion appear to have been associated with a reduction in the amount of connective tissue which normally separates the individual developing tooth germs.
The occurrence of fused and supernumerary molars in a less well known laboratory rodent, the rice rat, has been described briefly by Griffiths & Shaw (1961), and by Shaw, Griffiths & Osterholtz (1963). It may well be that the basis for these anomalies is similar to that discussed here for the tabby mouse.
SUMMARY
The development of the teeth of the tabby mouse has been studied and an attempt has been made to explain aspects of the dental abnormalities in terms of a single primary effect of the mutant gene, a partial suppression of the growth and differentiation of dental epithelium. Such an explanation is consistent with the retarded growth and lack of differentiation of the coat.
ft has been postulated that the level of this suppression varies in intensity at different stages of development in parallel with the observed effects on the developing hair follicles, and that the final outcome is dependent on an interplay of the suppressive influence and interaction between the developing teeth.
‘Twinning’ of the lower molars was found to be due to the de novo development of a supernumerary tooth from a normal anterior extension of the dental lamina. Evidence for this in the upper molars was not complete, although observations here were not inconsistent with the lower jaw findings. There was no evidence of division of a first molar germ into two at any stage.
ft seems most likely that the rare composite teeth observed in cases of ‘incomplete twinning’ are produced by fusion of the supernumerary with the adjacent germ of the normal series. Direct evidence for this was found in the upper incisors, and indirect evidence was found in upper and lower molars. Supporting evidence from other sources has been cited.
RÉSUMÉ
Aspects du syndrome ‘tabby-crinkled-downless’
I. Le développement des dents ‘tabby’
Le développement des dents de souris ‘tabby’ (tigré) a été étudié et on a tenté d’expliquer divers aspects des anomalies dentaires en termes d’un effet primaire unique du gène mutant, à savoir une suppression partielle de la croissance et de la différenciation de l’épithélium dentaire. Une telle explication s’accorde avec le retard de croissance et l’absence de différenciation du pelage.
On a postulé que le niveau de cette suppression varie en intensité à différents stades du développement, parallèlement aux effets observés sur les follicules pileux en cours de développement, et que le résultat final dépend d’une réaction réciproque de l’influence suppressive et de l’interaction entre les dents en cours de développement.
On a trouvé que la duplication des molaires inférieures était due au développement ‘de novo’ d’une dent surnuméraire à partir d’une expansion antérieure normale de la lame dentaire. La réalité de ce phénomène pour les molaires supérieures n’est pas évidente, quoique les observations faites ici ne soient pas en contradiction avec les résultats obtenus pour la mâchoire inférieure, fl n’est pas évident qu’un germe molaire primaire se soit divisé en deux à un stade quelconque.
Il para ît très vraisemblable que les rares dents composites observées dans les cas de duplication incomplète soient produites par fusion du germe surnuméraire avec le germe adjacent de la série normale. Une preuve directe de ceci a été trouvée pour les incisives supérieures, et une preuve indirecte l’a été pour les molaires supérieures et inférieures. On a cité les preuves à l’appui pour d’autres origines.
Acknowledgements
I am grateful to Professor D. S. Falconer for suggesting the investigation and for his interest and valuable advice during the work, to Professor C. H. Waddington for laboratory facilities, and to the Nuffield Foundation for financial support.
REFERENCES
EXPLANATION OF FIGURES
Unless otherwise stated the left of each illustration is anterior and the right is posterior.