aspects of the tabby—crinkled—downless syndrome · the teeth of tabby and crinkled mice have...

/ . Embryo!. exp. Morph. Vol. 22, 2, pp. 181-205, September 1969

Printed in Great Britain

Aspects of the tabby—crinkled—downless syndrome

I. The development of tabby teeth

By J. A. SOFAER1

The Institute of Animal Genetics, West Mains Road, Edinburgh

The sex-linked gene tabby, Ta (Falconer, 1953), and two autosomal mimics oftabby, 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 denti-tion of the mouse. Studies on the coats of tabby and crinkled mice point to atimed gene effect causing suppression of formation of new hair follicles between12£ and 17 days of gestation and again from birth onwards, with a reduction inthe rate of growth of the follicles that do form (Falconer et al. 1951). Associatedwith this is a reduction in hair calibre and a lack of differentiation of the coatinto hair types (Griineberg, 1966&). A model to explain the timed action of thetabby gene has been proposed by Dun (1959).

The teeth of tabby and crinkled mice have been described in detail by Griine-berg (1965, 1966 a), and a comparative study of the effects of two alleles oftabby, Ta and Ta°, crinkled and downless, has been made by Sofaer (1969). In allmutant homozygotes and tabby hemizygotes incisors may be reduced or absent.The first and second molars are generally reduced and their morphology ischaracteristic. Third molars are often absent. The dentitions of heterozygotesfor each of the genes may contain normal teeth, frankly mutant teeth, and teethcombining characteristics of both the normal and mutant phenotypes. All threetypes of tooth may be present in the same animal. A further feature of the hetero-zygote dentition is the rare occurrence of an additional molar tooth. Griineberg(.1966 a) has called this phenomenon' twinning' and has described three categories:

(i) Overt twinning, where there are four molars in a row instead of the usualthree. The normal first molar is represented by two twin teeth, the anterior ofwhich tends to be the smaller.

(ii) Concealed twinning, which is similar to overt twinning except that thethird molar is absent. There are therefore, only three teeth in the row, as in thenormal mouse, but the first two can usually be diagnosed as twins with reasonablecertainty on the basis of the appearance of twins in the overt cases.

(iii) Incomplete twinning, which is recognized by the presence of additional1 Author's address: National Institute of Dental Research, National Institutes of Health,

Bethesda, Maryland 20014, U.S.A.

182 J. A. SOFAER

cusps and roots, and by anteroposterior elongation and pinching in of the sidesof the first molar crown. In one case described the twins had separate crowns,but there was a single root that was common to both.

Griineberg (1966a) suspected that twinning may also take place in homo-zygotes and tabby hemizygotes. Examples of twinning in homozygotes andhemizygotes, including incisor twinning, have been found both in the embryo-logical material which will be described presently, and amongst the adultdentitions examined by Sofaer (1969).

The present investigation is concerned with the development of tabby (7a)teeth only, with particular reference to the phenomenon of twinning. There is noreason to suppose that the development of the teeth of Tac, crinkled, or downlessmice is fundamentally any different, so conclusions drawn here could be appliedwith equal confidence to all the genes. An attempt has been made to explaindental aspects of the syndrome in the light of what is known of the developmentof the coat so that both tooth and hair defects can be considered in terms of theprinciple of 'unity of gene action' (Griineberg, 1943a).

MATERIALS AND METHODS

The A strain background has been found to favour the expression of incisorabnormality in tabby hemizygotes (Griineberg, 1965), as well as the expressionof molar abnormalities in heterozygotes (Sofaer, 1969). Material for sectioningwas accordingly obtained as follows. A strain males mated to A strain femalesprovided a control group of litters. A strain males mated to homozygous tabbyfemales provided litters of mixed heterozygotes and tabby hemizygotes. Themajority of homozygous tabby mothers were from stock, but a few were theresult of one or two crosses to the A strain. It was originally intended to usethese latter animals exclusively, but poor fertility made this impossible. Themajority of litters examined were therefore heterozygous for the A strain back-ground, but a few were nearly homozygous. There were no obvious differencesbetween these two types of litter.

Animals were caged one male to a maximum of three females. No sucklingfemales were used. Matings were examined for births and females were examinedfor 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 post-partum litters were used for which plug dates were not known. Birth was thentaken as the criterion of age and was taken to have occurred at 20 days. (Of the25 post-partum litters collected for which plug dates were known, one was bornon day 18, twelve on day 19, eleven on day 20, and one on day 21). The ages oflitters collected before birth were checked by examination of the externalfeatures of the embryos (Griineberg, 19436). All animals of the A strain litters,but heterozygotes only of the mixed litters, were checked in this way.

Tabby tooth development. I 183

Tabby hemizygotes and heterozygotes of 13-day Jitters were separated on thebasis of presence or absence of the postorbital tubercles. These are the firstsigns of the developing postorbital vibrissae which are very nearly always absentin hemizygotes and present in heterozygotes. There was no difficulty in separa-ting the two types at this stage. For classification of older individuals additionalcriteria were adopted: the degree of eruption of body hairs; the number ofsupraorbital 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, slowgrowing, atypical fibre at this site. Such fibres are lost in the hair of the fullygrown coat. Fibres of this type were found in the present material but wereeasily distinguishable from those of heterozygotes. The additional use of theother criteria at this stage made the possibility of misclassification very remote.

Table 1. The numbers of animals of each genotype sectioned at each stage,followed in parentheses by the numbers of litters from which they were taken

Stage

13 days15 days17 days19 days21 days23 days25 days27 days29 days

A strain

5(3)5(3)5(3)5(2)5(3)2(2)2(1)3(1)

0

Genotype

Ta +

5(2)5(4)9(3)7(3)9(3)5(3)2(1)5(3)5(2)

Ta

5(2)5(3)5(3)5(3)5(3)4(2)5(3)4(2)5(2)

All individuals were classified prior to fixation after examination under adissecting microscope. The 13- and 15-day embryos were fixed whole. Seventeen-day embryos were decapitated and the heads only were fixed. The classificationof these embryos was checked again after fixation and prior to further proces-sing. Individuals of 19 days and older were decapitated and the heads wereskinned before fixation. Classification of these animals could therefore not bechecked subsequently. There were very few cases where classification was indoubt. These were mainly instances where a postorbital fibre was present on oneside but not on the other. Animals of this type were rejected. Examination of theprepared 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 de-calcified in 5 % nitric acid. All material was embedded in paraffin wax, seriallysectioned at 10 // in the sagittal plane, and stained with haematoxylin and eosin.

184 J. A. SOFAER

A total of 127 animals from sixty-five litters were prepared and examined. Thenumbers of each genotype sectioned at each stage are shown in Table 1.

RESULTS

These will be considered in four sections: incisors, lower first and secondmolars, upper first and second molars, and third molars. The findings in thecontrol 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 notconsidered here.

At 13 days there was no definite difference between the tooth rudiments ofTa and control animals except that, on average, the Ta rudiments were probablya little smaller. At 15 days a definite difference was apparent. The Ta toothgerms were obviously smaller than the controls and were barely invaginated.(Fig. 1, compare A and B).

EXPLANATION OF FIGURESLL labial lamina D dentineDL dental lamina PA pre-ameloblastsIE internal enamel epithelium E enamelEE external enamel epithelium S supernumerary tooth germ0 odontoblasts

Unless otherwise stated the left of each illustration is anterior and the right is posterior.

FIGURE 1.

A. Control lower incisor at 15 days. The tooth germ is in the early bell stage withearly differentiation of the internal and external enamel epithelia.B. Tabby hemizygote lower incisor at 15 days. The downgrowth of labial lamina iscomparable with the control, but almost no invagination of the tooth germ hastaken place.C. Control lower incisor at 19 days. Dentine formation has started and the pre-ameloblasts are well differentiated.D. Tabby hemizygote lower incisor at 19 days. An example of a more or less welldifferentiated tooth germ of abnormal size and shape.E. Tabby hemizygote lower incisor at 19 days. A poorly differentiated examplewith degenerating internal enamel epithelium and abnormal odontoblasts.F. Tabby hemizygote lower incisor region at 19 days, showing an undifferentiatedlower incisor rudiment (indicated by the arrow).G. Tabby hemizygote lower incisor at 27 days. A poorly differentiated examplewhich has grown and maintained its structure.H. Tabby hemizygote lower incisor region at 27 days, showing retained remnantsof degenerating dental epithelium (indicated by the arrow).


0-5 mm

186 J. A. SOFAER

0-5 mm


There was a striking difference in intensity of abnormality between upper andlower 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. Bycontrast there was a wide range of expression in the lowers varying from nearnormality to degeneration. From 19 days three distinct categories of abnormallower incisor germ were discernible:

(i) More or less well differentiated though variable in size and shape (Fig. 1D,compare with control C).

(ii) Poorly differentiated (Fig. 1 E), in which the internal enamel epitheliumshowed signs of degeneration and where the odontoblasts were abnormal. Noenamel but some dentine was formed. Such partially differentiated germsincreased in size up to the latest stage examined (Fig. 1G).

(iii) Undifferentiated (Fig. IF), in which the dental epithelium showed nosign of morphodifferentiation or further histodifferentiation and appeared to beundergoing degeneration. Epithelial remnants were retained up to the lateststage examined (Fig. 1H).

Table 2 shows the relative frequencies of these categories of abnormal lowerincisor germ at different stages.

In the upper jaw the relatively small size of the Ta germs was maintained atall stages and was associated with delayed histodifferentiation (compare Figs.2A and B).

It can therefore be concluded that, in Ta animals, growth and histodifferentia-tion of developing incisor germs may be retarded; that in more severely affectedcases, found only in the lower jaw, the internal enamel epithelium is the firsttissue to suffer degeneration; and that in the most severely affected cases there is

FIGURE 2

A. Control upper incisor at 17 days.B. Tabby hemizygote upper incisor at 17 days. The bell is smaller and histo-differentiation 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 thenormal anterior extension of dental lamina (indicated by the arrow).E. Tabby heterozygote at 17 days with the first molar sectioned lingually. There is alarge bud of dental lamina anteriorly (indicated by the arrow).F. The same example as in E, sectioned further buccally to show the maximumdiameter of mx 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 bythe arrow).H. The same example as in G, sectioned further buccally to show the maximumdiameter of m1 and m2, which are smaller than in the heterozygote shown in F, andmuch smaller than in the control.

188 J. A. SOFAER

Table 2. The numbers o/Ta lower incisor germs in three categories ofabnormality observed at different stages

Stage

19 days21 days23 days25 days27 days

Total

Welldifferentiated.Variable sizeand shape

50253

15

Category

Poorlydifferentiated

101215

Undiffer-entiated

410534

26

a complete lack of differentiation and epithelial growth very nearly, if notcompletely, ceases.

2. Lower first and second molars

Both the heterozygote and hemizygote groups showed differences from thecontrols and are therefore both considered here.

At 13 days there were no detectable differences between the tooth rudimentsof Ta, Ta + and control animals. At 15 days differences became apparent. Atthis and subsequent stages Ta tooth germs were generally smaller than thecontrols and more bulbous in shape. Small size was sometimes associated with

FIGURE 3

A. Control lower first molar at 19 days.B. The same example as in A, sectioned further buccally and further posteriorly toshow the maximum diameter of m2.C. Control lower first molar at 19 days sectioned lingually to show the normalanterior extension of dental lamina at this stage (indicated by the arrow).D. Tabby heterozygote at 19 days with mx sectioned lingually. There is a smallsupernumerary germ anteriorly with its own laminal connexions.E. The same example as in D, sectioned further buccally and further posteriorly toshow the maximum diameter of m1 and m2, which are smaller and less well differen-tiated than in the control.F. The same animal as shown in D and E, but the opposite side. There is considerableepithelial downgrowth anteriorly (indicated by the arrow). Comparison with theopposite side suggests that this was an unsuccessful attempt to form a supernumerarytooth germ.G. Tabby hemizygote at 19 days, showing the maximum diameter of an anteriorsupernumerary germ. The first molar is sectioned rather lingually.H. The same example as in G, sectioned further buccally and further posteriorly toshow the maximum diameter of mx and m2.

190 J. A. SOFAER


delayed histodifferentiation. Similar but less severe abnormalities were presentin some heterozygotes. Examples of interaction between developing first andsecond molars were observed. Poor development of nij was sometimes associa-ted with an enlarged m2 in which differentiation was sometimes more advancedthan in the control m2. However, m2 never appeared to be as advanced as m^ Atno stage was there any evidence of division of the first molar germ into two ineither Ta+ or Ta animals.

A feature of the control animals was a small extension of the dental laminaanteriorly from the point of origin of the first molar germ, and somewhatlingually (Figs. 2D; 3C). In a few Ta+ and Ta animals there was proliferationof this extension of lamina to form an epithelial downgrowth anterior to thedeveloping m1 (Figs. 2E, G). In some of these cases a supernumerary toothgerm was formed (Fig. 3D, G) and in others the epithelial downgrowth appearedto regress (Fig. 3F; Fig. 4 A, C). There was evidence of interaction between thisepithelial downgrowth and the developing m1 and m2, whether or not a super-numerary germ was formed. The presence of a potential or developing super-numerary germ was associated with a small mx and a small m2 (Fig. 2, compareF, H with control, C; Fig. 3, compare E, H with control, A, B). Cases ofdegenerating epithelial downgrowths showed signs of the same interactionthough to a lesser extent (compare Fig. 4A, B with Fig. 3G, H, and control,A, B).

Whether the most anterior germ was a first molar or a supernumerary wasdecided 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

FIGURE 4

A. Tabby hemizygote at 19 days, showing an anterior downgrowth of dental laminawhich suggests an unsuccessful attempt to form a supernumerary tooth germ.B. The same example as in A, sectioned further buccally and further posteriorly toshow the maximum diameter of m: and m2.C. Tabby heterozygote at 21 days with the first molar sectioned lingually. There is adegenerating downgrowth of dental lamina anteriorly (indicated by the arrow).D. Tabby hemizygote at 23 days, showing the laminal connexions of an anteriorsupernumerary with mt sectioned lingually. Dentine formation in these two teeth isabout equally advanced.E. The same example as in D, sectioned further buccally and further posteriorly toshow the maximum diameter of m2, which is much smaller and less advanced than them2 of the opposite side (see F), and possibly would not have progressed to form atooth.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 mx and m2.Enamel formation in nij is much further advanced than in m2, but mx is muchsmaller than m,.

192 J. A. SOFAER

more normal (e.g. Fig. 3D with F; Fig. 4E with F); and of the affected animalwith 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 moreadvanced than the m1 it preceded, although after 19 days histodifferentiation inthese two teeth appeared to be about equally advanced (Fig. 3D, G; Fig. 4D).It was also considered that mx would always be in a more advanced state ofhistodifferentiation than m2. However, mx and m2 were sometimes observed to beof almost equal size, and in one case the tooth taken to be m1 on the basis of thethickness of its enamel and dentine was considerably smaller than m2 (Fig. 4G).

Table 3. The total numbers of Ta+ or Ta lower first molars examined at eachstage (N), the numbers of cases where there was proliferation of the anteriorlamina without supernumerary tooth germ formation (P), and the numbers of caseswhere a supernumerary germ was found (S).

Stage

11 days19 days21 days23 days25 days

Total

N

171418104

63

To +

P

31100

5

S

02001

3

N

910108

10

47

To

P

11000

2

s01010

2

Table 3 shows the total numbers of developing Ta + and Ta first molarsexamined at different stages, the numbers of cases where proliferation of theanterior lamina without supernumerary tooth germ formation was observed,and the numbers of cases where a supernumerary germ had become established.

F I G U R E 5

A. Control upper first molar germ at 15 days, showing the maximum concavity ofthe bell.

B. The same example as in A, sectioned further buccally to show the maximumheight of the buccal margin of the bell (indicated by the arrow).

C. Tabby heterozygote at 15 days, showing the maximum heights of the buccalmargins 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 andm2. The total anteroposterior length of these three germs is similar to that of thenormal m1 and m2 in D.

F. Tabby heterozygote at 23 days, showing an anterior supernumerary with m1, m2,and the rudiment of m3.


0-5 mm

J E E M 22


3. Upper first and second molars

Abnormalities of the upper moJars were less striking than those of the lowers.Just as in the lowers, there was no evidence of division of a first molar germ intotwo. However, only one example of what appeared to be early supernumerarydevelopment was found (Fig. 5; compare C with control, A, B). Amongstindividuals of the more advanced stages there were two examples of establishedsupernumerary teeth (Fig. 5E, compare with control, D; Fig. 5F). These threecases were all in the Ta + group. No upper supernumeraries were observed inthe Ta group.

The 'rampart' of the tabby upper second molar (Griineberg, 1965) has beenregarded as a reaction to the small size of m1. Figure 6 A, B shows the differencein size between normal and tabby upper first molars at 17 days. The rampartstarts as an anterior outgrowth which is first noticeable at 19 days (Fig. 6D;compare with control, Fig. 6C), and which subsequently becomes bent occlu-sally 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 appearsto be at least partially frustrated by lack of space.

4. Third molars

A difference between rudiments which were presumed to be destined forregression and those which looked as if they would form teeth started to bedetectable at 25 days and was definite at 27 days. The rudiments which weredestined for regression did not invaginate to form bells. No cases of regressionwere found in the controls, though absence of lower third molars does occur inthe A strain at a low frequency. No bell was formed by any of the Ta m3 rudi-ments at 27 and 29 days. About half the Ta m3 rudiments had formed bells atthese stages. Most of the Ta + m3 and all of the Ta+ m3 rudiments had formed

FIGURE 6

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 thedeveloping rampart (indicated by the arrow).E. Control upper second molar at 21 days.F. Tabby hemizygote upper second molar at 21 days, showing further developmentof the rampart (indicated by the arrow). Histodifferentiation appears to be a littlemore advanced than in the control.G. Control upper second molar at 23 days.H. Tabby hemizygote upper second molar at 23 days, showing further developmentof the rampart (indicated by the arrow). Histodifferentiation is more advanced thanin the control.

13-2

196 J. A. SOFAER

FIGURE 7

Lower third molarsA. 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 molarsG. 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.


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 thirdmolars in CBA and crooked tail mice.

DISCUSSION

1. General observations

In tabby hemizygotes the general effect on the developing tooth germsappeared 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

FirstI suppression phase

Follicleformation

Secondsuppression phase

^=Observation point

Fig. 8. The relationship of the developmental sequence of the lower teeth to thephases of hair-follicle formation and suppression. The arrows represent the timetaken for the course of development of each tooth from the appearance of a definitiveepithelial bud to the first appearance of calcified dentine, observations being madeevery 2 days from day 13. No example of a supernumerary tooth was found at the21-day stage. The point of the S (supernumerary) arrow has been arrived at by inter-polation from 19- and 23-day examples. I = incisor.

was formed, and sometimes there was an intermediate condition where somedentine but no enamel was formed. In the case of the molars there was no evi-dence to suggest that enamel formation is ever prevented or that a first molar isever completely suppressed. It did seem likely that complete suppression couldbe the rare fate of some lower second molar germs (Fig. 4E). Regression of thirdmolar germs was a frequent occurrence. Similar but less severe effects wereobserved in the molars of some heterozygotes.

'Overt twinning' in the lower jaw was found to be produced by the de novodevelopment of a supernumerary tooth from an overgrowth of a normal

198 J. A. SOFAER

anterior extension of dental lamina. Direct evidence for this in the upper jaw waslimited, though observations here were not inconsistent with the lower jaw find-ings. There was no evidence of division into two of any first molar germ, eitherupper or lower, at any stage. As the failure of third molar rudiments to form bellswas observed many times it is reasonable to assume that'concealed twinning'doesoccur. Examples of developing supernumerary teeth were found in both upperand lower jaws of heterozygotes, but in the lower jaw only of hemizygotes.

The picture formed is therefore one of a generalized partial suppression ofgrowth and differentiation of dental epithelium with occasional localized pointsof 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 thedevelopment of the coat, and what has been observed in the fully formed denti-tion. A diagrammatic representation of the developmental sequence of relation-ships of the teeth of the lower jaw and the phases of hair follicle suppression isshown in Fig. 8.

The first period of hair follicle suppression, from 12£ to 17 days, is just thatduring which the first molar develops from a small bud of epithelium to anearly stage of morphodifferentiation and histodifferentiation. At 1.7 days, the endof this suppression phase and the beginning of the phase of follicle formation,definite signs of overgrowth of the anterior extension of dental lamina were ob-served (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 whichhistodifferentiation was almost as advanced, if not equally advanced, as in the firstmolar posterior to it (Fig. 3D, G). Subsequently, the various stages of histo-differentiation appeared to proceed together in the supernumerary and first molargerms.

2. Stabilization of length of the tooth row

The interpretation offered for these observations is based on the premise thatthere is a tendency for the length of the tooth row to be stabilized. Because of itsretarded growth the first molar fails to occupy all the space allotted to it. As aconsequence there is overgrowth of the dental lamina to form an additionaltooth germ to take up the vacant space.

Perhaps it would be better to say that, at least during the developmentalphase, the dental lamina is under a growth pressure which is inhibited whennormally developing tooth germs have become established. Poor growth ofdeveloping 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 anexplanation is consistent with the hypothesis that developing organs specificallyinhibit like differentiation of the surrounding tissues (Rose, 1952, 1957).Evidence in support of this hypothesis has been provided by Saetren (1956) andby Clarke & McCallion (1959a, b).

The formation of a supernumerary tooth at the anterior end of the molarrow 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 goodevidence that in the reverse situation a negative response can also occur, namelythe reduction in size and eventual complete suppression of the third molar withincreasing size of the first two (Griineberg, 1951; Grewal, 1962; Van Valen,1962). Van Valen (1962) also cites evidence in favour of the existence of suchsize interactions in a number of different developing systems.

The first obvious signs of laminal overgrowth were observed at 17 days—theend of the suppression phase and the beginning of the follicle formation phase.During the follicle formation phase rapid development of the supernumerarygerm occurred. It is difficult to avoid the conclusion that this reaction was aresponse to relaxation of suppression. Such relaxation would no doubt affectthe first molar as well as the dental lamina, and this could explain why epithelialdowngrowths which failed to produce supernumerary germs were found. Asudden increase in size of the first molar germ could presumably prevent adowngrowth from developing further. However, there must be some differencein sensitivity to the suppressive influence between the laminal cells and those ofthe first molar germ. If there were not, no supernumerary downgrowths woulddevelop. It is suggested that this difference in sensitivity is associated with thedegree of differentiation of the two groups of cells, the less well differentiatedcells of the dental lamina being more ready to react by proliferation as relaxa-tion of suppression becomes more complete. The fact that the differentiatinginternal enamel epithelium is the first tissue to suffer degeneration as abnor-mality of the lower incisors increases, is evidence in favour of such a differentialsensitivity.

The second molars, also in a less differentiated state than the first, wouldsimilarly be likely to react more readily to a relaxation of suppression. Thiswould then be the basis of the general size interaction observed between first andsecond molars, especially noticeable in cases where no supernumerary waspresent. More specifically, it would explain the origin of the rampart of m2. Theexistence of these size relationships in the fully formed dentition was recognizedby Griineberg, (1965).

If this interpretation of the observations is correct, then it is basically the sizeof the developing first molar at and before 17 days which controls the ultimateform of the whole molar row. The final size of the first molar is not a goodindication of its status at 17 days, as recovery or further suppression could takeplace after 17 days and before its final form is decided by the onset of hardtissue formation. A slight difference in size between left and right first molars at17 days could result in the successful formation of a supernumerary tooth germon one side but the suppression of a potential counterpart on the other. Thussmall differences in local conditions at a critical stage of development could beresponsible for formidable asymmetry in the adult dentition.

200 J. A. SOFAER


3. Incomplete twinning

'Overt twinning' and 'concealed twinning' have already been discussed butno explanation has so far been given for 'incomplete twinning'. If the extrateeth found in the first two cases arise independently, then in the third the rarecomposite teeth observed must be the consequence of fusion rather than ofincomplete fission. Hitchin & Morris (1966) showed that fusion of the developingincisors of the dog, or connation as they called it, is related to the persistence ofdental lamina between two adjacent incisor germs. Rapid growth of adjacentgerms was thought to cause the external enamel epithelium to be stripped off thepersisting interdental lamina. As a result, the stellate reticulum of the two germsbecomes confluent, their internal enamel epithelia come into contact, andfusion takes place. In addition to connation of two incisors of the normalseries there were examples of connation of a first incisor with a supernumerarytooth. Figure 9A shows a case already illustrated in a different section (Fig. 5E).The external enamel epithelium between the supernumerary and first molar hasjust become separated from the underlying dental lamina. This illustration iscomparable with one of those of Hitchin & Morris, though in their case separa-tion was more extreme.

It seems probable that separation of the external enamel epithelium frompersisting interdental lamina would not only be a function of rapid growth ofadjacent tooth germs, but also of their proximity. The more tightly squeezedtogether the developing germs the greater the likelihood of epithelial stripping.Fusion would then be more likely to occur in the presence of a supernumerarytooth, as a greater than normal number of germs are then growing and competingfor room in a restricted space.

In trials made prior to the main investigation some of the material wassectioned transversely. Figure 9B-D are of a tabby hemizygote at 21 days.

FIGURE 9

A. Tabby heterozygote at 19 days (the same example as in Fig. 5E, in a differentsection). The external enamel epithelium between the supernumerary and first molargerms is becoming separated from the underlying lamina (indicated by the arrow).B. Tabby hemizygote at 21 days. Transverse section through the upper right incisorregion. 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 aconnexion between the pulp cavities of the two germs.E. A fully formed composite upper right incisor from a tabby homozygote at4 weeks of age.F. Complete separation between a supernumerary and an upper right incisor in atabby hemizygote, viewed from the buccal surfaces.G. Lingual view of a composite lower right first molar from a downless homozygote.

202 J. A. SOFAER

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 theirinternal enamel epithelia in intimate contact (Fig. 9B). Further posteriorly onthe 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, whereasthe posterior end is the youngest region where proliferation continues throughoutlife. The case just described must therefore have started out as two separategerms which fused subsequently. An example of a fully formed upper incisor ofthis sort is shown in Fig. 9E. It can be appreciated that once such a tooth hasbeen subjected to wear the nature of its origin would be obscured. Figure 9Fshows a case where separation between supernumerary and normal incisor hasbeen maintained.

A similar argument can be used to explain the origin of composite molars withseparate crowns and common roots. The crown develops before the root, so ifthe crowns are separate and the root common there must originally have beentwo germs which fused after formation of the crowns was complete. Such a caseis illustrated in Fig. 9G.

Further evidence for the origin of fusion being associated with restriction ofspace comes from the study of artificially induced malformations. Knudsen(1965a, b, 1966 a, b) made a detailed study of the dental malformations associ-ated with exencephaly induced in mice by teratogenic agents. There were variousdegrees of fusion of the two incisors within each jaw, and also intermediatecases where the future pulp cavities of the two germs were separate but theirstellate reticulum was confluent. Upper incisor fusion was very much morecommon than lower incisor fusion. Ritter (1963) induced lower incisorfusion, and fusion of the lower molars of one side with those of the other,by ^-radiation. These mandibular fusions were associated with mandibularmicrognathy. Knudsen (1966tf) reported on the molar malformations ofexencephalic embryos. There were amazing cases of fusion of upper molargerms with lower molar germs on the same side. All these cases offusion appear to have been associated with a reduction in the amount ofconnective tissue which normally separates the individual developing toothgerms.

The occurrence of fused and supernumerary molars in a less well knownlaboratory rodent, the rice rat, has been described briefly by Griffiths & Shaw(1961), and by Shaw, Griffiths & Osterholtz (1963). It may well be that the basisfor these anomalies is similar to that discussed here for the tabby mouse.

SUMMARY

1. The development of the teeth of the tabby mouse has been studied and anattempt has been made to explain aspects of the dental abnormalities in terms ofa single primary effect of the mutant gene, a partial suppression of the growth


and differentiation of dental epithelium. Such an explanation is consistent withthe retarded growth and lack of differentiation of the coat.

2. It has been postulated that the level of this suppression varies in intensity atdifferent stages of development in parallel with the observed effects on thedeveloping hair follicles, and that the final outcome is dependent on an inter-play of the suppressive influence and interaction between the developing teeth.

3. 'Twinning' of the lower molars was found to be due to the de nowdevelopment of a supernumerary tooth from a normal anterior extension of thedental lamina. Evidence for this in the upper molars was not complete, althoughobservations here were not inconsistent with the lower jaw findings. There wasno evidence of division of a first molar germ into two at any stage.

4. It seems most likely that the rare composite teeth observed in cases of'incomplete twinning' are produced by fusion of the supernumerary with theadjacent germ of the normal series. Direct evidence for this was found in theupper incisors, and indirect evidence was found in upper and lower molars.Supporting evidence from other sources has been cited.

RESUME

Aspects du syndrome 'tabby-crinkled-downless\I. Le developpement des dents 'tabby''

1. Le developpement des dents de souris 'tabby'' (tigre) a ete etudie et on atente d'expliquer divers aspects des anomalies dentaires en termes d'un eflfetprimaire unique du gene mutant, a savoir une suppression partielle de la crois-sance et de la differenciation de l'epithelium dentaire. Une telle explications'accorde avec le retard de croissance et l'absence de differenciation du pelage.

2. On a postule que le niveau de cette suppression varie en intensite adifferents stades du developpement, parallelement aux effets observes sur lesfollicules pileux en cours de developpement, et que le resultat final dependd'une reaction reciproque de l'influence suppressive et de ^interaction entre lesdents en cours de developpement.

3. On a trouve que la duplication des molaires inferieures etait due audeveloppement 'de novo' d'une dent surnumeraire a partir d'une expansionanterieure normale de la lame dentaire. La realite de ce phenomene pour lesmolaires superieures n'est pas evidente, quoique les observations faites ici nesoient pas en contradiction avec les resultats obtenus pour la machoire inferieure.II n'est pas evident qu'un germe molaire primaire se soit divise en deux a unstade quelconque.

4. II parait tres vraisemblable que les rares dents composites observees dansles cas de duplication incomplete soient produites par fusion du germe surnu-meraire avec le germe adjacent de la serie normale. Une preuve directe de cecia ete trouvee pour les incisives superieures, et une preuve indirecte l'a ete pourles molaires superieures et inferieures. On a cite les preuves a l'appui pourd'autres origines.

204 J. A. SOFAER

I am grateful to Professor D. S. Falconer for suggesting the investigation and for hisinterest and valuable advice during the work, to Professor C. H. Waddington for laboratoryfacilities, and to the Nuffield Foundation for financial support.

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{Manuscript received 2 December 1968)

aspects of the tabby—crinkled—downless syndrome · the teeth of tabby and crinkled mice have...

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