ABSTRACT
The mutant twirler, symbol Tw, of the house mouse, Mus musculus, was first recognized by the ‘waltzing’ behaviour which the heterozygotes show. It was later found to affect the morphology of the inner ear and, when homozygous, to cause death of new-born animals through harelip and cleft palate.
This paper describes the behaviour and ear defects of heterozygotes and the appearance of homozygotes. It also describes the genetic tests carried out to determine the mode of inheritance.
DESCRIPTION
Heterozygotes
Twirler heterozygotes show head-shaking in a horizontal plane, combined often with circling, and less often with vertical head-shaking. Postural reflexes are abnormal. For example, normal mice of about a week old, if held up by the tail, respond to this change of position by extending the back and stretching the forelimbs forward. Twirler mice, on the other hand, flex the back, tuck the head under, and draw the limbs back. The ‘landing reaction’, in which the forelimbs are extended powerfully forwards in response to a downward falling movement, is also absent. In non-twirlers it develops during the third week of life.
Most twirlers react normally to sound. In animals where no reaction to sound can be observed it is difficult to tell whether the animals are truly deaf or are merely failing to stop circling long enough for a reaction to be seen.
The growth of young animals is usually normal although a few twirlers lag behind their normal sibs in growth. Adults, on the other hand, tend to become obese. This obesity may become obvious at any age from 3 months onwards, but some twirlers live out a normal breeding life and remain slim. The fat is found in the usual sites for deposition of fat in the mouse. Subcutaneous fat is particularly obvious in the inguinal and neck regions and between the shoulder blades. The ‘hibernating gland’ between the shoulder blades seems normal in size. In the abdomen fat surrounds the kidneys, ovaries, and testes, and fills the mesentery.
Both sexes are fertile but become sterile if obesity sets in. Some twirlers make good parents but some animals of both sexes are liable to attack and kill the young at any time during the first week of life, more especially after being disturbed in some way, e.g. by being given a clean cage. Male twirlers, if allowed to grow up together, will often begin to fight when about 2 months old to such an extent that they must be either separated or destroyed.
Homozygotes
Animals considered to be homozygous have shown either cleft lip and palate or cleft palate only. Of 30 such animals observed at birth, 13 showed cleft lip and palate and 17 had normal lips with cleft palate. Among the animals with cleft lips the defect was bilateral in 7 cases, on the left only in 2 cases, and on the right in 2 cases (Text-fig. 1). In the remaining 2 cases the side affected was not noted. Cleft palate was usually median or bilateral, these two categories not being separable, but in 2 cases it occurred on the left only and in 2 on the right only. One of the left-sided clefts accompanied a left-sided cleft lip and in the other unilateral cases the lip was not cleft.
Heads of new-born mice, a, normal; b and c, twirler homozygotes, b shows a left-sided cleft lip and c a bilateral cleft lip. In b the left and in c both maxillary processes have failed to grow to the midline, leaving the medial nasal processes exposed.
All homozygotes died within 24 hours of birth. The more highly affected ones died of respiratory difficulty. When removed from the mother by Caesarean section animals with cleft palate and harelip could be stimulated to breathe regularly by the same massage and pinching which would stimulate normal mice. When the stimulation was stopped, however, and the animals were left to breathe spontaneously they gradually became cyanosed. If again stimulated, so that they squeaked and gasped, they would become pink once more, but when the stimulation was stopped they again became cyanosed and died within a few hours. Animals without harelip and with a relatively narrow cleft in the palate were able to breathe normally, but if placed with a foster mother they died within 24 hours, presumably from starvation. Unaffected sibs born by Caesarean section and placed with foster mothers fed and grew normally. Some animals with harelip and cleft palate swallowed large amounts of air into the stomach and intestines. It is not clear whether this air was swallowed in attempts to suck or to breathe.
The inner ears of homozygotes are abnormal but have not yet been fully investigated.
EAR DEFECTS OF HETEROZYGOTES
Methods
The ears of twirler heterozygotes were studied by means of whole mounts of the bony labyrinth and of serial sections of the complete inner ear.
To prepare whole mounts of the bony labyrinth, part of the skull including the labyrinth was fixed in 70 per cent, alcohol for some days, macerated in 1 per cent, potassium hydroxide until the adherent soft tissue became transparent, dehydrated in alcohol, and cleared in benzyl alcohol. No stain was used. The bony labyrinth was then dissected away from the surrounding bone.
For sections of the inner ear 3-to 4-week-old mice were fixed by the injection of Bouin’s fluid into the aorta. The inner ear was removed and further fixed in Bouin’s fluid, decalcified with 2 per cent, nitric acid in 70 per cent, alcohol, and embedded in 3 per cent, celloidin and paraffin by the methyl benzoate-celloidin technique. Transverse sections were cut at 7-10μ, and stained with haematoxylin and eosin.
Observations
(a) Whole mounts
Text-fig. 2 shows some examples of abnormalities found in the bony labyrinth. The typical defect consisted of reduction or absence of the horizontal canal, accompanied by absence of otoliths, and uneven outlines of the vertical canals. The cochlea appeared normal.
Camera lucida drawings of the bony labyrinths of twirler heterozygotes, showing increasing grade of defect. a differs from normal only in the uneven outline of the vertical canals, while f lacks otoliths, has no visible horizontal canal, and shortened and branched vertical canals, AVC, anterior vertical canal; c, cochlea; HC, horizontal canal; o, otolith; PVC, posterior vertical canal, × 10.
Camera lucida drawings of the bony labyrinths of twirler heterozygotes, showing increasing grade of defect. a differs from normal only in the uneven outline of the vertical canals, while f lacks otoliths, has no visible horizontal canal, and shortened and branched vertical canals, AVC, anterior vertical canal; c, cochlea; HC, horizontal canal; o, otolith; PVC, posterior vertical canal, × 10.
The degree of defect varied considerably from one animal to another. At the least the horizontal canal was present and only slightly reduced in length, as in Text-fig. 2a, and the otoliths were present, although sometimes consisting of fewer crystals than normal. Some ears had one otolith present and one absent. Ranging from this slight defect there were progressive stages of reduction of the horizontal canal, accompanied by complete loss of otoliths, and by increasing abnormality of the vertical canals. The minimal defect of the vertical canals was unevenness of outline. The unevenness then increased to form projections from the walls of the canals. In the most extreme cases the projections formed complete branches or duplications which ran from the canal to the utriculus or the crus commune (Text-fig. 2 c, f). The arcuate fossa, in which the floccular lobe of the cerebellum normally lies, might be more or less obliterated by branches from the anterior vertical canal. Or the fossa might be smaller than normal as a result of reduction in length of the vertical canals.
(b) Sections
Serial sections were used to study the histology of the membranous labyrinth. Eighteen ears of 9 twirlers were examined and compared with 7 ears of normal sibs.
In the utriculus and sacculus the otoliths were usually lacking (Plate), but in the position in which they would normally be found there was some material which showed the same staining properties as, and was considered to represent, the organic matrix of the otolith. The neuro-epithelium of the maculae over which the matrix lay showed no abnormality and the remainder of the utriculus and sacculus appeared normal. When otoliths were present they were in some cases thinner than usual. Of the 18 ears examined, 9 lacked both otoliths, 7 had a normal saccular otolith, in 3 cases accompanied by a thin utricular otolith, and the remaining 2 ears had a thin saccular otolith only.
In the horizontal canals both the crista and the morphology of the canals were abnormal. The canal itself was always shortened and sometimes its bony capsule was not separated from that of the utriculus, so that in preparations of the bony labyrinth it would have appeared that the horizontal canal was lacking (Text-fig. 3). In fact, none of the 18 ears examined entirely lacked a horizontal canal or ampulla. The ampulla, probably in association with the shortening of the canal, was somewhat most posterior in position than in normal mice, and the crista lay lateral to rather than anterior to the utricle macula. It was always abnormal in shape. Normally the horizontal crista forms an upfolded ridge of epithelial cells extending across the ventroposterior wall of the ampulla. In twirlers the epithelium forming the crista was folded inwards rather than up, forming a pit instead of a ridge, in the ventromedial part of the ampulla (Plate, fig. A). The histological differentiation of the neuro-epithelial cells seemed normal, and covering material which probably represented the cupula was present.
Camera lucida drawings of transverse sections of the ears of a normal mouse (a) and a twirler heterozygote (b). In b the horizontal canal is shortened so that it has no separate bony capsule, and there is a canal diverticulum which, in other sections, could be seen to run from the anterior vertical canal to the utriculus. AVC, anterior vertical canal; CD, canal diverticulum; HC, horizontal canal; u, utriculus. × 20.
Camera lucida drawings of transverse sections of the ears of a normal mouse (a) and a twirler heterozygote (b). In b the horizontal canal is shortened so that it has no separate bony capsule, and there is a canal diverticulum which, in other sections, could be seen to run from the anterior vertical canal to the utriculus. AVC, anterior vertical canal; CD, canal diverticulum; HC, horizontal canal; u, utriculus. × 20.
The ampullae and cristae of the vertical canals were normal, but the lumen of the canals themselves varied in diameter, as was to be expected from the unevenness of outline seen in whole mounts of the bony labyrinth. In some cases the lumen extended into the diverticula of the canals (Text-fig. 3), but in other cases where the diverticula were narrower they contained only a solid core of connective tissue cells, with no canal lumen.
In all except two animals the cochlear duct was completely normal. In one of these two animals the left cochlear duct was normal but the right was highly hydropic, the organ of Corti was disorganized, and the stria vascularis apparently absent. The sacculus in this ear contained some form of pink-staining precipitate. In the other animal, a litter-mate of the first, both cochlear ducts were affected. The basal region appeared normal but the apical region was hydropic, with the organ of Corti abnormal. In the right ear the stria vascularis seemed absent from this region; in the left ear it was normal.
In some twirler ears the lumen of the endolymphatic duct appeared wider than in the normal sibs, suggesting some excess of endolymphatic fluid. It is not clear whether this was of much significance, however, as the lumen may vary in normal animals and the distension in twirlers was slight.
GENETICAL STUDIES
The mutation occurred spontaneously in a crossbred multiple recessive stock homozygous for the genes a, b, cch, d, s, se, the first twirlers found being two females which occurred in two successive litters of 8 born to a certain pair. Later studies showed that these females must have been heterozygous for a dominant gene, and hence it may be presumed that the mutation that gave rise to them occurred in the germ-line of one or other parent.
Of the two original twirlers one failed to breed and the other produced 11 offspring by her brother, 4 twirler and 7 normal. Four normal sisters of the twirlers were mated to the sire or a brother and produced a large number of young, all normal. This preliminary evidence suggested that the new character was due to a dominant gene, a hypothesis which was confirmed by further work. When twirlers were mated together the ratio of twirler to normal offspring was closer to a 2 :1 than a 3 : 1 ratio, suggesting death of homozygotes before classification.
Single-factor ratios
The numbers of twirler and normal offspring obtained in various types of cross are shown in Table 1. Twirler heterozygotes were outcrossed to linkagetesting stocks and to various inbred strains and the Tw + × + + class in the table includes these outcrosses and also the subsequent backcrosses of Tw + offspring to the linkage stocks. The animals mated in the Tw + × Tw + class were the progeny of outcrosses.
After Tw + × + + matings the proportion of twirler offspring is significantly less than the expected half (P < 0·01); and with Tw + × Tw + matings the ratio of twirler to normal offspring is less than the expected 2 :1 but not significantly so (0·05 < P < 0·1). Such a deficiency could be caused by incomplete penetrance of the twirler gene in the heterozygous state or by death of twirler offspring before classification. From observation of the animals either explanation seems possible. The grade of behaviour defect in twirler heterozygotes is variable and it is possible that some low-grade animals have passed undetected. On the other hand, highly affected twirlers are sometimes small and thin at weeks, when classification was usually made, and it is possible that some highly affected animals died before this age. In either case the deficiency is small; if incomplete penetrance is the cause then the penetrance may be estimated from the outcrosses as 2 × 46611024 or 91·0 per cent., and if low viability is responsible for the deficiency, then the relative viability of twirlers may be estimated as 466/558 or 83·5 per cent. The degree of deficiency in the intercrosses is similar to that in Tw+ × + + matings and the penetrance may be estimated as (84 × 3)/(2 × 142) or 88·7 per cent, or the viability as 84/2 × 58 or 72·4 per cent.
Homozygotes
In order to test the hypothesis that no TwTw young live, twirler young of intercross matings were tested for the possibility of their being TwTw by mating to unrelated normal animals. Of twenty-four such animals tested all produced at least one normal offspring and were taken to be Tw + . The numbers of twirler and normal offspring resulting from these tests are shown in Table 1. The proportion of twirlers is somewhat higher than in the outcrosses, which suggests that some of the tested animals may in fact have been homozygous TwTw but, owing to incomplete penetrance of twirler, did not produce all twirler offspring. On the basis of the outcrosses and intercrosses such animals would be expected to throw about 90 per cent, of twirler offspring. In fact none threw ratios of twirler to normal at all unexpected for the 1:1 of heterozygotes, so that there is no positive evidence that TwTw animals ever live and breed.
To find the missing homozygotes Tw + females were mated to Tw + males and killed at 14-17 days’ gestation for examination of the foetuses (Table 2, series 1). Roughly one-quarter (15 out of 69) of the young showed harelip, and these animals were taken to be the missing homozygotes. Later, a second series of embryo counts was made to check that no harelip young occurred after matings of Tw + × + +. This was in fact confirmed (Table 2, series 2a). Simultaneously another series of Tw-i-x Tw+ matings was made. For this series twirlers from a different outcross from those in the first were used, and females were opened at days’ gestation. Among the progeny of the Tw + × Tw + matings of this series (Table 2, series 2b) there were some foetuses which had cleft palate without cleft lip as well as those with cleft lip and cleft palate. Even so, however, the proportion of abnormals fell short of the expected 25 per cent. In yet another series, in the course of linkage tests, twirler females mated to twirler males were allowed to litter and the young were examined at birth for harelip and cleft palate. In this group (Table 2, series 3) the proportion of putative homozygotes was again roughly a quarter. The explanation of the shortage in the second series is not clear; as only five dead embryos were found it cannot be supposed that the missing abnormal foetuses had died. Incomplete penetrance of the homozygous phenotype is a possibility; in such a case tests of the twirler young of intercrosses should have revealed some living homozygotes. Nine of the 24 young tested came from this stock, and none gave any suspicion that they were other than heterozygotes. Thus, while it may be accepted that the animals with harelip and cleft palate represent the twirler homozygotes, it is not known why the expected proportion is not always found.
Linkage tests
The results of linkage tests with Tw are shown in Table 3. In the cross with dreher, dr, twirlers could be distinguished from drehers by observing the deafness of the latter, but among the drehers the twirler drehers were not distinguished. Similarly, in the cross with jerker, je, twirlers could be distinguished from jerkers by their ability to hear, but in this case twirlers could also be distinguished within the jerker class by observing the morphology in preparations of the ear.
In the tests of Tw with ax the linkage x2 is highly significant and no Tw + axax young were found. The greater part of the data, however, are from matings of the type Tw + + ax × + + + ax (CM) and from this type of mating the Tw + axax young would be the only known recombinant type. It is possible that these twirler ataxies would either die or not be distinguishable from either twirler or ataxia alone, thereby giving a false impression of linkage. The hypothesis of a true linkage is favoured, however, on two grounds. Firstly, the proportions of the three types of young found in the progeny of these mixed crosses are in agreement with those expected on the hypothesis of linkage and differ from those expected on other hypotheses. Secondly, among the offspring of matings of the type Tw+ +ax xTw+ + ax not only twirler ataxic but also normal animals would be recombinants and, in agreement with the hypothesis of linkage, none have so far been found among the small number of young examined.
It may therefore be accepted that twirler is closely linked to ataxia. Ataxia has previously been shown to segregate independently of marker genes in most of the known linkage groups (Lyon, 1955). Hence Tw and ax belong to a new group, for which the number XV is proposed. As no recombinants have yet been found the observed recombination between the two genes is 0 per cent.
Possible allelomorphism of twirler with similar mutants
There are many known mutants of the mouse which produce ‘waltzing’ behaviour similar to that of twirler. For most of these, however, the discovery of the linkage relations of Tw rules out the possibility of it being a new allelomorph of an already known gene. As Table 3 shows, direct tests of twirler with dreher and jerker gave no evidence of allelomorphism or linkage. The six ear mutants listed below are known to be linked to the genes shown, with which Tw shows no sign of linkage.
Allelomorphism with waltzer, v, or varitint-waddler, Va, can similarly be ruled out as these two mutants show no linkage with ax (Lyon, 1955), with which Tw is closely linked. Twirler has not yet been tested for allelomorphism with zigzag, Zg (Lyon, unpublished).
DISCUSSION
The linkage of twirler with ataxia means that there are now sixteen known linkage groups in the mouse, autosomal linkage groups I-XV and the sex chromosomal group XX. As group XV is so short, however, no known recombinants having yet been found, it could represent a remote part of an already known group; a point which will only be decided by future linkage tests with new mutants. Since the mouse has twenty pairs of chromosomes there now remain at least four chromosomes not marked by linkage groups, or more than four if some of the known groups are not really independent.
In its morphological effects twirler is interesting as being an addition to the list of mouse mutants which affect the inner ear. Grüneberg (1956), in reviewing the knowledge of these mutants, divides the majority of them into two classes: those in which the labyrinth shows morphological abnormalities and those in which degenerative changes occur in a morphologically normal labyrinth. Six or more mutants are now known in each group, in the first of which twirler clearly belongs. The mutants of this group now provide valuable material for study of both the physiology and the embryology of the ear. In Table 4 the defects found in each of these mutants are tabulated according to severity. Twirler occupies an intermediate position. Less affected are pallid, in which the only ear defect is absence of otoliths, and zigzag, in which the otoliths are normal but the horizontal canals are absent (Lyon, unpublished). The mutants twirler, dreher, and kreisler appear to form a series of increasing grade of defect, and embryological studies aimed at finding some common origin for their abnormalities would be valuable.
Nothing is yet known of the basis of the pleiotropy of twirler. In heterozygotes there is the combination of obesity with ear defect, and in homozygotes cleft palate, cleft lip, and ear defect are combined. Cleft lip and palate are well known in the inbred strain A (Reed, 1936). More recently cleft palate without cleft lip has been produced in mouse foetuses by treatment of pregnant females with cortisone (Fraser & Fainstat, 1951). It has also been reported briefly as occurring in mice homozygous for the mutant ur (Gluecksohn-Waelsch & Kamell, 1955). No link with ear defect is yet apparent. Hereditary obesity of the mouse has also been reported more than once. The obesity of mice carrying the mutant yellow, Ay, has been known from the early days of mouse genetics. More recently the mutant obese, ob, has been found (Ingalls, Dickie, & Snell, 1950), and obesity of unknown genetic cause has been reported in the NZO strain (Bielschowsky & Bielschowsky, 1956). In both these types of obesity there is hyperglycaemia, although the underlying hormonal or metabolic causes are thought to be different. The obob mice are sterile, as are obese twirlers. Again no link with ear defect is obvious.
SUMMARY
Twirler, Tw, is a new spontaneous mutant of the house mouse which, when heterozygous, causes abnormal behaviour, including circling, head-shaking, and absence of postural reflexes, which can be attributed to morphological defects of the inner ear. Homozygotes have cleft lip and palate in addition to ear abnormality, and die soon after birth. The ear defects of heterozygotes include absence of otoliths and reduction and malformation of canals. The animals are not deaf and the cochlea appears normal. Twirler is closely linked to ataxia, ax, this being the first linkage of a new linkage group, number XV.
ACKNOWLEDGEMENTS
The author is grateful to Miss M. L. Court for histological assistance and drawing, and to Mr. E. Lucas for photography.
REFERENCES
EXPLANATION OF PLATE
Fig. A. Transverse section of ear of twirler heterozygote showing the utriculus and horizontal ampulla. In the utriculus the otolith granules are lacking. In the ampulla the crista is malformed, being infolded to form a pit rather than a ridge, × 90.
Fig. B. Transverse section of the utriculus of a normal mouse showing otolith granules, × 90.
Fig. C. Section of the horizontal ampulla of a normal mouse showing the normal shape of the crista, × 90.
Fig. D. Higher magnification of the abnormal crista shown in Fig. A. × 290.
Fig. E. Higher magnification of the utricular macula shown in Fig. A. × 290.
Fig. A. Transverse section of ear of twirler heterozygote showing the utriculus and horizontal ampulla. In the utriculus the otolith granules are lacking. In the ampulla the crista is malformed, being infolded to form a pit rather than a ridge, × 90.
Fig. B. Transverse section of the utriculus of a normal mouse showing otolith granules, × 90.
Fig. C. Section of the horizontal ampulla of a normal mouse showing the normal shape of the crista, × 90.
Fig. D. Higher magnification of the abnormal crista shown in Fig. A. × 290.
Fig. E. Higher magnification of the utricular macula shown in Fig. A. × 290.