T W O N E W M U T A N T G E N E S I N T H E I-~OUSE ~ t O U S E

BY I-fANS Gtf(JNEBEI~G, Depco't.me~t of B.fov~etry, U~~h~e.rsity Co~ege, Lo;~cZo't~

(With Three Text-figures)

INTI¢ODUCTION

The two genes to be described in this paper have been cliseovered in the mouse colony of the Pathology Laboratory, ~ioant Vernon ItospRa], Northwood, Middlesex. One of them, fidget, is undoubtedly a new gent. The other, hydrocephalusog, resembles in many ways two other hydrocephalus genes described in the mouse; there are, however, certain differences which suggest that the gent, if nee a new locus, may at least turn out to be a newallelomorph of one of the other two genes ; a decision will require crosses which cannot be carried ou~ at present.

A. ]?m era'

This gent was discovered in 19-'1-1 in an stbino strain which is supposed to have been a pure line at one time; as it has been bred in a~ unoontroll6d manner for some years, it is now probably quite heterogeneous ; it certainly also segregated for the gent for macroeytle anaemia (IV, 'dominan~ spotting'}. Fidgets have been observed in the strain for some time past according to the laboratory stewards.

The. most obvious manifestation of 5he gent, for which we propose the name )g@et (symbol fi), is a derangement of behaviour: which is in some ways comparable to that of Japanese waltzers, shaker-i, shaker-2 a~d jerker (see Grtineberg, The Ge~,~eZics of~he Morose, pp. 125-35). Affected animals shake their heads; but while the four-mutants mentioned show head-shaking mainly in dorso-veatraI direction; the head being thrown backwards in rapid succession, and sometimes kept up in a ' sniffing' atti tude for an instan% the head movements of fidgets are nearly always from side to side; sometimes the head is shake~ only a few times~ the excursions fo the right and left being comparatively- small; some- times the shaking eontimtes at a rapid rate for some time; one fidget, shook its head about 95 times in I rain., i~terrupting the she -1dug two or three times for a moment. The head movements tend to be most marked when the mouse is walking about and generally active; during periods of quiet the sha'ldng tends to decrease, axld none i.~ noticeable when the animal is asleep. Like the genes of the waltzer-shaker group, fidgets sometimes run in circles; the circling is, kowever, not as marked as i~ waltzers; fidgets are more apt to circle in ~he confmed space of theb cages than on a table. One and the same fidget may circle clockwise or counterclockwise, though one direction seems to be favoured in most individuals. When normal mice are gently ]~fted from the table by their tails, they stretch ont and try toreach the tabie with their forefeet; when that proves impossible, they turn sideways and try to climb up the hand that.is, holding them. ~embers of the waltzer- shaker gToup under these conditions throw their bodies about with vioten~ j erldng move- :ment.s without any pnrposefrd co-ordination, but fidgets behave much like normal mice. Severe signs of lack of orientation are, however, shown in water. Normal mice] like most mammals, are able %0 swim in a perfectly eooorclinated way; when gently put into the

~ANS G ~ E ~ E R G 23

Water, the bead iS lifted big11 up and the mouse usually manages to keep its back dry; the front legs are held dose to the body, and the hind Iegs are used for padclling, When a £dget is similarly put ~.nto the water, it at. once seems to lose all tense of dh"eetion ; instead of keeping on top, it gycates n~ad'ly about, mostly under water, and on.ly speedy rescue can prevent it from clrowni~g, k. similar inability t0 swim is known in Japanese waltzers; the other three members of that group have apparently not yet been tested as to their ability to swim.

}~hile all members of the Waltzer-shaker group become deaf early in life, this is no~ the case wi~lz.fi'dgets. Young fidgets round about 3 weeks of age are actually hypersensitive ~o sounds and often jump into the air in response to moderate noises. Later this hyper- sensitivity is followed by a period in which only very drastic stimuli, like hand-clapping, will elicit a respons% and from the age of 3-4 months onwards no definite auditory reaction hag been observed ; the animals are either very hard of hearing or qu!te deaf.

The heed-shaking can sometimes .be observed ~s early as 3 or 4 days after birth; a reliable classification is, however, not possible anvil the ~nima.ls are about g, fortnight old, and repeated classifications have generally been carried out up to the age of 3 weeks. There is never any doubt at to whether a mouse is a fidge~ or not; hence normal overlaps are not likely to oeem'. -Dtrcing the first week or two, fidgets ortega fall Back i~ develop- ment, so ~hat tt~ey sometimes weigh only half as much a.s their normal sits; during this period they are apt to fall victims to interenrlgent diseases. Later, however, they usually make Up leeway, and in adult life most fidgets are quite sturdy mice. Males seem to be fgirly reliable breeders, while some females are apparently sterile; others produce an occasional IJtter, which they fail to rear; but one ~dget female, after eating her first littdr, has reared three subsequen.t litters snceessfully. A stock can easily be maintained in baekcross fashion, always mating rid.get males to ~heir heterozygous sisters.

We bs..ve t,o mention another disorder of ~.d.ge~,s, which la.as not yet been s~udied in det~aiL Soon after the eyes have opened, some discharge t¥om the eonjunctis~a occurs, and the eyelids tend to stick together ; most fidgets have one or bot.h eyes permanently closed t~roaghou.t life.' Some observations with the slit-lamp show that small blisters o:f the corneal epithelium are ]?resent in the early stages; these tend to burst a~d. coalesce, and larger ulcers may be formed; vascu].arization of Lhe cornea, occurs in later stages~,~nd cement opacities, sometimes covering tl~e whole cornea, are ecru.men.

While the :features mentimmd so far are regularly found in all fadgets, polydaetylJsm of t)le hind legs, involving the ~rst bee, lia, s been observed in only a minority oi' the animals ; a.s the re]ation of this trait to fidget is not yet, quite clear, it, will be discussed below after the report on the genetic hasis of ~d.get.

(;'thebes. A ficlget alb{no male, subsecl~ently proved t,o be of the oonsti~,nbion f i / . f i ; c/c; d/d,; ae/a/, was on,crossed to a Serm.tg CBA ~m.na].e and{ produced three normal off~ spriu.g, o~.m male and two females. This .F~ trio produced ll~I yonng--85, normals and 26 ~idgets. Other known heterrhzygotes mated ineer se produced 67 normds and 23 fidgets ; one pair of heterozygo~,es (recognized. as such by the progeney) had 32 ztormal and 9 fidget young. At%tether, these 17, rantings produced 187 normaIs and 58 fidgets, a close approxi- niation to a 3 : ?. ratio.

-'-k~. Pz :fidget male wa, s crossed to five of h~s norms,] sisters; all :fi.ve of them proved heterozygo.tes, producing 7, 10, 12, 16 an(l 16 young respectively; of these 33 were normals and 28 fidgets, a close approximation to a ,~ : 1 ratio, disregarding tlle s].ight selection in

24 T w o %ew ,muta.nt genes in the ho,ztse ~?~o~tse

~hese da.ta. Thus _Y~ and backcross segregations ag,~'ee in showing dmt fidge~ is d~ie to a single recessive gent without complications due to st]entire elimination or normal over- lapping.

The arigi.nM ~d.get rome introduced the reeessives o, d and c~ e into the cross, the CBA female the eorrespon.ding normal alldomorphs. Tke joint segregations of fidge~ with these genes (coupling F,, generations) are as follows:

+ + -!-fi z + ~fg '£o~M Albinism ~4 18 14 8 I11 Dilution 57 14 I7 4 92 Agougi 53 11 21. 7 92

In no o~zse is th.ere a signi£aeant deviation 5'ore free assortmen% @rough the data on c a~d f i deviate from free segregation in the dbection expected in ease of linkage.

.Polydcectglis~n. I t was mentioned al)ove that some animals sinewed polydactylism of tlxe ~rs~ toe on the hind feet. The condition is v~rigble (see Fig. 1). Sometimes there is only a doubling uf the diitM pad of the toe, wi~h or w}ghou~ an extra claw; i~ extreme cases

c

1% L I{ L

Fig. 1. Bit~t~l'~l pol2d~etylism in ~ b~ekeross fid~et~ ,~'~1 d~ys old, ~nd polyd~c~y.lism of the r ight foot in a haekeross ~dget, 15 days old; ~he extra toe in the I~ ter mouse ea.rr[ed a f t a t nM1 rather tha~ ~ claw. X- ray photographs of the older ani~aM showed tha t on the left foot the first metatarsal was thicker t h a n usua l >roxZm~lly, and forked in the distal qualger; on ~he right foot the first metatarsM was not not iceably ~hiekened and probably not forked distMly. X-ray p h o t o ~ a p h s of the younger mouse were not ddstinet enough for an interpretation.

the extra toe is completely free and then qsuMly l~rger than the normal first toe, Altogether seventeen polydactylous mice have been observe(l; in ~2 matings eleven fidgets and one noT.real were affected (Kve on the right side, roar on the left side, and three bi]aterally); in the backcross rantings, three fidgets were affected on the right side, and one bilaterMly. In addition one polydaetylous mouse (right side) was obs&ved in. a m~ting between two normal -/~2 .mice, which produced ~wenty-nin'e normal (non-fidget) young; on the 12th day there was only a duplication of the distal pad of the toe; on the 19th day the con- dition was 'scarcely visible'; on ~he 27th day its existence w~s ~very do, ubtfnI ' ; and at t!_~ age of 3 months no sign of polydaetylism was left. To judge from this ease, a slight doubling of the toes ma,y tend to disappear with grow.~h, and the :C~st classification .must always be made at Nrth.. NTarked cases, whio'.~ are in the majority, ~how no such tendency.

25

The incidence of polydacgylous mice am.ongs~ fidgets and normals is set out below in a 2 x 2 table. Treatment by the exact method shdws q, hat the probability of a chance dexqa-

Normal Fidget Total- _Normal 247 71 318 Polydaetylou~ g 15 17 Tota,l 249 86 335

tion from proportionality as great as, or greater than., that observed is very. small (P=¢-5 x 10-a). Hence there cannot be any doubt th-at polyd.aetylism and fidget are .somehow,dated; there are sevsrM genegb situations which might account for the observed facts:

(1) The pdydaetylism obsegved in the two normal mice n a y have been independent of that associated with fidget; this assumption 'seems unlikely.

($) The presence of po!ydaetylism in the two normal mice may have been due to semi- dominance=of ~he f i gene. Unfortunately it is unknown whether ~hese two animals were in fact tieterozygotes. F thi s interpretation is eo~:reet~ the case would be analogous to the nettro-muscdar -kinkiness ofthe taft associated with the recessive gene for short-ear (se), which is incompletely penetrant and apparently sometimes semi-dominant.

(3) k gene for polydactylism with low penetrance, and linked to fi, may have bee~ responsible; in this case the 15olycta.ctylous non-fidgets Would liave been crossovers. I do not regard this hypothesis as likely.

(~) A gene for polydaotytism with low penetrance, and ~ot linked to, but. favoured in i~s mamfestation by the presence of, fg may have been concerned. The scanty data avail- able are insufficient to decide betwee~ these possibilities.

We have to give the reasons why we regard fidget as a new gene. Wabzer, shaker-l, shaker~2 and jerker differ from ~qdget by the dbeedon of thehead-shaMng, by the ear]y onset of deafness, the absence of eye complications and of polydactylism, and by tl~e absence of a phase of retarded growth; allelomorpJxism wibh shaker-] is also excluded by different ]i~kage relations. The c:ireler strain briefly described by Laanes& NaeDowe]l (1935) shows cbcling without deafness; moreover, circlets are l?erm.anen~l) ~ hypersensitive to sound, while :fidgets are so 0nly for a short period; eircIers also do not shake their heads, ~or have eye or foot complications been mentioned; the onset of circler is mac]< later i~ life, normal overlaps are common, and the whole condition is probably due t'o two genes. Hence on close:,: inspection fidget is quite different from all these nervous disorders. Still more different are two other mutants with ehoreic behaviour; one of these is shaker-short which is stone-deaf from the beginning and has cerebral hernias and tail anomalies; similarly, kinky tail may show nervous derangemen.t; this gene is dominant and lstl.m] an ho:mozygons condition and produces obvious taft an,ms]ice.

The comparison of fi.dget with the nervous disorders described in the mouse leaves no doub~ th.at this gene Js distinct, from all the described routs.Jets. The pecu.liar combination of e:ffecl,s presents a fascinating problem f6r deve].opmental studies.

~ . ~YD~OCEPHALUS-~

Inthe summer 9f 1941, a female of the ' mixed' stock produced a litter of unknown p~ternity whieh co~ltained six normal and two very underdeveloped ianndiced young; they suffered from an extreme .mierocytie anaemia with Jo.ig[{ ]eueoeytosis, apparengy of a haemolytic kind; the liver was pale and of a buff eolon~. The occurrence of two,similm: young in the

26 T w o 'n,e,~z, ,m,~ta~zt ge,~es i~~ tt~e ]~o,~c,ge 'mo,m~e

litter suggested a genetic bacI<ground for the anomaly; the titter was unfortunately, not reared; so the .female was outcrossed to a Strong UB~I male in order to recover the supposed recessive from the JP~. Thirty-three normal -Yl young were produceql. Thirteen pairs o f / ~ animals produced between nineteen anti eighty-two young each, 502 yonr~g. in all; none was janndiceft. If the original female was,heterozygous for the supposed recessive, one-half of her offspring should have inherited the gone, and one quarter of the F 1 matings should have segregated; die probakglity of obtaining thirteen non-segregating marinas is 0-05)4. As nei.ther the interchange of partners between some of the f~'l pairs, nor various J£~ marinas has i.ed to the recovery of jaundiced young, ie seems unlikely dmt I have fallen a victim go the vagaries of sampling; perhaps it was a ease of mistaken identity of the mother, who came from a cage containing several females.

Kowever, the unsuccessful outcross led to the discovery of another gone for which the female was heterozygous. Five of th.e thirteen/~1 madngs segregated for a gone prod.acing hydrocephalus, and several .Y~ matings have since been added. In their general appearance hych'ocephalic young (to be designated as 5Sroeei)I~cd'us-3 ; symbol }Y 3) are very similar to the hydrocephalus-], described by Clark (1932, I93~) and to hydrocephalus-2 described by Zimmermann 0933). Extreme manifestation of hydrocephalus~3 is shown in Figs. 2 and 3; the cranial capacity of the normal skull was 0-40 c.e., that of the abnormal skull 0.88 e.e. k hy&'oeephalus-3 male which was less extreme and killed whet± 58 days old had:a cranial capacity of 0-73 s.c., its normal brother one of 0-~4 c.c. The form of the dome-shaped head, both in the living mouse and in the skull, is very similar to the illus- trations given by Zimmermann. The same seems to be the case with the gross anatomical structure of the brain, which shows an enormous dilation of the Iateral ventricles, with extreme thinning of the pallium, tn albinos the head may be almost transparent. The basal ganglia of the brain are much less affected than the pallium. The haemorrhages into the pia-arachnoid space mentioned by Clark (193~) for i~?]-I are common in far progressed cases of hyde. Taken as a whole, the three genes seem to be extraordinarily similar in their effects. There are, however, a number of differences which seem to &ifferentiate ~y-3 from ]~y-1 and 5y-2.

Ha-1 can often be recognized at birth;/~y-3 manifests itself later; the ear~iest tentative classification, was successful in a 5_-day-old litter, but usually the abnormality of the l~ead develops din'lug the second week. Occasionally the development of the anomaIy is later still. In one ease a young mouse which looked quite ~ormal at the age of 2I. days had developed hycboeephalus when next inspected 11 days later. The general ~'owth of t~y-3 is usually retarded towards the end of the first week, and heavily abnormal animals never reach normal size again.; they rarely live to the age of $ months, most of them dying when they are about 4-6 weeks old. Towards the end )~,y-3 young become increasingly inactive, Iose weigh~ and eventually die in an emaciated condition; possibly Clark's suggestion thaz hydroeephalic young become unable to find food and drink may account for this final dseline. On account of its later on<st, by-3 is more similar to l~y-2 than to by-1.

A peculiarity of h~-3 young not mentioned for either ]~y-1 or ]~y-2'is diseha,rge from the nose. Sometimes this is only sligh< the nostriIs looking reels% or there being dried scabs of mucus. Often, particularly ai'ter the animals have been handled for .some time, the discharge becomes so heavy that they blow bubbles from their nostrils. In smears the discharge contains a few squamous epithelia and some polynmrphonuelear leucocye.es; in addition, a gram-poSitive coccus, a gram-negative (colifomn) bacillus and a gramTositfve

27

spore~forming bacterium with subterminal spores have been observed, none of them in gre~t numbers; the spore-ibrming bacterium is probably a member of the B. subti~is group and presumably derived from the nesting material {hay). The smears do not give %he impression, as if the discharge was caused by the micro organisms encountered in it.

The degree of hydrocephalus varies greatly. From the most extreme-forms, tike the animal shown .in {he illustrations, a gTadual transition occurs to forms where classification in %he living anima.1 Becomes m~oertain. In segregaling rantings there occur some young which are retarded in g r o ~ h like the madifes~ hydroeeptsali, ~nd which, also show nasal discharge, without ~he head shape being appreciably abnormal. -Sometimes the skinned skull shows a slight bulging which was not recognizable in the living animal; sometimes the skinned s ta l l appears normal. In two or ~hree such ammals the post.-mortem examina- tion has revealed t h e existence of a milder form of internal hydrocephalus, which was apparently insu~eien% to blow up the skull. The fate of these doubtful animals varies. Some of them never reach normal size again and die like mani%st hydrocephali; others

gig. 2. Crown view of a by-3 skull (right} a~ld of t&e skull of a normal brother. 39 da.ys otd.

Fig.3. Lateral views of the sa.me skulls; Ay-3 bdow.

eventually make up the-leeway, the dischm'ge from the nose gradually subsides, and later oll they are indistinguishable from normal ndoe in external al~peara:~.oe. Whether such animals may breed, is uncertain. One male reached fall adub size (30.6 g. at the age of 207 days) and looked per£ecCly healthy; however, he failed to produce offspring with a ~ormal sister who shared his cage for 5 months ; as ~he females of {1.~e stock were all very prolige= he was prol)a,l)ly steri.le.

The segregations so far obtained are givez~ in Table it. Iiz addition ~he]:e was cue smMI mating of se~:en nohnals and otto 7ty-3. The ~donbtful' animals a,re individuals which were retarded, in developmm~t and ].tad discharge .Item the nose w~thout having a defini.tely hydroeepha,lic head shape. I+a will be seen tl.m.~ the undoubtedly hycbocephalic young are signifmant, ly less tha,n a quarter (17-73 ± 2-11%). Probably most or all ogthe 'doubtfuIs ' are hT/~g/~e.y-3; if they are added, to the fully mamifest hydrocephali, a precise 9,: 1. ratio is obtaiued. }Iowever, it is possible tha t this grmtp contained a few mice ~:etarded for di/~.'erent reasons; also, it is possible tha t some homozygotes arc so little a[i['ected that they are missed altogether mxd classified as normals. Xowever this m@" he, the conclusion

28 :Two "new 7nutant genes in the house ,mouse

seems justified that we are dee,ling wit, h a single recessive gone of varia.ble m~mifestation, which produces hydrocephalus visible ~n ehe living mouse in about 7]. % of the homo. zygotes, and nfin.or changes (retardation of development, discharge fl'om the nose, mild

Table 1. £'egregation of by-3 i~fatlng NormM Doubtful hy-3 Tota l

1 15 3 1 1.9 2 40 6 9 55 3 63 3 16 82, 4= 55 9 12 76 5 41 7 I3 61 6 2-t 2 7 33 7 37 0 1.0 47 8 42 1 7 50

To~ul 317 31 75 423

in~ernal hydrocephalus) in mos~ or all of ~he remaining 29 %. The occurrence of mildly affected animals in ]z.y-1 h~s been briefly mentioned by Clark; whether.they uIe ever as nearly norma,1 as many of the 'donbtNls' in 7zy-3 is not quite' clear from the pubIished account. Zimm.ermann men~ions no normal overlaps in by-2.

~U_'K~Jf3. I{ Y

I. Fidget is a mew recessive gene with regular manifestat%n and nearly normal vi~bility under laboratory conditions. 3:ffected animals shake their heads in the horizontal plane and often run in circles. A period of hypersensitivity ~o sounds early in life is followed by deafness or a condition approximating ;leafness. Lesions of the cornea are regularly found. Polyd~ctylism of the hind fee~ was observed in fifteen out of eighty-six fidge~s; its rare occurrence in normal segregants may be due ~o occasional semi:dominance of this trait. Fidge~ males usually breed welt, whiIe ~dget females are unreliable mothers .

2. tIy&roeephalus-3 is a recessive gone which overlaps normM if classification is tassel on living animals. The onset of clinical signs !s usually early in the second week; bu~ occasionally much later, tIeavily affected animals are greatly retarded in development and rarely reach the age of two months. A feature of hydrocephalie young is nasal dis- charge, which is also found in mild eases, which have no recognizable skM1 anomaly, though they are- regarded in development~ Such young often survive ~o reach fult adul~ size ; whether 5hey may become sexualIy functional is nnknown.. The genetic relations of hy-3 to by4 and by-2 remain ~o be ele~red up by crosses.