&p.1:Abstract Small eye (Pax6Sey) is a semi-dominant muta-tion affecting development of the eyes, brain and nasalstructures. The mutant phenotype arises from defectswithin the Pax6 gene and several mutant alleles havebeen identified. A previous study reported thatPax6Sey/Pax6Sey homozygotes, in a random-bred stock,had a median cartilaginous rod-like structure in the nasalregion and 80% had supernumerary upper incisor teeth.In this study we show that supernumerary upper incisorteeth and a previously unreported nasal capsule-derivedcartilaginous ‘spur’ occur in compound heterozygousPax6Sey-Neu/Pax6Sey and homozygous Pax6Sey/Pax6Sey fe-tuses from several strains of mice. The frequencies of theabnormal phenotypes were not related to allele type butshowed variable penetrance, which was dependent on ge-netic background. The median nasal cartilaginous rod-like structure was present in all homozygous small eyefetuses. The Pax6Sey/Pax6Sey homozygote may provideinsight into the complex gene interactions involved ineye, nasal and craniofacial morphogenesis.

&kwd:Key words Small eye · Pax6 · Sey · Upper incisor teeth ·Cartilaginous rod · Mouse · Genetic backgound · Fetus&bdy:

Introduction

The semi-dominant mouse small eye gene (Pax6Sey; pre-viously Sey) causes early postnatal lethality (with anoph-thalmia, brain and nasal abnormalities) in homozygotesand microphthalmia in heterozygotes. The mutation re-

sponsible for the small eye phenotype affects the Pax6gene on chromosome 2 (Hill et al. 1991; Ton et al.1992). Pax6 is a member of the paired-box gene familyof transcription factors (Stuart et al. 1994) and severaldifferent alleles have been identified in the mouse. Theseinclude two spontaneously-arising alleles, Pax6Sey (Rob-erts 1967) and Pax6Sey-Dey(Theiler et al. 1978, 1980; Da-visson 1986); three radiation-induced variants, Pax6Sey-

1H, Pax6Sey-2Hand Pax6Sey-3H(Hogan et al. 1986; Cattan-ach et al. 1996) and an ethylnitrosourea-induced muta-tion Pax6Sey-Neu(Favor et al. 1988; Hill et al. 1991). Allof these mutations produce a characteristic microphthal-mic heterozygous phenotype and are homozygous lethal.

The Pax6Sey and Pax6Sey-Neualleles used in this studyare both caused by single base pair changes within thePax6gene. The Pax6Seymutation results in a translationproduct that contains the paired box domain but is trun-cated prior to the homeobox region. The Pax6Sey-Neumu-tation causes abolition of a splice site in the serine/threo-nine-rich domain at the 3’ end of the mRNA transcript(Hill et al. 1991). Although mRNA is produced, both ofthese single base pair mutations almost certainly result innon-functional translation products.

It has been proposed that the primary eye and nasaldefects in the homozygous small eye mouse are due todefective induction of lens and nasal placodes, the pro-spective regions of surface ectoderm of the head fromwhich lens and nasal tissues will form (Hogan et al.1986; Davidson and Hill 1994; Grindley et al. 1995).Chimaera studies have shown that cells that are unable toproduce functional Pax6protein fail to contribute to thelens or nasal epithelium, implying that the effect of Pax6in these tissues is cell autonomous (Quinn et al. 1996).These studies also revealed roles for Pax6 in the opticcup (neural retina and retinal pigment epithelium). How-ever, other craniofacial abnormalities in the homozygousPax6Sey/Pax6Seyfetus have been less well characterised.

Homozygous Pax6Sey/Pax6Sey fetuses with both acomplete and partial lateral duplication of the upper inci-sors were recently reported by Kaufman et al. (1995).These fetuses also had a midline cartilaginous rod-like

J.C. Quinn · J.D. WestCentre for Reproductive Biology,Department of Obstetrics and Gynaecology,University of Edinburgh, 37 Chalmers Street,Edinburgh EH3 9EW, UK,Tel.: +44-131-229-2575; Fax: +44-131-229 2408

M.H. Kaufman (✉)Department of Anatomy, University of Edinburgh,Medical School, Teviot Place, Edinburgh EH8 9AG, UKTel.: +44-131-650 3113; Fax: +44-131-650 6545&/fn-block:

Anat Embryol (1997) 196:311–321 © Springer-Verlag 1997

O R I G I N A L A RT I C L E

&roles:Jane C. Quinn · John D. West · Matthew H. Kaufman

Genetic background effects on dental and other craniofacialabnormalities in homozygous small eye (Pax6Sey/Pax6Sey) mice

&misc:Accepted: 28 April 1997

structure protruding between the two maxillae in the ab-normal nasal region, and this appeared to be an anterior-ly-directed extension of the chondrocranium.

The present histological study was undertaken to testwhether the supernumerary upper incisor teeth and thecraniofacial abnormalities reported by Kaufman et al.(1995) were consistent features of the homozygous Pax6mutant genotype or were dependent on the genetic back-ground.

Materials and methods

Mice

The original small eye mutation (Roberts 1967) arose in a stockcalled “CSR” (Roberts 1966) and was subsequently outcrossed.The small eye strain used in the study by Kaufman et al. (1995)was maintained at the Institute of Cell, Animal and Population Bi-ology, University of Edinburgh. This strain was of heterogeneouscoat colour and was obtained from Dr. Ruth Clayton. The presentgenetic background of this strain is unclear but it may contain ele-ments of C57BL/Fa, JU/Fa and JBT/Jd as well as the original CSRstock (Pritchard et al. 1974). This stock was maintained by ran-domly intercrossing heterozygotes and will be identified as“MHK”.

Four more small eye strains were included in the histologicalanalysis. Strains CBA/Ca-Seyand CBA/Ca-SeyNeu (heterozygousfor Pax6Seyand Pax6Sey-Neu alleles, respectively, on a predominant-ly CBA/Ca background) were obtained from the MRC Human Ge-netics Unit. Neither strain can be considered to be fully congenicon the CBA/Ca inbred strain (pedigree records are not available)but the two strains had been maintained by crossing heterozygotesto CBA/Ca mice for over 4 years (CBA/Ca-Sey) and over one year(CBA/Ca-SeyNeu) respectively, before the start of the experiment.The Pax6Sey/+ stock was obtained from Dr. Ruth Clayton by theMRC Human Genetics Unit in 1990 and maintained by crossingPax6Sey/+ heterozygotes to inbred CBA/Ca mice. The Pax6Sey-Neu

allele arose on a (C3H × 102)F1 genetic background at the Institütfür Genetik, Neuherberg, Germany and was obtained from Dr.Jack Favor by the MRC Human Genetics Unit in the summer of1993, where it was maintained by crossing Pax6Sey-Neu/+ heterozy-gotes to inbred CBA/Ca mice. Homozygous fetuses were pro-duced by CBA/Ca-Sey × CBA/Ca-Sey and CBA/Ca-SeyNeu × CBA/Ca-SeyNeu crosses initiated in the Centre for Repro-ductive Biology in January 1995. Pax6Sey-Neu/Pax6Sey fetuses wereproduced from crosses initiated in September 1994 between CBA-SeyNeuand another small eye stock, SEYTG.

SEYTG is homozygous for the reiterated β-globin transgeneTgN(Hbb-b1)83Clo (abbreviated to Tg) on chromosome 3, derivedfrom “strain 83” (Lo 1986; Lo et al. 1987). The original “strain83” or “β83” was generated on a C57BL/6J × SJL/J genetic back-ground and our stock was derived from some imported into theUK by Professor David Whittingham, on a B63H × CD1 back-ground (Wood 1990). Strain TGB was derived from crosses be-tween “strain 83” and (C57BL/Ws × CBA/Ca)F1 hybrids set up in1992 (Keighren and West 1994). After selection for Tg/Tg homo-zygotes, TGB was maintained as a random-bred, closed colony.The SEYTG stock (Pax6Sey/+; Tg/Tg) was derived from matingsset up in June 1993 between CBA/Ca-Sey/+ heterozygotes andTGB mice.

Pax6 maps approximately 26cM proximal to the agouti locuson chromosome 2 (Davisson 1986; Hogan et al. 1986, 1987).Strain CBA/Ca is homozygous agouti (A/A), strain C57BL/Ws ishomozygous non-agouti (a/a), TGB mice may be non-agouti (a/a)or agouti (A/a or A/A) and the CBA/Ca-Sey/+ mice were allphenotypically agouti (A/A or A/a). Each first generation matingbetween CBA/Ca-Sey/+ and TGB produced some non-agouti, het-erozygous small eye offspring (Pax6Sey a/+ a) and Pax6Sey segre-gated preferentially with a. Thus, the alleles for Pax6Sey and a

must have been introduced in coupling from CBA/Ca-Sey/+ mice,which were Pax6Seya/+ A. Progeny were backcrossed to TGB fortwo further generations before resultant Pax6Sey/+ , Tg/Tg animalswere used to establish the SEYTG stock. The SEYTG stock wasthen maintained by crossing SEYTG animals (Pax6Sey/+ , Tg/Tgand a/a, A/a or A/A) to agouti TGB mice (+/+ , Tg/Tgand A/a orA/A). The SEYTG stock continued to segregate at the agouti locusand separate agouti (A/a or A/A) and non agouti (a/a) sublineswere established.

Fetal dissections and analysis

Pax6Sey/+ or Pax6Sey-Neu/+ small eye heterozygotes were mated toanimals of the appropriate genotype and mating was confirmed bythe presence of a vaginal plug the following morning. This wasdesignated E0.5 days. Pregnant females were killed by cervicaldislocation on days E16.5 to E18.5. Fetuses were dissected fromthe uterus into cold phosphate buffered saline (PBS) on ice and ex-amined under a Wild M5 dissecting microscope.

Homozygous Pax6Sey/Pax6Sey, Pax6Sey-Neu/Pax6Sey-Neuor com-pound heterozygous Pax6Sey-Neu/Pax6Sey fetuses produced in thedifferent matings were easily identified at dissection by the ab-sence of eyes and characteristic craniofacial phenotype of fore-shortened upper jaw associated with a protruding tongue. Fetusesof desired phenotype were decapitated and their heads fixed inBouin’s fixative overnight. Samples were embedded in paraffinwax, serially sectioned at 7µm in the coronal plane and stainedwith haematoxylin and eosin. Slides were examined using a LeitzDiaplan light microscope.

In addition 19 MHK (Pax6Sey/Pax6Sey) neonates (postnatal dayP1.0) from the original study by Kaufman et al. (1995) were re-considered in this study. For ease of description, both theE16.5–E18.5 fetuses and P1.0 MHK strain neonates will be re-ferred to as fetuses.

Statistical analysis

Statistical analysis comparing the frequency of supernumeraryupper incisor teeth and the presence of cartilaginous ‘spurs’ in thedifferent genotype and strain groups was performed by Fisher’sexact test on an Apple Macintosh computer using the statisticalpackage “Statview 4.1” (Abacus Concepts, Berkeley, Calif.USA).

Results

A total of 46 homozygous (Pax6Sey/Pax6Seyor Pax6Sey-Neu/Pax6Sey-Neu) and compound heterozygous (Pax6Sey-Neu/Pax6Sey) fetuses were examined for the presence of super-numerary upper incisor teeth and a median cartilaginousnasal rod. The fetuses were separated into four groups bystrain and genotype: 12 CBA/Ca-Sey (Pax6Sey/Pax6Sey),11 CBA/Ca-SeyNeu (Pax6Sey-Neu/Pax6Sey-Neu), 14 SEYTG(Pax6Sey/Pax6Sey) and 9 Pax6Sey-Neu/Pax6Seycompound het-erozygotes from (CBA/Ca-SeyNeu × SEYTG)F1 matings.Of the 14 Pax6Sey/Pax6Sey, SEYTG fetuses examined, sev-en were from the non-agouti subline and seven were fromthe agouti subline.

In all of the small eye homozygous or compound het-erozygous fetuses and neonates examined, developingmolar dentition was present appropriate to the develop-mental age. All three of the abnormal features describedbelow were present before E16.5, so differences in de-velopmental age could not account for any differences in

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the frequencies of the abnormalities among the differentstrains of mice that were studied.

Incidence of supernumerary upper incisor teeth

The results of histological observations are shown in Ta-bles 1 and 2. No supernumerary upper incisor teeth wereobserved in any of the 12 CBA/Ca-Sey(Pax6Sey/Pax6Sey)or 11 CBA/Ca-SeyNeu (Pax6Sey-Neu/Pax6Sey-Neu) fetusesexamined (Fig. 1c,d). Of the seven SEYTG(Pax6Sey/Pax6Sey) fetuses from the agouti subline, three(43%) possessed supernumerary upper incisor teeth. Twofetuses possessed one extra upper incisor tooth posi-

tioned lateral to the normal left upper incisor. The thirdfetus possessed two supernumerary incisors, giving a to-tal of four; one supernumerary tooth was slightly smallerthan normal and located lateral to the normal left incisor,while the right supernumerary tooth was considerablysmaller than normal, resembling no more than a toothbud. Of the seven SEYTG (Pax6Sey/Pax6Sey) fetuses ex-amined from the non-agouti subline, only one possesseda single supernumerary upper incisor tooth (14%), thisbeing located lateral to the normal left incisor.

Of the nine Pax6Sey-Neu/Pax6Sey fetuses examined, two(22%) possessed a single supernumerary upper incisortooth. In both cases this extra tooth was situated lateralto the normal left incisor. In one fetus the supernumerary

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Table 1 Number and percentage of fetuses in each strain and genotype group examined exhibiting supernumerary upper incisor teeth,cartilaginous ‘spurs’ and a median nasal cartilaginous rod&/tbl.c:&tbl.b:

Strain Genotype No No. (%) of fetuses with featureoffetuses Supernumerary Cartilaginous Median cartilaginous

upper incisors ‘spurs’ nasal rod

MHK Pax6Sey/Pax6Sey 20b 16 (80)b 12 (63)c 20 (100)bSEYTG (A/?)a Pax6Sey(A or a)/Pax6Sey(A or a) 7 3 (43) 6 (86) 7 (100)SEYTG (a/a)a Pax6Seya/Pax6Seya 7 1 (14) 0 (0) 7 (100)CBA-Sey Pax6Sey/Pax6Sey 12 0 (0) 4 (33) 12 (100)(CBA-SeyNeu×SEYTG)F1 Pax6Sey-Neu/Pax6Sey 9 2 (22) 2 (22) 9 (100)CBA-SeyNeu Pax6Sey-Neu/Pax6Sey-Neu 11 0 (0) 3 (27) 11 (100)

a SEYTG (a/a) fetuses are from a/a×a/a matings. SEYTG (A/?)fetuses are from A/?×A/? matings where A/? is either A/A or A/a.Consequently SEYTG (A/?) fetuses may be A/A, A/aor a/a at theagouti locusb Since Kaufman et al. (1995) was published, the histological sec-tions from one of the fetuses reported has been misplaced, there-

fore, data pertaining to number of fetuses with supernumeraryteeth and median cartilaginous nasal rods is taken from the origi-nal publication whilst number of fetuses with cartilaginous ‘spurs’considers only 19 of the original 20 fetusesc Data from analysis of 19 extant fetuses&/tbl.b:

Table 2 Table showing number of supernumerary upper incisor teeth and cartilaginous ‘spurs’ possessed by each fetus examined forall genotype groups studied (Extra teethnumber of supernumerary upper incisor teeth, Spursnumber of cartilaginous ‘spurs’)&/tbl.c:&tbl.b:

Genotype: Pax6Sey Pax6Sey(A or a) Pax6Seya Pax6Sey Pax6Sey-Neu Pax6Sey-Neu

Pax6Sey Pax6Sey(A or a) Pax6Seya Pax6Sey Pax6Sey-Neu Pax6Sey

Strain: MHK SEYTG (A/?) SEYTG (a/a) CBA/Ca-Sey CBA/Ca-SeyNeu (CBA/Ca-SeyNeu×SEYTG)F1

Fetus Extra Spurs Extra Spurs Extra Spurs Extra Spurs Extra Spurs Extra Spursno. teeth teeth teeth teeth teeth teeth

1 2 2 2 0 1 0 0 1 0 1 1 02 2 2 1 1 0 0 0 1 0 1 1 03 2 1 1 1 0 0 0 1 0 1 0 24 2 1 0 2 0 0 0 1 0 0 0 15 2 0 0 1 0 0 0 0 0 0 0 06 2 0 0 1 0 0 0 0 0 0 0 07 2 0 0 1 0 0 0 0 0 0 0 08 2 0 – – – – 0 0 0 0 0 09 2 0 – – – – 0 0 0 0 0 0

10 1 2 – – – – 0 0 0 0 – –11 1 2 – – – – 0 0 0 0 – –12 1 1 – – – – 0 0 – – – –13 1 1 – – – – – – – – – –14 1 1 – – – – – – – – – –15 1 1 – – – – – – – – – –16 1 0 – – – – – – – – – –17 0 2 – – – – – – – – – –18 0 1 – – – – – – – – – –19 0 0 – – – – – – – – – –

incisor was of normal size and shape, but in the secondfetus the extra tooth appeared smaller than its normalcounterpart. The development of both supernumeraryteeth was retarded and the connection between the devel-oping tooth and the oral epithelium was still evident.This appearance confirmed that the normal incisor andthe supernumerary incisor were both derived from inva-ginating oral epithelium rather than the supernumerarytooth having budded from the lateral aspect of the nor-mal incisor tooth.

Of the 20 MHK (Pax6Sey/Pax6Sey) fetuses reported inthe original study (Kaufman et al. 1995), five (25%)possessed a single supernumerary tooth lateral to thenormal left upper incisor (Fig. 2 g, h; Fig. 4b), and two(10%) showed a single supernumerary tooth lateral tothe normal right upper incisor tooth, only four fetusespossessing the normal complement of two upper incisorteeth (see Fig. 4a). However, in nine fetuses (45%) acomplete duplication of the upper incisor teeth was ob-served with a supernumerary incisor tooth located later-

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23

4

1 5

5a b

6

c d

6

77

Fig. 1a, b Representative co-ronal section through the fron-to-nasal region of a Pax6Sey/+(strain MHK) postnatal dayP1.0 control mouse. Note thepresence of the following: 1 vi-brissae, 2 cartilaginous nasalcapsule, 3 cartilaginous nasalseptum, 4 nasal (olfactory) cav-ity, 5 upper incisor teeth.c, d Close to symmetrical coro-nal sections through the fronto-nasal region of (c) an E17.0 ho-mozygous Pax6Sey-Neu/Pax6Sey-

Neu fetus (strain CBA-SeyNeu)and (d) a postnatal day P1.0homozygous Pax6Sey/Pax6Sey

mouse (strain MHK). Note thepresence in both (c) and (d) ofa median cartilaginous rod-likestructure (6), two upper incisorteeth and, in fetus d, two‘spurs’ (7). Close-up views ofthe fronto-nasal region of thefetus illustrated in 1c are shownin Figs. 2a–c, and those of themouse illustrated in 1d areshown in Figs. 3d–f. All sec-tions illustrated in Figs. 1–4were stained with haematoxylinand eosin. a, c, d× 25; b × 40&/fig.c:

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cb

d fe

a

g h

Fig. 2 Representative intermittent serial sections through thefronto-nasal region of an E17.0 Pax6Sey-Neu/Pax6Sey-Neufetus fromstrain CBA-SeyNeu (a–c) and two day P1.0 postnatalPax6Sey/Pax6Seymice from strain MHK (d–f; g–h). The fetus illus-trated in a–c possessed two upper incisor teeth; no cartilaginous‘spurs’ were present. The mouse whose fronto-nasal sections are

illustrated in d–f possessed three upper incisor teeth, the ‘spur’(arrow) being present on the same side as the supernumerary up-per incisor tooth. While the latter is not observed in this series ofsections, it is shown in Fig. 4b. The mouse illustrated in g and hpossessed a left-sided supernumerary upper incisor tooth, but inthis instance a single ‘spur’ was present on the right side. × 63&/fig.c:

al to each normal upper incisor (Fig. 4c). Only one ofthe supernumerary teeth examined in this series ap-peared to be less well differentiated and smaller than itsnormal counterpart.

No teeth, whether supernumerary or normal, showedany evidence of pathology or abnormality of the enamel,dentine or pulp tissues where present. All fetuses exam-ined possessed the normal number of upper and lowermolars and lower incisor teeth.

Pairwise comparisons (Table 3) indicated that the ge-netic background modulated the occurrence of supernu-merary upper incisor teeth in the Pax6Sey/Pax6Seyfetuses.The frequency of fetuses with supernumerary teeth wasnot significantly different in the agouti and non-agouti

sublines of SEYTG (3/7 versus 1/7). However, severalother differences were significant (Table 3) and this im-plies that genetic background affects the penetrance ofthis phenotype. The penetrance was highest in MHK fe-tuses (16/20; 80%) and lowest in CBA/Ca-Sey fetuses(0/12; 0%). No supernumerary teeth were found amongPax6Sey/Pax6Sey or Pax6Sey-Neu/Pax6Sey-Neu genotypeswhen they were on a similar CBA/Ca genetic back-ground. Although homozygous Pax6Sey-Neu/Pax6Sey-Neufe-tuses were not tested on other genetic backgrounds, theoccurrence of extra teeth in Pax6Sey-Neu/Pax6Sey fetusessuggests that the two alleles produce similar abnormali-ties. No extra teeth were found in serial sections of10 Pax6Sey/+ heterozygotes (6 fetal and 4 newborn) fromthe MHK stock (Kaufman et al. 1995), so this is not adominant effect of the Pax6Seyallele.

The median cartilaginous nasal rod

All 46 fetuses examined in this study (Pax6Sey/Pax6Sey,Pax6Sey-Neu/Pax6Sey-Neu and Pax6Sey-Neu/Pax6Sey) pos-

316

a

d

b

e

c

f

Fig. 3a–f Representative intermittent serial sections through thefronto-nasal region of two postnatal day P1.0 Pax6Sey/Pax6Seymice(strain MHK) both of which possessed two ‘spurs’ (arrows). Thesections illustrated in (a–c) are from a mouse which possessed twoupper incisor teeth, whilst those illustrated in (d–f) are from amouse which possessed two supernumerary upper incisor teeth.The latter are illustrated in Figure 4c. × 63&/fig.c:

sessed a median nasal cartilaginous rod (Tables 1, 2;Figs. 1c, d, 2–4). Thus, this phenotype was fully penet-rant on all genetic backgrounds studied. No evidence ofany nasal or eye tissues were observed in any of thesmall eye homozygous or compound heterozygous fe-tuses examined.

Presence of cartilaginous ‘spurs’

A previously unreported phenotype was observed in thePax6Sey/Pax6Sey, Pax6Sey-Neu/Pax6Sey-Neu and Pax6Sey-Neu/Pax6Sey fetuses. Ectopic cartilaginous ‘spurs’, derivedfrom the antero-lateral aspect of the nasal capsule, werepresent in some fetuses. At their origin, the ‘spurs’ ap-peared to have a central core of cancellous bone; moredistally, a central cartilaginous core was present sur-rounded by a fibrous connective tissue capsule (Figs. 2d–h, 3 a–f).

The ‘spurs’ extended forwards from the level of thenormal incisor teeth towards the front of the snout, andmeasured up to 250µm in length. In the normal wild-type fetus, the nasal capsule surrounds the developingnasal tissues and septum, the area of the upper mandiblein which the incisors are developing being outside thisdiscrete nasal area (Fig. 1a,b). However, in thePax6Sey/Pax6Sey homozygous fetus, the “nasal” capsulesurrounds the entire area of the developing incisor teeth,which are embedded in cancellous bone (Fig. 1c,d). The‘spurs’ are not dental derivatives, as cartilage is neverpresent in the developing tooth. ‘Spurs’ were presentunilaterally or bilaterally in Pax6Sey/Pax6Sey, Pax6Sey-Neu/Pax6Sey-Neuand Pax6Sey-Neu/Pax6Sey fetuses and in all butone of the strains examined (Tables 1, 2) but were notobserved in any wild type mice or in their small eye het-erozygous littermates.

Comparisons of the incidence of cartilaginous ‘spurs’between different strains are shown in Table 3. No sig-nificant difference was found between Pax6Sey/Pax6Sey

and Pax6Sey-Neu/Pax6Sey-Neu fetuses on a similar geneticbackground. However, as was observed for supernumer-ary teeth, genetic background appeared to influence thepenetrance of the cartilaginous ‘spurs’ and there weresome statistically significant differences betweenPax6Sey/Pax6Seyfetuses from different strains.

The penetrance was highest in MHK (Pax6Sey/Pax6Sey)fetuses (12/19; 63%) – significantly higher (P = 0.0064)than for fetuses from the non-agouti subline of SEYTG(0/7; 0% penetrance). A significant difference betweenagouti and non-agouti SEYTG sublines suggests thatthey differed for genes that affect the penetrance of carti-laginous ‘spurs’ (Table 3) but there are insufficient datato test whether the region of chromosome 2 close to theagouti locus is likely to be involved.

The stocks with the highest frequency of cartilaginous‘spurs’ (MHK and the agouti subline of SEYTG) alsohad the highest frequency of supernumerary upper inci-sor teeth (Tables 1, 2) which suggests that the penetranceof these phenotypes are affected by the same genes. Thepresence of cartilaginous ‘spurs’ was not, however, de-pendent on the presence of a supernumerary tooth as‘spurs’ were present unilaterally or bilaterally in fetusesthat possessed no supernumerary teeth (Table 2; Figs. 2d–h, 3 a–c). Moreover, division of the 19 MHK strainmice into three groups, according to the number of su-pernumerary teeth (0, 1 or 2), did not reveal a paralleltrend in either the presence of cartilaginous ‘spurs’ (2/3;

317

a

b

c

Fig. 4 Representative coronal sections through the fronto-nasalregion of three postnatal day P1.0 Pax6Sey/Pax6Sey mice (strainMHK) that possessed either (a) the normal complement of two up-per incisor teeth, (b) a single supernumerary upper incisor, or (c)two supernumerary upper incisor teeth. × 63&/fig.c:

6/7; 4/9) or the mean number of ‘spurs’ (1.0; 1.1; 0.7).When a single supernumerary tooth was present, the su-pernumerary tooth and ‘spur’ were not always on thesame side (Fig. 2, g, h).

Variation in the number of supernumerary teeth or car-tilaginous ‘spurs’ (Table 2) implies that these phenotypesshow variable expressivity (1 or 2) as well as variablepenetrance (presence or absence). Therefore, geneticbackground, but not allele type, appears to be affectingboth the penetrance and expressivity of supernumeraryupper incisor teeth and cartilaginous ‘spur’ formation inPax6Sey/Pax6Sey, Pax6Sey-Neu/Pax6Sey-Neu and Pax6Sey-Neu/Pax6Seyfetuses. In contrast, in all small eye homozygotesand compound heterozygotes studied, regardless of ge-netic background, the median cartilaginous rod was al-ways present. However, as none of these phenotypes werepresent in Pax6Sey/+ heterozygotes or wild-type litter-mates (Fig. 1a,b), all these morphological abnormalitiesare causally related to a loss of functional Pax6product.

Discussion

Pax6effects on craniofacial development

Kaufman et al. (1995) reported that 80% of homozygousPax6Sey/Pax6Seyfetuses examined possessed supernumer-ary upper incisor teeth, with 45% of fetuses showing afull duplication of the upper incisors. In most cases, su-pernumerary teeth were of similar size to their normalcounterparts. The incidence of supernumerary upper in-cisor teeth that we observed in our crosses was consider-ably lower (0–43%). No supernumerary teeth were ob-served in homozygous small eye offspring from theCBA/Ca-Sey or CBA/Ca-SeyNeu strains. Although thevery high incidence of complete upper incisor duplica-tion reported by Kaufman et al. (1995) was not emulatedby the other stocks, all fetuses examined had a medianrod-like cartilaginous structure.

The presence of aberrant cartilaginous craniofacialstructures in Pax6Sey/Pax6Seyhomozygous fetuses may beconsequential to the loss of Pax6 function. The entirefrontofacial/nasal region in the Pax6Sey/Pax6Sey homozy-gous fetus is grossly abnormal. The absence of nasalstructures clearly has a dramatic effect on the morpho-genesis of the whole frontofacial/nasal region, with theupper incisors forming within a mass of cancellous bone.The severe degree of retrognathia observed inPax6Sey/Pax6Seyfetuses reflects a deficiency of the frontalpart of the maxillae and pre-maxilla. We therefore sug-gest that the median nasal cartilaginous rod and cartilagi-nous ‘spurs’ observed in the homozygous fetuses mayrepresent manifestations of inappropriate tissue specifi-cation related to deformity of the upper jaw resultingfrom loss of nasal derivatives.

Our results show that genetic background affects boththe penetrance and expressivity of Pax6Seywith respect tothe number of upper incisor teeth and cartilaginous‘spurs’ in Pax6Sey/Pax6Sey homozygotes. However, thepresence of the median cartilaginous rod-like structure isfully penetrant and was characteristic of both types ofhomozygous small eye fetuses and the compound hetero-zygote. The novel finding reported here of cartilaginous‘spurs’ originating from the lateral aspect of the abnor-mal nasal capsule adds further complexity to thePax6Sey/Pax6Sey homozygous phenotype. The abnormalfacial phenotypes seen are likely to be a consequence ofboth direct and indirect effects of the loss of the func-tional Pax6product in a variety of different tissues.

Pax6mutants in other species also result in facial ab-normalities. Rat fetuses homozygous for rSey (the rathomologue of small eye) have bilateral facial clefting(Matsuo et al. 1993; Fujiwara et al. 1994). However, al-though a limited delay of palatal fusion has been report-ed in homozygous Pax6Sey/Pax6Seymouse fetuses (Kauf-man et al. 1995), cleft palate and facial clefting havenot been observed in any mouse small eye strains todate.

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&/tbl.b:Table 3 Significance of genetic background and Pax6genotype on incidence of morphological abnormalities&/tbl.c:&tbl.b:

Strains of mice compared Proportion of fetuses with the morphological feature (positive: negative ratio)

Strain 1 Strain 2 Supernumerary upper incisor teeth Cartilaginous spurs

Strain 1 Strain 2 P* Strain 1 Strain 2 P*

Pax6Sey/Pax6Seyon different genetic backgroundsSEYTG (A/?) SEYTG (a/a) 3:4 1:6 0.56 6:1 0:7 0.0047MHK SEYTG (A/?) 16:3 3:4 0.057 12:7 6:1 0.37MHK SEYTG (a/a) 16:3 1:6 0.0022 12:7 0:7 0.0064MHK SEYTG (total) 16:3 4:10 0.0031 12:7 6:8 0.30MHK CBA/Ca-Sey 16:3 0:12 <0.0001 12:7 4:8 0.15CBA/Ca-Sey SEYTG (A/?) 0:12 3:4 0.036 4:8 6:1 0.057CBA/Ca-Sey SEYTG (a/a) 0:12 1:6 0.37 4:8 0:7 0.25CBA/Ca-Sey SEYTG (total) 0:12 4:10 0.10 4:8 6:8 0.70

Different Pax6 alleles on a similar genetic backgroundCBA/Ca-Sey CBA/Ca-SeyNeu 0:12 0:11 >0.99 4:8 3:8 >0.99

* Significant P values from Fisher’s exact tests are shown in italics&/tbl.b:

PAX6, the human homologue of mouse Pax6, is locat-ed in a syntenic region on chromosome 11p13 (Jordan etal. 1992; Ton et al. 1992; Glaser et al. 1994; Hanson andvan Heyningen 1995; Mirzayans et al. 1995). Althoughocular disorders in human PAX6heterozygotes have beenwidely reported, homozygotes are rare. A suspected ho-mozygous human fetus was reported by Hodgson andSaunders (1980) which lacked not only the eyes and nosebut also the adrenal glands. The absence of nasal bonesand defects of the parietal bones was noted at necropsybut no deformities of the palate or dentition were record-ed (S.V. Hodgson, K.E. Saunders, personal communica-tion to M.H.K.). More recently, a human homozygotewas reported that was phenotypically similar to themouse small eye homozygous fetus (Glaser et al. 1994).This individual exhibited anophthalmia, absence of ol-factory lobes, a high arched palate and micrognathia.

A number of other genes have been identified thatplay a role in morphogenesis of bone and cartilage in thehead region including Msx1and Msx2(MacKenzie et al.1991a,b, 1992; Jowett et al. 1993; Satokata and Maas1994) human MSX1(Vastardis et al. 1996), several Hoxgenes (Lufkin et al. 1992; Gendron-Maguire et al. 1993;Rijli et al. 1993) and Otx2 (Simeone et al. 1992a,b;Matsuo et al. 1995). Most strikingly, a median nasal car-tilaginous rod-like structure, similar to that observed inthe small eye homozygote fetus, was present in Otx2-/-

fetuses lacking nasal and forebrain tissues (Matsuo et al.1995). The presence of a median nasal cartilaginous rodboth in the Otx2-/- and all of the small eye homozygousfetuses supports the suggestion that it is a secondary con-sequence of the absence of frontofacial structures. Fewmouse mutants have been identified that exhibit abnor-malities of tooth development but exencephalic p53-defi-cient mice (Armstrong et al. 1996) and homozygous tet-raploid mouse embryos (Kaufman and Webb 1990;M.H.K., unpublished observation) have abnormal upperincisor dentition. Aberrant dentition was also seen in ex-encephalic mouse fetuses induced by prenatal exposureto hypervitaminosis A and trypan blue (Knudson 1965,1966).

The nature of the interactions between Pax6and othergenes involved in craniofacial morphogenesis is still un-clear. Pax6is involved in its own transcriptional regulation(Plaza et al. 1993) and is also able to initiate the transcrip-tion of other genes (Chalepakis et al. 1994; Cvekl et al.1995). Indirect evidence of Pax6 interaction with Msx1 inPax6Sey/Pax6Seymice has been reported, and there is someindication, from aberrant expression of Msx1 in the nasalregion in Pax6Sey/Pax6Sey embryos, that Msx1may be in-volved in positional specification of both presumptive na-sal and tooth regions (Grindley et al. 1995).

Genetic background effects on Pax6phenotypes

Heterozygous effects of loss of Pax6 function have beenwell documented in small eye strains of various geneticbackground (Roberts 1967; Clayton and Campbell 1968;

Pritchard 1973; Pritchard et al. 1974; Hogan et al. 1988).Clayton and Campbell (1968) commented on the asym-metry and extreme variation of the size and histologicalphenotype of the eye, variable effects on the skull andbrain, and the absence of the optic chiasma in some het-erozygotes. Pritchard (1973) showed that the size of theeye and the incidence of cataracts varied in Pax6Sey/+heterozygous mice when crossed onto the inbred strainsC57BL/Fa, JU/Fa and JBT/Jd. Overall, the Pax6Sey/+phenotype showed lowest penetrance on the JBT/Jd ge-netic background and small eyes were most often ob-served on the JU/Fa background. Conversely, cataractswere less obvious on the JU/Fa background than on theC57BL/Fa background but this may partly reflect the dif-ficulty in identification of small cataracts in eyes of thealbino JU strain. According to Jordan et al. (1992), thehuman PAX6 disorders aniridia and Peters’ anomalymost closely resemble the mouse Pax6Sey/+ ocular phe-notypes when they are expressed on outbred Swiss or in-bred CBA genetic backgrounds.

As in the mouse, mutations within human PAX6causea spectrum of ocular phenotypes, ranging from mild cat-aracts to severe iris hypoplasia with retinal abnormalities(Glaser et al. 1994; Fantes et al. 1995; Mirzayans et al.1995; Azuma et al. 1996). Different PAX6 mutationsmay result in different ocular phenotypes if, for example,some have residual transcriptional activity (Glaser et al.1994) but there is not always a direct correlation betweenthe phenotype and the severity of the genetic lesion.Variable expressivity was reported for a family with aninherited human eye disorder that was subsequentlyfound to be caused by a PAX6 mutation (Hittner et al.1982; Martha et al. 1994). Other familial differences inphenotype, resulting from heterozygosity for the samePAX6genomic defect, have been reported (Hanson et al.1994) and such cases of variable expressivity and pene-trance are probably attributable to genetic backgroundeffects.

Evidence from mouse and man suggests that Pax6 isinvolved in a cascade of gene functions affecting numer-ous aspects of frontofacial/nasal and cranial morphogen-esis, as well as eye and nasal development. Variability ofphenotype in the human PAX6/+ heterozygote, thePax6Sey/+ heterozygous mouse and the Pax6Sey/Pax6Sey

homozygous fetus can be conveniently attributed to ge-netic background effects. However, it is unclear how theloss of Pax6 function combines with the effects of otherunknown genes to generate the different phenotypes de-scribed. The small eye mouse may be a useful model foranalysis of the complex interactions that occur betweengene families involved in patterning and development ofthe vertebrate head.

&p.2:Acknowledgements We thank Denis Doogan, Maureen Ross,Jim Macdonald (CRB) and Paul Rooney (Ashworth Laboratory,University of Edinburgh) for expert mouse husbandry. We alsothank Dr. R.E. Hill for kindly providing the CBA-Seyand CBA-SeyNeu founder stocks and for helpful comments on the manu-script. J.C.Q. is grateful to the Faculty of Medicine, University ofEdinburgh for a Ph.D. studentship and J.D.W. is grateful to theWellcome Trust for financial support.

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