insertional mutation on mouse chromosome 18 with vestibular and

Copyright 0 1994 by the Genetics Society of America

Insertional Mutation on Mouse Chromosome 18 With Vestibular and Craniofacial Abnormalities

Chao-Nan Ting,**’.‘ David Kohrman,**‘ Daniel L. Burgess,* Ann Boyle? Richard A. Altschuler,* G. Gholizadeh? Linda C. Samuel~on,*’~ Wonhee Jang* and Miriam H. Meisler*

*Department of Human Genetics, University of Michagan, Ann Arbor, Michigan 48109-0618, TKresge Hearing Research Institute, University of Michigan, Ann Arbor, Michigan 48109-0506 and *Department of Genetics, Yale University,

New Haven, Connecticut 06510 Manuscript received June 25, 1993

Accepted for publication September 28, 1993

ABSTRACT A dominant mutation was generated in transgenic mice as a consequence of insertional mutation.

Heterozygous mice from transgenic line 9257 (Tg?257) are hyperactive with bidirectional circling behavior and have a distinctive facial appearance due to hypoplasia of the nasal bone. Morphological analysis of the inner ear revealed asymmetric abnormalities of the horizontal canal and flattening or invagination of the crista ampullaris, which can account for the circling behavior. The sensory epithelium appeared to be normal. The transgene insertion site was localized by in situ hybridization to the B 1 band of mouse chromosome 18. Genetic mapping in an interspecific backcross demonstrated the gene order centrome~e-Tg?~’~-8.8 f 3.4-Grl-1, Egr-1, Fgf-1, Apc--14.7 f 4.3-Pdgfr. The phenotype and the mapping data suggest that the transgene may be inserted at the Twirler locus. Homozygosity for the transgene results in prenatal lethality, but compound heterozygotes carrying the Tw allele and the transgene are viable. The function of the closely linked ataxia locus is not disrupted by the transgene insertion. This insertional mutant will provide molecular access to genes located in the Twirler region of mouse chromosome 18.

M OLECULAR analysis of mutant mice has be- come an important method for identifying

genes involved in mammalian development (REITH and BERNSTEIN 1991). The roles of growth factors, receptors and transcriptional regulators in normal development have been revealed by analysis of spontaneous mutants such as undulated (BALLING et al. 1 988), small eye (HILL et al. 199 l), Steel (reviewed in WITTE 1990), Patch (STEPHENSON et al. 199 l), short ear (KINGSLEY et al. 1992) and Snell dwarf (CAMPER et al. 1990; LI et al. 1990). The molecular defects in these spontaneous mutants were identified by posi- tional cloning and by their genetic proximity to candidate genes. Developmental mutations caused by insertion of foreign DNA provide another mode of access: by use of the inserted sequence as a molecular probe for direct isolation of the mutated gene (CON-

This approach has been successfully applied to the isolation of novel genes required for development from transgenic insertional mutants (WOYCHIK et al. 1990; WEIHER et al. 1990; HODCKINSON et al. 1993; HUGHES et al. 1993).

The Twirler mutation, which produces craniofacial abnormalities, was described by LYON (1958). Mice

STANTINI et al. 1989; GRIDLEY 199 1 ; MEISLER 1992).

MC 5041, Chicago, Illinois 60637. ’ Present address: Division of Cardiology, University of Chicago, 0509

‘ The first two authors contributed equally to this work. ’ Present address: Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109-0622.

Genetics 136: 247-254 (January, 1994)

heterozygous for the spontaneous allele, Tw, have a complex phenotype that includes circling behavior, hyperactivity and early onset obesity. The circling behavior is the consequence of abnormal structural development of the crista ampullaris in the inner ear. Tw/Tw homozygotes are born with cleft palate f cleft lip and do not survive. The Twirler locus has been mapped to proximal chromosome 18 (LYON 1958; LANE et al. 198 1). In this paper we describe a transgene induced insertional mutation that exhibits many of the phenotypic features of Twirler mice. The transgene insertion may have generated a new allele of Twirler, and thus provide molecular access to this developmentally important gene. Alternatively, Twirler and TZz5’ may represent mutations in two closely linked genes that are both required for craniofacial development.

MATERIALS AND METHODS

Animals: The origin of transgenic line 9257 was previously described (TING et al. 1992). A 25-kb ApaI/MluI fragment containing a human salivary amylase gene was microinjected into fertilized mouse eggs from matings between (C57BL/6J X CSH)FI individuals. Transgenic animals were identified by PCR of tail DNA using the human amylase primers HTA-C and HTA-N (TING et al. 1992). The line was maintained by crossing with strain C57BL/6J.

C57BL/6J and SPRET/Ei mice were obtained from the Jackson Laboratory, Bar Harbor, Maine. Tw/+ mice carrying the original Twirler mutation were kindly provided by MARY LYON, Medical Research Council, Harwell. Hetero-

248 C.-N. Ting et al.

zygous axJ/+ mice carrying the ataxia allele were obtained from GLENN RADICE, Massachusetts Institute of Technol- ogy, Cambridge, Mass.

Histology: Seven heterozygous transgenic mice and five C57BL/6J mice were heavily anesthetized with chloral hy- drate and perfused through the heart with phosphate buffer followed by 4% paraformaldehyde in phosphate buffer. Intrascalar perfusion of the same fixative through the round window was followed by immersion in fixative for 16-20 hr, one rinse with phosphate buffer, intrascalar osmication for 1 hr with 1% osmium in phosphate buffer through the round window and another buffer rinse. Inner ears from four transgenic and three normal mice were dissected and drilled to expose the semicircular canals and assessed under a Wild dissection microscope. All inner ears (dissected and nondissected) were decalcified for 1 week in 2% EDTA, dehydrated through a graded series of alcohols and embedded in plastic (EmGed 81 2). Six-micron sections were cut through the vestibular and auditory portions of the inner ear and treated with Richardson’s stain. Sections were assessed under normal and differential interference contrast optics on a Leitz photomicroscope.

Pulsed field gel electrophoresis: Spleen DNA was prepared in agarose blocks and digested with restriction en- zymes as described (SAMBROOK, FRITSCH and MANIATIS 1989). Digested blocks were electrophoresed on 1 % gels at 200 V for 24 hr, using a BioRad CHEF DR I1 apparatus with 40-100 sec pulse ramp. Gels were stained in 0.5 rg/ ml ethidium bromide and destained in water. Fragments were transferred to a nylon filter and hybridized with radi- olabeled cosmid clone N2 (GUMUCIO et al. 1988). In situ hybridization: Metaphase chromosomes were pre-

pared from spleen cells from a heterozygous transgenic animal after culture for 2 days in the presence of concana- valin A, as previously described (BOYLE, B A L L A R D ~ ~ ~ WARD 1990). Cosmid N2 (GUMUCIO et al. 1988) was labeled by nick translation with biotin-1 1-dUTP (BRIGATI et al. 1983). The L1 banding probe KS13A (FANNING 1983) was labeled with digoxigenin-1 1-dUTP. Fluorescence in si tu hybridization was carried out exactly as described (BOYLE et al. 1992). All image acquisition and processing was done on a Macin- tosh IIci computer. Images were merged and 24-bit pseu- docolored using custom software developed in DAVID WARD’S laboratory (T. RAND, unpublished data). For additional details on image generation see BOYLE et al. (1992). Images were photographed directly from the computer screen.

Genetic mapping: To generate the interspecific backcross, heterozygous transgenic mice were crossed with SPRET/Ei and female transgenic offspring were back- crossed to C57BL/6J. This backcross has been used for mapping loci on chromosomes 3 and 6 (BAIN et al. 1993; BARROW et al. 1993). DNA was prepared from newborn offspring by the salting out method of MILLER, DYKES and POLESKY (1988). Southern blotting was carried out as previously described (BARROW et al. 1993). For resolution of the 12-kb and 14-kb SphI fragments detected with the flanking probe, electrophoresis was carried out on 0.4% agarose gels (PFGE Agarose, Boehringer Mannheim).

Restriction fragment length polymorphisms were identified by Southern blot analysis of genomic DNA from strains C57BL/6J, C3H/HeJ and SPRET/Ei after digestion with a variety of restriction endonucleases. Blots were hybridized with the following isolated plasmid inserts: a 1.3-kb EcoRI fragment from mouse Apc cDNA clone 5 1 B (SU et al. 1992); an 0.7-kb EcoRI-Hind111 fragment from mouse Egr-1 cDNA clone pRSV3.1 (SUKHATME et al. 1988); an 0.475-kb EcoRI- XbaI fragment from mouse Fgf-1 cDNA clone fgfa(a1) (HE- BERT et al. 1990); a 1-kb Hind111 fragment from rat Grl-1

TABLE 1

Dominant inheritance of circling behavior in line 9257

Transgenic offspring

Nontransgenic offspring

Cross No. % circling No. % circling

1. Tgl+ X +/+ 53 77 86 0 2. Tgl+ X Tg/+ 23 78 5 0

Transgenic mice were crossed with C57BL16J. Inheritance of the transgene was assayed by PCR of genomic DNA.

cDNA clone pRM16 (MIESFELD et al. 1984); and a 1 .2-kb HincII fragment from mouse Pdgfrb cDNA clone pGR102 (YARDEN et al. 1986).

Cloning of flanking DNA: Genomic DNA from a transgenic heterozygote was digested with MboI, size selected by sucrose gradient centrifugation, and cloned into a cosmid vector. Nitrocellulose filter replicas of the library were screened by hybridization (SAMBROOK, Fritsch and MANIA- TIS 1989) using two transgene specific probes. Sixty-eight transgene-positive clones were probed with labeled mouse DNA to identify clones that also contain mouse repeated sequences; Cosmid AT 1 was found to contain approximately 15 kb of mouse DNA and 20 kb of transgene DNA.

RESULTS

Behavioral abnormalities in transgenic line 9257: Transgenic line 9257 was generated by microinjection of fertilized mouse eggs with a human genomic fragment (TING et al. 1992). The founder animal was crossed with strain C57BL/6J to generate the N1 generation. Three N 1 individuals demonstrated behavioral abnormalities including bidirectional circling and hyperactivity. This phenotype has persisted through nine generations of breeding. In a swimming test, affected mice are unable to remain at the surface of the water for more than 1-2 min, while normal mice can d o so for at least 5 min.

More than 20 lines expressing similar constructs have been studied [TING et al. ( 1 992) and unpublished data]. None of the other lines exhibit behavioral abnormalities, indicating that the circling phenotype is a characteristic of the specific insertion site in line 9257.

Autosomal dominant inheritance of circling behavior: The data are consistent with autosomal dominant inheritance of the circling phenotype with approximately 80% penetrance (Table 1). In the cross between heterozygous transgenic mice and C57BL/ 6J, the proportion of transgenic offspring was significantly lower than 50% (53/139, P C 0.01). However, in three other crosses described below, with SPRET/ Ei, ax/+ and Tw/+ mice, the proportion of transgenic offspring was close to 50%, suggesting that the viability of Tg/+ mice is influenced by genetic background.

Recessive lethality of the transgene mutation: Cos- mid clone A T 1, containing the junction between the transgene and flanking mouse DNA, was isolated from a library of heterozygous DNA by hybridization with

Mouse Craniofacial Mutation 249

+ @){

23 -

f o m

9.4 -

6.6 -

FIGURE 1.-Detection of a transgene junction fragment by Southern blotting. Genomic DNA was digested with SphI and fragments were separated by electrophoresis on an 0.4% agarose gel. Filters were probed with a 1-kb subclone (2B2) of cosmid A T l . Left, Hind111 fragments from bacteriophage X (kb): arrow, 16 kb transgene junction fragment.

both transgene and mouse genomic DNA probes. Subclones of AT1 containing nonrepetitive mouse DNA were used for RFLP analysis of the [(Tg/+ X SPRET/Ei)F, X C57BL/6J] backcross described below (not shown). The lack of recombination between AT1 and the transgene (0/46) is consistent with der- ivation of the mouse DNA in AT1 from the transgene insertion site. A 1-kb subclone of AT1 hybridized with a 12-kb SphI fragment in genomic DNA from strain C57BL/6J and an additional 14-kb fragment in heterozygous transgenic mice (Figure 1, arrow). The transgene specific 14-kb SphI fragment is contained within clone ATl .

T o determine whether transgenic homozygotes are viable, 5 litters containing 33 fetuses were collected from the cross Tg/+ X Tg/+ at embryonic day 17.5. N o evidence of resorbed embryos was observed. Gen- otypes were determined by analysis of the Sphl poly- morphism. We detected 8 individuals homozygous for the 12-kb C57BL/6J allele, 25 heterozygotes with 12- kb and 14-kb hybridizing fragments, and 0 homozygotes for the 14-kb transgene allele. The data are inconsistent with the 25% frequency of homozygotes that is expected for a nonlethal mutation (x’ = 16.5, P < 0.00 1). The observed proportion of heterozygotes and homozygotes does not differ significantly from the 2:l ratio expected if the transgenic mutation is a recessive lethal (x2 = 1.23, P > 0.25).

Abnormalities of the semicircular canals in transgenic mice: The bony labyrinth of the inner ear was dissected from four transgenic animals and three normal C57BL/6J animals. A dramatic reduction of the horizontal canal was evident in all the transgenic animals (Figure 2, A and B). These four samples and additional nondissected inner ears from three transgenic and two normal mice were assessed with 6-pm plastic sections through the vestibular and auditory sensory epithelium. Sections through the vestibular apparatus showed normal utricle and sacculi with nor-

mal maculae and otoliths in all of the transgenic animals. Abnormalities were evident in the crista ampullaris, particularly of the horizontal canal. The sensory epithelium of the crista ampullaris normally has a convex shape, overlying an evagination (Figure 2C). The cristae of the horizontal canal of the seven transgenic animals lacked the usual evagination. Instead, the epithelium was either flattened or invaginated (Figure 2, D-F). In the posterior and anterior canals, flattening was only occasionally observed and invagination was never seen. Despite the abnormal appearance of the crista, the sensory epithelium including the vestibular hair cells was normal (Figure 2D). The overlying cupula was sometimes underdeveloped and in other cases appeared normal. The flattening of the crista and underdevelopment of the horizontal canal are sufficient to account for the circling behavior of the transgenic mice.

Auditory sensory epithelium and general anatomy of the cochlea appeared normal. The presence of a normal Preyer’s startle reflex suggested that the circling mice are not deaf. This was confirmed by measurement of auditory brain stem responses, which were normal (data not shown). Brain histology did not reveal any obvious neuroanatomical abnormalities (R. Albin, unpublished data).

Craniofacial abnormalities: Transgenic mice can be recognized by their abnormal facial appearance resulting from marked hypoplasia of the nasal bones (Figure 3). The length of the nasal bones in transgenic animals is approximately 75% that of nontransgenic littermates, and overall skull length is reduced by 10%. Abnormalities of the maxilla, premaxilla, nasal septum, turbinates and frontal bones are also present.

Structure of the transgene insert: The microinjected transgene in line 9257 was a linear 25-kb human ApaI/MZuI genomic fragment (TING et aZ. 1992). T o analyze the structure of the insert, which segre- gates as a single locus, genomic DNA was prepared from transgenic mice and analyzed on standard South- ern blots. Comparison of the hybridization intensity of transgenic DNA with copy number standards indicated that the transgene was present in more than 20 copies (data not shown). High molecular weight DNA was prepared and digested with ApaI, which does not cut within the transgene. The resulting fragments were separated by pulsed field gel electrophoresis and hybridized with a transgene-specific probe (Figure 4). No hybridization was observed with nontransgenic C57BL/6J DNA (lane 1). Intensely hybridizing fragments of 280 kb and 500 kb were detected in transgenic DNA (lanes 2 and 3). A weakly hybridizing fragment of 800 kb was also generated under partial digestion conditions (lane 2). The data are consistent with a tandem array containing between 20 and 32 copies of the full length transgene. The internal ApaI site may have been generated by a single “head-to-


E

Cochlea

C

FIGURE 2.-Anatomical assessment of semicircular canals. (A) Exposed semicircular canals from transgenic mice (left and right) and normal littermate (center). The horizontal canal (H) is missing from the transgenic mice, while anterior (A) and posterior (P) canals have a normal appearance. (B) Diagrammatic representation of semicircular canals. (C) Cross section through the crista ampullaris of the horizontal canal of a normal animal showing the characteristic evagination. (D) Cross section through the crista ampullaris of the horizontal canal from a transgenic animal. (E, F) Sections through the crista ampullaris of the semicircular canal from a second transgenic animal, showing invagination of the crista (arrow). Magnification lOOX to 300X.

head” alignment of transgenes within a larger “head- to-tail” array, or by incorporation of exogenous DNA within the array. In situ hybridization of the transgene to the B1

region of chromosome 28: To determine the chromosomal location of the insert, metaphase chromosomes were prepared from cultured spleen cells and hybridized with a biotin-labeled transgene probe. The hybridization solution also contained a digoxygenin- labeled L1 repetitive sequence probe that generates a unique Giemsa-like banding pattern for each mouse chromosome (BOYLE, BALLARD and WARD 1990). Flu- orescent visualization of the probes demonstrated the presence of the transgene insert in the B1 region of

mouse chromosome 18 (Figure 5). A total of 57 metaphase spreads were examined and 54 provided a strong signal on chromosome 18. (The other three metaphases were partial spreads.) Fourteen metaphase spreads were imaged and used to make the band assignment.

Linkage to genetic markers in the proximal region of chromosome 18: To locate the transgene insert on the meiotic map of chromosome 18, an interspecific backcross was generated by crossing mice from line 9257 with strain SPRET/Ei. Transgenic female F1 animals were then mated to strain C57BL/6J. Ge- nomic DNA from backcross offspring was analyzed by PCR to identify transgenic mice. Five genes on

Mouse Craniofacial Mutation 25 1

TABLE 4

Genetic markers on chromosome 18

Locus Enzyme C57BLl6J SPRET/Ei

+/+ Tg/+

?

d

FIGURE 3.-Craniofacial abnormalities in heterozygous transgenic mice. D o r s a l view of skulls prepared from littermates at 10 weeks of age. Tg/+, transgenic heterozygotes: +/+, nontransgenic controls.

1 2 3

1125-

1020 - 850 - 700 - 630 - 580 - 460 - 290 - 245 -

- 800 =

370 -

FIGURE 4.-Pulsed field gel electrophoresis of transgenic DNA. Genomic DNA was digested with Apal and electrophoresed on a 1% agarose gel using a BioRad CHEF DRll apparatus. Fragments were transferred to a nylon filter and hybridized with the transgene probe. The molecular weight standards at the left (kb) correspond to S. cerrvizicre chromosomes. Lane 1, C57BL/6J complete digest; lane 2. line 9257 partial digest: lane 3. line 9257 complete digest.

proximal chromosome 18 were typed by Southern blotting to detect unique SPRET/Ei alleles (Table 2). Haplotypes for the backcross progeny are presented in Figure 6A. The data demonstrate that the transgene is located proximal to the other markers typed (Figure 5B, left). The position of the transgene is very close to that of the Twirler mutation (Figure 5B, right), which has been mapped relative to the cen- tromere of chromosome I8 (BEECHEY et al. 1980; LANE et al. 198 1).

The cytological locali7ation of the 9257 transgene to band B1 and its genetic locali7ation provide the first link between the genetic and physical maps of the proximal region of chromosome 18 (LYON and KIRBY

Generation of mice carrying both the transgene and the Tw allele: To test the viability of compound heterozygotes for the two mutations, we crossed het-

1993).

Apc Pst I 6.7.4.2. 3.4, 2.5. 2.4. 1.5 3.4. 2.5.2.1 1.8, 1.6

Fgf-I Taq I 4.7, 3.1. 1.8 5.54.7.1.4 Grl-1 Taq I 11.8.6. 3.4. 1.4 6.1, 2.7. 1.4 Pdgfrb Taq I 11.2.5, 2.1

Loci were mapped by RFLP analysis as described in MATERIALS AND METHODS. The unique Mw sprrrus fragments that were typed in backcross animals are underlined. The alleles in strain CSH/HeJ were identical to those described for C57Bl./fiJ.

&-I Pst I 2.6 -

"

- "

- 17.2.5.2.1

TABLE 3

Identification of compound heterozygotes

Circlcn/total ofTspring

genotype of F, offspring Transgenic Nonrransgenic Apparent No. of test-cross

TglTw I . 13 414 719 2. 9 313 516 3. I 1 617 214

Total 33 1311 4 14/19

Tg/+ 1. 17 315 011 2 2. 14 515 019 3. 12 415 017 4. 12 414 018 n. 12 818 014

7. R 214 014 8. 6 112 014

6. 9 515 014

Total 90 32/38 0152

Eleven transgenic F, mice from the cross (Tw/+ X Tg/+) were test crossed to strain C57BL/6J. F, mice were classified as TglTw if they generated nontransgenic offspring with circling behavior. FI mice whose nontransgenic offspring did not exhibit circling behavior were classified as Tg/+.

erozygous transgenic mice (Tg/+) with heterozygous Twirler mutants (Tw/+). Among 139 offspring, 62 carried the transgene (44%). The phenotypes of transgenic offspring were not more extreme than the heterozygous parents. Examination of 12 fetuses on day 18 of gestation detected no resorbed embryos and no cases of cleft palate.

To determine whether any of the transgenic F1 animals carried Tw, a test cross to C57BL/6J was carried out. Offspring were examined for the presence of the transgene and for circling behavior. If there were any FI animals of genotype Tg/Tw, they were expected to generate transgenic offspring that are circlers and also nontransgenic offspring that are circlers due to inheritance of the Tw allele. Three of the 11 F1 animals tested were of this type (Table 3). For the other eight F1 animals, all of the nontransgenic offspring were noncirclers. These FI were classified as Tg/+. The penetrance of circling in both classes of offspring was consistent with the previous estimates.

The results demonstrate that mice carrying both the Tw allele and the transgene insert can be viable


FIGURE 5.-ln situ hybridization to metaphase chromosomes lo- calizes the transgenic insert to band B1 of nlouse chromosome 18. Metaphase chromosomes from a transgenic heterozygote were prepared from cultured spleen cells. Chromosonles were hvbridi7ed with a biotin-labeled 11uma11 amylase transgene probe and a digos- ygenin-labeled L I repeat probe, as described in MATERIALS AND

METHODS. The biotinvlated probe was detected wi th rhodamine- avidin. The digoxygenin probe $vas detected with FIT(:-laheletl antidigoxigenin Fab fi-agment (Boehringel- Mannheim). Chromo- sonles were counterstained wi th DAI'I. The DAPl and L1 images are displayed i n green and the transgene hybridiration signal is red. As indicated by the partial karyotype at the top of the figure. the chromosomes c o n t a i ~ ~ a single insert of~hurnan D N A that is l o c a l i d to band B1 of nwuse chron1osome 18.

and fertile. The number of offspring analyzed is not sufficient to determine whether viability of the compound heterozygotes is reduced.

Complementation of the ataxia mutation: Ataxia is a recessive mutation causing progressive paralysis that maps approximately 0.5 cM from the Twirler locus, with a reported recombination frequency of 6/ 1206 (LYON 1955, 1958, 1975). A complementation test was carried out by mating ax/+ females with Tg/+males and scoring progeny for the ataxia phenotype. Paralysis of the hind limbs in axlax mice is first noticeable at 2-3 weeks of age and progresses to near immobility at 1-2 months (LYON 1955). We examined 47 progeny, including 24 transgenic animals. None of the animals exhibited any sign of paralysis when observed for a minimum of 2 months after birth. This complementation of the ataxia phenotype by the transgenic chromosome demonstrates that the transgene does not affect expression of the nearby locus ( P < 0.001).

DISCUSSION

The interesting characteristics of this transgenic mutant include failure of normal morphological de-

B

FIGURE 6,"Linkage analysis. (A) Haplotypes of backcross progeny from the cross (C57BL/6J-9257 X SPRET/Ei)FI X C57BL/6J. Each column represents the haplotype of the chromosome inherited from the F I parent. The number of mice with each haplotype is given at the bottom of each column. Fgf-1, 01-1 and Pdgfrb were typed on the entire backcross DNA panel: Apc and Egr-1 were typed on all backcross progeny with recombination in the interval between the transgene and Pdgfrb. Haplotypes were inferred by assuming the absence of double crossovers. Solid symbols, SPRET/Ei; open symbols, C57BL/6J. (B) Genetic map of chromosome 18 derived from our data (left) and the consensus map (DAVISSON andJoHNsoN 1992) (right).

velopment of the cristae ampullaris and shortening of the membranous and bony labyrinth. The observed asymmetry of left and right canals is sufficient to account for the circling behavior of transgenic mice, and perhaps for the hyperactivity as well. The appearance of the ampullae in transgenic and Twirler mice suggests that the production of extracellular material may be defective in the mutant. The affected struc- tures of the inner ear and cranium develop during the period between embryonic day 11-1 6 . A defective bone morphogen, growth factor or related receptor that is normally present during this period of development is an attractive model to account for the multiple cranial defects.

The phenotypes of mice heterozygous for the 9257 transgene and for the Twirler mutation are compared in Table 4. The similarities include the virtually identical appearance of the cristae ampullaris (compare this paper and LYON 1958). Three features specific to Twirler are the loss of otoliths, early onset obesity and the birth of homozygotes with cleft palate. The extensive similarities in phenotype together with the genetic localization to the same chromosome region make it likely that the two mutations have altered at least one gene in common. In view of the complex phenotypes, both mutations could represent contiguous gene syn- dromes affecting two or more adjacent genes. An-

Mouse Craniofacial Mutation 253

TABLE 4

Phenotypes of mice heterozygous for TC” and Twirler mutations

W 2 ” Twirler

Hearing Normal Normal Horizontal canal Shortened Shortened Cristae ampullaris Flat or inverted Flat or inverted Otoliths Normal Missing Cochlea Normal Normal Facial bones Hypoplastic Normal Behavior Circling, non- Circling, non-

swimmer, swimmer, hyperactive hyperactive

Obesity Normal Early onset

Penetrance of circling 80% 90% Chromosomal map Proximal Chr Proximal Chr

position 18 18 Homozygosity Prenatal lethal Day 1 lethal;

cleft palate

obesity

other possibility is that Twirler and 7’$,,’ contain mutations in two distinct but closely linked genes, both of which are required for craniofacial development. This could account for the complementation (survival) observed in compound heterozygotes. De- termination of the molecular relationship between these two mutations is a goal of our future work.

According to the classification scheme proposed by Steel and Bock (1985) for inner ear mutants of the mouse, the insertional mutation produces a mild mor- phogenetic defect with limited structural abnormalities and no apparent effect on hearing. More than 40 circling mutations have been mapped in the mouse genome (GREEN 1989). Many of these cause extensive defects in the cochlea and bony labyrinth. None of these mutants has yet been characterized at the molecular level.

The mutation described here is one of a growing number of insertional mutations in transgenic mice. The reported frequency of mutations in transgenic mice is 3-5% (MEISLER 1992). Two other insertional mutants with circling behavior have been described, the dominant mutation Wocko with extreme disor- ganization of the vestibular apparatus (CRENSHAW et al. 199 1) and the recessive mutation chakragati, which appears to have a central nervous system defect (RATTY et al. 1990, 1992). In view of the large number of transgenic lines that are currently being generated, transgenic insertions are likely to be an increasingly useful resource for investigation of morphogenesis of the inner ear as well as other developmental processes.

We thank MARY LYON for providing Twirler mice and GLENN RADICE for providing mice carrying the ataxia mutation. We are grateful to ROGER ALBIN for histological analysis of the brain of transgenic mice and DAVID F. DOLAN for measurement of auditory brain stem response. We thank J. ESCOBEDO, K. YAMAMOTO, V. SUKHATME and J. HEBERT for providing probes. Supported by

USPHS grant GM24872 (M.H.M.) and HG00272 to DAVID C. WARD, Yale University. C.N.T. received a predoctoral fellowship from the Michigan Center for Cancer Research. D.C.K. is the recipient of National Research Service Award DC00109 and sup- port from the Developmental Biology Training Grant (HD07247). D.L.B. is a predoctoral fellow of the University of Michigan Ge- netics Training Program (GM07544).

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Communicating editor: R. E. GANSCHOW

insertional mutation on mouse chromosome 18 with vestibular and

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