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TEKATOLOGY 47:595-602 (19931 Expression of the IV (Reversed and/or Heterotaxic) Phenotype in SWV Mice W.M. LAYTON, M.W. LAYTON. MICHAEL BINDER, DAVID M. KURNIT, ANDRZEJ J HANZLIK, MARGARET VAN KEUREN, AND FRED G. BIDDLE Department of Anatomy, Dartmouth Medical School, Hunocw, Neu' Humpsh,ire iW.M.L., M,W.I,,, M.R.) 03755; Departments of Pediatrics (A.J.H..D.M.K., M.V.K.) and Human Genetics (D.M.K.I.Howard Hughes Medicat Institute ('D.M.K., A.J.H., M.V.K.1, Urtiucrsity of Michignri Medical School, Ann Arbor, Michtgan 48109; and Depurtnrt.nt uf Pcdiutr University of Calgary, Calgary, Alberta, Canada T2N 4N1 IF.G.B.1 ABSTRACT Approximately 50% of iuliu mice have situs incersus (mirror image reversal of viscera) and 40% have heterotaxia (anomalous arrangement of viscera). The occurrence of heterotaxia is independent of situs. Using the cross-intercross breeding system to put the iv gene on the SWV background, an occasional presumed i u l i mouse was found that had a n IV (situs inucrsus and/or heterotaxic) phenotype. Testcrosses of these reversed animals indicated an iui t genotype. Since iv is linked tightly t o Igh-C on chromosome 12, we inferred the genotype with a polymorphism of Igh-C demonstrated using the polymerase chain reaction (PCR). This confirmed them to be ivi + . The expression of the IV phenotype in animals heterozygous for the tu gene may be due t o an interaction of iv with a n autosomal recessive gene found in SWV. We have not found the IV phenotype in heterozygous iui + mice following placement of the it, gene on six other inbred strains. Rarely, we also found that presumed SWV + + mice had the IV phenotype. Test matings of these phenodcviants, corroborated by PCR, have confirmed them to be + I + . Although the phenotypes of the affected SWV + I + and ivl + mice resembled those found in iviiu mice, the occurrence of situs inversus and heterotaxia were not independent of each other, and most, of the SWV mice with the IV phenotype had heterotaxia with situs solitus. This infrequent. dominant expression of the iv gene has so far only been seen when iv is on the SWV background. These findings are consistent with the idea that this phenomenon is due to the interaction of the iv gene with another gene found so far only in the SWV strain. 1993 Wiley-Liss, Inc. ( The autosomal recessive gene iu (Hummel and Chapman, '59), which maps to chromosome 12 (Brueckner et al., '89), causes situs inuersus (mirror image reversal of placement of organs) in mice. One half of iuliu mice have situs inuersus and approximately 40% have some sort of heterotaxia, a n abnormal arrangement of organs in relation to each other. Heterotaxia occurs independently of visceral situs in ivliv mice, so that the frequency of heterotaxia in mice with situs inversus is equal to its frequency in those with situs solitus (normal placement of organs). In mouse strains examined previously, ivl + heterozygotes have demonstrated normal laterality indicating that Q 1993 WILEY-LISS, INC ~ 8 the iu gene is a loss-of-function allele: in the absence of iu gene activity in the ii)!ii$ mouse, laterality determination does not occur resulting in the random determina t ion ' of visceral .situs (Layton, '76; Kurnit et al., '87). Heterotaxia in the context used here can be considered as residual situs inriersus in a mouse with situs solitu's, or residual s i t u s solitus in one with situs inr?ersus.Occasionally, thc heterotaxia is so marked that the predominant situs cannot be determined; Received January 30, 1992: accepted Januar? 12 1993 596 W.M. LAYTON ET AL. iv donor swv Generation Cycle i1 discard - +/+ iv/+ iv/iv iv/iv 112 SOL 1/2 IV +I+ l2 discard Fig. 1. Crossintercross mating plan used to place the iu gene on the SWV background. The solid black circles indicate iuiiu animals with situs inuersus and the hatched circles indicate iuliu animals with situs solitus. these cases are categorized as situs ambiguus. Most cases of situs ambiguus are in mice with the situs of the thorax discordant with that of the abdomen. Heterotaxia occurs in a number of reproducible patterns, the majority of which have been described by Hummel and Chapman ('59) and Layton ('78). During a n attempt to put the iu gene on various inbred backgrounds using a crossintercross system of mating (Green, '81; Fig. l ) , we encountered apparent partial dominant expression of the iu gene on the SWV background. In the cross-intercross mating system, a G1 (Fl) generation is produced from the cross iuliu X + I + to yield iul+ heterozygotes. Members of G1 are crossed among themselves to produce a G2 generation. Reversed G2 mice, presumably reflecting a n iuliu genotype, are then crossed with +I+ mice to produce G3 iui+ mice for a second cycle. This process is repeated, usually for 10 cycles (20 generations). The even numbered generations contain one-quarter iuliu mice, approximately one half of which should have situs inuersus. The odd numbered generations are all iul+ and should have the wild-type phenotype. This expected result was the case with six of the seven inbred strains; however, for the SWV strain, a reversed pup (based on location of the stomach) was found in G7. Since the albino SIV (iuliu) mice were housed in the same room as the albino SWV, this unexpected finding was attributed to a mating error and the study was begun again. However, in the repeat study another reversed pup was found in G3 and still others in succeeding odd-numbered generations, which we shall call the G-odd effect. There are two possible explanations for these findings: 1)One of the parents was a SIV (iuiiu)mouse instead of SWV (+I + 1, so t h a t the reversed G-odd mice were actually iuliu. 2) The iu gene is dominant on the SWV background, but with reduced penetrance. IV (REVERSED) PHENOTYPE IN SWV MICE Breeding tests to infer genotype are difficult to interpret in these animals because of reduced penetrance of the IV phenotype and the high rate of lethal congenital heart malformations associated with the IV phenotype (Layton, '78). Recently, we have demonstrated that SWV and SIV are polymorphic at the Zgh-C locus on chromosome 7 2 (Hanzlik e t al., '90). Since theZgh-C locus is linked tightly to zu, this has made i t possible to distinguish if the reversed G-odd mice are id+ or iuliu. Below are summarized genetic analyses that support the hypothesis that SWV has a n unlinked autosoma1 recessive gene that interacts with iv to cause expression of the IV phenotype in iul + heterozygotes with reduced penetrance. MATERIALS AND METHODS Non-inbred iuliv mice were obtained originally from Dr. Katherine Hummel of The Jackson Laboratory. These were kept in a small closed colony and eventually an inbred strain was produced by brother-sister mating for 20 generations. This strain is called SIViLay provisionally (SIV in this paper). Inbred strains AiJ, AKRIJ, BALHlcJ, CBAiJ, C57BLi6J, and DBAl2J were obtained from The Jackson Laboratory and SWV from F.G.B. At weaning, all mice were classified and tagged by ear punch. Newborn pups were examined for the location of the milk spot, which is the white milk-filled stomach viewed through the translucent abdominal wall. Those mice with a right-sided milk spot (and thus situs irzuersus) were marked by clipping the tip of the tail. The attempt to inbreed the iv gene on the SWV background using a cross-intercross system (Fig. 1)was started three times (experiments 1 , 2 , and 3). Although none of the experiments went the 20 generations required for inbreeding, all experiments were consistent with the unanticipated results reported herein. During experiment 1, the identification of IV phenotype was based on the position of the milk spot. During experiment 2, examinations became more thorough. In addition to using the milk spot location to identify the IV phenotype in living pups, most of the animals were autopsied excepting those that were too autolyzed or lost due to maternal cannibalism. These two methods of identifying affected mice are not equivalent. The use of the milk spot only identifies animals with a right-sided stom- 597 ach and thus misses cases that have a leftsided stomach with heterotaxia. However, because of the selective loss of pups with the IV phenotype prior to weaning, autopsies done after the perinatal period miss mice with the IV phenotype that have died (usually as a result of heart malformations). Since G5 of experiment 2, all wild-type SWV ( + i + 1 mice have been autopsied. In order to test the hypothesis that the iv phenotype in G-odd iui+ mice is due to interaction of the iu gene with another autosoma1 gene, two sets of experiments were done: 1. If the reversed G-odd mice were genetically different from the non-reversed G-odd mice, the incidence of reversal and heterotaxia and their association wit,h each other in the offspring of rkversed G-odd parents would be different from that in the offspring of non-reversed G-odd parents. This experiment was set up prospectively and G-odd parents of generations G-5 to G-7 were used. Results of 102 of 805 autopsies of offspring of non-reversed G-odd parents were not used in the tabulated results of this experiment because the parents of these mice, although not reversed, were found a t autopsy t o have the iv phenotype. 2. Two backcrosses were set up (see Fig. 3). BC1 should show the G-odd effect since approximately li4 of its members are heterozygous for iu and homozygous for the putative "hi" gene that is hypothesized t o interact with i u i + t o result in the ii, phenotype. None of these offspring can be iuizu. RBCl should not show the G-odd effect since no animals would be iui + , hi/hi. However, approximately half of them will be iuliv so that about 114 should be reversed. To infer the genotype of these mice a t t,he iv locus, we took advantage of the tight linkage of Igh-C with iu (Brueckner et al., '89; Hanzlik et al., '90) and of a polymorphism of Igh-C that enabled us to differentiate SWV from SIV. This was based on the difference in the length of a segment ofIgh-C, which in t u r n was due t o a variable number of tandem dinucleotide repeats of (A,C) and/or (C,T) in this segment (Hanzlik et al., '90). This difference was demonstrated using PCR. The segment from SWV was longer than that from SIV (Hanzlik et a]..'90). This "PCR test" first became available during G9 of the third experiment and was used subsequently. Details about the primers and 598 W.M. LAYTON ET AL. the conditions used for the PCR, as well as the mapping of iu close to Igh-C ( 1 recombinant out of 201 animals examined) are given in Hanzlik et al. (’90). RESULTS In all three experiments attempting to place the iu gene onto the SWV background, we continued to find mice with the IV phenotype in odd numbered generations. Hereafter, we shall call this the “G-odd effect.” This unexpected finding does not appear to be the result of a breeding error; testcrosses of two reversed G5 mice with C57BLl6-ivliu mice resulted in 4123 and 4/24 reversed pups, respectively. This is consistent with the 25%incidence of reversal expected in a n ivi+ x iuiiv mating and not with the 50% expected if both G5 animals were iuliu. The PCR test established the i d + genotype of 16 G9 animals with the IV phenotype from experiment 3; one animal was homozygous for the upper (SWV) allele, presumably representing a recombinant between the iu and Igh-C loci (Fig. 2; Hanzlik et al., ’90). The incidence of the IV phenotype a s shown by reversal a t birth and the phenotype at autopsy is given in Table l. The incidence of this G-odd effect was independent of the sex of the reversed parent and was found with approximately equal frequency in males and females. The proportion of mice with the IV phenotype that had heterotaxia alone was much higher in the G-odd mice than in ivliv mice as most of the affected G-odd mice had situs solitus (Table 2). This is in contrast to SWV iuliu mice, in which situs inversus and situs solitus are equally frequent in heterotaxic mice. In 424 G1 mice, there were no cases of reversal at birth and only a single instance of IV phenotype. Note that this finding rules out a n anomaly a t the iv locus in the SWV mouse, as the compound heterozygote created in G1 essentially shows no IV phenotype. Assuming that the single case of the IV phenotype in G1 was a phenodeviant (which is seen in SWV mice; vide infra), we hypothesized that the increasing frequency of the G-odd effect with G-number was the result of interaction of iv with another recessive gene carried by SWV which we shall call “hi.” In a n effort to evaluate the h i gene hypothesis further, we performed three breeding experiments that were consistent with this hypothesis: 1) We found that there was a significantly higher frequency of IV phe- 4 2 7 0 bp 4 2 4 0 bp Fig. 2. Determination of status a t the iu locus using the closely-linked Zgh-C locus. We demonstrated previously that thelgh-C locus is closely linked to iu and that the SIViLay (original) genotype could be distinguished from the SWV genotype at the Zgh-C locus (Hanzlik et al., ’90).Using the oligonucleotides described in Hanzlik et al. (’901,we amplified via PCR (Saiki et al., ’85) the intervening region of the lgh-C locus that includes a n (A,C)- (T,C)-repeat stretch. Following arnplification, the product was electrophoresed through a 2% Nusieve + 0.54 agarose (Seakem) gel, stained with ethidium bromide, and photographed. This protocol distinguished between the SWV + and the SIV/Lay (original iu) chromosomes, with the SWV allele being larger than the SIViLay allele. Using this analysis, we confirmed that a rare reversed SWV mouse indeed had a SWV + / i genotype (lane 2). Using this analysis, we also confirmed that three heterozygous reversed F9 mice were id+ (lanes M),as expected from the breeding analysis. Other mice were subjected to this analysis with the same result (data not shown). This analysis was consistent with the breeding experiments. + notype in the offspring of reversed G-odd mice than in the offspring of non-reversed G-odd mice (Table 2, cf. rows labeled “G-Odd Inverted x G-Odd Inverted” and “G-Odd Solitus x G-Odd Solitus”). This difference was consistent with the hypothesis that such reversed animals were homozygous for a second (“hi”) gene. 2 ) Offspring of the backcross BC1 [(SWV x SIV) x SWV] (Fig. 3), which should contain no iuiiv mice but N ( R E V E R S E D ) PHENOTYPE IN SWV 599 MICE TABLE 1. Incidence of situs inversus at birth and the iv phenotype at autopsy in odd and even gen.erations of S W V 1+ I+ 1 X SIV livliv) cross-intercross matings (Fig. 1)’ Generation G3 G5 G7 G9 Odd generations total G2 G4 G6 G8 E v e n generations total Sctus inuersus at birth Number affectedltotal (R) Litters 17 36 38 28 119 3/176 101283 241399 101271 4711129 (1.7) 13.5) (6.0) (3.7) (4.2) 25 63 48 46 182 401276 831674 551530 411279 21911759 (14.5) (12.3) (10.4) (14.7) Phenotype at autopsy Number affectedkotal (‘XI (50) (17 11 61121 33/193 311339 211226 911879 (9 1) (9 31 (10 41 (16 8 ) (13 6) (15 11 111 1) (13 9) 38!226 791581 731482 441395 234/1684 (11 2) ’These data are from experiments 2 and 3. TABLE 2. Patterns of N nhenotwe’ Phenotvue of offsurine Situs inuersus IV phenotype IV phenotype Autopsies Group Non inbred-iuliu’ C57BLl6 Lay-iuiiu SIVLay-iuliu Total-iuiiu G-Odd ( G 3 4 9 ) G-Even (G2-G8) G-Odd INV X G-Odd INV G-Odd SOL X G-Odd SOL Backcross (BC1) Backcross (RBCI) Heterotaria IV phenotype # # (91 # I%)~ # (%)% 950 842 290 2082 754 1632 94 703 944 258 638 582 206 1426 78 231 24 85 (67.2) (69.1) (71.0) (68.51 484 359 150 20 127 328 355 113 796 73 167 (51.41 (61.01 (54 91 (10.3) 19 64 (2.0) (24.8) (75.91 (61.7) (72 8 ) (69.61 125.6) 155.0) (58.3) (48.2) (21.1) (14.21 (25.51 (12.1) 993 14 41 4 39 160.9) 17 65 19 40 I55 81 (93 61 (72 3 1 I70 8, (76 5) (10001 (62 51 ’Results of third experiment only. G-Odd are litters in odd-numbered generations (G-Odd all have an tv + genotype1 G-Even are litters in even-numbered generations (G-Even are 114 iuku, 1/2 i d + , and 114 f: + genotype).G-Odd INV x G-Odd INV are mice that are the offspring of parents with situs inversus of G-Odd generations (the parents’ genotype was zu! - I, G-Odd SOL i G-Odd SOL are mice that were offspring of parents with situs solitus of G-Odd generations (the parents’ genotype was m + I . Backcross IBC11 were V and 112 + / genotype. Reciprocal backcross mice that were the product of (SWV x SIV!Lay) x SWV and thus BC1 are 112 ~ L + (RBC1)were mice that were the product of ISWV x SIV/Lay) x SIVLay and thus RBCl are 1!2 iu!+ and 1!2 i r h genotype ‘From data in Hummel and Chapman (‘59) and Layton (’761. 3Number reversed a t autopsy as a percentage of number with IV phenotype. ‘Number with heterotaxia as a percentage of number with IV phenotype. - 114 of which should be ivl + hilhi, showed a low incidence of IV phenotype (Table 2). It is noteworthy that all of the affected mice had heterotaxia and virtually all had situs solitus. I n this respect, they resembled the affected G-odd mice rather than the “conventional” iviiu mice (non-inbred iuliv, C57BLi 6Lay iviiv, and SIViLay iuliu). These two backcrosses were originally done before the PCR test was available. Because of the importance of BC1, this was repeated and all seven of the mice with the IV phenotype that resulted from this backcross were iui + as indicated by PCR. 3) Affected offspring of the reciprocal backcross [(SWV x SIV) x SIVl; (RBC1 in Table 2 and Fig. 3) resembled affected iviiv mice: when compared with affected G-odd mice they had a relatively high incidence of situs inversus and low incidence of heterotaxia (Table 2). We also encountered a few instances of the IV phenotype (most frequently heterotaxia with situs solitus) in putative SWV +i+ mice, We started our SWV colony in 1976; we examined 1,351 newborn pups cursorily and autopsied a relatively small number of weanlings and adults without finding the IV phenotype. However, since 1983, when we realized that there might be a n association between the SWV background and reversal, we have examined all newborn SWV pups carefully and have done autopsies on virtually all SWV mice. During this period, we have encountered three in- 600 W.M. LAYTON ET AL. swv SIV + I + , hilhi iv/iv,+l+ I Fl DISCUSSION iv/+, hi/+ *iv/+ { hilhi backgrounds (AIJ,AKWJ, BALBlcJ, CBAIJ, C57BL/6J, and DBA/2J), we examined 209 G-odd litters containing 1,179 pups at birth. None of these pups had the IV phenotype as demonstrated by a right-sided milk spot. iv/+ hi/+ { hi/+ ivliv i +/+ Fig. 3. Mating plan for backcrosses BC1 and RBC1. In BC1, 114 of the mice are hypothesized to be i u l + hiihi and thus to include the mice with the IV phenotype (asterisked). However, none of them are iuiiu, so the IV phenotype cannot be the result of homozygosity for iu. In RBC1, 1/2 of the resultant animals are iviiu and 1/2 iui + . However, none of them are hilhi, so that none of the iv/ i- mice would be expected to show the IV phenotype. stances of situs inversus in 1,587 newborn SWV pups and six cases of heterotaxia with situs solitus in 1,250 autopsies. This gives a n 0.3% incidence of the IV phenotype in SWV i I + mice. Recently, using PCR to detect the Zgh-C allele, we tested two of these SWV +I+ animals with the IV phenotype and have confirmed that these cases are not due to accidental crosses to SIV (Fig. 2). Unfortunately, those + / + mice that have heterotaxia with situs solitus cannot be ascertained prior to autopsy, and thus cannot be used for breeding tests. Recently, however, a male SWV + / + mouse (identification number 10VU41) was born with a reversed milk spot. His parents had another pup with heterotaxia and situs solitus and a pair of littermates of his parents also had a pup with situs solitus and heterotaxia. Breeding tests of this male and his close relatives have been uniformly negative. Matings with his mother and sisters produced only normal offspring. Matings between his siblings have also failed to produce any pups with the IV phenotype. At autopsy, this mouse had situs ambiguus and the PCR test was consistent with a + i + genotype a t the iu locus. Putting the iv gene on six other inbred Tests for polymorphism at the Zgh-C locus demonstrated that the instances of 1V phenotype found in SWV ivl+ and + i + mice were real and not the result of mistaking the ivliu albino parent for one that is + i + . In contrast to iviiv mice, those showing the G-odd (n greater than or equal to 3) effect and phenodeviant SWV + / + mice most often have heterotaxia with situs solitus (Table 2). In G1, the genotype of all animals is heterozygous a t both the IV and HI loci, so little reversal is expected. This appears to represent a graded response; the mildest expression of the phenotype is heterotaxia alone. Next most frequent is heterotaxia with situs inversus. Situs inversus without heterotaxia is rare in SWV mice. The G-odd effect is due to a n interaction of the iu gene with the SWV genome, possibly with a hypothetical mutant gene that we shall call “hi.” We propose t h a t both iu and hi are loss of function mutations. The iv gene product enforces normal coordination of the sense of asymmetry among asymmetrical structures and sets a switch that determines global situs. Without the iv gene product the coordination of asymmetry is imperfect, resulting in heterotaxia, and the situs switch is set randomly. Although the hi gene product acts in a manner similar to that of iu i t is weaker and acts mostly t o enforce coordination of asymmetry. In its absence (in the SWV mouse) there are rare instances of heterotaxia, a few of which have situs inversus. However, in the absence of the hi gene product, iv acts as a dominant trait due to haploinsufficiency . In such a case the resulting phenotype is most likely to consist of heterotaxia, less often of heterotaxia with situs inversus, and rarely of situs inversus alone. A number of phenodeviant traits, such a s the cases of IV phenotype described here, have been found in various inbred mouse strains. For example, lateral cleft lip, open eyelids at birth, and atrial septa1 defects are found in the A strain (Kalter, ’68; Nora et al., ’68).In the C57BLi6 strain, microphthalmia or anophthalmia is relatively common (Kalter, ’79). This strain also has a IV (REVERSED) PHENOTYPE IN SWV MICE low incidence of ventricular septa1 defects (Nora et al., '68). As we have documented, stochastic effects may play a n important role in these cases (Kurnit et al., '87). The penetrance and expression of single gene-determined phenotypes can be changed by genetic background (Juriloff et al., '87). For example, the first arch (fur) mutation is lethal when homozygous due to abnormal development of structures derived from the maxillary process of the first branchial arch (Juriloff and Harris, '83). The far mutation occurred in the BALBicGaBc strain; heterozygotes (far/ ) in this strain are normal, but in the ICRiBc strain most heterozygotes have some expression of far such a s disrupted patterns of mystacial vibrissae (8081, hemifacial deficiencies (40%1),and cleft palate (20%) (Juriloff et al., '87). Breeding studies suggest the change from recessive to partial dominance of the fur mutation is a n effect of genetic background due to a small number of loci rather than due to isolalleles or a difference between BALBicGaBc and ICR/Bc in the normal wild-type ( + ) alleles at the fur locus (Harris and Juriloff, '89). Another mutation, Dactylaplasia (Due)is a dominant mutation that causes absence of the median digits of all four limbs and is a recessive lethal due to unknown causes (Chai, '81). The phenotype of dactylaplasia in D a d + heterozygotes depends on homozygosity for a n unlinked recessive modifier (mdacl rnduc); a dominant supressor ( + / + or mdad + ) causes Dad + heterozygotes to develop phenotypically normal digits. The incidence of some phenodeviant traits can be changed by altering the intrauterine environment. The incidence of situs inuersus, heterotaxia, and heart malformations in the Non Obese Diabetic (NOD) strain of mice is affected by the presence and severity of diabetes in the dam during early pregnancy (Morishima et al., '91). The incidence of the IV phenotype varied from .05% in the offspring of non-diabetic dams to 31% in those from dams that were diabetic during the first 3 days of pregnancy. Similar to the findings reported here for SWV, in the NOD mouse situs inversus without heterotaxia was rare. Most cases of the IV phenotype consisted of heterotaxia with situs solztus. There is some indication of clustering of the IV phenotype in the SWV iuiiu mouse, which suggests a n environmental effect. Thus stochastic, genetic, and/or environ- + 60 1 mental factors may play a role in the expression of the IV phenotype. Because the discovery of heterotaxia requires a detailed post mortem examination, it may be more prevalent in laboratory mice than is generally recognized. For example, the WBiReJ strain of mice have a variety of azygous drainage patterns of the thorax, some of which are similar to those found in the IV phenotype iBiddle e t al., '91). Determination of laterality therefore involves at least two loci, viz., IV and HI. The finding that putative + i + h i!hi SWV mice only rarely show the IV phenotype but that i d + hi/hi SWV mice more often show the IV phenotype indicates that the HI locus interacts with the IV locus to effect laterality. ACKNOWLEDGMENTS This work was supported by a grant from the NIH (IIL 377031, by the Howard Hughes Medical Institute, and by the New Hampshire AHA. DMK is an Investigator, AJH is an Associate, and MVK is a Research Specialist of the Howard Hughes Medical Institute. LITERATURE CITED Biddle, F.G., J.D. Jung, and B.A. Eales (19911 Genetically-determined variation in the azygous vein in the mouse. Teratology, 44:675-683. Brueckner, M., P. D'Eustachio, and A.L. Horwich i 19891 Linkage mapping of a mouse gene, iu, that controls left-right asymmetry of the heart and viscera. Proc. Natl. Acad. Sci. U.S.A., 86:5035-5038. Chai, C.K. (1981) Dactylaplasia in mice: A two-locu:: model for development anomalies. J. Hered., 72.234237. Green, E.L. (1981) Genetics and Probability in Animal Breeding Experiments. New York, Oxford University Press. Hanzlik, A.J., M. Binder, W.M. Layton, L. Rowe. 11. Layton, B.A. Taylor, M.M. Osemlak, J.E. Richards. D.M. Kurnit, arid G.D. Stewart (19901 The murine situs inversus viscerum iiv) gene responsible for visceral asymmetry is linked tightly to the fgh-Ccluster on chromosome 12. Genomics, 7:389%393. Harris, M.J., and D.M. Juriloff (1989)Test of the isoallele hypothesis at the mouse first arch ( f a r )locus. J. Hered., 80:127-131. Hummel K.P., and D.B. Chapman (1959) Visceral inversion and associated anomalies in the mouse. J . Hered., 502-13. Juriloff, D.M., and M.J. Harris (19831Abnormal facml development in the mouse mutant first arch. J. Craniofac. Genet. Dev. B i d ?3:317-337. Juriloff, D.M., M.J. Harris, and U. Froster-Iskenius (1987) Hemifacial deficiency induced by a shift in dominance of the mouse mutation far: A possihie genetic model for hemifacial microsomia. J . Craniofac. Gen. 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