Human Molecular Genetics, 2000, Vol. 9, No. 13 1937-1942
© 2000 Oxford University Press
The X-linked mouse mutation Bent tail is associated with a deletion of the Zic3 locus
Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA, 1Department of Pathology, University of Texas Health Sciences Center, Houston, TX 77030, USA and 2Children’s Research Institute and Departments of Pediatrics and Pathology, The Ohio State University, Columbus, OH 43221, USA
Received 2 March 2000; Revised and Accepted 16 June 2000.
DDBJ/EMBL/GenBank accession nos AZ044550–AZ044554.
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ABSTRACT |
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Bent tail (Bn) is a spontaneous, semi-dominant mutation on the mouse X chromosome that produces tail deformities and, rarely, open neural tube defects. Analysis of 292 normal male and affected male and female progeny from an intraspecific back-cross involving Bn supports a gene order of cen–DXMit89–18.5 ± 2.3 cM–DXMit166–1.4 ± 0.7 cM–Bn–1.0 ± 0.6 cM–DXMit140 –4.8 ± 1.3 cM–DXBay6–tel. A high frequency of sex chromosomal non-disjunction, unrelated to the Bn mutation, was also identified in the background strain. Refined genetic and physical mapping of the Bn critical region demonstrate that the mutation is associated with a <170 kb submicroscopic deletion that includes the anonymous microsatellite marker DXMit208 as well as the entire Zic3 locus. Human mutations in ZIC3 are associated with left–right axis malformations (MIM 306955, 208530, 207100). Abnormalities of abdominal and thoracic situs were also detected in viable Bn males and females. The presence of anal and spinal abnormalities in some of the human patients and the deletion of Zic3 in Bn mice support a key role for this gene in neural tube development and closure.
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INTRODUCTION |
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Neural tube defects (NTDs) are one of the most common human congenital malformations, second only to congenital heart disease in terms of overall frequency. Although chromosomal, monogenic and teratogenic causes of human NTDs are recognized, the majority are multifactorial in etiology (reviewed in ref. 1). It has been estimated that genetic factors account for ~60% of the risk for human NTDs. The introduction of prenatal screening and periconceptual folic acid supplementation have resulted in a dramatic decline in the incidence of NTDs (2); however, the basic mechanisms involved in their pathogenesis remain largely unknown.
To help to identify genes that may play a role in the pathogenesis of human NTDs, many researchers have examined mouse mutants that exhibit NTDs as part of their phenotype. Numerous spontaneous mouse mutants with NTDs have been described, and many additional mutations have recently been generated through the technology of transgenic mutagenesis and homologous recombination (3). Mouse models of NTDs share many features with their human counterparts. These include the location of the defects, possible mechanisms of pathogenesis related to intermittent sites of neural tube closure and variable expressivity that is based on genetic background and sex (3,4). The shorter gestation period in the mouse compared with human is usually reflected in the preservation of brain tissue for cranial malformations, and the resulting defect is exencephaly. Lower NTDs may present as tail defects in the mouse and are represented by mutations such as vestigial tail (vt), fused (Fu) and Bent tail (Bn). Some mouse mutants display both upper and lower NTDs, and some are associated with malformations of other organ systems. In particular, eye, vertebral/rib and limb malformations are commonly associated with mutants that display NTDs.
The X-linked Bn mutation was isolated in 1952 among offspring of an outbred Namru strain female by a bald (hrba) male (5). A single allele exists. Affected hemizygous males and homozygous females have short, kinked tails, occasional exencephaly or sacral NTDs, and reduced viability and fertility on the inbred Bn background. Heterozygous females have variable, milder tail abnormalities (5) (Fig. 1a). The presence of an interfrontal bone in the skulls of affected males and females has also been described (6). The Bn locus has been regionally mapped in several phenotypic crosses to the middle third of the mouse X chromosome, close to trembly-like (Tyl) (7,8). Lack of penetrance in affected Bn females is common, complicating these genetic mapping attempts. We demonstrate here refined genetic mapping of Bn using molecular markers and further provide evidence that the mutation is caused by a submicroscopic deletion that includes Zic3, a gene whose human ortholog has been previously associated with malformations of left–right asymmetry and with NTDs.
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RESULTS |
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Genetic mapping of Bn
To map Bn, an intraspecific back-cross was established between heterozygous Bn/+ females and C3H/HeJ males. A prior attempt to generate a back-cross using Mus castaneus males resulted in no phenotypically affected animals in >40 F1 females examined, supporting the influence of modifier loci on the expression of the tail defects. Affected F1 females from the Bn x C3H cross were identified at weaning by the presence of one or more kinks in their tail and crossed to normal males from the inbred Bn stock. Normal appearing female progeny were excluded from the analysis because of the reported (5) and observed incomplete penetrance (Fig. 1a).
A total of 292 N2 progeny from the back-cross were analyzed with the microsatellite markers DXMit89 and DXBay6 (Fig. 1b), and fine mapping was performed using 79 animals that had recombination events between these markers. Pedigree analysis of these recombinants supports a gene order of cen–DXMit89–18.5 ± 2.3 cM–DXMit166–1.4 ± 0.7 cM–Bn–1.0 ± 0.6 cM–DXMit140–4.8 ± 1.3 cM–DXBay6–tel, where the numbers represent recombination frequencies calculated in centiMorgans ± a standard error (SE). Complete mapping data for the cross are available through the Mouse Genome Database (http://www.informatics.jax.org/ ; accession no. J62602).
During these analyses, we observed several males that had inherited both a Bn strain and C3H allele at two or more loci tested. The presence of a Y chromosome was confirmed in each case by PCR genotyping using primers for the Smcx locus that detect different sized fragments from the X and Y chromosome (H. Willard, personal communication). Possible explanations for the occurrence of heterozygous males included duplication of a portion of the X chromosome or meiotic non-disjunction. Further analysis using additional polymorphic microsatellite markers spanning the X chromosome supported the latter since large portions of or the entire X chromosome contained both alleles in all of the animals (Table 1). Based on the pattern of cross-overs in some of these heterozygous males, it was clear that both a maternal and paternal X chromosome had been inherited, consistent with paternal non-disjunction. Since the father’s X chromosome always contained a normal Bn allele, the non-disjunction is almost certainly unrelated to the mutation and is a characteristic of the background strain. Complete karyotype analysis of two Bn females demonstrated a 39,X karyotype in one and a normal karyotype with a normal X chromosomal banding pattern in the second, suggesting that the non-disjunction probably affects both males and females. From the number of heterozygous back-cross males, the frequency of non-disjunction in this strain is estimated to be at least 4.4%.
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Identification of a submicroscopic deletion associated with the Bn mutation
To identify additional polymorphic molecular markers within the Bn critical region, DNA from affected males was examined with 20 microsatellite markers (DXMit165, 47, 83, 192, 225, 226, 208, 22, 107, 23, 159, 91, 68, 193, 108, 86, 142, 141, 74 and 127) localized on the X chromosomal consensus map between 14 and 20 cM (9). Although the microsatellite marker DXMit208, localized at 16.5 cM, was not polymorphic between the C3H and Bn strains, it was found to be deleted in hemizygous Bn males (Fig. 2a). None of the other 19 anonymous markers from the region was found to be deleted (data not shown). Subsequently, additional genes from the region were screened by PCR for their presence or absence in Bn males. These included Hprt, Fgf13, Tnfsf5 (formerly Cd40l) and Zic3. All of these genes produced a normally sized PCR product with the exception of Zic3 which was also deleted in Bn males (Fig. 2a). Southern analysis using several genomic probes from the Zic3 locus confirmed that the gene was deleted completely in Bn males. Further, there was no expression of the Zic3 locus in brain, heart or testis of an affected adult Bn male as examined by RT–PCR (data not shown).
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To determine the extent of the deletion in Bn, four bacterial artificial chromosomes (BACs) were identified by screening two genomic BAC libraries with DXMit208 and Zic3. All of the BACs identified contained both markers. Based on PCR and hybridization of end probes generated from three of these BACs, a physical map of the region deleted in Bn alleles was constructed (Fig. 2b). As in human, the Zic3 gene spans <10 kb of genomic DNA. The Bn deletion is estimated to be between 60 and 170 kb in size based on the amplification pattern of genomic DNA prepared from affected Bn males using primers for the loci DXChri1–DXChri5 that represent BAC clone end sequences (Fig. 2b).
As part of the efforts of the Human Genome Project, two overlapping P1-derived artificial chromosome (PAC) clones containing the human ZIC3 locus and spanning 246 kb have been completely sequenced and assembled (GenBank accession nos AL035443 and AL022576). There are no known genes or expressed sequence tags (ESTs) within the 37 kb of assembled sequence 3' of ZIC3 and the closest gene or EST 5' is the P21-RAC1 (RAS-related C3 botulinum toxin substrate 1) locus, at a distance of 124 kb. P21-RAC1 is an intronless transcript encoding a protein of 192 amino acids that functions as a small GTP-binding protein and is a member of the Rho family of proteins (10,11). Recently, a chick ortholog of P21-RAC1 was shown to be expressed during neural development (12). Using PCR primers designed from the murine R21-RAC1 cDNA sequence (GenBank accession no. AW318995), we determined that this gene was not deleted in Bn males (Fig. 2a).
Phenotypic characterization of situs defects in Bn mice
Mutations in the human ZIC3 locus have been identified in sporadic males with left–right axis malformations, as well as in several families with similar malformations and X-linked patterns of inheritance (13,14). All of the affected males had situs ambiguus with complex congenital heart disease, abnormalities of lung lobulation, and abnormal placement or structure of the stomach, liver and/or spleen. Therefore, we examined viable Bn males and females, in comparison with normal male littermates, for any abnormalities of left–right axis determination. In the inbred Bn stock, situs ambiguus, in which individual organ position is randomized, was identified in >50% of surviving Bn males and in 38% of Bn females examined (Table 2 and Fig. 3). One Bn female also had an abnormality of lung lobulation, although this finding was not statistically significant (Fig. 3D). Anomalous vessels, including aortic arch abnormalities or congenital heart defects, were not detected after careful thoracic dissection. However, their occurrence would often be expected to cause pre- or perinatal lethality and, hence, not be present in surviving affected animals.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have demonstrated that the murine Bent tail mutation is associated with a submicroscopic deletion of the X chromosome that encompasses the Zic3 locus. Zic3 is a C2H2 zinc-finger transcription factor that is homologous to the Drosophila odd-paired gene (15). Its cellular targets remain unknown although it is expressed in embryonic mesoderm by 7 days post-conception in the mouse and at later stages (days 9.5–11.5) in the developing central nervous system, eye, dermomyotomes, limb bud and tail bud (16).
The Bn mutation was originally identified based on its tail phenotype. In addition to left–right axis malformations, 8 of 18 affected human males with ZIC3 mutations had anal anomalies, and three additional affected males had lumbosacral spine defects, including sacral agenesis in two and an open NTD in the third (13,14). We have now detected abnormalities of situs, similar to those found in human ZIC3 patients, in affected Bn males and females from the original inbred stock. There is also a significant difference between the number of affected and normal males in our back-cross (P < 0.01, 
2 analysis), raising the possibility of pre- or perinatal loss of some more severely affected males, perhaps with lethal situs abnormalities. It is also interesting to speculate about possible species-specific differences in the penetrance of tail and spinal defects versus cardiac and situs anomalies between human and mouse. Examining the effects of modifier loci on the Bn phenotype by placing the mutation onto different genetic backgrounds may begin to address this issue.
During our genetic mapping studies, we detected a high frequency of sex chromosomal non-disjunction in the background strain on which the Bn mutation arose. The X-linked, semi-dominant mouse mutation patchy fur (Paf) maps to the boundary of the pseudoautosomal region and is associated with an increased rate of sex chromosome aneuploidy, perhaps as a result of a small chromosomal rearrangement that impairs X-Y pairing (17). Hunt and Eicher (18) generated a strain of males that produce >50% XY sperm. Mice carrying Robertsonian translocations also show high frequencies of autosomal non-disjunction (19). The Bn background strain may, thus, prove useful as another model to study mammalian non-disjunction.
In human, the region immediately surrounding ZIC3 is gene-poor, and it is possible that no additional genes are within the murine deletion. Our finding of left–right axis malformations in Bn mice, in addition to tail defects, is consistent with the phenotype of ZIC3 mutations in human patients and suggests that deletion of Zic3 is responsible for most, if not all, of the phenotypic features of this mutant.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mouse strains, crosses and genetic mapping
The Bn strain was obtained from the Jackson Laboratory (Bar Harbour, ME). It has been maintained by brother–sister matings between phenotypically affected heterozygous females and normal males from the original Bn stock. For genetic mapping, heterozygous Bn females were crossed to C3H/HeJ males (Jackson Laboratory), and resultant phenotypically affected F1 females were back-crossed to normal appearing males from the inbred Bn stock. Microsatellite markers were purchased from Research Genetics (Huntsville, AL) and PCRs performed as recommended by the manufacturer.
Physical mapping of Bn
Southern hybridizations of Bn and normal genomic DNA were performed as described (20) on SacI- or EcoRI-digested DNA using DNA probes from the 5', middle and 3' portions of the murine Zic3 gene (S.M. Purandare et al., submitted). Genomic PCR for Zic3 utilized forward primer 5'-ATTGCTGCAAGTGCCGGGAACT-3' and reverse primer 5'-ACTCACATCTCCTATTTGTAG-3' from the 3'-untranslated region and cycling parameters of 94, 55 and 72°C for 30 s each for 35 cycles. For P21-Rac1, the PCR primers were forward primer 5'-GCCAATGTTATGGTAGATGG-3' and reverse primer 5'-CAGGACTCACAAGCGAAAAGC-3'. PCR was performed for 35 cycles with denaturation at 93°C for 1 min, annealing at 57°C for 1 min and extension at 72°C for 1 min. BACs were obtained by PCR screening a total mouse genomic BAC library (Research Genetics) with DXMit208 or by hybridization screening of the RPCI23 library (21) using a mouse Zic3 cDNA probe. BAC DNA was prepared using a NucleoBond Plasmid kit (Clontech, Palo Alto, CA) and end sequencing performed with left and right vector-specific primers on an ABI377 automated sequencer (PE Biosystems, Foster City, CA). Primer sequences and PCR conditions for the BAC end loci DXChri1–DXChri5 (GenBank accession nos AZ044550–AZ044554) have been entered into the Mouse Genome Database. For pulsed field gel analysis, 1 µg of BAC DNA was digested with NotI or EagI (New England Biolabs, Beverly, MA) and electrophoresis performed on a CHEF Mapper XA System (Bio-Rad, Hercules, CA) as recommended by the manufacturer. Digested DNA was transferred to Sure Blot Nylon Membranes (Intergen, Purchase, NY) and Southern hybridization performed as described (22) with radiolabeled vector-specific or gene probes.
Phenotypic characterization
Affected Bn males and females and normal male littermates from the inbred stock, 2 weeks to 5 months of age, were sacrificed and abdominal and thoracic viscera examined using a dissecting microscope (magnification 1–5x) (Zeiss, Thornwood, NY) with assistance from an experienced perinatal pathologist (T.F.). Cardiac anatomy was ascertained by tracing the pattern of normal blood flow prior to sectioning of the heart to look for internal malformations.
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ACKNOWLEDGEMENTS |
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The authors thank Dr Rosemary Elliott for sharing unpublished genetic mapping data, Dr Eva Eicher for helpful discussions, Dr Joan Durbin and Ms Brenda Van Dyke for help with photography and Mr John Hayes for assistance with statistical analyses. This work was supported by funds from Children’s Research Institute to G.E.H. DNA sequences were determined with the help of the core facility at Children’s Research Institute which is supported in part by NIH grant HD34615.
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FOOTNOTES |
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+ These authors contributed equally to this work
§ To whom correspondence should be addressed at: Children’s Research Institute, 700 Children’s Drive, Room W403, Columbus, OH 43205, USA. Tel: +1 614 722 2848; Fax: +1 614 722 27116; Email: hermang@pediatrics.ohio-state.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 Botto, L.D., Moore, C.A., Khoury, M.J. and Erickson, J.D. (1999) Neural-tube defects. N. Engl. J. Med., 341, 1509–1519.
2 American Academy of Pediatrics Committee on Genetics (1999) Folic acid for the prevention of neural tube defects. Pediatrics, 104, 325–327.
3 Harris, M.J. and Juriloff, D.M. (1999) Mini-review: toward understanding mechanisms of genetic neural tube defects in mice. Teratology, 60, 292–305.[Web of Science][Medline]
4 Neumann, P.E., Frankel, W.N., Letts, V.A., Coffin, J.M., Copp, A.J. and Bernfield, M. (1994) Multifactorial inheritance of neural tube defects: localization of the major gene and recognition of modifiers in ct mutant mice. Nature Genet., 6, 357–362. [Web of Science][Medline]
5 Garber, E.D. (1952) Bent-tail, a dominant, sex-linked mutation in the mouse. Proc. Natl Acad. Sci. USA, 38, 876–879.
6 Johnson, D.R. (1976) The interfrontal bone and mutant genes in the mouse. J. Anat., 121, 507–513.
7 Lyon, M.F. (1966) Order of loci on the X-chromosome of the mouse. Genet. Res., 7, 130–133.[Web of Science][Medline]
8 Sweet, H.O. Bronson, R.T., Harris, B.S. and Davisson, M.T. (1990) Trembly-like (Tyl)–a new X-linked mutation in the mouse. Mouse Genome, 87, 112.
9 Denny, P., Haynes, A.R., Masson, W., Laval, S.H., Boyd, Y., Disteche, C. and Elliott, R.W. (2000) Chromosome X (http://www.informatics.jax.org/ ).
10 Didsbury, J., Weber, R.F., Bokoch, G.M., Evans, T. and Snyderman, R. (1989) Rac, a novel ras-related family of proteins that are botulinum toxin substrates. J. Biol. Chem., 264, 16378–16382.
11 Drivas, G.T., Shih, A., Coutavas, E., Rush, M.G. and D’Eustachio, P. (1990) Characterization of four novel ras-like genes expressed in a human teratocarcinoma cell line. Mol. Cell. Biol., 10, 1793–1798.
12 Malosio, M.L., Gilardelli, D., Paris, S., Albertinazzi, C. and de Curtis, I. (1997) Differential expression of distinct members of Rho family GTP-binding proteins during neuronal development: identification of RaclB, a new neural-specific member of the family. J. Neurosci., 17, 6717–6728.
13 Ferrero, G.B., Gebbia, M., Pilia, G., Witte, D., Peier, A., Hopkin, R.J., Craigen, W.J., Shaffer, L.G., Schlessinger, D., Ballabio, A. et al. (1997) A submicroscopic deletion in Xq26 associated with familial situs ambiguus. Am. J. Hum. Genet., 61, 395–401.[Web of Science][Medline]
14 Gebbia, M., Ferrero, G.B., Pilia, G., Bassi, M.T., Aylsworth, A.S., Penman-Splitt, M., Bird, L.M., Bamforth, J.S., Burn, J., Schlessinger, D. et al. (1997) X-linked situs abnormalities result from mutations in ZIC3. Nature Genet., 17, 305–308.[Web of Science][Medline]
15 Aruga, J., Nagai, T., Tokuyama, T., Hayashizaki, Y., Okazaki, Y., Chapman, V.M. and Mikoshiba, K. (1996) The mouse zic gene family. Homologues of the Drosophila pair-rule gene odd-paired. J. Biol. Chem., 271, 1043–1047.
16 Nagai, T., Aruga, J., Takada, S., Gunther, T., Sporle, R., Schughart, K. and Mikoshiba, K. (1997) The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation. Dev. Biol., 182, 299–313.[Web of Science][Medline]
17 Korobova, O., Lane, P.W., Perry, J., Palmer, S., Ashworth, A., Davisson, M.T. and Arnheim, N. (1998) Patchy fur, a mouse coat mutation associated with X-Y non-disjunction, maps to the pseudoautosomal boundary region. Genomics, 54, 556–559.[Web of Science][Medline]
18 Hunt, P.A. and Eicher, E.M. (1991) Fertile male mice with three sex chromosomes: evidence that infertility in XXY male mice is an effect of two Y chromosomes. Chromosoma, 100, 293–299.[Web of Science][Medline]
19 Everett, C.A., Searle, J.B. and Wallace, B.M. (1996) A study of meiotic pairing, non-disjunction and germ cell death in laboratory mice carrying Robertsonian translocations. Genet. Res., 67, 239–247.[Web of Science][Medline]
20 Angel, T.A., Faust, C.J., Gonzales, J.C., Kenwrick, S., Lewis, R.A. and Herman, G.E. (1993) Genetic mapping of the X-linked dominant mutations striated (Str) and bare patches (Bpa) to a 600-kb region of the mouse X chromosome: implications for mapping human disorders in Xq28. Mamm. Genome, 4, 171–176.[Web of Science][Medline]
21 Osoegawa, K., Tateno, M., Woon, P.Y., Frengen, E., Mammoser, A.G., Catanese, J.J., Hayashizaki, Y. and de Jong, P.J. (2000) Bacterial artificial chromosome libraries for mouse sequencing and functional analysis. Genome Res., 10, 116–128.
22 Faust, C.J. and Herman, G.E. (1991) Physical mapping of the loci Gabra3, DXPas8, CamL1 and Rsvp in a region of the mouse X chromosome homologous to human Xq28. Genomics, 11, 154–164.[Web of Science][Medline]
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