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Localization of am3 using EL Congenic Mouse Strains
T. Shimizu1,*,
J. Han1,
Y. Asada2,
H. Okamoto1 and
T. Maeda1
1 Department of Pediatric Dentistry, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan; and
2 Department of Pediatric Dentistry, Tsurumi University School of Dental Medicine, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan;
Correspondence: * corresponding author, takehiko{at}mascat.nihon-u.ac.jp
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ABSTRACT
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EL/Sea mice have 100% incidence of the absence of third molars (M3). Our previous linkage analysis using EL/Sea and MSM/Msf mouse strains provides statistical evidence of a major locus for the absence of M3, designated am3, of EL/Sea at the middle region of chromosome 3. To obtain independent evidence for linkage and more precisely determine the location of the am3 locus, we generated EL/Sea congenic strains for am3 in which the restricted interval of chromosome 3 of EL/Sea was replaced by an MSM/Msf-derived homologue. EL/Sea congenic mice that were either heterozygous or homozygous for the MSM/Msf-derived interval exhibited a significant decrease in the incidence of the absence of third molars, confirming previous genome scan results. These results confine the am3 locus to an approximately 4.4-cM region, and demonstrate that other unmapped genes are also involved in the absence of M3 in EL/Sea mice.
Key Words: hypodontia • congenic mouse strain • gene mapping
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INTRODUCTION
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Hypodontia or oligodontia is seen in more than 49 genetic syndromes or occurs as an isolated sporadic or familial trait (Stockton et al., 2000). Although genetic mutations in MSX1 (Vastardis et al., 1996; van den Boogaard et al., 2000; Jumlongras et al., 2001; Lidral and Reising, 2002) and PAX9 (Stockton et al., 2000; Frazier-Bowers et al., 2002) have been associated with familial tooth agenesis, the causes of various forms of tooth agenesis remain unknown (Nieminen et al., 1995; Arte et al., 1996; Goldenberg et al., 2000; Scarel et al., 2000).
In mice, the third molar (M3) is the most frequently absent tooth. Reported frequency of absent M3 is 3% for CBA/J mice, and 2% for A/J mice (Murai, 1975). In mutant mouse stocks, crinkled, tabby (Miller, 1978), downless, and sleek (Sofaer, 1977) genes, which affect the morphological structure of teeth, strongly affect the absence of M3. However, in such mutants, the absence of M3 is part of the pleiotropic phenotypes that are analogous to human hypohidrotic ectodermal dysplasia. EL/Sea (EL) mice have 100% incidence of the absence of M3 without abnormal crown shapes of other molars or any generalized anomalies of appearance (Asada et al., 2000). Absence of teeth in EL mice is influenced by major gene effects with recessive transmission, as indicated by crosses with the wild-type mouse strain MSM/Msf, and a genome scan of F2 progeny from intercrosses between EL and MSM mice provides evidence suggesting that alleles on chromosome 3 (Chr 3) cause the absence of teeth in EL mice (Nomura et al., 2003).
In the present study, to obtain independent evidence for linkage to Chr 3 for the absence of M3 (absence of the third molar, allele symbol am3), and to define the location of the locus more precisely, we produced EL congenic mice virtually identical to standard EL mice, with the exception of replacement of the selected interval on Chr 3, which carries the am3 allele, with the corresponding chromosomal segment of the MSM mouse. To examine effects of the EL am3 allele in an MSM genetic background, we also generated a group of EL congenic strains that carry the restricted EL-derived homologue containing the am3 allele in an MSM background.
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MATERIALS & METHODS
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Mice
EL/Sea (EL) mice were obtained from Seac Yoshitomi Ltd. (Fukuoka, Japan). MSM/Msf (MSM) mice, which are a wild-type strain derived from Mus musculus molossinus progenitors, were obtained from the animal facilities at the National Institute of Genetics (Mishima, Japan). All procedures were approved by the Institutional Animal Care Committee.
Genotyping of Backcross Progeny
For each generation of backcross mice, genomic DNA was extracted from the tail (Laird et al., 1991) and genotyped by polymerase chain-reaction (PCR) with the use of MIT (Massachusetts Institute of Technology) primers. DNA amplification by PCR and gel electrophoresis of PCR products were performed as previously described (Shimizu, 1999). Lef1 and Egf, as candidate genes for am3 (Nomura et al., 2003), were used for genotyping of backcross mice. Short tandem-repeat polymorphisms between EL and MSM mice in the 5' untranslated region of Lef1 (Ensembl Gene ID, ENSMUSG00000027985) and in the intron region between exons 15 and 16 of Egf (Ensembl Gene ID, ENSMUSG00000028017) were detected by DNA sequencing, and were then used for PCR typing. The following oligonucleotides were used for PCR amplification: Lef1, 5'- GGAGGCTGCATAGATTCACTC-3' and 5'-CTCAACCCCT CCCCTCAAGTC-3'; and Egf, 5'-GACCCCTAAAGGG TTTTTGC-3' and 5'-GGAGGAGGAACAGGTTGAGG-3'.
Production of EL-1 Congenic Mice for the am3 Allele
To generate EL-1 congenic mice that contain an MSM-derived interval for the am3 allele on Chr 3 in an EL genetic background, we first mated EL females with MSM males, and F1 (EL x MSM) males were then backcrossed to EL females to produce N2 mice. Subsequent backcrosses were performed with EL males or females and the appropriate backcross progeny, based on genotyping results. N2 mice were screened for genotypes at the D3Mit106 and D3Mit260 microsatellite loci flanking the approximately 18-cM interval, which has previously been defined as a candidate region for am3 (Nomura et al., 2003). All marker positions and cM distances were obtained from the Mouse Genome Database (MGD). Congenic mice are usually regarded as established at the N10 stage. In this study, we used the speed congenic method, which can halve the time required (Markel et al., 1997; Wakeland et al., 1997). N2 mice heterozygous at the two microsatellite loci were further screened for genotypes at a total of 58 MIT markers spanning the autosomes (Nomura et al., 2003). One N2 mouse, which had the most recipient loci, was backcrossed to EL mice to produce N3 animals. This backcross cycle was repeated six times to obtain N7a mice that were heterozygous (E/M) at loci from D3Mit56 to D3Mit260 (region 1) and N7c mice that were homozygous for the EL allele (E/E) at region 1. We selected a N6 mouse to generate N7b mice, which were E/M at loci from D3Mit56 to D3Mit290 (region 2), and N7b mice were intercrossed to generate N7bF2 mice, which were homozygous for the MSM allele (M/M) at region 2. From the N7 generation, two mice, each carrying a recombination event between D3Mit56 and D3Mit290, were selected to produce N8a and N8b animals, respectively. N8a mice were E/M at the loci of D3Mit216 and D3Mit290 (region 3), and N8b mice were E/M at the loci of D3Mit56 and D3Mit106 (region 4). To obtain N9 mice, we backcrossed to EL mice an N8 mouse containing a smaller MSM-derived interval than N8a mice. N9b mice were E/M at the loci of D3Mit216 and D3Mit13 (region 5), and N9a mice were E/M at D3Mit290 (region 6). N9c mice, which were E/E at regions 5 and 6, were produced as controls. N8a and N9a mice were intercrossed to generate N8aF2 and N9aF2, which were M/M at regions 3 and 6, respectively. All mice in each congenic line were typed for markers on chromosome 3.
Production of EL-2 Congenic Mice for the am3 Allele
The EL-2 congenic mice, which contain an EL-derived interval with am3 in an MSM genetic background, were produced by repeated backcrossing to the MSM strain and selection for EL alleles by genotyping at each generation. The MIT markers used for production of EL-1 congenic mice were used for the speed congenic method. A backcross cycle of six iterations was performed to obtain N7d mice E/M at loci from D3Mit103 to D3Mit260 (region 7) in an MSM genetic background and N7e mice (control) that were M/M for the target interval. N7dF2 mice, which were E/E at region 7, were generated from mating between N7d mice.
Mutation Analysis for Candidate Genes for am3
For mutation analysis of the coding sequence of Egf and Lef1, as candidate genes for am3 (Nomura et al., 2003), exons 1 to 24 of Egf and exons 2 to 13 of Lef1 from EL and control mice (MSM and C3H/J) were amplified by PCR with use of the primers shown in Table 1 . DNA amplification was performed according to the same PCR procedure that was used for PCR genotyping. PCR products were directly sequenced with the use of the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Tokyo, Japan) and an ABI PRISMTM 310 Genetic Analyzer (Applied Biosystems, Tokyo, Japan). Sequences obtained were verified against the sequences in the Ensemb Exon Report (Lef1, ENSMUSG00000027985; Egf, ENSMUSG00000028017) and the sequences of controls.
Evaluation of Frequency of the Absence of M3
N7, N8, and N9 mice were killed under anesthesia at 6 to 8 wks of age. The heads were soaked in 1% KOH at 45°C for 48 hrs, and the soft tissue was removed. The upper and lower M3 were observed under a dissection microscope (20 magnification). In N7, N8, and N9 generations of EL-1 congenic mice, the frequency of absence of 2 or more M3 was evaluated based on the frequency of the EL strain (100%), by the chi-square test.
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RESULTS
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Analysis of the EL-1 Congenic Mice for the am3 Allele
We generated 9 groups of EL-1 congenic mice (Fig. 1). Genotyping of the N7 mice showed that, except for the am3 interval, they were E/E at all polymorphic markers, which were distributed throughout the autosomes. Table 2 shows the distribution of numbers of absent M3 in the EL-1 congenic and parental mice. We found that 38 of the 50 EL mice were missing all M3, and the remaining 12 mice lacked 2 or 3 M3. N7c, N8c, and N9c mice, which were E/E for the am3 allele, showed 100% incidence of the absence of 2 or more M3. N7a mice, which were E/M at region 1 (Fig. 1), showed a significant decrease in incidence of missing 2 or more M3 (9.1%), compared with EL homozygous littermates and the EL parental strain. N7b mice, which were E/M at region 2 (Fig. 1), and N7bF2 mice, which were M/M for this interval, showed a significant decrease in the incidence of missing 2 or more M3 (52.6% and 30%, respectively). N8aF2 mice, which were M/M at region 3 (Fig. 1), exhibited complete rescue from the dental phenotype of EL. N8b mice, which were E/M at region 4 (Fig. 1), and N9b mice, which were E/M at region 5 (Fig. 1), did not exhibit the rescued am3 phenotype, excluding these sections as candidates for the am3 allele. N9a mice, which had a smaller heterozygous region than N8a mice, which were E/M at region 6 (Fig. 1), and N9aF2 mice, which were M/M at region 6, showed positive results (57.1% and 0% incidence of missing 2 or more M3, respectively). Region 6 contained Lef1 and Egf as potential candidate genes for am3. In N7b, N7bF2, N8a, N8aF2, N9a, and N9aF2 mice, the absence of upper M3 was rescued at a higher frequency than the absence of lower M3 (upper M3:lower M3 = 162:100,  2 = 14.7, p < 0.01). There was no difference between the right and left sides in incidence of appearance of M3 (right:left = 131:131).
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Figure 1. Diagram of chromosome 3 for EL-1 congenic mouse strains. Genotypes at microsatellite, Lef1, and Egf loci, spread over the am3 interval, and frequencies of the absence of 2 or more third molars (M3) in EL-1 congenic mice are presented. The chromosomal position of the am3 interval was determined by our previous study (Nomura et al., 2003). Map positions of markers and genes were obtained from the Mouse Genome Database, the Jackson Laboratory. Numbers in parentheses represent the numbers of the mice obtained.
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Analysis of the EL-2 Congenic Mice for the am3 Allele
We generated 3 groups of EL-2 congenic mice by repeated backcross to the MSM strain (Fig. 2). Genotyping of the N7 generation of EL-2 congenic mice showed that they were M/M at all markers other than the am3 interval, throughout the autosomal genome. N7e mice, which were M/M in the region surrounding the am3 locus, exhibited 100% incidence of the presence of all M3. N7d mice, which were E/M at region 7 (Fig. 2), and N7dF2 mice, which were E/E for this interval, also exhibited 100% incidence of the presence of all M3.
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Figure 2. Diagram of chromosome 3 for EL-2 congenic mouse strains. Genotypes at microsatellite, Lef1, and Egf loci, spread over the am3 interval, and frequencies of the presence of all M3 in EL-2 congenic mice are presented. The chromosomal position of the am3 interval was determined by our previous study (Nomura et al., 2003). Map positions of markers and genes were obtained from the Mouse Genome Database, the Jackson Laboratory. Numbers in parentheses represent the numbers of the mice obtained.
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Mutation Analysis of Egf and Lef1 as Candidate Genes
We detected single-nucleotide polymorphisms in exons 19 and 21 of Egf between EL mice and the Ensemb Exon Report. However, comparison of the coding sequence of Egf and Lef1 between EL and both controls demonstrated complete concordance.
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DISCUSSION
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Congenic breeding strategies are becoming increasingly important as complex trait linkages are identified (Markel et al., 1997). An effective strategy for defining the am3 allele is to produce congenic strains for the previously linked chromosomal region. The present finding that the am3 phenotype was rescued in N7a mice confirms the accuracy of previous mapping results. By comparing genotypes and phenotypes of EL-1 congenic mice, we clearly mapped the am3 locus within an approximately 4.4-cM region flanked by D3Mit13 and D3Mit125. However, replacement of the mutation by the normal allele at am3 did not completely abolish the incidence of missing M3 in EL-1 mice. Therefore, other genes with incomplete penetrance, which are still present within the EL genetic background, must be involved in the loss of M3. In contrast, we expected that EL-2 congenic mice, which had an EL-derived homologue containing the am3 allele in an MSM background, would exhibit the absence of M3, but we did not find hypodontia in N7d or N7dF2 mice. This suggests that the loss of teeth in EL mice requires effects of not only the am3 allele on chromosome 3 but also other allele(s). Nomura et al.(2003) reported statistical evidence that other loci in chromosome 16 are involved in M3 agenesis. Thus, there is a need for production of congenic lines for these loci to assess their genetic contributions to the absence of M3.
In the present analysis of the difference in frequency of missing 3 or all M3 between E/M and M/M at the am3 locus (i.e., N7b and N7bF2, N8a and N8aF2, and N9a and N9aF2), the M/M genotype at am3 appears to be more protective than the E/M genotype against the absence of M3. We presume that am3 has a somewhat dominant effect, although F1 (EL x MSM) mice did not show hypodontia.
We examined differences in effects of am3 between the maxilla and mandible. In the EL-1 congenic mice, the absence of upper M3 tended to be rescued at a greater frequency than the absence of lower M3 by an MSM-derived chromosome. This finding suggests that although the am3 allele influences both upper and lower M3, the EL am3 allele affects the upper M3 more strongly than the lower M3, and that other gene(s) have additive effects that contribute to the total absence of M3 in the EL strain.
Lef1 and Egf genes, as candidate genes for am3 (Nomura et al., 2003), were found to be located within region 6 (Fig. 1 ). Previous analysis of Lef–/– embryos indicates that the absence of LEF1 results in a complete lack of tooth development (Kratochwil et al., 2002). Antisense oligomers to Egf mRNA block the initiation of odontogenesis in mandibular explants of embryonic mice (Kronmiller et al., 1991). Therefore, we performed mutation analysis of Egf and Lef1 as strong candidates for am3. However, we found no mutations in the coding sequence of these two genes in EL mice. This finding suggests that Egf and Lef1 are not responsible for M3 agenesis (although sequences of the intron and regulatory region were not analyzed), and suggests that a novel gene between the D3Mit13 and D3Mit125 loci is involved in tooth agenesis in the EL strain.
Efforts are now being made to generate additional EL congenic strains with a smaller MSM-derived interval, to localize the am3 locus more precisely. Cloning of these causative genes in mice may help to elucidate the underlying mechanisms of hypodontia in humans, because mouse and human genes are highly syntenic.
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ACKNOWLEDGMENTS
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This investigation was supported by a Grant-in-Aid for Young Scientists (B) 15791227 from the Ministry of Education, Culture, Sports, Science and Technology, and by a Nihon University Research Grant for Assistants and Young Researchers 03-126.
Received for publication April 26, 2004.
Revision received January 7, 2005.
Accepted for publication January 18, 2005.
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REFERENCES
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- Arte S, Nieminen P, Pirinen S, Thesleff I, Peltonen L (1996). Gene defect in hypodontia: exclusion of EGF, EGFR, and FGF-3 as candidate genes. J Dent Res 75:1346–1352.
- Asada Y, Shimizu T, Matsune K, Shimizu K, Suzuki Y, Takamori K, et al. (2000). Absence of the third molars in strain EL mice. Ped Dent J 10:19–22.
- Frazier-Bowers SA, Guo DC, Cavender A, Xue L, Evans B, King T, et al. (2002). A novel mutation in human PAX9 causes molar oligodontia. J Dent Res 81:129–133.
- Goldenberg M, Das P, Messersmith M, Stockton DW, Patel PI, D’Souza RN (2000). Clinical, radiographic, and genetic evaluation of a novel form of autosomal-dominant oligodontia. J Dent Res 79:1469–1475.
- Jumlongras D, Bei M, Stimson JM, Wang WF, DePalma SR, Seidman CE, et al. (2001). A nonsense mutation in MSX1 causes Witkop syndrome. Am J Hum Genet 69:67–74.[CrossRef][Medline]
[Order article via Infotrieve]
- Kratochwil K, Galceran J, Tontsch S, Roth W, Grosschedl R (2002). FGF4, a direct target of LEF1 and Wnt signaling, can rescue the arrest of tooth organogenesis in Lef1(–/–) mice. Genes Dev 16:3173–3185.[Abstract/Free Full Text]
- Kronmiller JE, Upholt WB, Kollar EJ (1991). EGF antisense oligodeoxynucleotides block murine odontogenesis in vitro. Dev Biol 147:485–488.[CrossRef][Medline]
[Order article via Infotrieve]
- Laird PW, Zijderveld A, Linders K, Rudnicki MA, Jaenisch R, Berns A (1991). Simplified mammalian DNA isolation procedure. Nucleic Acids Res 19:4293.[Free Full Text]
- Lidral AC, Reising BC (2002). The role of MSX1 in human tooth agenesis. J Dent Res 81:274–278.
- Markel P, Shu P, Ebeling C, Carlson GA, Nagle DL, Smutko JS, et al. (1997). Theoretical and empirical issues for marker-assisted breeding of congenic mouse strains. Nat Genet 17:280–284.[CrossRef][Medline]
[Order article via Infotrieve]
- Miller WA (1978). The dentitions of tabby and crinkled mice. In: Development, function and evolution of teeth. Butler PM, Joysey KA, editors. London: Academic Press, pp. 99–109.
- Murai M (1975). A genetic study on the development of the lower molars and mandible in mice: change of genetic and environmental effects in the course of pre- and postnatal morphogenesis. Jpn J Genet 50:73–90.
- Nieminen P, Arte S, Pirinen S, Peltonen L, Thesleff I (1995). Gene defect in hypodontia: exclusion of MSX1 and MSX2 as candidate genes. Hum Genet 96:305–308.[Medline]
[Order article via Infotrieve]
- Nomura R, Shimizu T, Asada Y, Hirukawa S, Maeda T (2003). Genetic mapping of absence of the third molars in EL mice to chromosome 3. J Dent Res 82:786–790; erratum in J Dent Res 82:934.
- Scarel RM, Trevilatto PC, Di Hipolito O Jr, Camargo LE, Line SR (2000). Absence of mutations in the homeodomain of the MSX1 gene in patients with hypodontia. Am J Med Genet 92:346–349.[CrossRef][Medline]
[Order article via Infotrieve]
- Shimizu T (1999). Mapping of a gene causing mouse gutter-shaped tooth root to chromosome 5. Arch Oral Biol 44:917–924.[Medline]
[Order article via Infotrieve]
- Sofaer JA (1977). The teeth of the "sleek" mouse. Arch Oral Biol 22:299–301.[CrossRef][Medline]
[Order article via Infotrieve]
- Stockton DW, Das P, Goldenberg M, D’Souza RN, Patel PI (2000). Mutation of PAX9 is associated with oligodontia. Nat Genet 24:18–19.[CrossRef][Medline]
[Order article via Infotrieve]
- van den Boogaard MJ, Dorland M, Beemer FA, van Amstel HK (2000). MSX1 mutation is associated with orofacial clefting and tooth agenesis in humans. Nat Genet 24:342–343.[CrossRef][Medline]
[Order article via Infotrieve]
- Vastardis H, Karimbux N, Guthua SW, Seidman JG, Seidman CE (1996). A human MSX1 homeodomain missense mutation causes selective tooth agenesis. Nat Genet 13:417–421.[CrossRef][Medline]
[Order article via Infotrieve]
- Wakeland E, Morel L, Achey K, Yui M, Longmate J (1997). Speed congenics: a classic technique in the fast lane (relatively speaking). Immunol Today 18:472–477.[CrossRef][Medline]
[Order article via Infotrieve]
Journal of Dental Research, Vol. 84, No. 4,
315-319 (2005)
DOI: 10.1177/154405910508400404
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ABSTRACT
INTRODUCTION