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Localization of am3 using EL Congenic Mouse Strains

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;

^* corresponding author, takehiko{at}mascat.nihon-u.ac.jp

	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

	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.

	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 PRISM^TM 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.

	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).

View larger version (44K): [in this window] [in a new window]   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.

View larger version (30K): [in this window] [in a new window]   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.

	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.

	ACKNOWLEDGMENTS

 
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 April 26, 2004; Last revision January 7, 2005; Accepted January 18, 2005

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