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Department of Genetics,3 Curriculum in Genetics and Molecular Biology,4 Departments of Nutrition,5 and Cell and Molecular Physiology,6 Carolina Center for Genome Sciences,7 Lineberger Comprehensive Cancer Center,8 University of North Carolina, Chapel Hill, North Carolina 27599-7264
Genetics Division,9 Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115
ABSTRACT
When females of the DDK inbred mouse strain are mated to males of other strains, 90–100% of the resulting embryos die during early embryonic development. This DDK syndrome lethality results from incompatibility between an ooplasmic DDK factor and a non-DDK paternal gene, which map to closely linked loci on chromosome 11. It has been proposed that the expression of the gene that encodes the ooplasmic factor is subject to allelic exclusion in oocytes. Previous studies have demonstrated the existence of recessive modifiers that increase lethality in the C57BL/6 and BALB/c strains. These modifiers are thought to skew the choice of allele undergoing allelic exclusion in the oocytes of heterozygous females. In the present study, we demonstrate the presence of modifiers in three Mus musculus domesticus wild-derived strains, PERA, PERC, and RBA. These modifiers completely rescued DDK syndrome lethality. We mapped the major locus that is responsible for rescue in PERA and PERC crosses to proximal chromosome 13 and named this locus Rmod1 (Rescue Modifier of the DDK Syndrome 1). Our experiments demonstrate that PERA or PERC alleles at Rmod1 rescue lethality independently of allelic exclusion. In addition, rescue of the lethal phenotype depends on the parental origin of the Rmod1 alleles; transmission through the dam leads to rescue, while transmission through the sire has no effect.
developmental biology, early development, embryo, oocyte, ovum
DDK syndrome is a polar embryonic lethal phenotype that disrupts early mouse development in crosses involving the DDK inbred strain [1–3]. When DDK females are crossed to males of many other inbred strains, 90–100% of the embryos die [2]. In contrast, both the reciprocal cross (non-DDK female x DDK male) and the intra-strain cross (DDK female x DDK male) are fully viable [2]. Developmental abnormalities, such as cell death and cleavage defects, have been observed as early as the 2-cell stage [2, 4]. However, most of the affected embryos arrest and begin to degenerate between the morula and peri-implantation stages [2, 4]. Investigations into the cause of lethality have revealed the presence of low intracellular pH, defective gap junctional communication [5, 6], and defects in trophoblast formation [7]. However, the direct physiological cause has not yet been determined.
Ooplasm transfer, RNA microinjection, pronuclear transfer, and somatic cell nuclear transfer studies have demonstrated that the lethality results from an incompatible interaction between a factor present in DDK oocytes and a non-DDK paternal genome [3, 8–11]. Pronuclear transfer studies have also revealed a parent-of-origin effect, such that the lethal interaction is only observed between the DDK ooplasm and a non-DDK paternal pronucleus but not between the DDK ooplasm and a non-DDK maternal pronucleus [3, 9]. Recently, it has been shown that the incompatible interaction between the ooplasm and the paternal genome is recapitulated in cloned embryos, whereas the parent-of-origin effect is not, which indicates that the epigenetic mark that discriminates between maternal and paternal pronuclei is not retained in somatic cells [11].
The original genetic model of DDK syndrome postulated that two tightly linked genes were responsible for the embryonic lethality, a paternal gene and a gene that encodes the maternal DDK factor [12]. Subsequent studies have confirmed that these two components are non-allelic and lie within the Ovum mutant (Om) locus on mouse chromosome 11 [13–18]. We have recently reduced the interval that contains the paternal gene to a 23.2-kb region in the Schlafen gene cluster [18]. High-resolution mapping was possible because compatible and incompatible alleles in the paternal gene are equally represented among a wide variety of inbred strains. Our studies indicate that the mutation responsible for the incompatible paternal allele most likely arose within the Mus musculus domesticus lineage, and that the incompatible paternal allele is carried by many common laboratory strains [18]. Furthermore, the incompatible allele at the paternal gene of the DDK syndrome is highly penetrant; the fate of an embryo derived from an oocyte that carries the maternal DDK factor depends exclusively on whether it inherits a compatible or incompatible paternal allele at the paternal gene [13, 18].
In contrast, identification of the gene that encodes the maternal DDK factor has proven remarkably difficult, in part due to the fact that DDK is the only strain known to carry the incompatible allele at the gene encoding the maternal factor [18]. Furthermore, previous studies have indicated that the maternal contribution to the DDK syndrome acts as an incompletely penetrant maternal effect, whereby approximately 50% of the embryos survive in crosses between hybrid F1 females and incompatible BALB/c or C57BL/6 (B6) males [3, 19]. In addition, survival is independent of the maternal allele inherited at Om. In other words, approximately half of the surviving embryos inherit a maternal B6 allele at Om (OmB6) and half inherit a maternal DDK allele (OmDDK). It has been concluded that embryo survival depends on the genotype of the dam at Om and, therefore, it is a maternal effect [19]. It has also been proposed that the maternal effect is incompletely penetrant, as only half of the embryos were affected in these crosses [19]. To account for the incomplete penetrance, we proposed that the gene that encodes the maternal DDK factor undergoes random allelic exclusion, such that only one of the two alleles present is expressed in a given oocyte. This would generate two classes of oocytes in (B6 x DDK)F1 females, one class that expresses the DDK allele and dies upon fertilization by B6 sperm, and another class that expresses the B6 allele and survives [19].
The allelic exclusion hypothesis is supported by recent studies demonstrating the existence of modifiers of the DDK syndrome that are recessive and unlinked to Om [20–23]. In these experiments, incompatible males were backcrossed to heterozygous OmDDK/OmB6 or OmDDK/OmBALB/c females that had different proportions of their genomes derived from DDK and either B6 or BALB/c. Overall, there was a strong correlation between the level of embryonic death and the proportion of the dam genome that was of B6, BALB/c or DDK origin. This correlation was attributed to the presence of modifiers in these strains that could increase (B6 and BALB/c) or decrease (DDK) the fraction of embryos that died due to the DDK syndrome. Most importantly, these effects were only observed in crosses that involved females that were heterozygous at Om. Therefore, we and others have proposed that these modifiers act through allelic exclusion by skewing the choice of the allele to be expressed at the gene that encodes the maternal factor [20–23]. Allelic exclusion is thought to be important in the regulation of expression of a growing number of genes in mammals [24–28]. It has also been implicated in the etiology of human disease [29–31]. However, the molecular mechanism of allelic exclusion is not yet understood.
Wakasugi has shown that although NC males are incompatible with DDK females, crosses between NC males and (NC x DDK)F1 females are fully viable [12]. These data suggest to us the presence of modifiers of the DDK syndrome in the NC strain that rescue lethality in a dominant manner. The objectives of this study were to confirm the existence of rescue modifiers of the DDK syndrome, to map the loci needed for rescue, and to characterize their modes of action. We report the genetic characterization of modifiers found in two wild-derived inbred strains, PERA and PERC, which are able to rescue the lethal phenotype of the DDK syndrome. These parent-of-origin modifiers act in a manner that is independent of allelic exclusion.
The mouse strains, C57BL/6J, 129X1/SvJ, BALB/cJ, DBA/2J, PERA/EiJ, PERC/EiJ, RBA/DnJ, WSB/EiJ, SKIVE/EiJ, and CAST/EiJ, were originally obtained from the Jackson Laboratory (Bar Harbor, ME). JF1/Ms and DDK/Pas are maintained by Terry Magnuson and Fernando Pardo-Manuel de Villena, respectively, at the University of North Carolina at Chapel Hill. The DDK/Pas strain was originally obtained from Charles Babinet of the Institut Pasteur (Paris, France). The phylogenetic origins of these strains have been previously described [32]. All animal protocols were approved by the Institutional Animal Care and Use Committees of the University of North Carolina at Chapel Hill.
Mating Schemes and Reproductive Performance
For all crosses described in this study, the dam is listed first and the sire second, unless otherwise indicated. F1 hybrid females were generated by crossing DDK/Pas males to C57BL/6J, 129X1/SvJ, DBA/2J, BALB/cJ, JF1/Ms, CAST/EiJ, WSB/EiJ, PERC/EiJ, PERA/EiJ, RBA/DnJ or SKIVE/EiJ females. PERC/EiJ males were also crossed to DDK/Pas females to generate reciprocal F1 females. The reproductive performance of (C57BL/6J x DDK/Pas)F1 x C57BL/6J and (C57BL/6J x DDK/Pas)F1 x DDK/Pas crosses has been described previously [17–20]. F2 males were generated in a (PERC/EiJ x C57BL/6J)F1 intercross. Male offspring were selected based on their genotypes at Om and Rmod1. All crosses involved females between the ages of 2 and 10 mo and males between the ages of 2 and 12 mo. Cages were checked daily for the presence of newborn pups and litter size was recorded to avoid bias due to postnatal lethality. The reproductive performance of each cross was estimated using the litter size, as described previously [17, 20]. We accounted for average litter sizes of zero by dividing the sum of the average litter size of a female by the number of females. Experimental crosses involved a dam and sire with incompatible alleles at the gene encoding the maternal factor and the paternal gene, respectively. All control crosses had a compatible combination of alleles at Om. The level of rescue was determined by comparing the distribution of litter sizes of experimental crosses to that of control crosses. Under the null hypothesis that these two values are equal, significance was calculated using the Wilcoxon signed-rank test (JMP 6 release 6.0; SAS Institute, Cary, NC).
All physical positions of markers are based on the Ensembl v31 of the NCBI m33 mouse genome assembly [33]. DNA extraction from tail biopsies and genotyping by PCR amplification and gel electrophoresis were all performed as described previously [18, 34]. Oligonucleotide primers for microsatellite markers [35] were either purchased from Integrated DNA Technologies or Invitrogen Research Genetics. Alleles at Om were inferred from the genotypes at markers within (D11Spn31 or D11Spn78) or flanking (D11Mit35 and D11Mit33) the 632-kb candidate interval for the maternal gene [18]. Marker D13Mit135 was used to infer the genotypes at Rmod1.
A whole genome scan was performed on 36 heterozygous OmDDK/OmB6 G2 females that had C57BL/6J-DDK/Pas-PERA/EiJ mixed backgrounds and 39 heterozygous OmDDK/OmB6 G2 females that had C57BL/6J-DDK/Pas-PERC/EiJ mixed backgrounds. DNA samples were extracted from tail biopsies of these females using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma). Genotypes were obtained by three methods: 1) In an initial whole genome scan, we genotyped 394 SNPs using Sequenom MassARRAY technology followed by refinement on chromosome 13 by genotyping 34 additional SNPs using Sequenom Iplex technology [36]; 2) an additional genome scan in the PERC crosses with a panel of 768 SNPs genotyped by Illumina (San Diego, CA) using the BeadArray technology; and 3) microsatellite genotyping using PCR and acrylamide gel electrophoresis was used to fill in large gaps between markers. Supplemental Table 1 (available online at www.biolreprod.org) lists the informative markers (152 and 479 markers for PERA/EiJ and PERC/EiJ crosses, respectively) and their physical and genetic positions. Marker coverage spanned 20 chromosomes. Markers on chromosome 11 were excluded from this analysis because all the females used were selected to be OmDDK/OmB6. Additional markers were excluded on the basis of: 1) our inability to score genotypes in more than 50% of the samples, 2) failure of the assay to recognize both possible genotypes, 3) the presence of impossible genotypes, and 4) genotypes that created impossible recombination events between closely linked markers. On average, markers were spaced every 3 cM (range 0–25 cM) in PERC/EiJ crosses and every 10 cM (range 0–35 cM) in PERA/EiJ crosses.
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The genetic distances of informative SNPs included in this analysis were inferred using closely linked microsatellite markers for which the genetic distance from the centromere was determined previously [37] (Supplemental Table 1, available online at www.biolreprod.org). We used the average litter size as the phenotype for QTL mapping. Similar results were obtained when the median litter size was used. Linkage analyses were performed using a non-parametric model (an extension of the Kruskal-Wallis test) within the R/qtl software program [38, 39]. Compared to the parametric model within R/qtl (expectation-maximization), this approach yielded more conservative linkage values and was better suited to our data because the average litter sizes were not normally distributed [38, 39]. Using 1000 permutations within R/qtl, we determined the genome-wide significance thresholds as: 2.48 in PERC crosses and 2.31 in PERA crosses (P = 0.05); and 3.05 in PERC crosses and 3.31 in PERA crosses (P = 0.01). The percentage variance given by each QTL was estimated using the fitqtl function of R/qtl [38, 39]. Markers D13Mit135 and rs13479321 were used to infer the effects of Rmod1 and Rmod2.
A Sensitized Screen Reveals Modifiers That Completely Rescue DDK Syndrome Lethality
To identify strains that carry dominant modifiers that rescue DDK syndrome lethality, we screened a panel of F1 hybrid females that were generated by crossing females from a variety of strains to DDK males. We determined the presence of modifiers by comparing the reproductive performance of crosses between these F1 hybrid females and B6 males (experimental crosses) to the reproductive performance of viable control crosses. All F1 hybrid females tested in experimental crosses carry one DDK allele at Om. Therefore, in the absence of rescue, mating between F1 females and B6 males should result in a significant reduction (~50%) in reproductive performance in comparison to viable control crosses [2, 12, 17, 20]. This expectation was fulfilled in crosses involving the following types of F1 females: (CAST/Ei x DDK)F1, (SKIVE x DDK)F1, (129X1 x DDK)F1, (C57BL/6 x DDK)F1, (BALB/c x DDK)F1, (JF1 x DDK)F1, (DBA/2 x DDK)F1, and (WSB x DDK)F1 (crosses 7–14 and 17–21 in Fig. 1). In contrast, there were no significant differences between the reproductive performances of control and experimental crosses involving three types of F1 females: (PERC x DDK)F1, (PERA x DDK)F1 and (RBA x DDK)F1 (crosses 1–6 and 15–16 in Fig. 1). In these crosses, the ratio between the average litter size in experimental and control crosses was approximately one (Fig. 1), which demonstrates extensive rescue of the lethal phenotype. We conclude that rescue is due to the presence of genetic modifiers in these three strains. It is noteworthy that (RBA x DDK)F1 females had small litter sizes in both the experimental and control crosses (crosses 15 and 16, Fig. 1). The RBA strain carries the Rb(4.12)9Bnr Robertsonian translocation [40]. Females heterozygous for Robertsonian translocations are known to have reduced litter sizes due to the improper segregation of chromosomes during meiosis [41–44]. We conclude that the reduced litter size in the RBA crosses is unrelated to DDK syndrome lethality. Unfortunately, the limited phenotypic range of these crosses creates difficulties for accurate determinations of the presence and extent of rescue. Therefore, we have only pursued the genetic analyses of the modifiers present in the PERC and PERA strains.
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A Major Modifier Locus Maps to the Proximal Portion of Chromosome 13
The similarity between the reproductive performances of experimental and control crosses involving (PERC x DDK)F1 and (PERA x DDK)F1 females is consistent with complete rescue of the lethal phenotype (i.e., no embryos die from the DDK syndrome; compare cross 3 to cross 4 and cross 5 to cross 6 in Fig. 1). Three possibilities may account for complete rescue: 1) mitochondrial inheritance; 2) a single nuclear locus acting as a maternal effect; and 3) multiple unlinked loci that independently rescue the lethal phenotype. The nuclear loci may be linked or not linked to Om. To discriminate between these possibilities, we tested whether the rescue phenotype segregated among OmDDK/OmB6 G2 female offspring of experimental crosses (Fig. 2). We analyzed the reproductive performances of crosses between B6 males and 39 OmDDK/OmB6 G2 females with B6-DDK-PERC mixed backgrounds and 36 OmDDK/OmB6 G2 females with B6-DDK-PERA mixed backgrounds (Fig. 2a). Figure 2b shows the wide variation in reproductive performance among these G2 females, including some that were fully viable. Given that all of these females had identical OmDDK/OmB6 genotypes and carried PERC or PERA mitochondrial genomes, we conclude that the loci responsible for this variation in phenotype are neither mitochondrially inherited nor closely linked to Om.
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The distribution of the average litter size shown in Figure 2b suggests the involvement of few modifiers with major effects on rescue rather than many QTLs with small effect sizes. Therefore, we performed a whole genome scan on this panel of females, to map the PERC and PERA rescue modifiers. We identified a locus on proximal chromosome 13 that was significantly linked to the variation in reproductive performance (LOD scores: 4.2 and 5.3, PERC and PERA crosses respectively; Fig. 3). For both PERC and PERA, the maximum LOD score was observed at microsatellite marker D13Mit135. As expected, rescue was associated with the presence of a PERC or PERA allele at this locus in the dam. When G2 females were partitioned according to their genotypes at D13Mit135, two distinct phenotypic classes emerged. D13Mit135 DDK/D13Mit135B6 females had average litter sizes of 3.6 ± 0.4 and 2.7 ± 0.3 in PERC and PERA crosses, respectively (filled arrows in Fig. 2b). The average litter size of individual D13Mit135DDK/D13Mit135B6 females ranged from 0 to 5.6 (filled circles in Fig. 2b). In contrast, D13Mit135PERC/D13Mit135B6 and D13Mit135PERA/D13Mit135B6 females had average litter sizes of 7.0 ± 0.4 and 7.5 ± 0.4, respectively (open arrows in Fig. 2b). The average litter size of individual D13Mit135PERC/D13Mit135B6 and D13Mit135PERA/D13Mit135B6 females (open circles in Fig. 2b) ranged from 0 to 10 and from 4.5 to 9.3, respectively (note that there is a single D13Mit135PERC/D13Mit135B6 female with a litter size of zero; Fig. 2b). The fact that none of the D13Mit135DDK/D13Mit135B6 females had an average litter size consistent with extensive rescue indicates that a PERC and PERA allele at a locus closely linked to D13Mit135 is necessary for complete rescue. The overlapping linkage peaks and the correlation between rescue and PERC and PERA alleles at D13Mit135 (Fig. 3) suggest that this major modifier locus is shared by both strains. We have named this locus Rmod1, for Rescue Modifier of the DDK Syndrome 1.
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A second locus on chromosome 7 is also significantly associated with rescue, but only in the PERC crosses (LOD = 2.9; Fig. 3a). The maximum LOD score is observed at SNP marker rs13479321. At this locus, the DDK allele is associated with rescue. We have named this locus Rmod2, for Rescue Modifier of the DDK syndrome 2.
Rescue is Independent of Allelic Exclusion
To test whether rescue of lethality by PERA and PERC alleles at Rmod1 depends on allelic exclusion, we analyzed the reproductive performances of 22 OmDDK/OmDDK F2 females crossed to B6 males. These females were generated by (PERC x DDK)F1 or (PERA x DDK)F1 intercrosses (Fig. 4a). Figure 4b shows that there was extensive phenotypic variation in females with either type of genetic background (PERC or PERA). In fact, on average, these females had significantly higher reproductive performance than reported previously for lethal crosses [17, 20]. Given that these females were homozygous at Om, and that rescue by skewed expression of a gene subject to allelic exclusion necessarily requires heterozygosity at that locus, we conclude that rescue of lethality by these modifiers does not require allelic exclusion.
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Parent-of-Origin-Dependent Rescue of Lethality by PERA or PERC alleles at Rmod1
The experiments described in the previous sections demonstrate that the presence of PERC or PERA alleles at Rmod1 in the female germline leads to rescue of DDK syndrome lethality. Next, we examined whether the presence of Rmod1 in the male germline was also able to rescue the embryonic lethality. Previous studies have shown that PERC males carry compatible alleles at the paternal Om locus [18]. In contrast, PERA males carry incompatible alleles [18]. Testing for the presence of rescue requires the sire to have at least one incompatible allele at the paternal gene. Therefore, we generated four types of males, carrying one or two incompatible alleles at Om and one or two PERC or PERA alleles at Rmod1 (Table 1). The reproductive performances of these males mated to (B6 x DDK)F1 females were as predicted from their genotypes at Om and were not affected by the presence of PERC or PERA alleles at Rmod1. For example, experimental crosses 4 and 5 in Table 1 had average litter sizes consistent with approximately 50% lethality, as expected in these types of crosses in the absence of rescue. Note that the average litter sizes of these two crosses are not significantly different from that of semilethal control cross 2 but are significantly different from that of viable control cross 1 (Table 1). Likewise, crosses 6 and 7 had average litter sizes consistent with 25% lethality, as expected in these types of crosses in the absence of rescue. Again, compare these average litter sizes with the average litter size of control cross 3 (Table 1).
Finally, embryonic lethality caused by DDK syndrome is expected to be restricted to embryos that inherit an incompatible paternal allele at Om [18]. Therefore, there should be transmission ratio distortion against incompatible paternal alleles among the surviving embryos of sires that are heterozygous for compatible and incompatible alleles at Om. On the other hand, there should be a proportional reduction in the distortion levels in the presence of a given level of rescue. We determined the transmission ratio in the progeny of such males in our experimental crosses (crosses 6 and 7 in Table 1). Overall, we observed strong transmission ratio distortion against the incompatible paternal allele at Om (164 offspring inherited incompatible alleles, 365 inherited compatible alleles; Table 1). In these two crosses combined, 31 ± 2% of the progeny inherited the incompatible paternal allele compared to 34.5 ± 3% in control cross 3 (Table 1). In other words, the level of distortion was greater in the experimental crosses than in the control cross, which is in complete opposition to expectations in the presence of rescue. Therefore, we conclude that neither the PERC nor the PERA alleles at Rmod1 are able to rescue lethality when transmitted through the paternal germline. It is important to point out that the effect of transmission of a paternal allele at Rmod2 was not tested in our crosses.
DDK syndrome is an early embryonic lethal phenotype that has been used to study interactions between the ooplasm and the maternal and paternal genomes [3, 9, 11]. These interactions are essential for early embryonic development in mammals, a stage that is supported by ooplasmic factors. Previous studies have shown that variation in the genetic composition of oocytes can have dramatic effects on early development [45–51]. In the present study, we demonstrate the existence of modifiers that act during early development to rescue the DDK syndrome lethality and that have a parent-of origin effect. Two previous studies support the existence of rescue modifiers [12, 20]. In 1974, Wakasugi provided the first evidence of dominant rescue modifiers on the NC strain background [12]. His experiments showed that crosses between incompatible NC males and (NC x DDK)F1 females are fully viable. However, the possibility of rescue modifiers in the NC strain was not discussed in that report and further investigations were not pursued. More recently, we have proposed that the DDK strain carries recessive rescue modifiers [20]. However, a subsequent study failed to replicate those results [23].
Of the eleven strains we screened for modifiers, four are M. m. domesticus (PERC, PERA, RBA, and WSB), one is a hybrid between M. m. domesticus and M. m. musculus (SKIVE), one is M. m. molossinus (JF1), one is M. m. castaneus (CAST), and four are classical inbred strains (B6, 129X1, BALB/c, and DBA/2) [32, 37]. Given that M. m. domesticus is the subspecies that has contributed the most to the genomes of classical inbred strains [52] and NC is a classical inbred strain derived from Japanese fancy mice [53], we conclude that four of the nine M. m. domesticus-like strains tested carry modifiers that rescue the lethality in a dominant manner in crosses involving F1 hybrid females. In contrast, none of the three strains derived from other subspecies carry these modifiers. This finding suggests that the rescue allele probably arose within the M. m. domesticus lineage. Our data show that these rescue alleles are widespread within the M. m. domesticus lineage because they are found in strains derived from natural populations of mice from different geographical locations (RBA from Switzerland and PERC and PERA from Peru). However, these alleles have not been fixed in the M. m. domesticus lineage (i.e., WSB does not rescue; Fig. 1). These conclusions expand on an emerging theme in DDK syndrome research, namely, that inbred strains derived from natural populations are valuable tools for testing, and in some cases, rejecting longstanding hypotheses [23, 18].
Our linkage analyses identified two loci that are significantly associated with rescue of the DDK syndrome: Rmod1 on proximal chromosome 13 and Rmod2 on chromosome 7 (Fig. 3). We conclude that while PERA or PERC alleles at Rmod1 are necessary for complete rescue, they are not sufficient for complete rescue in crosses between B6 males and OmDDK/OmB6 G2 females (Fig. 2). This conclusion is based on the fact that the combined average litter size and the range of average litter sizes of OmDDK/OmB6, Rmod1PERC/Rmod1B6 and OmDDK/OmB6, Rmod1PERA/Rmod1B6 G2 females in experimental crosses (mated to B6 males) were smaller than those of control crosses (mated to DDK males, data not shown). The combined average litter size in experimental crosses was 7.2 ± 0.3 and ranged from 0 to 10, while the combined average litter size in control crosses was 10.8 ± 0.3 and ranged from 10.5 to 11.
The results of crosses involving OmDDK/OmDDK F2 females confirm the critical role of the Rmod1 locus in the rescue phenotype. The reproductive performance of OmDDK/OmDDK F2 females was directly correlated to the number of PERA or PERC alleles that a female carried at Rmod1 (range 0–2; Fig. 4b). In fact, homozygous OmDDK/OmDDK, Rmod1PERC/Rmod1PERC and OmDDK/OmDDK, Rmod1PERA/Rmod1PERA F2 females had reproductive performances that were comparable to those of viable control crosses (Fig. 4b). These data suggest that rescue alleles at the Rmod1 locus may have a dosage effect on the extent of rescue from the DDK syndrome.
The presence of a DDK allele at the second modifier locus, Rmod2, also appears to be necessary but not sufficient for complete rescue in the PERC crosses. G2 females with a PERC allele at Rmod2 had an average litter size of 3.9 ± 2.3, and G2 females with a DDK allele at Rmod2 had an average litter size of 6.9 ± 1.9. Rmod2 had no significant effect on litter size in the PERA crosses. The fact that no linkage was detected between rescue and Rmod2 in the PERA crosses (Fig. 3b) may be a reflection of differences in marker density between the two crosses or a consequence of the small sample size. Alternatively, it may be due to the smaller effect of Rmod2 on the rescue phenotype or differences in the combination of alleles present in the four strains involved in our crosses. We tested whether there was an interaction between rescue alleles at Rmod1 and Rmod2. In the PERC crosses, Rmod1 and Rmod2 appeared to act independently of each other and to have additive effects on the rescue phenotype. For example, G2 females with a PERC allele at Rmod1 and a DDK allele at Rmod2 showed the best reproductive performance at 7.8 ± 1.3 (in fact, complete rescue is never observed without this combination of alleles). The worst reproductive performance was observed in females with a DDK allele at Rmod1 and a PERC allele at Rmod2, 3.1 ± 1.8. Finally, G2 females with DDK alleles at Rmod1 and Rmod2 and G2 females with PERC alleles at Rmod1 and Rmod2 had intermediate reproductive performances of 4.6 ± 1.1 and 5.1 ± 2.4, respectively.
In conclusion, we have mapped two major modifiers that explain to a significant extent the observed variation. Rmod1 accounts for 43% and 73% of the variance in average litter size observed among G2 females in PERC and PERA crosses, respectively. Rmod2 alone accounts for 35% of the variance in average litter size in the PERC crosses, while Rmod1 and Rmod2 combined in an additive model account for 60% of the variance in PERC crosses. The remaining variation may be due to other QTLs with smaller additive or epistatic effects. Given the small sample sizes in our mapping experiments, it is likely that such QTLs are present but remain undetected. It is also possible that the remaining phenotypic variance is due to genetic and/or environmental effects on reproductive performance that are unrelated to the DDK syndrome.
There is indirect but compelling evidence that the gene that encodes the maternal factor undergoes allelic exclusion in the oocyte [19–23]. Based on the results of our crosses between OmDDK/OmDDK F2 females and B6 males, we conclude that rescue by PERC or PERA alleles at Rmod1 does not require allelic exclusion at the gene encoding the maternal factor (Fig. 4). We have also shown that rescue of lethality requires the presence of PERC or PERA alleles at Rmod1 in the maternal germline. In contrast, transmission through the paternal germline has no discernible effect. This extreme gender dichotomy must reflect differences in the expression patterns of maternally and paternally inherited alleles at modifier loci, including Rmod1. The interaction between the maternal DDK factor and the incompatible paternal allele has been shown to affect embryo development as early as the 2-cell stage [2, 4]. If rescue requires expression of the PERC and PERA alleles at Rmod1 prior to this lethal interaction, then a paternally inherited allele may be expressed too late to be effective. Likewise, if Rmod1 is a gene that is only expressed in the female germline or is subject to genomic imprinting, a paternally inherited allele would be unable to rescue. It remains to be determined whether PERC or PERA alleles at Rmod1 rescue lethality by inhibiting the incompatible interaction between the maternal DDK factor and the incompatible paternal gene or by providing a factor necessary for normal embryonic development that is absent in incompatible crosses.
ACKNOWLEDGMENTS
We are grateful to Terry Magnuson for providing us with the JF1/Ms inbred mice, and we thank Timothy A. Bell, Andrew Bolton, Kyle Gaulton, and Clemencio Salvador for providing technical assistance.
FOOTNOTES
1Supported in part by grants from the National Science Foundation (MCB-0133526 to F.P-M.V.) and the National Institutes of Health (U01HD43430 to D.R.B.). 
Correspondence: 2Fernando Pardo-Manuel de Villena, Department of Genetics, CB#7264, 103 Mason Farm Road, University of North Carolina-Chapel Hill, Chapel Hill, NC 27599-7264. FAX: 919 966 3630; e-mail: Fernando{at}med.unc.edu
Received: 23 August 2006.
First decision: 27 September 2006.
Accepted: 12 October 2006.
REFERENCES
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