The Collaborative Cross as a Resource for Modeling Human Disease: CC011/Unc, a New Mouse Model for Spontaneous Colitis

Inflammatory bowel disease (IBD) is an immune-mediated condition driven by improper responses to intestinal microflora in the context of environmental and genetic background. GWAS in humans have identified many loci associated with IBD, but animal models are valuable for dissecting the underlying molecular mechanisms, characterizing environmental and genetic contributions and developing treatments. Mouse models rely on interventions such as chemical treatment or introduction of an infectious agent to induce disease. Here, we describe a new model for IBD in which the disease develops spontaneously in 20-week-old mice in the absence of known murine pathogens. The model is part of the Collaborative Cross and came to our attention due to a high incidence of rectal prolapse in an incompletely inbred line. Necropsies revealed a profound proliferative colitis with variable degrees of ulceration and vasculitis, splenomegaly and enlarged mesenteric lymph nodes with no discernible anomalies of other organ systems. Phenotypic characterization of the CC011/Unc mice with homozygosity ranging from 94.1 to 99.8% suggested that the trait was fixed and acted recessively in crosses to the colitis-resistant C57BL/6J inbred strain. Using a QTL approach, we identified four loci, Ccc1, Ccc2,Ccc3 and Ccc4 on chromosomes 12, 14, 1 and 8 that collectively explain 27.7% of the phenotypic variation. Surprisingly, we also found that minute levels of residual heterozygosity in CC011/Unc have significant impact on the phenotype. This work demonstrates the utility of the CC as a source of models of human disease that arises through new combinations of alleles at susceptibility loci.Electronic supplementary materialThe online version of this article (doi:10.1007/s00335-013-9499-2) contains supplementary material, which is available to authorized users

A Diallel of the Mouse Collaborative Cross Founders Reveals Strong Strain-Specific Maternal Effects on Litter Size

Reproductive success in the eight founder strains of the Collaborative Cross (CC) was measured using a diallel-mating scheme. Over a 48-month period we generated 4,448 litters, and provided 24,782 weaned pups for use in 16 different published experiments. We identified factors that affect the average litter size in a cross by estimating the overall contribution of parent-of-origin, heterosis, inbred, and epistatic effects using a Bayesian zero-truncated overdispersed Poisson mixed model. The phenotypic variance of litter size has a substantial contribution (82%) from unexplained and environmental sources, but no detectable effect of seasonality. Most of the explained variance was due to additive effects (9.2%) and parental sex (maternal vs. paternal strain; 5.8%), with epistasis accounting for 3.4%. Within the parental effects, the effect of the dam’s strain explained more than the sire’s strain (13.2% vs. 1.8%), and the dam’s strain effects account for 74.2% of total variation explained. Dams from strains C57BL/6J and NOD/ShiLtJ increased the expected litter size by a mean of 1.66 and 1.79 pups, whereas dams from strains WSB/EiJ, PWK/PhJ, and CAST/EiJ reduced expected litter size by a mean of 1.51, 0.81, and 0.90 pups. Finally, there was no strong evidence for strain-specific effects on sex ratio distortion. Overall, these results demonstrate that strains vary substantially in their reproductive ability depending on their genetic background, and that litter size is largely determined by dam’s strain rather than sire’s strain effects, as expected. This analysis adds to our understanding of factors that influence litter size in mammals, and also helps to explain breeding successes and failures in the extinct lines and surviving CC strains

A Multi-Megabase Copy Number Gain Causes Maternal Transmission Ratio Distortion on Mouse Chromosome 2

<div><p>Significant departures from expected Mendelian inheritance ratios (transmission ratio distortion, TRD) are frequently observed in both experimental crosses and natural populations. TRD on mouse Chromosome (Chr) 2 has been reported in multiple experimental crosses, including the Collaborative Cross (CC). Among the eight CC founder inbred strains, we found that Chr 2 TRD was exclusive to females that were heterozygous for the WSB/EiJ allele within a 9.3 Mb region (Chr 2 76.9 – 86.2 Mb). A copy number gain of a 127 kb-long DNA segment (designated as responder to drive, <i>R2d</i>) emerged as the strongest candidate for the causative allele. We mapped <i>R2d</i> sequences to two loci within the candidate interval. <i>R2d1</i> is located near the proximal boundary, and contains a single copy of <i>R2d</i> in all strains tested. <i>R2d2</i> maps to a 900 kb interval, and the number of <i>R2d</i> copies varies from zero in classical strains (including the mouse reference genome) to more than 30 in wild-derived strains. Using real-time PCR assays for the copy number, we identified a mutation (<i>R2d2^WSBdel1</i>) that eliminates the majority of the <i>R2d2^WSB</i> copies without apparent alterations of the surrounding WSB/EiJ haplotype. In a three-generation pedigree segregating for <i>R2d2^WSBdel1</i>, the mutation is transmitted to the progeny and Mendelian segregation is restored in females heterozygous for <i>R2d2^WSBdel1</i>, thus providing direct evidence that the copy number gain is causal for maternal TRD. We found that transmission ratios in <i>R2d2^WSB</i> heterozygous females vary between Mendelian segregation and complete distortion depending on the genetic background, and that TRD is under genetic control of unlinked distorter loci. Although the <i>R2d2^WSB</i> transmission ratio was inversely correlated with average litter size, several independent lines of evidence support the contention that female meiotic drive is the cause of the distortion. We discuss the implications and potential applications of this novel meiotic drive system.</p></div

Transmission ratios in the progeny of <i>R2d2</i>^{<i>WSB/notWSB</i>} heterozygous F1 hybrid sires and dams.

<p>Transmission ratios in the progeny of <i>R2d2</i>^{<i>WSB/notWSB</i>} heterozygous F1 hybrid sires and dams.</p

<i>R2d</i> maps to a 9.3 Mb candidate interval.

<p>CC and DO mice were crossed to generate G1 dams, which were then crossed to FVB/NJ sires to determine the TR in their progeny. Each G1 dam carries a chromosome that is recombinant for the WSB/EiJ haplotype (shown under the heading <i>cis</i>) and a non-WSB/EiJ chromosome (the haplotype on the homologue is shown at far right under the heading <i>trans</i>). Dams with the same diplotype in the central region of Chr 2 were grouped together to define ten unique diplotypes. The aggregate number of WSB/EiJ and non-WSB/EiJ alleles transmitted by dams of each diplotype are shown for dams A) with TRD and B) without TRD. Significance of TR deviation from Mendelian expectation of 0.5 was computed using one-sided binomial exact test (<i>p</i>-value). The contribution from the eight founders of the CC and DO are shown in different colors. Thick purple bars indicate the extent of WSB/EiJ contributions, and thin bars indicate the extent of contributions from all other strains. The black box indicates the boundaries of the <i>R2d</i> candidate interval as determined by the region that is WSB/EiJ in all dams with TRD.</p

TRD at <i>R2d2</i> requires the combined action of meiotic drive and embryonic lethality.

<p>Relationship between maternal TR and average litter size (top panels) and average number of offspring inheriting alternative alleles at R2d2 (bottom panels) for A) DO G13 dams, B) DO G16 dams, C) G3 dams in the D0-G13–44 pedigree and D) (NZO/HILtJxWSB/EiJ)F1 dams. Top panels: gray circles are dams without TRD (A, B, D) or having the low-copy allele (C); blue circles are dams with TRD (A, B, D) or having the high-copy allele (C). For each point, bars show standard error for TR (horizontal) and average litter size (vertical). Dotted lines show mean litter sizes for each type of female. Red line shows a linear fit to TR and average litter size. Bottom panels: left and right pairs of boxplots show average number of offspring per litter in females without and with TRD (A, B, D) or having the low- and high-copy allele (C) that inherit a WSB/EiJ (purple) or non-WSB/EiJ (gray) allele. Females with a mutant <i>R2d2</i>^<i>WSB</i> allele are excluded. Note that there are significantly more WSB/EiJ offspring of dams with TRD in F1 hybrid dams than in DO without TRD.</p

Mapping the causal locus for maternal TRD in a family segregating for a copy-number variant at <i>R2d2</i>.

<p>A) Pedigree of DO-G13–44xCC cross. Female DO-G13–44, mother of the G3 dams phenotyped for TR, is segregating for a copy-number variant at <i>R2d2</i>. G3 dams inheriting the maternal WSB/EiJ haplotype associated with the high-copy allele (<i>R2d2</i>^<i>WSB</i>) are colored black; those inheriting the WSB/EiJ haplotype associated with the low-copy allele (<i>R2d2</i>^{<i>WSBdel1</i>}) are colored red. Genotypes at marker chr2:85.65Mbp is denoted -/- (homozygous non-WSB), +/- (heterozygous WSB/EiJ) or +/+ (homozygous WSB/EiJ). ΔC_t, normalized cycle threshold by TaqMan qPCR assay; TR, transmission ratio, denoted as count of progeny inheriting a WSB/EiJ allele: count of progeny not inheriting a WSB allele; the paternal haplotype at chr2:83.6 Mb as determined by genotypes from the MegaMUGA array using the standard CC abbreviations is shown, A = A/J, E = NZO/HILtJ, ? = haplotype unknown. B) Distribution of ΔC_t values among 27 G3 dams. Points are colored as in panel A. C) TR among 27 G3 dams partitioned according to copy-number (CN) haplotype at <i>R2d2</i>. Points are colored as in panel A. D) QTL scan for TRD, treated as a binary phenotype, in 25 G3 dams genotyped with MegaMUGA. Only the maternal signal from Chr 2 is shown. Grey dashed line indicates threshold for significance at <i>α</i> = 0.01 obtained by unrestricted permutation. Candidate interval for <i>R2d</i> is shaded yellow. E) Empirical cumulative distribution of both maternal and paternal LOD scores genome-wide, with <i>α</i> = 0.01 significance threshold indicated by grey dashed line.</p

Linkage mapping localizes <i>R2d2</i> to a 900 kb region in Chr 2.

<p>A) Distribution of sum-intensity for the 34 probes in <i>R2d</i> present on the Mouse Diversity Array (MDA) for mice with a non-recombinant CAST/EiJ haplotype (green), a non-recombinant WSB/EiJ haplotype (purple) and non-CAST/EiJ/non-WSB/EiJ haplotypes (grey) is shown at the top of the panel. The sum intensity and recombinant haplotypes in six mice defining the boundaries of copy-number gain in the CAST/EiJ strain are shown below. B) Distribution of sum-intensity across three probes in <i>R2d</i> on the MegaMUGA array for mice with non-recombinant CAST/EiJ haplotype (green), a non-recombinant WSB/EiJ haplotype (purple) and non-CAST/EiJ/non-WSB/EiJ haplotypes (grey) is shown at the top of the panel. The sum intensity and recombinant haplotypes in six mice defining the boundaries of copy-number gain in the WSB/EiJ strain are shown below. C) QTL scan for the <i>R2d2</i> copy number gain using MDA sum-intensity as the phenotype in 330 CC G2:F₁ mice. D) QTL scan for the <i>R2d2</i> copy number gain using MegaMUGA sum-intensity as the phenotype in 96 (FVB/NJx(WSB/EiJxPWK/PhJ)F1)G2 offspring. E) Superposition of LOD curves from panels (C) and (D) on chromosome 2. The <i>R2d2</i> candidate interval is shaded in yellow.</p